Doctor of Philosophy (Md) School of Pharmacy

Development, safety and efficacy evaluation of actinic damage retarding nano-pharmaceutical treatments in oculocutaneous albinism.

J. M Chifamba (R931614G) B. App Chem (Hons), M Phil (Upgraded to D.Phil.), Dip QA, Dip SPC, Dip Pkg

Thesis submitted in fulfilment of the requirements for the degree of DOCTOR OF PHILOSOPHY (MD)

Main Supervisor: Prof C. C Maponga 1 Associate Supervisor: Dr A Dube (Nano-technologist) 2 Associate Supervisor: Dr D. I Mutangadura (Specialist dermatologist) 3, 4

1School of Pharmacy, CHS, University of Zimbabwe, Zimbabwe 2School of Pharmacy, University of the Western Cape, South Africa 3Fellow of the American academy of dermatology 4Fellow of the International academy of dermatology

SCHOOL OF PHARMACY COLLEGE OF HEALTH SCIENCES

This work is dedicated to the everlasting memory of my dearly departed father, his scholarship, mentorship and principles shall always be my beacon.

Esau Jeniel Mapundu Chifamba (15/03/1934-08/06/2015)

ACKNOWLEDGEMENTS

“Art is I, science is we”, Claude Bernard 1813-1878

Any work of this size and scope inevitably draws on the expertise and direction from others. I therefore wish to proffer my utmost gratitude to the following individuals and institutions for their infallible inputs and support.

My profound appreciation goes to Prof C C Maponga and Dr A. Dube for crafting, steering and nurturing my interests and research pursuits in nano-pharmaceuticals. Indeed, I have found this discipline to be most intellectually fulfilling. I wish to further acknowledge the mentorship, validation and insight into dermato-pharmacokinetics from the specialist dermatologist Dr D I Mutangadura. Many thanks go to Prof M Gundidza for the contacts, guidance and refereeing in research methodologies, scientific writing and analytical work. I wish to state my Indebtedness to Paidamoyo Kurauvone and the albino welfare organisations in Zimbabwe for embarking on this journey with me and giving me a personal insight into living positively with Albinism.

I would also like to acknowledge the related work on skin permeation, spectrophotometry and dermato-pharmacokinetics tape stripping studies done by A O Gamer, Tokumura F et al, Karin Sperling-Vietmeier, and J Lademann et al, which served as efficacy assaying and study guides. I wish to appreciate the assistance and facility from the University of Zimbabwe, School of pharmacy and the HIT, Pharmaceutical technology department. I wish to acknowledge my indebtedness to the sponsorship and financial support by HIT, the University of Zimbabwe research grants and the ICF grant from the Government of Zimbabwe. For instrumental analysis and skin diffusion studies, many thanks go to the Unilever RIC, Croda International ltd Research Laboratories, Mining Research Institute, TRB and Silchem pharmaceuticals for equipment, materials and analytical techniques.

Your contributions to this work can never be justly accentuated enough.

J M Chifamba

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PUBLICATIONS AND PRESENTATIONS ON RESEARCH-WORK

Selected publications from this work in refereed journals

1. Chifamba J, Dube A and Maponga CC: Ex-vivo penetration of nanometric ZnO and TiO2 across actinically damaged porcine skin: Development of an albinistic skin protection treatment. Int J Pharm Sci Res 2015; 6(6): 2339-48.doi: 10.13040/IJPSR.0975-8232.6 (6).2339-48. 2. Chifamba J, Dube A and Maponga CC: Investigation of In-vivo penetration and distribution of nanometric TiO2 in tropical albinistic skin by sequential adhesive tapes stripping. Int J Pharm Sci Res 2015; 6(10): 4181-89.doi: 10.13040/IJPSR.0975-8232.6(10).4181-89

Selected media coverage on this research-work

1. ZTV main news, feature on albinistic treatment development: 15 April 2014: 2. The Herald newspaper, feature article on albinistic treatments: August 2014: 3. Chronicle newspaper: feature article on albinistic treatments: 20 April 2014: 4. Africa on line news, feature article on albinistic treatments: April 2014 5. Indian online news, feature on albinistic treatments: April 2014 6. ZTV main news, feature on Nanomedicine: 07 August 2014: 7. Sunday Mail Newspaper, feature on herbal medicines: 14 September 2014.

Selected conference and symposia presentations on research-work

1. Zimbabwe Pharmacists council conference, feature presentation titled: Nanotechnology and herbal based skin treatments: Victoria Falls, March 2015 2. NUST multidisciplinary research conference, paper presentation titled: Emerging technologies impact on dermatological treatments: Victoria Falls November 2014 3. IPSF, feature presentation titled: Emerging technologies in the pharmaceutical industry and novel product development: Victoria Falls June 2014 4. ZPSA annual conference, feature presentation titled: nanotechnology and pharmaceutical product development: ZIPAM march 2014 5. RIE SET, paper presentation and research exhibition titled: Breakthrough in Albinistic treatments: Harare September 2014 6. RIE-SET, paper presentation and research work exhibition titled: Nanotechnology and Indigenous Knowledge systems in skin care week: Harare September 2013 7. Parliamentary portfolio committee on Health presentation titled: Breakthrough in Albinistic treatments: Harare April 2014

8. Parliamentary portfolio committee on Higher and tertiary education, presentation titled: Nanotechnology implementation for socio-economic transformation in Zimbabwe on research-work: Harare August 2014

ABSTRACT

Introduction: There are at least 17 000 Persons living with albinism (PLWA) in Zimbabwe. Oculocutaneous albinism (OCA) is a congenital amelanistic pigmentation disorder that affects all known vertebrates and has no known cure. Melanogenesis is the body’s primary protection from actinic damage, which summarizes all the acute and chronic solar induced adverse dermatological conditions. This impairment therefore makes PLWA highly susceptible to all forms of this damage. Problem statement: Commercial products for actinic damage in PLWA are not readily available. Chemical sunscreens used by PLWA are ineffective and do not treat symptoms of actinic damage. The possible use of promising broad spectrum physical sunscreens in albinistic treatments is hindered by their opaque and un-aesthetic nature. Research hypothesis: A treatment based on nanometric TiO2 and ZnO incorporating the active extracts of A. excelsa, T. emetica and M. flabellifolia will be aesthetic, efficacious and safe in retarding and alleviating all forms of actinic damage in PLWA. Research aims: To develop albinistic actinic damage treatments, using nano TiO2 and ZnO as sun-blocks and incorporating selected herbs. The dermato-pharmacokinetics, stability, efficacy, toxicity and aesthetics of the resultant formulation on albinistic skin types were also investigated in this study. Materials and methods: Emulsion formulation was done according to FDA-CFSAN, COLIPA, and OECD mandated technical guidelines and testing methods. Formulation skin sensitivity were evaluated through Draize ocular and skin sensitivity tests as well as in-vivo patch tests guided by OECD 428/404 technical guidelines and opinion SCCNFP 0750/03. Percutaneous absorption and albinistic skin dermato-pharmacokinetics were evaluated ex-vivo using Franz diffusion tests and sequential adhesive tape stripping respectively according to OECD guidelines 428 and SCCNFP opinions as well as related work done by A O Gamer and Diembeck et al as guides. Analysis for Ti and Zn were done by ICP-AES and Flame AAS respectively. Efficacy and SPF testing was done as per FDA–CFSAN, Colipa and OECD M389/EN mandated test methods. Principal Results: SPF 16, aesthetic and stable emulsions were formulated. Negligible irritation indices for the treatment were recorded for Draize and human patch testing. No percutaneous absorption was observed for ex-vivo diffusion tests and sequential tape stripping tests. Different skin reservoir properties were observed at different skin sites Conclusions: The studies demonstrate, direct evidence that neither Zn nor Ti can penetrate actinic damaged skin regardless of anatomical site and that albinistic dermato-pharmacokinetics are depended on anatomical region and extent of UVR exposure. The high extraction yields and the phyto-constituents of the selected herbs show a correlation with the traditional uses of the plants in traditional medicine. All sensitivity tests showed negligible irritation potential. Based on the foregoing, it is concluded that, incorporation of nanometric TiO2, ZnO and herbs in treatments to retard actinic damage in PLWA is feasible, aesthetic, efficacious, and commercializable and does not pose any health risk.

CONTENTS

INTRODUCTION ...... 1 1.1 HUMAN ALBINISM ...... 2 1.1.1 UVR and sunscreens ...... 4 1.1.2 Natural protection against UVR ...... 7 1.1.3 Sunscreens and Sun filters for UVR ...... 8 1.1.4 Physical sunscreens ...... 9 1.1.5 Geographical UVR variations impact on cosmeceuticals ...... 10 1.1.6 Nano-cosmeceuticals and nanomaterials ...... 14 1.1.7 Nanometric sunscreens ...... 14 1.1.8 Botanical active ingredients ...... 16 1.2 PROBLEM STATEMENT ...... 17 1.3 RESEARCH HYPOTHESIS ...... 18 1.4 RESEARCH AIMS ...... 18 1.4.1 Specific objectives ...... 19 2. LITERATURE REVIEW ...... 20 2.1 THE HUMAN SPECIES ...... 21 2.1.1 Genetic and anatomical diversity of cosmeceutic concern ...... 22 2.1.2 Geographic anatomic variation of cosmeceutical concern...... 22 2.1.3 Racial diversity of cosmeceutical concern ...... 23 2.1.4 The Human Skin ...... 24 2.1.5 Dermis ...... 30 2.1.6 Hypodermis ...... 31 2.1.7 Human pigmentation ...... 31 2.2 GENETIC PIGMENTATION DISORDERS ...... 34 2.2.1 Amelanistic Albinism ...... 35 2.2.2 Ocular albinism (OA) ...... 37 vii

2.2.3 Oculocutaneous albinism (OCA) ...... 39 2.2.4 Forms of OCA ...... 39 2.2.5 Albinism genetics ...... 41 2.2.6 Diagnosing and treatment of Albinism ...... 42 2.3 DIAGNOSIS OF SKIN CONDITIONS ASSOCIATED WITH ALBINISM ...... 43 2.3.1 Solar Induced complications in albinism ...... 45 2.3.2 Principles of Dermatological treatments ...... 46 2.4 PERSONAL CARE PRODUCTS ...... 47 2.4.1 Cosmetics ...... 47 2.4.2 Drugs ...... 48 2.4.3 Cosmeceuticals ...... 49 2.4.4 Emerging technologies in Cosmeceuticals ...... 50 2.4.5 Nanotechnology ...... 51 2.4.6 Nano-medicine ...... 52 2.4.7 Nano-Cosmeceuticals ...... 52

2.4.8 Nano scale Titanium dioxide (TiO2) (CAS number 13463-67-7) ...... 54 2.4.9 Nano scale zinc oxide (ZnO) (CAS number 1314-13-2) ...... 55 2.4.10 Botanicals in cosmeceuticals...... 65 2.4.11 Plant Oils ...... 66 2.4.12 Essential oils ...... 67 2.4.13 Fixed oils ...... 68 2.4.14 Trichilia emetica ...... 68 2.4.15 Aloe excelsa ...... 72 2.4.16 Myrothamnus Flabellifolia ...... 74 2.5 FORMULATION OF DERMATOLOGICAL AND COSMECEUTICAL PREPARATIONS ...... 75 2.5.1 Regulations that affect cosmeceutical formulation development ...... 76 2.5.2 Formulation of emulsion sunscreen dosage forms...... 77 2.5.3 Emulsion technology ...... 78 2.5.4 Emulsifiers used in Cosmeceuticals...... 79 2.5.5 Emollients used in cosmeceuticals...... 80 2.5.6 Functional ingredients ...... 83 viii

2.5.7 Aesthetic ingredients ...... 85 2.6 EFFICACY AND STABILITY TESTING OF SUNSCREEN EMULSIONS ...... 87 2.6.1 Sunburn Protection Factor (S.P.F) testing ...... 88 2.6.2 Franz diffusion studies ...... 89 2.6.3 Franz diffusion experimental design and equipment ...... 90 2.6.4 Tape stripping technique for dermato-pharmacokinetics ...... 91 2.6.5 Draize Skin sensitivity and ocular testing ...... 92 2.6.6 Anti Draize tests arguments ...... 92 2.6.7 Pro Draize tests proponents ...... 93 2.6.8 Stability testing ...... 93 2.6.9 Stability tests types ...... 94 2.6.10 Physico-chemical stability tests ...... 94 2.6.11 Instrumental and Classical product Analysis ...... 96 3. MATERIALS AND METHODS ...... 101 3.1 RESEARCH FACILITIES, ETHICS AND ANIMAL TESTING APPROVALS ...... 102 3.2 NANO-MATERIALS TOXICITY AND SAFETY INVESTIGATIONS FOR POTENTIAL USE ON ALBINISTIC SKIN 102

3.2.1 Ex-Vivo safety investigation of nanometric TiO2 and ZnO permeation through porcine skin, using vertical, static, Franz diffusion cells ...... 102 3.2.2 In-vivo Ti dermato-pharmacokinetics across albinistic skin investigation by sequential adhesive tape stripping ...... 110 3.3 BOTANICAL ACTIVES PREPARATION FOR THE ALBINISTIC TREATMENT DEVELOPMENT ...... 118 3.3.1 Fixed oil extraction from Trichilia emetica (Banket mahogany) seed kernels ...... 118 3.3.2 Extraction of Aloe excelsa gel matrix ...... 120 3.3.3 Extraction of Myrothamnus flabellifolia by steam distillation ...... 125 3.4 ALBINISTIC ACTINIC DAMAGE RETARDING TREATMENT EMULSION DEVELOPMENT ...... 127 3.4.1 Principles and guidelines ...... 127 3.4.2 Formulation protocol ...... 129 3.4.3 Treatment physico-chemical and stability tests ...... 131 3.5 PRODUCT SENSITIVITY TESTING ...... 135 3.5.1 In-vivo sensitivity testing: Draize skin sensitivity test ...... 135

1.1.1 Product ocular sensitivity test: Low volume eye test (LVET) ...... 138 3.5.2 In-vivo Human sensitivity tests: Patch testing ...... 141 3.5.3 Ti and Zn assay in finished product ...... 144 3.5.4 In-Vitro SPF determination of final albinistic treatment ...... 145 4. RESULTS...... 146 4.1 EXPERIMENTAL STUDIES RESULTS ...... 147

4.1.1 Ex-Vivo safety investigation of nanometric TiO2 and ZnO permeation through porcine skin, using vertical, static, Franz diffusion cells ...... 147

4.1.2 In-vivo TiO2 dermato-pharmacokinetics investigations across albinistic skin by sequential adhesive tape stripping ...... 154 4.2 PRODUCT SENSITIVITY TESTING ...... 158 4.2.1 In-vivo sensitivity testing: Draize skin sensitivity test ...... 158 4.2.2 LVET ocular evaluations ...... 159 4.2.3 In-vivo Human sensitivity tests: Patch testing ...... 159 4.2.4 Formulation development of the albinistic treatment ...... 161 4.2.5 Extraction of Aloe excelsa gel matrix ...... 162 4.2.6 Extraction of Myrothamnus flabellifolia by steam distillation ...... 166 4.3 ALBINISTIC ACTINIC DAMAGE RETARDING TREATMENT EMULSION DEVELOPMENT ...... 167 4.3.1 Treatment formulation protocol ...... 167 4.3.2 In-Vitro SPF determination of SG3 albinistic treatment ...... 172 5. DISCUSSION ...... 175 5.1 MATERIAL SAFETY INVESTIGATION ...... 176

5.1.1 Ex-Vivo safety investigation of nanometric TiO2 and ZnO permeation through porcine skin, using vertical, static, Franz diffusion cells ...... 176

5.1.2 In-vivo TiO2 dermato-pharmacokinetics investigations across albinistic skin by sequential adhesive tape stripping ...... 180 5.2 PRODUCT SENSITIVITY TESTING ...... 184 5.2.1 Draize skin sensitivity ...... 184 5.2.2 Low volume eye test (LVET) irritation tests ...... 186 5.2.3 In-vivo Human sensitivity tests: Patch testing ...... 187 5.3 FORMULATION DEVELOPMENT OF THE ALBINISTIC TREATMENT ...... 189 5.3.1 Extraction of Trichilia emetica fixed oil ...... 189 x

5.3.2 Extraction of Myrothamnus flabellifolia essential oil ...... 190 5.3.3 Extraction of Aloe excelsa gel ...... 191 5.3.4 Formulation protocol ...... 192 5.3.5 Emulsion stability ...... 195 5.3.6 Treatment SPF ...... 196 5.3.7 Treatment functionality and aesthetics ...... 201 5.3.8 Active ingredient assay ...... 202 5.3.9 Treatment pH, functionality and aesthetics ...... 202 5.3.10 Nanocosmeceuticals ...... 203 5.3.11 Perceived health and safety risk of nanomedicine ...... 204 5.3.12 Regulation of nanotechnology ...... 205 5.3.13 Study limitations and recommendations for further studies ...... 206 6. CONCLUSIONS ...... 208 6.1 EXPERIMENTAL STUDY CONCLUSIONS ...... 209 6.1.1 Nanometric Zn and Ti safety risk ...... 209 6.1.2 Albinistic anatomical skin profiles and Ti dermato-pharmacokinetics ...... 209 6.1.3 Skin sensitivity tests ...... 210 6.1.4 Plant extracts ...... 210 6.1.5 Panel evaluations and product costing ...... 211 6.2 OVERALL CONCLUSION ...... 212 6.3 SIGNIFICANCE OF THE STUDY ...... 212 7. REFERENCES ...... 213 8. APPENDICES ...... 225

LIST OF FIGURES

Figure 0-1; Pre-pubescent female living with albinism (source, Zimbabwe Albino Society Picture Gallery: 2014) ...... 2 Figure 0-2; Radiation spectrum highlighting Ultraviolet radiation spectrum section (Source UVR media- resources-ordp-.com) ...... 5 Figure 0-3: Erythermal damage experienced by an albinistic research participant between 9:30 am and 11am on 09/03/14 in Harare, Greendale ...... 12 Figure 0-4 : Onset of UVB Damage on an overcast day in Raffingora Zimbabwe on 16/01/2013 ...... 13 Figure 0-5: Actinic damage experienced on an overcast day after applying recommended chemical sunscreens in Raffingora, Zimbabwe on 16/01/13 ...... 13 Figure 2-1: The three basic racial groups (A) Mongoloid (Liu Yi Fei) (B) African (Lupita Nyong’o) (C) Caucasian,(Cate Blanchet) (Source: http://www.vogue.com/13257111) ...... 24 Figure 2-2: Cross section of the skin (source https://www.skin-conditions.knoji.com)...... 26 Figure 2-3: Cross section of the stratum corneum (Adapted from Heather Baron MD Homepage 2007) 27 Figure 2-4: Eumelanin (left) and pheomelanin (right). (Source: Wakamatsu K, Ito S, (2003)) ...... 33 Figure 2-5 : General Hypo-Pigmentation disorders affecting people (Source: Walden: 2015) ...... 35 Figure 2-6: African Albinistic females (Source: https://www.albinos in Zimbabwe: 2015) ...... 35 Figure 2-7: A normal skin type boy with OA (Source, Zimbabwe Albino Society photo gallery: 2014) ...... 37 Figure 2-8: A young female adult, with OCA albinism and corrected eye sight. (Source: research participant picture taken by researcher) ...... 39 Figure 2-9: Young male displaying ACA 1a symptoms (Source: Zimbabwe Albino society photo gallery: 2014) ...... 40 Figure 2-10: Female displaying OCA 2 symptoms (Source: Zimbabwe Albino Society Photo Gallery: 2014) ...... 41 Figure 2-11: Albinistic male with neoplasms: In Albinism, cutaneous conditions often occur with complications (Source: https://theafrikamarket.com) ...... 43 Figure 2-12: The diﬀerent light scattering of bulk and nano-sized ZnO clusters (Adapted from (Filipponi L, Sutherland D: 2013)...... 62 Figure 2-13: Illustration of the band model for semiconductors like Zn and Ti ...... 64 Figure 2-14: Artificially propagated Trichilia plant and the leaves and fruits of the plant (Pictures taken by author February 2014) ...... 69 Figure 2-15: Trichilia emertica seeds and the trichlin A chemical structure (source: Pooley E :1993) ...... 72 Figure 2-16: The author illustrating the unique height of naturally growing Aloe excelsa at Khami ruins: 2014 ...... 72 Figure 2-17: Picture showing the Inflorescence and short curved aloe excelsa raceme (picture by author: 2014) ...... 73

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Figure 2-18: Myrothamnus Flabellifolia in (left) the hydrated state and (right)) the dehydrated state ( pictures by author: 2014) ...... 74 Figure 3-1: Selected anatomical sites for sequential tape stripping: (a) the inner forearm, (b) outer forearm of the same arm and (c) the forehead of an albinistic volunteer showing the anatomical site based variations in actinic damage on the same individual ...... 115 Figure 3-2: Adhesive tape stripping methodology on an albinistic forearm: (a) demarcation of the selected site with an adhesive tape template (b) application of test material on site (c) spreading the cream (d) removal of surface formulation after 15 minutes with cotton swab (e) pressing of adhesive tape over selected site with glass rod and a paper shield (f) removal of the adhesive tape using tongs 117 Figure 3-3: Belvedere area where Trichilia emetica was collected (adapted from www.geohack.com) . 119 Figure 3-4: Lake Mutirikwi and Great Zimbabwe area (adapted from www.geohack.com) ...... 122 Figure 3-5: Extraction of M. Flabellifolia using Clavenger type apparatus in (UZ School of Pharmacy laboratory) ...... 127 Figure 4-1: Porcine skin 1 before (a) and after (b) simulated actinic damage ...... 149 Figure 4-2: Porcine skin 2 before (a) and after (b) simulated actinic damage ...... 149 Figure 4-3: Porcine skin 3 before (a) and after (b) simulated actinic damage ...... 149 Figure 4-4: Total Ti recoveries and distribution in dermatomes by ICP-AES ...... 152 Figure 4-5: Percentage nanometric and normal Ti recovered from epidermal tissue of 12 porcine dermatomes by ICP-AES...... 152 Figure 4-6: Total Percentage Ti recovered from 12 porcine dermatomes by ICP-AES analysis and Zn recoveries from 12 porcine dermatomes by Flame AAS analysis...... 153 Figure 4-7: Percentage Ti recoveries from all sites and skin profiles following application of 3.08% Ti cream ...... 156 Figure 4-8: Average percentage Ti recoveries from all three sites from all participants according to number of tape strips.\ ...... 157 Figure 4-9 Instrument MPF scans for Aloe excelsa cream ...... 165 Figure 4-10: Excel spread sheet close up of instrument MPF scans for aloe excelsa cream ...... 166 Figure 4-11: Application of macromolecular cream (L) and the nanometric cream (R) on a research participant ...... 168 Figure 4-12: SG3 lotion in its final packaging as compared to Nivea SPF 30 lotion ...... 171 Figure 4-13: Optometrics 290S instrument scans for the SG3 actinic damage treatment ...... 173 Figure 4-14: Amplified excel presentation of the actinic damage treatment MPF scans ...... 173

LIST OF TABLES

Table 2-1: Scientific classification of modern human beings (adapted from White et al: 2003) ...... 21

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Table 2-3: Product category designations of sunscreens ...... 89 Table 3-1: Reagents and starting materials for porcine permeation studies by Ti and Zn nanoparticles...... 104 Table 3-2: Investigation Materials for patch testing ...... 114 Table 3-3: In-vitro SPF testing starting materials ...... 122 Table 3-4: Actinic damage treatment Starting Materials ...... 128 Table 3-5: Draize irritation classification protocol ...... 136 Table 3-6: Draize Irritation response categories ...... 137 Table 3-7: Scale of weighted scores for grading the severity of ocular lesions (Draize et al 1944) ...... 140 Table 3-8: Treatment panel test score sheet ...... 143

Table 4-1: Optimized O/W emulsion containing nanometric TiO2 and ZnO ...... 148 Table 4-2: Nanometric Ti recovery from damaged and non-damaged porcine skin ...... 151 Table 4-3: Normal Ti recovery from damaged and non- damaged porcine skin ...... 151 Table 4-4: Average Ti recovery from 12 porcine permeation studies ...... 151 Table 4-5: Nanometric Zn recovery from damaged and non-damaged porcine skin ...... 153 Table 4-6: Normal Zn recovery from damaged and non- damaged porcine skin ...... 153 Table 4-7: Optimized O/W emulsion cream formulation for dermato-pharmacokinetics studies ...... 154 Table 4-8: Characteristics of the actinic damage retarding cream ...... 155 Table 4-9: Ti recoveries from inner volar forearm adhesive strippings in the three OCA human volunteers ...... 156 Table 4-10: Ti recoveries from upper forearm adhesive strippings in the three OCA human volunteers 156 Table 4-11: Ti recoveries from volar forehead adhesive strippings in the three OCA human volunteers 157 Table 4-12: Average Ti recoveries from all three sites from all participants ...... 157 Table 4-13: Irritation, erytherma mad oedema scores for rabbit skin sensitivity ...... 158 Table 4-14: Cutaneous patch reaction after application of treatment on albinistic skin ...... 160 Table 4-15: Cutaneous patch reaction after application of treatment to normal skin types ...... 161 Table 4-16: Physico-chemical analysis of Trichilia emetica ...... 162 Table 4-17: Fatty acid (%) composition of Trichilia emetica ...... 162 Table 4-18: Physico-chemical analysis of Aloe excelsa ...... 163 Table 4-19: Aloe excelsa leaf crude composition ...... 163 Table 4-20: optimized aloe excelsa base cream formulation ...... 164 Table 4-21: Aloe excelsa cream Analytical Report ...... 164 Table 4-22: Physico-chemical analysis of the Myrothamnus flabellifolia extract ...... 167 Table 4-23: Overall stability test results for the final 9 formulations ...... 168 Table 4-24: Costing formulations for the final 3 treatments ...... 170 Table 4-25: Comparative consumer panel test scores for SG3 and a commercial SPF 30 product ...... 171 Table 4-26: General comments from panellists on panel test product use scoring ...... 171 Table 4-27: Active ingredient assay for Ti and Zn by ICP-AES ...... 174

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LIST OF APPENDICES

Appendix 1 Publication 1, from the Journal of Pharmaceutical Science and research

Appendix 2 Publication 2, from the Journal of Pharmaceutical Sciences and research

Appendix 3 Signed consent form for research participants

LIST OF ABBREVIATIONS

A. excelsa Aloe excelsa AAS Atomic absorption spectrum BCOP Bovine Cornea Opacity Test BSE Bovine spongiform encephalitis BTC Behind the counter medications CANSA Cancer South Africa CAS Chemical Abstracts service CFSAN Centre for food safety and applied nutrition CHS Chediak-Higashi Syndrome CIR Cosmetic ingredient review COLIPA European Cosmetic Toiletry and perfumery association CTFA Cosmetics toiletries and fragrances association EDTA Ethylene Di-amine tetra-acetic acid EEC European Economic Community EU European Union FDA Food and drug administration GMS Glycerol Monostearate HLB Hydrophilic lipophilic balance HPS Hermansky-Pudlak Syndrome IARC International Agency for Research on Cancer ICE Isolated Chicken Eye ICP-AES Inductively Coupled Plasma-Atomic emission spectrum IKS Indigenous knowledge systems INCI International Cosmetic Ingredient JREC Joint Research and Ethics Committee M. flabellifolia Myrothamnus Flabellifolia MED Minimum erythermal dose

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MEDPS Minimum erythermal dose, protected skin

MEDUS. Minimum erythermal dose, unprotected skin NLC Nano-structured lipid carriers OA Ocular albinism OCA Oculocutaneous albinism OECD Organization for economic Cooperation and development OTC Over the counter medications PBS Phosphate buffer saline PCD Product category designations PII Primary Irritation Index PII Primary Irritation Index PLWA Persons living with albinism POM Prescription only medications PPD Persistent pigmentation darkening SABS South African Bureau of standards SAZ Standard association of Zimbabwe SCCNFP European Scientific Committee on Cosmetics and Non-food Products SLN Solid lipid nanoparticles SPF Sunburn Protection Factor SPI Score of Primary Irritation SPI Score of Primary Irritation T. emetica Trichilia emetica TEWL Trans-epidermal water loss Ti Titanium UVR Ultraviolet radiation WHO World Health Organization Zn Zinc

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CHAPTER ONE

INTRODUCTION

“It is difficult to say what is impossible, for the dream of yesterday is the hope of today and the reality of tomorrow”

Dr Robert H Goddard 1882-1945

1.1 HUMAN ALBINISM

Albinism, also called achromia, is a heterogeneous group of congenital dermatological disorders, characterised by a marked reduction in melanogenesis. The anomaly manifests through a whole spectrum of pigmentation levels from comprehensive, partial to complete absence of melanin in the skin, hair and eyes (McGraw Hill: 2002).

The homeostatic processes of melanogenesis and keratinization are the only two ways in which the human body protects itself from actinic damage. Actinic damage refers to the solar Ultraviolet Radiation (UVR) induced dermatological complications, including sunburn, inflammation, photo-aging, solar keratosis, solar urticaria and various skin cancers (Carden S.M: 2002). People living with albinism (PLWA) are therefore prone to all kinds of actinic damage (Figure 1-1).

Figure 0-1; Pre-pubescent female living with albinism (source, Zimbabwe Albino Society Picture Gallery: 2014)

Pigmentation errors involving the melanocytes of the hair, skin and eyes are medically referred to as oculocutaneous albinism (OCA), while those errors primarily localized to the eyes are described as ocular albinism (OA) (Richard D et al: 2006). This congenital impairment in melanogenesis makes albinistic persons highly susceptible to actinic damage. There are various forms of albinism and the exact causes of some have not yet been scientifically identified. Mutations in six genes have been reported to be responsible for the different types of both OCA and OA (Richard D : 2006).

There is still no cure or treatment specifically developed for PLWA and no meaningful scientific work has been carried out to establish the racial skin morphological differences between Caucasoid (white), Mongoloid (Asian) and Negroid (black) PLWA which could assist in the development of treatments for the condition (Carden S M et al:

2002). Albinism affects people of all ethnicities but its prevalence has both racial and sexual biases (Machipisa L: 2004). The condition is most prevalent in Sub Saharan

African black females and even though global statistics estimate that albinism affects one in 17 000 people worldwide, in Zimbabwe, it affects 1 in every 600 people

(Machipisa L: 2004).

In Sub-Saharan Africa (Zimbabwe in particular), both the sun and the society are quite hostile to PLWA. The sub-tropical sun causes actinic damage to their melanin deficient skin, including wrinkles, lentigines, actinic keratoses and epitheliomata which may shorten their life expectancy. Most ethnic people erroneously still attribute albinism to retribution from wrathful gods and origins beyond human comprehension (Machipisa L:

2004).

There is no treatment that can replace the lack of melanin or impairment in melanogenesis which causes the various skin problems afflicting albinistic persons.

Abstinence from outdoor activities and the use of sunscreens are the only available treatments to ease the symptoms of albinism and mitigate the UVR induced effects of actinic damage (Raymond D : 2005).

1.1.1 UVR and sunscreens

UVR is part of the electromagnetic spectrum. It has wavelength shorter than that of visible light but longer than X- rays. The wavelength is nanometric, ranging from 10nm to 400nm. The photon energies range from 3 ev to 124 ev (Gardner R: 2010). The sun emits ultraviolet radiation at all wavelengths including the extreme ultraviolet where it crosses into the disposition of X-rays at 10 nm. The sun’s emissions in the lowest UVR bands; the UVA, B and C are of interest in this study as these are the UVR bands commonly encountered from various sources on earth. The UVR spectrum is composed of UVA (400-315nm), UVB (315-280nm) and UVC (280-10nm). (Figure 1-2)

Figure 0-2; Radiation spectrum highlighting Ultraviolet radiation spectrum section (Source UVR media-resources-ordp-.com)

The principle that applies to all magnetic waves, that is ‘energy is inversely proportional to wavelength’, also applies to UVR. This entails that as the wavelength decreases, the energy and effects of UVR increase (Hockberger P. E: 2002).

Highly ionizing extreme UVR which is below 120 nm is blocked by the ozone layer in the stratosphere. It does not reach the ground in normal circumstances and therefore is of no consequence to human actinic damage in the practical product development sense.

It should be noted though that the entire UVR spectrum exhibit biological features of ionizing radiation, responsible for considerable damage to bio-molecular and biological systems than any other simple heating effects. Such detrimental activity is a result of the UVR photon’s capacity to alter biochemical bonds even in the absence of enough atom ionizing quantums (Matsimu Y: 2004).

On April 13, 2011, the International Agency for Research on Cancer (IARC) of the

World Health Organization (WHO) classified all categories and wavelengths of UVR as

Group 1 carcinogens. This is the highest level designation for all categories of cancer accelerators, implying, there is enough evidence to show that all wavelengths of UVR 5

can cause cancer in humans. As early as 1978, the ‘Proposed monographs for sunscreens’ by the FDA stated that overexposure to sunlight damages the skin and can lead to various skin lesions, solar keratoses, skin cancers and premature aging of the skin (Gardner R: 2010).

The toxic effects of UVR from the sun are of major concern for human skin in general and albinistic skin in particular. The effects of UVR can either be chronic or acute.

Damage to collagen fibres accelerates aging of the skin as seen on exposed albinistic skin areas such as the face, the neck and forearms. Both UVA and B have a capacity to destroy vitamin A (Richard D: 2006).

UVA radiation accounts for up to 95% of the total UVR reaching the earth depending on cloud cover and atmospheric conditions on the particular day. Though it is of lower energy compared to UVB, it can penetrate deeply into the skin dermis and beyond. UVA was thought to be less harmful in the past, but recent research shows that it is the biggest contributor to skin cancer through indirect DNA damage. The biological effects are cumulative and responsible for skin fragility, skin aging, excessive wrinkling, loss of elasticity, and is the suspected trigger of malignant melanoma (Matsimu et al 2010).

Most mutations caused by DNA damage carry a UVA signature that is commonly found in most, if not all albinistic skin cancers (Richard D: 2006).

UVA does not cause direct erythermal damage and inflammation, it is therefore not measured in the usual sunburn protection factor (SPF) testing on sunscreens. There is therefore no good clinical measurement for blockage of UVA radiation. Scientists blame the absence of UVA filters in sunscreens for the higher melanoma risk found in sunscreen users (A. S Dussert et al: 2005). 6

Essentially all UVC is blocked by the dioxygen (from 100-200nm), or by ozone (200-

280nm) in the atmosphere. The ozone blocks most of the UVB, however, UVA is hardly affected by the ozone layer and most of it reaches the ground (Beeson et al: 2008).

1.1.2 Natural protection against UVR

In genetically uncompromised skin, natural mechanisms exist for protection against

UVR to retard actinic damage regardless of skin tone. These include: melanisation due to radiation exposure and keratinisation to protect the basal layer (Burton J L: 1985).

The amount of melanin and the activity of melanocytes is a homeostatic process which increases when increased UVR onslaughts are detected. The photochemical properties for melanin make it an excellent photo protectant from both UVA and UVB. Melanin absorbs the UVR and dissipates it as harmless heat thereby blocking the UVR from damaging skin tissue. This occurs by means of a process called ‘ultrafast internal conversion’ which enables melanin to dissipate 99.8% of all absorbed UVR as heat

(Burton J. L: 1985). Melanin is not only a determinant of skin colour, it is also found in the hair, the iris of the eye, the striae vascularis of the inner ear and in brain tissue

(medulla, zona reticularis of the adrenal gland, the substantia nigra and the locus coerulus) (Hockberger P.E et al: 2002). This might explain the correlation between albinism and eyesight problems, albinism and deafness and albinism and other health complications.

The second natural mechanism of protection against UVR is keratinisation. During photo protection, an organic process which deposits keratin in cells to thicken the epidermis so as to protect the basal layer occurs. The result is a thick horny and rough 7

skin profile developing as a protective mechanism (Burton J L: 1985). This mechanism usually works in conjunction with melanisation in genetically uncompromised skin as a last resort to block solar assaults. To most observers, this keratinized skin is actinic aging. This is perhaps the only protective mechanism available to protect albinistic skin from UVR and explains the horny rough feel of all exposed albinistic skin.

1.1.3 Sunscreens and Sun filters for UVR

Franz Greiter an avid outdoor adventurer developed the first commercial sunscreen in

1938 for personal recreational purposes (Burnett M.E: 2011). Since then, considerable research work has been done to develop sun-blocks or sun-filters marketed as sunscreens to protect light skinned Caucasians from the damaging effects of UVR during limited exposure to sunlight mostly for sun bathing and vacation .This is largely product development targeted at normal healthy skin that needs a leisurely tan and protection from sunburn. Cosmetic companies are at the forefront for this commercially viable niche market for aesthetic product development research. The sunscreen products developed for aesthetic reasons on normal healthy skin are the only available treatments for albinistic persons facing short life expectancies because of actinic damage, dealing with searing pain from UVB onslaughts, nursing recurring skin lesions and contenting with permanently scaly rough keratinized skin. There is no commercially available medicinal sunscreen primarily developed to alleviate albinistic skin complications. Despite the dependency on them by albinistic persons, the ineffectiveness of these commercial sunscreens is apparent on PLWA within the tropics.

Sunscreens are cosmeceuticals that absorb or reflect some of the sun's UVR on exposed skin and protect it against actinic damage. Sunscreens can either be organic chemical UV filters that absorb light or physical inorganic metal oxides that reflect, scatter and absorb UV light (Norval M et al: 2009).

Most of the commercially available sunscreens are chemical sunscreens due to the unaesthetic nature of macromolecular physical sun-blocks. The use of chemical sunscreens is controversial for various reasons. Most do not block UVA radiation because their primary focus is in alleviating acute actinic damage from UVB, like sunburn. Generally, people using chemical sunscreens may be exposed to high UVA levels without realizing it (Norval M et al: 2009). The chemical UV filters absorb light energy and convert it into heat in the basal layer of the epidermis; the fate of the generated heat is unknown and is suspected to be responsible for cell death in the basal layer after prolonged use. Despite these and other concerns, the use of chemical sunscreens is widespread. Annex V11 of the Cosmetics Toiletries and Fragrance

Association (CTFA) cosmetic compendium provides a positive list of allowed UV filters and the concentrations at which they may be used (SPF Testing, FDA: 2009).

1.1.4 Physical sunscreens

Two inorganic materials commonly used as sunscreens are zinc oxide (ZnO) and titanium dioxide (TiO2). Zinc oxide is the broadest spectrum UVA and UVB reflector approved as a sunscreen by the FDA (Sheree et al: 2005). Titanium dioxide has widespread use in sunscreens and products for infants and people with sensitive skins.

When used as sunscreens, these physical sunscreens stay on the skin’s surface and 9

are not absorbed. They are therefore non-irritating non allergenic and non comedogenic. Their ability to scatter and reflect light is apparently also their ‘Achilles heel’ for commercial acceptability and use in all day sunscreens (Pinnel S R et al:

2000). When using physical sunscreens, protection and aesthetic aspects are directly related to particle size and mineral distribution on the skin. When physical sunscreens are applied to the skin, they leave a white tactile plastering that persists until washed off

(Gamer A. O et al: 2005). This makes the products unsuitable for all day use or consistent lifelong use by albinistic persons. However the nanometric analogues of these metallic oxides are transparent, non-tactile and aesthetic. The birth of novel sciences like nanotechnology and the subsequent ability to manipulate molecules has therefore ushered in new possibilities and opened up new frontiers in cosmeceutical product development which can effectively retard actinic damage in PLWA using the transparent nanometric versions of these materials to retard actinic damage.

1.1.5 Geographical UVR variations impact on cosmeceuticals

Apart from the radiation monitoring stations in Cape Town, Pretoria and Durban in

South Africa, observations from this study show that there are no monitoring stations elsewhere in Sub Saharan Africa. Given that protection from actinic damage should be based on expected radiation incidences and effects, this immediately casts affectivity doubt on products developed and tested in temperate areas like Europe and North

America on albinistic persons living in the tropics and sub-tropics like Zimbabwe.

UVR Intensity has a geographical bias; maximum UVR intensity is received between the

Equator and 30 degrees latitude North and South. UVA Intensity remains almost 10

constant from the equator to 60 degrees North and South latitude. During the winter months, very little UVB is detectable above 50 degrees latitude, The winter UVB levels in tropical countries is higher than the summer UVB levels in temperate areas (Gardner

R: 2009). Zimbabwe is a Sub-tropical country lying within 23.5 degrees from the equator which makes the country susceptible to serious UVR onslaughts throughout the year.

UVR is also affected by altitude, an increase in altitude of 1000 m results in a 15% increase in UVB while UVA remains practically constant. At the Dead Sea, which is

400m below sea level, UVB is almost absent. Zimbabwe is a plateau whose capital city,

Harare (17° 51′ 50″ S, 31° 1′ 47″ E) sits at 1480 m above sea level

(www.hararecity.co.zw). Geographically, the Harare climate is designated in the category of subtropical highland. Unlike UVA, The spectral irradiance of UVB decreases rapidly as the solar zenith angle increases. UVB incidence is therefore highly dependent on the time of day and position of the sun more than UVA (Gardner R: 2009). It is widely believed that the hottest time of the day in temperate areas is at noon in summer.

During the 2011 and 2012 summer season heat waves in Zimbabwe, the meteorological services in Zimbabwe confirm that the record breaking temperatures were however recorded between 2:30 pm and 3:00 pm (Zimbabwe Met office statement; ZBC

28/10/2011). During the summer months, temperatures rise rapidly and are almost unbearable by 10 am and persist until after 4 pm in the afternoon. Zimbabweans are therefore exposed to an average of 6 hours of intense actinic exposure in summer.

The amount and nature of UV radiation reaching the earth is also affected by mean day cloud cover measured in eights parts. The Belvedere meteorological department in

Harare uses a simple determination method whereby the sky is theoretically divided into 11

eight parts and a trained eye estimates how much of the eight segments have complete cloud cover. Unfortunately for albinistic persons the highest temperatures occur in the months with the lowest average cloud covers and actinic damage is therefore severe throughout the whole year (Figure 1-3).

Figure 0-3: Erythermal damage experienced by an albinistic research participant between 9:30 am and 11am on 09/03/14 in Harare, Greendale

UV radiation protection in sub-tropical countries like Zimbabwe should be done throughout the day and throughout the year by a wide spectrum sunblock for both UVA and B. The product has to be aesthetically pleasing to be suitable enough for all day use with sun protection for up to six hours if the product is going to protect PLWA.

During this study, a 21 year old albinistic female participating in the study received severe sunburn on an overcast rainy day in Raffingora, Zimbabwe while using normal recommended applications of a chemical sunscreen (Figure1-4 and 1-5).

Figure 0-4 : Onset of UVB Damage on an overcast day in Raffingora Zimbabwe on 16/01/2013

Figure 0-5: Actinic damage experienced on an overcast day after applying recommended chemical sunscreens in Raffingora, Zimbabwe on 16/01/13

The physical sun-blocks containing normal macromolecules of zinc oxide and titanium dioxide are anaesthetically pleasing. The insoluble sunscreens have to be suspended in greasy emulsions which leave an opaque white film after use that is unsuitable for all day use and has only been used by cricket players for limited times of exposures during matches. 13

1.1.6 Nano-cosmeceuticals and nanomaterials

Nanotechnology is a branch of science and engineering, which involves the creation of functional devices or materials in the nanometric range (generally 10-1000 nm) and the exploitation. (Gamer A. O et al: 2005). There are two main applications of nanotechnology in cosmeceuticals, the first, being the use of metallic oxide nanoparticles as UVR filters which can retain the UVR absorption and filtration capacities and appear transparent on the skin and therefore eliminate the white chalky appearance of their larger sized opaque analogues (Gamer A. O et al: 2005).

The second application is in achieving improved transdermal and cellular penetration.

Nano-structured lipid carriers (NLCs) have been identified as potential next generation cosmetic and pharmaceutical delivery agents. These particles can load high amounts of

UVR filters. They can protect the encapsulated UVR filters from degradation and can also be used for controlled delivery of the UVR filters into the skin over a prolonged period of time (Gamer A. O et al: 2005).

1.1.7 Nanometric sunscreens

Metallic oxides such as titanium dioxide and zinc oxide have been found to be highly protective against harmful UV rays because they mobilize electrons within their atomic structure while absorbing UVR without undergoing decomposition or degradation.

Unfortunately these two are totally insoluble in oil and water which makes it difficult to aesthetically incorporate them into cosmetic emulsions. They have scientifically been proved to offer a wider spectrum of photo-protection than many chemical sunscreens

(Gamer A. O et al: 2005). Large or normal size metallic oxide particles reflect ambient light and have an opaque appearance that users find unacceptable. Nano-sized particles are smaller than the wavelength of visible light and vanish when applied onto the skin. Due to this minute particulate size, nano-metallic sunscreens will also be more evenly spread on to the skin following natural skin contours and leaving fewer gaps

(Gamer A. O et al: 2005). Opaque molecules despite lacking cosmetic appeal have a reported trend towards lower and insufficient application rates. Nanoparticles of TiO2 and ZnO are transparent in formulations and on the skin surface. This transparency provides the cosmetic acceptability not achievable with larger particle formulations.

Nanoparticles will therefore resolve the problem of unsightly white film of traditional physical sunscreens and create a vehicle that is more transparent less viscous and blends into the skin more easily. Engineered metallic nanoparticles, exhibit enhanced physical, chemical and biological properties distinctly different from their bulk form because of their increased surface to volume ratio.

Nanotechnology is an emerging discipline and there is a gap between commercial product development and the nanotechnology research that produces new raw materials, new synthesis, new concepts and new applications. In terms of cosmetic product development, product efficacy and safety, they should be treated as new materials. There is need to understand better the compatibility, suitability, efficacy, safety and in use life cycle of the nanoparticles.

1.1.8 Botanical active ingredients

The use of plant extracts as active ingredients and lipid emollients has been the cornerstone of aesthetic alchemistry for aeons. Within the realms of recorded history, some of the earliest texts that have survived are entirely devoted to the therapeutic properties of botanical extracts (Bruneton J: 1995). The earliest records from Egypt

(Papyrus Ebers; 1550 BC), Sumer (3000 BC) and China (Emperor Shen Nung’s Pen

Tsao Ching plant remedies (2500BC) all provide examples of extensive use of botanicals in cosmeceuticals. Their complexity and wide extent is testament to a tradition as old as mankind (Bruneton J: 1995). The antiquity of these early works is indicated by superstitious beliefs which alluded that herbs were “made from the flesh of the gods” (Bruneton J: 1995). When it comes to botanicals, the Holy Bible is no exception. The book of Exodus is full of citations when Moses went up mount Sinai to be given therapeutic formulations for anointing oils for the alter and the body (Hebrew

Bible: Exodus 21-30). The same myths also pervaded throughout Africa up until modern times. African folklore is full of terrifying legends and horrifying curses from wrathful gods unleashed upon anyone who harmed plants of therapeutic essence (Mbuya

Chiparatani interview, registered Zimbabwean traditional healer: 2013).

With regards to the pharmacological activity and efficacy of indigenous plant extracts, the transition from myth to reality has been accomplished through the identification by modern chemistry of the active components of the various parts of any particular plant and the verification by medical science of the powers of soothing, emolliency and healing traditionally ascribed to that plant (Gundidza Interview: 2014). Consequently,

the 1990s saw a phenomenal resurgent interest in botanical extracts as active ingredients and emollients for cosmeceuticals. The sophistication and inquisitive nature of the modern consumers now demand safer products free from real and imagined allergens (Van Wyk B.E et al: 2009). The revival of herbal extracts in mainstream medicinal products was further accelerated by the poor image animal-derived extracts acquired during the same period, mostly due to reports regarding bovine spongiform encephalitis (BSE) (FDA/CFSAN: 2005). Plant extracts clearly became the active ingredients of choice for the ever growing multitudes of discerning patients.

Zimbabwean traditional medicine has employed various botanical extracts in crude form for the treatment of various skin ailments since time immemorial. Such plants include;

Zimbabwean Tree aloe (Aloe excelsa), Banket mahogany (Trichilia emetica) and the resurrection bush (Myrothamnus flabellifolia). These plants whose favourable properties have not been fully commercially exploited due to the lack of structured studies on them present interesting opportunities and potential use as primary pharmaceutical and cosmetic active ingredients if given applied product development research priority.

1.2 PROBLEM STATEMENT

Most commercially available chemical sunscreens introduced above are formulated for

UVB protection only and do not block UVA. The conventional micro-sized TiO2 and ZnO with broad spectrum sunscreen capabilities can cater for UVA, but unfortunately give an opaque un-aesthetically pleasing white, chalky, greasy and un-absorbable treatment which is unsuitable for all day, everyday use by albinistic humans. The commercially

available sunscreen formulations do not consider influence of altitude and latitude on actinic damage and geographic discrepancies in UVR incidences impact and damage.

Current sunscreens being given to albinistic persons in Zimbabwe do not contain any ingredients to assist in wound healing, anti-aging, inflammation and maintenance of the natural moisturising factor which are all symptoms of actinic damage found on almost all albinistic persons living within the tropics. There is therefore no effective all day treatment for albinistic persons living in the tropics. The ineffectiveness of current sunscreens is also due to the poor release profile of the active ingredients as well as the uncontrolled absorption which is high on application, but rapidly decreases afterwards, hence the recommendation for impractical frequent reapplication of sunscreens.

1.3 RESEARCH HYPOTHESIS

The formulation of a treatment incorporating nanoparticle sized TiO2 and ZnO in a cream base, incorporating Trichilia emetica, Aloe excelsa and Myrothamnus flabellifolia will be efficacious in blocking a wide spectrum range of both UVA and UVB and will give a safe, stable and affordable product for actinic damage in PLWA. TiO2 and ZnO will not penetrate normal and actinically damaged skin and will remain on the skin periphery to absorb, scatter and reflect UVA and UVB.

1.4 RESEARCH AIMS

The main aim of this research is to develop a nanotechnology based actinic damage retarding treatment for use by albinistic persons living within the tropics using

nanometric TiO2 and ZnO, incorporating the seed oil extract from Trichilia emetica, essential oil extract of Myrothamnus flabellifolia and the active extracts of Aloe excelsa and to evaluate the treatment physico-chemical parameters, skin dermato- pharmacokinetics and formulation toxicity and efficacy.

1.4.1 Specific objectives

i. To extract and characterise the active constituents of Trichilia emetica, Aloe excelsa and Myrothamnus flabellifolia plants. ii. To develop an SPF 15 (minimum) ternary phase O/W emulsion (lotion)

treatment base incorporating nanometric TiO2, ZnO and herbal remedies including Trichilia emetica, Aloe excelsa and Myrothamnus flabellifolia. iii. To evaluate the formulation physico-chemical parameters, emulsion stability, shelf life, aesthetics and functional parameters. iv. To investigate, ex-vivo, the treatment safety risk for systemic exposure across actinically damaged porcine skin. v. To investigate albinistic skin reservoir properties and the treatment dermato- pharmacokinetics across human albinistic skin. vi. To evaluate in-vitro the treatment SPF for UV-B vii. To investigate in-vivo the formulation skin sensitivity and toxicity using laboratory animals. viii. To evaluate product compatibility and aesthetics through albinistic persons panel tests

CHAPTER 2

2. LITERATURE REVIEW

“To be perfectly original, one should think much and read little, and this is impossible as one must have read much before one learns to think” Lord Byron 1788-1824

2.1 THE HUMAN SPECIES

The modern humans are the only surviving members of the hominin clade of the great apes family (Table 2.1). These great apes were distinguished primates which evolved the unique capability to stand in an erect posture and walk entirely on two limps (Wilson

D. E: 2005). Humans evolutionarily, separated from their DNA closely related knuckle walking relatives, chimpanzees and gorillas about 4800 million years ago. Apart from the larger brains, they have extricated themselves from the rest of the primates through adaptations in bipedalism, extended ontogeny and reduced sexual dimorphism. Human beings are the only extant subspecies that can make fires, cook food, clothe themselves and employ technologies and arts (Wilson D. E et al: 2005).

Table 2-1: Scientific classification of modern human beings (adapted from White et al: 2003)

Kingdom: Animalia

Phylum: Chordata

Class: Mammalia

Order: Primates

Family: Hominidae

Tribe: Hominini

Genus: Homo Species: H. sapiens Sub species H. sapiens-sapiens

Thanks to their more complex thought matrix, modern human beings have complex social structures too, characterised by cooperative and competitive social interactions, norms and rituals (Tattersall I, Schwartz J: 2009).

Their quest to comprehend the environment and manipulate any phenomena with a bearing on their lives led to scientific, religious and philosophical development.

2.1.1 Genetic and anatomical diversity of cosmeceutic concern

Through the lengthy evolutionary process, the modern human’s gene pool has remained considerably small pointing to the possibility of a single ancestor.

Scientifically, there is more genetic diversity among a chimpanzee clan found on a single hillside valley in the Congo than in the whole human species gene pool (McHenry

H.M: 2009). Anatomically the average human has more hair follicles on their body than any chimpanzee and over 100 times the number of sweat glands over their entire body.

The average human females weigh between 54-64 kg and stand at 1.6-1.7m tall. The average male counterpart weighs an average 74-83 kg and stands between 1.7-1.8m tall (McHenry H.M: 2009). Despite their common ancestry and small gene pool, there is considerable phenotype variation and diversity in this sub-specie (McHenry H.M: 2009).

Variations in human phenotypes include: Blood types, skin, eye and hair pigmentation, body structure and teeth sizes vary across the globe. These variations in anatomy and pigmentation appear to follow a geographic pattern (McHenry H.M: 2009).

2.1.2 Geographic anatomic variation of cosmeceutical concern

Humans are a largely cosmopolitan species that has made home to all regions and places of animal habitat including harsh deserts, rainforests and heavily polluted cities.

Their recent conquests and setting up of permanent bases in the freezing Antarctica

and Arctic circles has put a human foot print on the entire globe thereby proving unmatched adaptability by any species surviving or extinct (McHenry H.M: 2009).

Anatomical variation of cosmeceutical regard appears to have a geographical fingerprint. People in warm climates like the Sub Saharan African region are darker in pigmentation and have slender tall body structures. People living and originating from cold to very cold climates are shorter, stockier and fairer skinned. The amount and type of the natural pigment melanin in hair and skin differs accordingly (McHenry H.M: 2009).

2.1.3 Racial diversity of cosmeceutical concern

These geographic anatomical variations have given rise to three distinct racial groups which include Caucasians, Africans and the Orientals (Figure 2-1). The races are distinguished in terms of skin: colour, biochemistry and morphology. They also show considerable distinctions in hair type and morphology as well as consumer aspirations and grooming techniques (Begun, David R: 2010). The said differences have a huge bearing on cosmeceutical and pharmaceutical product efficacy and suitability.

Cosmeceutical formulators must take account of these anatomical discrepancies due to the fact that racial groups require specific products to address specific needs (Begun,

David R: 2010). Some cosmeceutical products and grooming techniques for

Caucasians are totally irrelevant or even harmful for Africans and vice versa.

Figure 2-1: The three basic racial groups (A) Mongoloid (Liu Yi Fei) (B) African (Lupita Nyong’o) (C) Caucasian,(Cate Blanchet) (Source: http://www.vogue.com/13257111)

2.1.3.1 Albinism in Sub-Saharan Africa

Albinism is not a sub-race in itself but a congenital recessive disorder that affects all known vertebrates. It therefore affects all the three distinct racial groups mentioned above. World statistics show that albinism affects 1 in every 17 000 people. However this prevalence is quite different in Zimbabwe and the Sub-Saharan African region where the condition is more prevalent and affects 1 in every 600 people (Machipisa L:

1995).

2.1.4 The Human Skin

The human skin is the largest and heaviest body organ. Accounting for up to 20% of the total body weight of humans, it covers a vast surface area of almost 2 m2 in adults

(Robinson et al: 2005). With an average pH of 5.5 in Caucasians and 5.9 in Negroids,

Schade and Marchionini in 1929 described the skin as an acid mantle that protects the internal organs from both environmental and pathogenic attacks. In humans, two 24

morphologically different types of skin exist on each individual, depending on the location on the body (Fitzpatrick et al: 2005). First, there is the non- hairy skin that occurs in palms and soles of the feet (also referred to as palmoplanter surfaces) and the second and most widespread is hair bearing skin found all over the human body. The later type has pilosebaceous units which consist of a hair follicle, sebaceous glands and other components (Fitzpatrick et al: 2005). The skin was erroneously widely believed to be impervious until recent times when Bos and Meinardi (2000) demonstrated that the skin can reasonably be permeated by external molecules that are smaller than 500 Da.

However, this selectively permeable barrier forms the curtain that protects our bodies from dehydration, physical damage as well as other diseases (Fitzpatrick et al: 2005).

Skin morphology, pH, moisture content and thickness vary over different regions of the human body, with the average full skin ranging from 0.05 – 2.00 mm in thickness. It is therefore not surprising that different regions of the body might be afflicted by different skin diseases and conditions. The inter-digitating skin waffle is a laminate of two distinct layers which consists of the epidermis, the dermis and under them is the subcutaneous fatty layer or hypodermis (Fitzpatrick et al: 2005). These layers are further subdivided into other morphologically distinct sub layers (Figure 2-2). The skin laminates are laden with appendages including hair follicles, endocrine and epocrine glands as well as sebaceous glands which play cardinal roles in skin function and integrity. Far from being just a covering for internal organs from the outside environment, the skin has a wide range of processes and functions that support human survival due to the various structures that impart defensive and homeostatic attributes (Burton J.L: 1985).

Consequently, the skin protects the internal organs from dehydration, injury, chemicals, 25

microorganisms and actinic damage. Through communication on changes in the external environment to the brain via sensors, the skin regulates body temperature and is also the synthesis site for vitamin D (Burton J. L: 1985).

Figure 2-2: Cross section of the skin (source https://www.skin-conditions.knoji.com).

2.1.4.1 The epidermis

This outermost layer of skin that separates us from the environment is regarded as the keratin bio-factory (Figure 2-2). New cells are continuously produced in the deep basal cell layer or stratum basale by mitosis. As the daughter cells migrate up, they become flatter as they reach the Stratum spinosum which is also known as the prickle cell layer.

In the granular layer or Stratum granulosum, the cells undergo a profound change which converts their contents to keratin. In this process, they lose their nuclei and consequently die. These flattened dead squames forming the stratum corneum are continuously abraded from the skin surface by physical and chemical trauma (Burton J

L: 1985:8). 26

2.1.4.2 Stratum corneum

Figure 2-3: Cross section of the stratum corneum (Adapted from Heather Baron MD Homepage 2007)

The essential part of the epidermis which is paramount to cosmeceutic product development is the Stratum Corneum (SC) (plural strata cornea) also referred to as the horny layer. This first line skin barrier is the outer most part of the epidermis and the contact point in topical applications. The SC is a congregation of layers of non- nucleated and non-morbid keratin filled cells. The SC was previously believed to be biologically inert. It was envisaged to be as a thin impervious laminate sheet enveloping the body, however it has been scientifically demonstrated that this less than 20 µm thick

SC has intricate and complex biological and chemical processes (Burton J L: 1985).

The illustration above (Figure 2-3) shows that the main building cells of the SC are the corneocytes, which are linked by the corneodesmosone. The corneocytes are surrounded by a tight lipid cornified envelope. These flattened cells that have migrated from the stratum granulosum encapsulate the natural moisturising factor (NMF). The

NMF is perhaps the most important attribute of skin integrity to cosmetic scientists (John

A. et al (1982). Corneocytes contain keratin but have no nuclei and cytoplasmic organelles. The SC is made up of 10 to 15 layers of corneocytes. This layering is reminiscent of the “brick and mortar model” (Naik et al: 2000). There are no phospholipids as compared to other biological membranes. The process that controls the 14 day migration of cells through the epidermis from proliferating keratinocytes towards the surface for ultimate desquamation, is paramount to the comprehension of pathological conditions such as wound healing and other skin lesions (Burton J L:

1985.9). Skin disorders are usually a culmination of failures in regulation of skin homeostasis due to various weaknesses in desquamation and in the formation of the cornified envelope (Ovaere P et al: 2009).

The cornified lipid envelope of the corneocytes forms the main barrier to penetration.

Permeation through the stratum corneum can only be achieved through the lipid based

“mortar” than through the corneocytes “bricks” (John A. et al (1982). The staggered brick and mortar arrangement complicates diffusion pathways through the SC. This enhances the barrier function of the skin (Naik et al: 2000).

2.1.4.3 Stratum granulosum (SG)

The SG lies just beneath the Stratum corneum or the Stratum lucidum in palmoplanter skin surfaces. The granular layer gets its name from the prevalence of protein laden keratohyalin granules (James et al: 2005). The cells at the transition point to the

Stratum corneum secrete lipid and protein containing lamellar bodies into the 28

intercellular space. The resultant hydrophobic lipid casing is the cornerstone of the skin's barrier properties. In this, transitional layer, cells lose their nuclei and organelles.

The granular cells transform into non-viable corneocytes, when they get to the Stratum corneum (Venus et al: 2010:469).

Apart from the keratohyalin granules in the keratinocytes, the cytoplasm also contains granules called Odland bodies (Naik et al: 2000). The keratohyalin granules are responsible for promoting hydration and cross-linking of keratin, which is of utmost importance in skin integrity and function. This layer forms an occlusive layer that prevents fluid loss from the underlying skin components (Venus et al: 2010).

2.1.4.4 Stratum spinosum

This layer is commonly referred to as the prickle cell layer due to sandwiched layers of desmosomes connected polyhedral cells which give prickly or spiny impressions when viewed microscopically (McGrath J.A et al: 2004). Langerhans cells are also resident in the Stratum spinosum (Venus et al: 2010:469).

2.1.4.5 Stratum basale

The basal layer which is also known as the Stratum germinativum is the deepest layer of the epidermis. This layer is one cell thick in normal healthy skin, it has however been found to be thicker with even up to three cell thicknesses in cases of some skin diseases that are characterised by hyperproliferation of the epidermis like psoriasis

(McGrath JA et al: 2004:3). The Stratum basale which borders the Stratum spinosum is

mostly made up of the stem cells of the epidermis, the keratinocytes. However it also has pigment producing melanocytes, Langerhans as well as Merkel cells. This epidermal base cell layer is the foundation which also lies adjacent to the dermis (Venus et al: 2010:469).

2.1.5 Dermis

The dermis and the epidermis together form what is scientifically known as the ‘cutes’.

Regarded as the ‘true’ skin, the dermis is composed of connective tissue including collagen fibres, elastin tissue, ground substance, fibroblasts, and other cellular components. The dermis is also host to sweat glands, sebaceous glands, and networks of blood vessels, lymphatic vessels and nerve endings (Lai-Cheong & McGrath:

2009:224). The superficial area close to the epidermis is known as the papillary region and the deeper thicker area is known as the reticular dermis (James et al: 2005).

The papillary region or Stratum papillare is composed of loose connective tissue. It has finger-like projections that extend towards the stratum basale which contain blood vessels as well as Meissner corpuscles (http://microvet.arizona.edu: 2012). The thick reticular region is made up of dense connective tissue. Its name is derived from the high dispersion of collagen, elastin, and reticular in it. These fibres are responsible for the strength of the dermis.

2.1.6 Hypodermis

The hypodermis (‘under the skin’) is not really a part of the true skin. It is mainly the adipose fatty tissue deposit under the ‘true’ skin. It is composed of lipocytes; fibroblasts as well as macrophages (Naik et al: 2000.

2.1.7 Human pigmentation

Human pigmentation is a combination of 4 colour tones (Greco G et al: 2011):

 Red tone due to oxygenated haemoglobin.

 Blue tone due to deoxygenated haemoglobin.

 Yellow tone due to carotenoids.

 Brown-Black tone due to melanin.

The condition of the stratum corneum also affects the colour of the skin. Skin pigmentation is therefore due to a balance of the four tones and the general condition of the stratum corneum. Since all human beings have basically the same haemoglobin, the blue and red tones are therefore almost standard in all people. The determinants of colour variations are therefore carotenoids and melanin. Discrepancies in carotenoids are mostly found in Mongoloids or the ‘yellow’ race. For Caucasians and Negroids, the primary determinants of skin and hair colour are melanin and melanogenesis (Greco G et al: 2011).

2.1.7.1 Melanin and melanogenesis

Melanin is a generic term ascribed to natural pigments that give colour to organisms.

The only living organisms in which melanin pigments have not been detected are arachnids. The process that produces melanin is referred to as melanogenesis and it occurs in specialised cells called melanocytes through the oxidation and subsequent polymerisation of tyrosine under the direction of the enzyme tyrosinase. Melanin is a non-refractive granular pigment with a diameter less than 800 nm. Three types of melanin have been characterised (Brenner M, 2008):

 Eumelanin; this is the most common pigment, a stable homo polymer of tyrosine

which has black and brown subtypes (Figure 2-4).

 Pheomelanin; An inferior red-brown polymer of tyrosine which has cysteine in

parts of its chain. Pheomelanin is responsible for freckles and red hair types and

is the primary pigment in nipples (Figure 2-4).

 Neuromelanin; found in parts of the brain and its functions which are little

understood appear not to be of any relevance to cosmeceuticals.

Melanin has the unique capacity to absorb UVR and harmlessly dissipate 99.9% of it through a process known as ‘ultrafast internal conversion’ (Brenner M: 2008). In general, human beings possess similar concentrations of melanocytes though the degree of their viability might vary. Since the aggregate pigment is built of monomer units, the variation of melanin types in humans is prevalent due to differing bonding

arrangements and the number of monomers. Eumelanin is the most prevalent form and therefore the form usually absent in albinistic humans (Brenner M, Hearing VJ: 2008).

Figure 2-4: Eumelanin (left) and pheomelanin (right). (Source: Wakamatsu K, Ito S, (2003))

In eumelanin the ‘COOH’ side group may sometimes just simply be an ‘H’. Eumelanin polymers therefore comprise either cross linked 5, 6 dihydroxyindole (DHI) or 5, 6 dihyroxyindole -2-carboxylic acid (DHICA) polymers. These polymers give rise to two eumelanin black and brown pigmentation, found in Negroids and Caucasians respectively (Castelvecchi D2007).

In pheomelanin the ‘COOH’ side group may also be substituted by a simple ‘H’.

Pheomelanin is a pink to reddish pigment which is usually abundant in tissues found in lips, nipples, vagina and penis. The chemical differences between pheomelanin and eumelanin are that, the presence of L-Cysteine in pheomelanin incorporates benzothiazine units in the oligomer structure instead of the DHI and DHICA in eumelanin (Castelvecchi D: 2007).

2.2 GENETIC PIGMENTATION DISORDERS

Pigmentation disorders or dyschromia can either be due to hyper or hypo pigmentation, they range from something as benign as freckles to fatal complex diseases like

Hermansky-Pudlak Syndrome (McGrath et al: 2004).

Leucism is the general condition affecting all vertebrates, characterized by a reduction in all pigmentation (not just melanin). Leucism, (Leukism) is a generic term that refers to the resultant phenotype from failures in differentiation or consequent migration of the pigment cells to the skin, hairs or feathers of all vertebrates in various ways (figure 2-7).

The absence of pigmentation can either be partial as in vitiligo or complete as in albinism (McGrath et al: 2004). Commonly mistaken for albinism, which only results from a defect in melanin, leukism is broader and affects all pigments (McGrath et al:

2004).

Figure 2-5 : General Hypo-Pigmentation disorders affecting people (Source: Walden: 2015)

2.2.1 Amelanistic Albinism

Figure 2-6: African Albinistic females (Source: https://www.albinos in Zimbabwe: 2015)

Amelanism (amelanosis) is a form of leukism, characterised by a lack of the pigment melanin. This genetic abnormality in melanogenesis affects all known vertebrates and the general phenotype of amelanistic individuals depend on the available non-melanin pigmentation remaining (Raymond E et al: 2005).

Albinism is the most common form of amelanosis (Figure 2-8). The condition has various other names including achromia, achromatosis or achromasia. The congenital disorder is identified by a complete or partial absence of the pigment melanin.

Organisms with complete absence of melanin are ‘albino’ and those with a partial absence of melanin are ‘albinoid’ (Carden SM et al: 1998). Apart from the epidermal manifestations, albinism has links with visual disorders including nystagmus, astigmus as well as photophobia. Rare syndromes like Hermansky Pudlak and Chediak–Higashi are associated with albinism (Gronskov K et al: 2007).

Two forms of human albinism have been characterised. The main form is the oculocutaneous albinism (OCA), which affects the hair, eyes and skin of affected individuals. The second form is ocular albinism (OA), which is limited to the eyes

(Gronskov K et al: 2007). Individuals with albinism have normal growth and development patterns since the condition in itself is not fatal. However, the lack of melanin makes People Living with Albinism (PLWA) defenseless against UVR, which has a capacity to cause various fatal melanoma and dermal complications (Carden S.M et al: 1998). PLWA have to deal with both acute and chronic actinic damage due to the absence of the natural skin UVR screen.

2.2.2 Ocular albinism (OA)

Figure 2-7: A normal skin type boy with OA (Source, Zimbabwe Albino Society photo gallery: 2014)

Under genetic direction, the human eye produces enough pigment to color the iris part of the eye, brown-black, blue or green so as to opacify the eye. Most albinistic eyes are however bluish, reddish to pink because of the absence of melanin (Figure 2-9). The observed color of eyes is the red retina seen through the iris. This apparent lack of pigment gives rise to various eye problems including photosensitivity and other unrelated conditions (Carden S.M et al: 1998). The human optical system depends to a large extend on melanin and its absence leads to disorders including the following

(Gronskov K et al: 2007):

 Abnormal crossing of optic nerves due to retinogeniculate projections misrouting

 Ocular stray light (photophobia) leading to suppressed visual accuracy

 Prevalent photo-induced retinal damage

 Foveal hypoplasia leading to a reduction in sight

The most common eye disorders found in PLWA, include, but are not limited to the following (Gronskov K et al: 2007):

 Astigmata; the cornea needs correction due to an irregular structure.

 Nystigmata; eyes appear shaky, due to sharp uncontrollable irregular motion of

the eyes in all directions.

 Nervous hypoplasia; Caused by poor sight and visual acuity due to an

underdevelopment of the optic nerve.

 Ambylopia: caused by poor visual acuity due to poor transmission of impulse to

the brain.

The human iris is a heavily pigmented structure, which limits the amount of light passing through to the retina by timeously contracting the pupil in the presence of bright light. In conditions where light is low, the iris allows more light to the retina by relaxing. In

PLWA, the iris does not have enough pigment to ensure process integrity. This results in photophobia and other complications especially symptoms of strabismus (Raymond E et al: 2005).

2.2.3 Oculocutaneous albinism (OCA)

Figure 2-8: A young female adult, with OCA albinism and corrected eye sight. (Source: research participant picture taken by researcher)

OCA is a congenital autosomal recessive pigment disorder affecting the skin, hair and eyes (Figure 2-10). There are at least nine different types of OCA. Four of the nine types have been scientifically, characterized.

2.2.4 Forms of OCA

Autosomal recessive disorders causing disruptions in melanogenesis lead to the following forms and types of albinism afflicting humans:

OCA 1: This form of albinism is due to tyrosinase gene alteration and occurs in two

variants. In OCA 1a, the individual fails to develop any perceivable pigment at all

(Figure 2-11). The condition is characterised by white hairs, translucent pale skin,

and poor vision. Due to the complete lack of melanogenesis even for wound healing,

individuals do not even develop freckles or pigmented lesions and spots due to

UVR. In OCA 1b, afflicted persons can tan and show limited pigment in the hair.

Other individuals are born with these conditions but gradually develop pigment as

they grow (Gronskov K et al: 2007).

Figure 2-9: Young male displaying ACA 1a symptoms (Source: Zimbabwe Albino society photo gallery: 2014)

OCA 2: This is the most prevalent form of albinism. It is due to the p gene mutation.

Pigment development is manifest in moles and freckles. Individuals are not as pale

as OCA1 but have fair skin, blonde to golden hair and sometimes even brown hair

(Figure 2-12) (Gronskov K et al: 2007). In Zimbabwe, phenotypes with yellow hair

and greyish eyes participated in this study.

Figure 2-10: Female displaying OCA 2 symptoms (Source: Zimbabwe Albino Society Photo Gallery: 2014)

OCA 3: This form is due to tyrosinase related protein-1 mutation. Affected

individuals tend to have red hair. The skin tends to be reddish brown and the eyes

are either blue or gray. Not much research work has been carried out on this form of

albinism (Gronskov K et al: 2007).

OCA 4: This form is also known as, the Japanese type, due to its prevalence in

Japan, accounting up to a quarter of all albinistic cases. In Africans OCA 4, is

distinguished from OCA 2 only through DNA tests (Gronskov K et al: 2007). The

phenotypic symptoms are similar to for OCA 2 and OCA 4.

2.2.5 Albinism genetics

OCA is a congenital condition from the inheritance of recessive genes passed from both parents of an individual. Some rare cases of OCA are inherited from a single parent or can result from genetic mutations. These gene mutations lead to alterations in

melanogenesis. The incidences of OCA inherited from only one afflicted parent and a normal parent are very low. However, due to the fact that mammals can be carriers of recessive genes which do not show any traits, the incidences of two non albinistic parents producing an albinistic offspring are probable (Figure 2-13) (Gronskov K et al :

2007).

2.2.6 Diagnosing and treatment of Albinism

Albinism diagnosis is established by clinical manifestations of amelasia of the eyes, skin and hair. Characteristic eye findings are used for OA. For both OCA and OA, molecular genetic testing can also identify individuals. Though in African populations, albinistic individuals can easily be visual diagnosed and appraised, the case is quite different in

Caucasian albinos. DNA testing is used to determine the exact type and form of the condition afflicting an individual. However, even though genetic testing can confirm albinism and the specific form or variety, it offers no medical assistance except in some non–OCA complications that contribute to albinism and other treatable diseases associated with OCA. There is no cure for albinism. Treatment of symptoms is done with limited success using various interventions. Treatments of ocular problems include visual habilitation and surgery to decrease nystagmus, strabismus and astigmus.

Success of treatment procedure depends on the individual and the type of condition.

Prescription eyeglasses, sunglasses, contact lenses, large print materials and specially designed reading lights are used to help individuals living with the condition.

For skin problems, since albinism itself is not a specific sickness or disease and there is no cure for it, PLWA should avoid direct contact with UVR and can reduce sunburn with varying degrees of efficacy using commercial sunscreens.

2.3 DIAGNOSIS OF SKIN CONDITIONS ASSOCIATED

WITH ALBINISM

Skin diseases (cutaneous conditions) generally affect the integumentary system. PLWA are severely prone to these dermatoses which include skin infections and skin neoplasms (Figure 2-13) (Lippens J et al: 2009).

Figure 2-11: Albinistic male with neoplasms: In Albinism, cutaneous conditions often occur with complications (Source: https://theafrikamarket.com)

The first scientific work dedicated to skin afflictions was probably done by the Italian

Geronimo Mercuariali when he completed the De Morbis Cutaneis (Diseases of the skin) compendium in 1652 (Fitzpatrick et al: 2005). (Burton J.L: 1985) estimates that

there are thousands of skin diseases afflicting human beings and he postulates that practically, we all suffer from at least one of these during our lifetime. Between 5 to 10

% of all medical conditions are skin diseases (Burton J L: 1985). Skin conditions differ from other conditions in that, patients have a psychological response to their disease since the skin is easily visible to others. The skin has social implications in a person’s self-image and is a well-known organ of sexual attraction and beauty. Socially there is a widely held belief that, the skin is the mirror of the soul and skin diseases have been demonstrated to be accelerated by emotional factors. The fear of contagion affects both patients and care givers probably as a relic from the days of the biblical Job and the castigated lepers in the bible (Hebrew Bible: Job1). Diagnosis and treatment of skin conditions is made more complicated by the fact that the patient can appraise their skin and unsuccessful treatments cannot be disguised (Burton J. L: 1985:3). The accurate diagnosis of skin conditions can only be done through physical examination of the skin and its appendages. The conditions mostly present with surface manifestations called lesions which are distinct and particular to a specific condition. Historical information is paramount in the diagnosis of cutaneous conditions; other important parameters include lesion morphology, configuration as well as distribution on the patient (Callen, Jeffrey:

2000). While on average, less than 10% of the human population suffer from cutaneous conditions, all albinistic persons suffer from debilitating and fatal skin diseases throughout their lives. Diagnosing and appraisal of albinistic skin is usually complicated by the fact that skin conditions in albinistic persons rarely occur singularly but they occur as exaggerated combinations of various afflictions.

2.3.1 Solar Induced complications in albinism

Albinistic persons suffer from various forms of actinic damage due to the compromised melanisation process. From the short cosmic rays right up to long low energy radio waves, the sun emits a continuous spectrum of electromagnetic radiation that is inimical to compromised skin. While normal healthy skin can absorb and safely dissipate most of the waves and prevent actinic damage, albinistic skin is badly affected even by low radiation waves (Burton J .L; 1985.157).

2.3.1.1 Acute effects

When albinistic skin is exposed to UVR, the latent period before erythema develops is very short. In normal healthy skin, even though melanin transforms up to 99.9% of the

UVR into harmless heat, the damage from the remaining small fraction of radiation is still enough to cause sunburn (John A. et al; 1982). The Minimal Erythermal Dose

(MED) used as a clinical measure of individual susceptibility to sunburn is greatly decreased in albinistic persons. Any dose, even below the MED in normal skin will cause oedema, blistering, nausea, headaches and even rigors in albinistic persons

(Burton J L: 1985). During recovery (which is much slower in genetically compromised skin), there is widespread desquamation and pruritus. The absorption spectrum of melanin is more biased towards UVB than UVA. The action spectrum of acute actinic damage is directly related to the absorption spectrum of melanin and it is therefore generally accepted that lack of melanisation is the cause of sunburn. In higher doses,

UVA can also cause erythema just like UVB (John A et al: 1982).

2.3.1.2 Chronic effect

UVR is responsible for a host of changes we associate with ageing, like solar elastosis and wrinkling. These changes are much more evident in tropical areas where UVR onslaughts are relentless throughout the year. Actinic keratoses affect almost all albinistic persons. They manifests as scaly lesions usually at the outer side of the arms, forehead and can virtually cover all exposed skin areas. These scaly plaques may itch or even ulcerate. Solar keratoses are generally referred to as pre-malignant because at least 20% of them may progress to squamous cell carcinoma (Prajapati V: 2008). They are found on almost all PLWA and may occur even on the lips

2.3.2 Principles of Dermatological treatments

Regardless of how benign it could be, most patients do not tolerate skin diseases. The runaway success of the cosmetic industry is clear testimony that even minor, real and imagined skin anomalies are not welcome. Any skin disease is visible and individual appraisal is simple and so, the patients are demanding. Treatment of dermatological conditions requires a holistic approach which includes the patient’s personality, occupation and social background. In most cases, how the patient uses treatments is just as important as what the treatment is. Self-medication is very common in dermatology and the use of multiple treatments is rife (Burton J.L: 1985.247).

Environmental factors play a big role and the effects of heat, allergens, detergents, type of clothing as well as personal hygiene are critical in successful treatment of

dermatological conditions. Most conditions come back after the treatment has run out due to the inadequacy of treatment regiments that do not take into account environmental factors.

Drugs for topical applications are usually carried in a base or vehicle. The choice of base determines whether ingredients will penetrate the skin or stay on the surface. At times, treatments are required to permeate the skin and frequently they are required to sit on the skin periphery. The choice of whether to formulate a powder, cream, an ointment, a suspension or a solution is paramount in product efficacy as well as acceptance by patients, which is critical in treatment adherence (Burton J L: 1985:250).

2.4 PERSONAL CARE PRODUCTS

2.4.1 Cosmetics

The legal regulatory frameworks from the Food Drug Administration (F.D.A) the E.E.C,

South African Bureau of Standards (SABS) and the Standards Association of Zimbabwe

(SAZ) define cosmetics by their intended or generally assumed use. The term is extended to include all materials that are used as ingredients or components in cosmetic products (Gardner R: 2010). The law therefore generally defines cosmetics as

"articles intended to be rubbed, poured, sprinkled, or sprayed on, introduced into, or otherwise applied to the human body, for cleansing, beautifying, promoting attractiveness, or altering the appearance” (FDA/CFSAN: 2005). This definition includes among other various products: body lotions and creams, facial treatments, deodorants and perfumes; color cosmetics, fingernail polishes, eye and facial makeup

preparations; hair care treatments, relaxers, permanent hair waves, hair colors; Oral care products, mouthwashes, breath fresheners and teeth whitening treatments

(COLIPA; 1997).

2.4.2 Drugs

The legal definition for drugs is clear and unambiguous. Regulatory bodies including the

FDA, EEC and the World Health Organization (WHO) define drugs by their intended use, as "(A) articles intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease and (B) articles (other than food) intended to affect the structure or any function of the body of man or other animals"(FDA/CFSAN: 2005). The regulatory bodies have requirements that pertain to drugs which do not apply to cosmetics, these include, registration requirements, labeling requirements, good manufacturing practice (GMP) requirements as well as distribution or dispensing requirements (FDA/CFSAN: 2005). All claims made have to be scientifically substantiated otherwise the drug will be regarded as misbranded.

A medication is a drug dosage form taken to mitigate symptoms of a medical condition or illness or to provide future benefits in preventing a medical condition (Aulton, (2007).

Local regulatory authorities or statutory bodies control the dispensing or retailing of drugs. Dispensing of drugs is regulated by these three categories;

 Prescription only medications (P.O.M) are given or prescribed by a medical

professional.

 Over the counter medications (O.T.C) are dispensed with little or no restrictions.

 Behind the counter medications (BTC) which can be dispensed by a pharmacist

only without a doctor’s prescription (EDLIZ; 2011).

2.4.3 Cosmeceuticals

Many products found in pharmacies meet both cosmetics and drugs definitions. This happens when the product has more than one intended use, for example, a deodorant is a cosmetic because its intended use is to mask body mal odors; whereas an antiperspirant is drug because it incorporates, salts that stop sweat secretion in the axilla. A deodorant antiperspirant is therefore a cosmetic – drug combination. The list of such products, referred to as cosmeceuticals, is long and it includes antidandruff shampoos, herbal dermatological creams, fluoride anti-cavity toothpastes and sunscreen products (DermIS: 2005).

This marriage between pharmaceuticals and cosmetics is not independently regulated.

Currently there is no legal definition of a cosmeceutical. Regulatory authorities have no provision for such hybrid products (FDA/CFSAN: 2005). If a product is launched with claims to affect the structure and function of the body, regulatory authorities will control it as a new drug. Despite the absence of dermatological research, which shows that topical cosmeceutical creams with bioactive ingredients have benefits beyond the traditional moisturizer, there are no legal restraints controlling their use and retail (Chen et al: 2005). Depending on the intended use, a cosmeceutical product will be regulated as either a drug or a cosmetic. A personal care product’s intended use might be established in a number of ways. Among them are the medical claims made by the product labeling, through advertising material, or in other promotional materials (CTFA; 49

1993). Product claims will cause a product to be regarded as a drug, even though the manufacturer might wish to market it as a cosmetic, as long as the intended use is to either treat or prevent disease or to otherwise affect structure or functions of the human body (FDA/CFSAN: 2005). Examples of such medical claims in cosmeceuticals include claims that products will restore hair growth, treat cellulite or varicose veins, or remove wrinkles.

Medicinal claims may also be insinuated by consumer perceptions that have long been established through a product’s traditional reputation (CTFA; 1993). For example, the perceived reputation of the therapeutic value of vitamins by consumers is long established even though there is no scientific evidence that vitamins through topical applications can nourish skin (FDA/CFSAN: 2005).

2.4.4 Emerging technologies in Cosmeceuticals

Emerging technologies are technical developments distinct from conventional technologies that transcend new boundaries with significant impact on society, individuals and industry. They are characterized by innovations that usher in competitive advantage and breakthrough solutions and improvements to conventional techniques, products and processes (Raysman R: (2002). These contemporary advances in various fields could be from theoretical exploratory research or applied commercial research.

Emerging technologies offer both incremental developments as well as ‘disruptive’ technological growth (Hung D: 2006)).

Emerging Technologies (ET) that are relevant to cosmeceuticals include nanotechnology, biotechnology, information and communication technology, cognitive 50

sciences as well as educational technology. These distinct emerging technologies have a tendency to evolve towards similar goals and have converging intersections. The term converging technologies is used to describe the dis-similar processes that tend to move towards strong interconnections through sharing resources as well as developing innovative efficiencies (Kaldis, Byron: 2010).

2.4.5 Nanotechnology

The American national nanotechnology Initiative defines this emerging technology as the manipulation as well as use of materials at both the atomic and sub-molecular level with dimensions sized below 100 nm (Bowman D: 2007). At this quantum realm the definition does not concentrate on one technological goal but focuses on wide and encompassing research categories and technologies that dwell on the enhanced capacities of materials that emerge below precise dimension thresholds. This manipulation of matter has found potential use as an emerging technology in various fields including, medicine, electronics, textiles and material sciences as well as many other un-related fields (Fritz A et al: 2010).

Nanotechnology (nanotech) as a breakthrough technology offers potential use and improvements in; best proven manufacturing practice, enhanced food technology and production, intelligent drugs and improved therapeutics, enhancement of energy systems and water purification systems. Automation of tasks, which were previously unimaginable, is possible with nanotech due to the reduced size of materials (Fritz

Allhoff et al: 2010). Techno Utopians view nanotech as the “good shepherd” that is critical to humanity’s future through offering betterment for the human condition. 51

However, like all new technologies much ethical debates have been raised on the perceived risks imposed by nanotech and other emerging technologies, which should be borne in mind by researchers, involved in this category as potential threats (Fritz A et al: 2010).

2.4.6 Nano-medicine

The application of nanotechnology for medical purposes is referred to as nanomedicine. The current applications range from nanomaterials in pharmaceuticals to biosensors and medical molecular nano-modelling (Bowman D: 2007). Research seeks to harness the unique properties of nanomaterials for a wide range of applications.

Since most biological molecules are also at the nano scale, the combination of nanomaterials with biotechnology and pharmaceutics has ushered in new frontiers in targeted drug delivery, intelligent drugs, advanced analytical techniques as well as improved diagnostic devices (Swenson S et al: 2005).

2.4.7 Nano-Cosmeceuticals

In cosmetics, the benefits of nanotechnology are currently being investigated for employment in a number of areas. The first is the potential use of nano-particles as cosmeceutical delivery vehicles. The newer structures discussed above including nanostructured lipid carriers (NLC) and solid lipid nanoparticles (SLN) are regarded as next generation delivery agents to replace liposomes. They offer better bioavailability, better active stability as well as improved skin hydration and controlled occlusion (Muller

RH et al: 2007). Van Hal et al demonstrated that estradiol could be transported across the impervious Stratum corneum after encapsulation with nanometric niosomes (Van

Hal et al: 1996).

The second area of interest is the use of nano-emulsions as highly metastable dispersions of nano-scale phases. These emulsions are translucent and have better shelf lives than ordinary emulsions due to their greater surface area for interface absorption (Jenning V, Gothla S: 2001).

Nano-crystals are being investigated in research work that has already been protected by patents. Nano-crystals can be used for the delivery of insoluble actives in cosmeceuticals to improve dermal availability (Petersen R: 2008).

The potential use of metallic oxides as non-opaque broad-spectrum sunscreens has also been investigated. The sun blocking effects of metallic oxides of titanium dioxide and zinc oxide have always been known to science. Their limited use is mostly seen in

(specially prepared) sport sunscreen cream preparations used for outdoor activities like cricket and mountain climbing. Their unaesthetic nature is apparent. The use of the metallic oxides in practical, all day creams have not been possible hitherto. Chance observation and experiments have revealed to science that at the nano scale metallic oxides of zinc and titanium are not opaque and interestingly do not lose their sunscreen effects. This realization throws in a new novel potential application in reformulation of sunscreens with nano-scale particles for use in situations that have not been possible before (Lewis N: 2009).

2.4.8 Nano scale Titanium dioxide (TiO2) (CAS number 13463-67-7)

Titanium dioxide is also known by the IUPAC name, titanium (iv) Oxide. TiO2 traditionally has conformational polymorphism and occurs naturally as rutile, anatase and brookite polymorphs. The metallic oxide is also known as Titania and the other added names designated to it are based on the three different polymorphs (Marchand R et al: (1980). This white odorless solid has a molecular mass of 79.9 gmol-1 and the anatase form has a specific gravity of 3.78 while the rutile form has a specific gravity of

o 4.23. The melting point for both polymorphs is very high at 1843 C. TiO2 is virtually insoluble in water and the three polymorphs have different refractive indices

(Greenwood et al: 1984).

Recently two additional high-pressure forms were added to the TiO2 bouquet of polymorphs after the discovery of an orthorhombic and monoclinic, crystalline forms in

Bavaria (Goresy E. l et al: 2001). The most widespread ore in the world from which TiO2 is sourced, is the ilmenite ore, followed by the rutile ore, which contains up to 98% TiO2.

When heated to temperatures beyond 600 oC, the metastable anatase and the brookite polymorphs will undergo conformational transformation to the rutile polymorph.

Synthetically, TiO2 can also be produced as three metastable polymorphs, which include tetragonal, monoclinic as well as orthorhombic forms (Goresy E. l et al: 2001).

There are various methods of producing TiO2, the most common being the production of synthetic rutile from ilmenite by mixing it with Sulphuric acid. The Becher® process may also be utilized to produce synthetic rutile from ilmenite. This method is much related to the chloride process whereby the crude TiO2 is reduced with carbon before oxidizing it

with chlorine to give titanium tetrachloride. After this carbo-thermal chlorination, the titanium tetra chloride undergoes distillation and subsequent re-oxidization to give pure titanium dioxide. These methods are for the production of normal macromolecular TiO2

(Goresy E. l et al: 2001).

2.4.8.1.1 TiO2 and UV blocking

Apart from its other many uses in the food, paint and detergents industries, TiO2 has cardinal importance in cosmeceuticals and general skincare as a sunscreen, opacifier, formulation thickener as well as a pigment for cosmeceutic use. TiO2 is available in varying sizes depending on the intended function (Lewis, Nathan: 2009).

TiO2 in both macro and nano particle size is an excellent physical sunblock characterized by high UVR absorbance, high refractive index and inability to withstand photo degradation and discoloration under UVR onslaught (Winkler, Jochen: 2003).

TiO2 in its various forms is therefore a sunblock of choice in skin protection. Research has shown that nano-scaled TiO2 provides the same protection as the macromolecule.

Sunscreens for sensitized skin and babies are often based on TiO2 and ZnO as these metallic oxides cause less irritation than other UVR absorbers (Lewis, Nathan: 2009).

2.4.9 Nano scale zinc oxide (ZnO) (CAS number 1314-13-2)

Zinc Oxide is also commercially known as zinc flower, zinc white or calamine. Virtually insoluble in water, ZnO is a white odourless solid with a specific gravity of 5.6, a molecular mass of 81.4 gmol-1 and a melting point of 1975 oC. The refractive index is high at 2.0041nD (Özgür, et al: 2005). ZnO occurs naturally as the mineral zincite, which 55

usually has manganese and other metallic impurities, which imparts a yellowish color to the natural mineral. Most of the commercially available ZnO is however, synthetic. This metallic oxide is thermo-chromic; it converts from pure white to yellow on heating and reverts upon cooling. The amphoteric oxide is soluble in most acids including hydrochloric acid (Craddock P. T: 2008).

ZnO is available in two crystalline polymorphs. Wurtzite is the hexagonal polymorph and

Zincblende is the cubic form. In industrial synthesis, there is a process preferential formation of Wurtzite over Zincblende (Wiberg E :2001).

2.4.9.1.1 ZnO nanostructures

ZnO can be synthesized as nanostructures with various morphologies. Nanoparticles are produced via a number of routes that include metalorganic vapor phase epitaxy, vapor-liquid-solid method, sol gel synthesis, atomic layer deposition and the chemical vapor deposition method. The morphology of the resultant nanoparticles is controlled by altering process parameters such as temperature, concentration and pH (Hua G et al:

2008).

Aqueous methods for producing nanostructures of ZnO are preferred because the processes can easily be scaled up due to their low cost and unhazardous nature. The morphology of the nanoparticles is easy to manipulate using this technique (Hua G et al:

2008).

2.4.9.1.2 Cosmeceutical Applications of ZnO

In both the crude and processed form, humans have always used ZnO compounds for eons as a medicinal ointment. Early Indian texts like the Charaka Samhita have accounts of the Pushpanjan (ZnO) as a salve for wounds and burns (Craddock; P. T. et al: 1998). The Greek 1st century physician Dioscorides and the Avicenna writings in

“The Canon of Medicine” (1025 AD) talk about ZnO as a treatment of choice for various skin conditions including skin cancer (Craddock P. T. (2008).

Currently, ZnO has remained very popular for skin conditions treatment. Calamine, a mixture of ZnO and 5% Fe2O3 is the base for calamine lotion widely used for topical treatments. Zinc oxide is still widely used in baby products for diaper dermatitis, in antiseptic ointments and as ZnO tape for athletes which prevents soft tissue damage during workouts (Agren, MS: 2009).

However, the use in sunscreens draws the most attention here. ZnO is the most photo- stable and broadest spectrum UVA and UVB sunblock approved for use by the FDA. As a physical sunscreen just like TiO2, ZnO is non-irritating, it is non-allergenic and has been shown to be non-comedogenic (Burnett M. E: 2011).

A number of researchers are also currently studying the effects of using nano scale ZnO in sunscreens after concerns that they may be slightly absorbed into the skin. Published studies have come up with conflicting conclusions on the systemic absorption of zinc oxide (Burnett M. E: 2011).

2.4.9.2 Interaction of materials with incident electromagnetic

radiation.

During interaction with Visible and UV radiation, ZnO and TiO2 display unique optical properties with regards to colour and transparency when nanosized compared to their bulk. The determinant of colour in materials is the nature of interaction between incident light and the material surface. What is perceived as colour in materials is the result of unabsorbed light wavelengths that lie within the visible section of the spectrum. All absorbed wavelengths will not be observed as colour and the consequent colour is from reflected light only (Filipponi L, Sutherland D: 2013). When incident light strike the surface of a material, various phenomena may occur and the light can either be transmitted, absorbed or reflected. The general equation that governs the fate of incident light is therefore:

I = T + A + R

Key: I stands for the Incident wave, T is for the transmitted, A for the absorbed and R for the reflected waves

However, as the size of the particles in materials is reduced in dimensions, a fourth new phenomenon called scattering (S) may also arise, which contributes to the perceived colour of materials (Filipponi L, Sutherland D: 2013).

2.4.9.3 Mechanisms of incident light interaction with materials

surfaces

Reflection (R): This is a process that occurs when incident light waves strike material surfaces, the incident waves do not penetrate the smooth material surface but are redirected and the direction is reversed back into the medium where the incident wave originated from. The geometric structure of the waves remains unaltered in any way

(Filipponi L, Sutherland D: 2013).

Absorption (A) : This process is not merely a redirection of incident rays but involves energy transformation. The mechanisms is based on the excitations of electrons to higher energy levels after absorption of specific wavelengths of incident radiation which is depended on the energy levels of a material and its chemical identity. This process is independent from particle size (Filipponi L, Sutherland D: 2013).

Transmission (T): This is the third phenomenon that occurs in both macromolecular and micronized substances interactive process with incident light. In this instance the incident light passes through the material, Transmission is further associated with absorption and is the fate of the incident light after S, A and R have occurred (Filipponi

L, Sutherland D: 2013).

Scattering (S): This is perhaps the phenomenon that is of most interest with regards to nanomaterials. The process occurs when radiation is incident on materials with size dimensions that are in the same scale with the radiation wavelength. This physical

process is affected by the cluster dimensions, and the refractive indices of both the cluster and the colloidal medium. This physical process is not associated with any energy transformations but is characterised by redirection of radiation in various directions (Filipponi L, Sutherland D: 2013).

In scattering there is no change in wavelength of incident radiation, it is rarely a single interaction of incident light with a particular surface but multiple interactions of a particular wave and various clusters characterised by multiple redirections by numerous clusters in the medium, a phenomenon referred to as multiple scattering. Scattering can also be ‘back’ and ‘forward’ scattering depending on the ultimate direction of the scattered waves (Filipponi L, Sutherland D: 2013).

The key point to note in scattering which can therefore be manipulated in nanosciences is that maximum scattering of incident radiation occurs at wavelengths that are double the cluster dimensions: for example, maximum scattering for a cluster measuring

100nm occurs at wavelengths measuring 200nm. The category and nature of wavelengths reflected is therefore depended on the cluster size of particles congregation in a material (Smijs T G, Pavel: 2011) (Filipponi L, Sutherland D: 2013).

2.4.9.4 Development of transparent products from opaque ZnO and

TiO2

The white tactile appearance of protective sunscreens based on metallic oxides of Zn and Ti is as a result of the scattering of light in the visible section of the spectrum.

Typical micronized commercialized ZnO and TiO2 used in sunscreens had been to the order of dimensions around 0.2micrometers or 200nm. As visible light interacts with clusters at 200nm, maximum scattering occurs at wavelengths twice the dimension of the clusters which is 400nm. Radiation wavelengths measuring 400nm fall within the visible section of the electromagnetic spectrum. All light is scattered at this dimension and therefore the sunscreens appear white (Filipponi L, Sutherland D: 2013).

However if the dimensions of the clusters are reduced from the commercial 200nm to

100nm (which falls within the nano scale range), the maximum scattered radiation wavelengths will correspondingly fall to 200nm. The result is that the same material which has been nanosized no longer has maximum scattering within the visible section but the UV section of the electromagnetic spectrum. The sunscreen therefore no longer appears white but transparent. Figure 2:12 depicts the scattering curves for 100 nm and

200 nm ZnO clusters as an illustration of this phenomenon (Detoni C.B et al: 2014).

Figure 2-12: The diﬀerent light scattering of bulk and nano-sized ZnO clusters (Adapted from (Filipponi L, Sutherland D: 2013).

2.4.9.5 UVR attenuation mechanisms of physical sunscreens

The UV attenuation properties of TiO2 and ZnO are complimentary, TiO2 is mostly a

UVB absorbing compound and ZnO is primarily best in UVA absorption.

The combination of these two in formulations as proposed in this development work therefore assures broad spectrum UV protection. Size related optical particle properties as well as the surrounding medium also affect UV radiation attenuation. Attenuation is also resultant from the reflection and scattering of UV and visible radiation (Detoni C.B et al: 2014).

ZnO and TiO2 both combine aspects of absorption reflection and scattering which give them the broad spectrum sunscreen effects. Paramount optical effects of the metallic oxides are the refractive indices in both the UV and the visible wavelength range. An 62

average refractive index (n) ranging from 3.6 to 4 has been reported for the different forms of rutile and anatase TiO2. An average refractive index of 2.4 has been reported for ZnO. The whiteness of these metallic oxides is partly due to these high refractive indices(Filipponi L, Sutherland D: 2013) (Detoni C.B et al: 2014).

Both materials are semiconducting materials, which means that they have electronic structures that are composed of bands of orbitals that are separated by energy band gaps devoid of molecular orbitals (Figure 2:13). Radiation absorption that supposedly equals the band gap between the valence and the conduction bands will result in the consequent excitation of valence band electrons to the conduction band leaving holes in the valence band. This resultant hole therefore represents localized electron vacancy in the particles. For rutile titanium, the band gap energy is approximately 3.03eV and for

Zinc it has been determined to be around 3.22Ev. Therefore as expected with the

3.1Ev band gap width, TiO2 particles will not absorb visible light but will effectively scatter and reflect it absorption occurs in the UV range. The TiO2 valence band accommodates many absorption possibilities whenever the energy absorption exceeds the band gap width. TiO2 will therefore absorb more in the UVB part of the spectrum while ZnO absorbs more in the UVA range. The Zinc energy gap coincides with 385nm, and therefore ZnO will absorb all radiation with wavelengths shorter than 385nm (Detoni

C.B et al: 2014).

Figure 2-13: Illustration of the band model for semiconductors like Zn and Ti

2.4.9.6 Side effects and toxicity of ZnO and TiO2

ZnO is generally recognized as safe (GRAS) by the FDA and its affiliates. On the other hand TiO2 is classified by the IARC as a group 2B carcinogen (which implies, possibly carcinogenic) based on a study which showed that very high concentrations of micronized TiO2 were linked to respiratory tract cancer in rats, Cytotoxicity and genotoxicity of both metallic oxides have been claimed in contradicting studies on their photo catalytic activity. Uchino et al and Sayes et al described the photo activity of TiO2 on Chinese hamster ovary (CHO) cells. The authors however needed considerably very high concentrations of 1g L-1 to achieve photo effects that were significant enough to be measured (Smijs T G, Pavel: 2011).

Studies focusing on the genotoxicity of TiO2 and ZnO vary considerably in terms of the significance and interpretation of the results. In most (if not all) of the studies, though the metallic oxides have been characterised, no attempt is made to correlate obtained toxicity data results to nano-particulate physicochemical characteristics. Even though a

number of authors have alluded to the fact that in surface chemistry, the size and shape of particles were determinants of material toxicity, they conclude that direct scientific proof of this was insufficient. Precise scientifically validated mechanisms related to TiO2 and ZnO toxicity (if any) remains generally unknown (Smijs T G, Pavel: 2011). All regulatory bodies including the FDA, the OECD and the EC-SCCP opinions generally recognize TiO2 and ZnO as generally safe and recommend their unlimited use as broad spectrum sun-blocks in cosmeceutical and pharmaceutical formulations (Smijs T G,

Pavel: 2011).

2.4.10 Botanicals in cosmeceuticals

By March 1998, a review of cosmetic formulations listed under the International cosmetics voluntary registration program (CVRP), revealed that of the 15774 reviewed formulations in that year, 6087 or 38% had botanical extracts as active ingredients

(FDA/CFSAN :2005). In the same year at least 205 different plant species extracts were on the recommended list by the CIR and in frequent use by leading personal care manufacturers (CTFA; 1993). Through the CIR, the CTFA annually reviews cosmetic ingredients for their safety and efficacy for inclusion or withdrawal from the continuously updated cosmetic handbook or the International Cosmetic Ingredients Dictionary

(INCID) (CTFA; 1993). Ingredients reviewed and adopted by the CIR are publicly gazetted and designated as International Cosmetics Ingredients (INCI) for the benefit of all the worldwide cosmetics manufacturers and they are allocated approval CAS numbers (CTFA; 1993).

2.4.11 Plant Oils

Oils and fats are nonpolar, neutral and hydrophobic chemical substances. The difference between oils and fats is the state at ambient temperatures. Oils are liquid and fats are semi solid to solid at room temperature. Oils generally have high carbon and hydrogen contents and have a slippery feel to touch. The solid form of hydrophobic substances with oil like properties is a wax (Harper Douglas “oil”; online dictionary on etymology: 2009).

The general definition of lipophilic substances incorporates various categories of compounds some of which are unrelated in stereochemistry properties, structure and application. Oils and fats are usually obtained from living organisms (plant and animals) as well as fossils in the case of petrochemical or mineral oils (Cooley: 2002).

These organic oils are produced by a diversity of flora and fauna through metabolic processes. The scientific term ‘lipid’ is used to refer to fatty acids and other hydrophobic chemicals often found as constituents of oils. Organic oils usually contain various other chemicals apart from lipids, including proteins and alkaloids (Cooley: 2002).

Different kinds of organic oil extracts have found use in cosmeceuticals and pharmaceuticals as either emollients or active pharmaceutical ingredients. These include essential oils, fixed oils and “mineral” oils which application depends on the chemical nature of the material being sought and the consequent use of the extracts.

2.4.12 Essential oils

A number of different commercial names, which include essential oils, volatile oils or ethereal oils, refer to volatile hydrophobic liquids containing aromatic chemical groups.

They may also be known as aetherolea, but mostly they may just be referred to as the oil extracts of the plant they are obtained from, such as “lavender oil”. The term

‘essential’ derives from the fact that the oils are characterized by distinctive essences of the specific plant. Despite public opinion and perception, essential oils are not in the real sense essential for health and they are not a specialized category for any medical or pharmaceutical purpose (Gilman et al: 1990).

The methods of extraction for obtaining essential oils include steam and hydro- distillation. Solvent extraction and cold expression can be used to draw them out of the plant parts that yield them (Gilman et al: 1990). Historically essential oils have been used for medicinal and culinary purposes. Ancient texts including the Hebrew Bible, the

Koran and ancient Indian writings all extol the virtues of aromatic volatile plant oils including Myrah, cinnamon, arnica and others. Evidence based medicine prefers not to generalize essential oils as a category but to refer to specific volatile compounds in their own rite. For example, methyl salicylate is referred to, as such, in medicine and not as

Wintergreen oil. Cosmeceutical use however prefers the opposite and commercially refers to the plant origin name due to the aura associated with mysterious oil ingredients and in pursuant of the ‘natural’ product image (Klaassen et al: 1991).

Resurgence in global interest in essential oils has been attributed to the rise in alternative medicines and its branches that include aromatherapy. Ibn al-Baitar, a 12th

century pharmacist and alchemist penned the earliest recoded technique of processing essential oils (Houtsma M: 1993).

2.4.13 Fixed oils

Fixed oils are triglycerides of vegetable or animal origin. These carrier oils also known as base oils are the non-volatile part of plant or animal extracts, which are oily. They are typically obtained from seeds, nuts or beans. These oils are referred to as fixed because they are non-volatile large molecules unlike essentials oils (Cooley: 2005).

The mixtures of both saturated and unsaturated fatty acids usually also carry vitamins and minerals and other medicinal components at trace levels (Gundidza et al 1993).

The most common means of extraction of fixed oils for pharmaceutical purposes are cold pressing and solvent extraction. Solvent extraction is only acceptable if the solvent can feasibly be all driven out after extraction to leave an unadulterated oil. Extracted fixed oils are subsequently refined so as to remove impurities, improve on color as well as odors. Refining will also preserve the oils from rapid aerial oxidation (Gundidza et al:

1993). Fixed oils have found extensive employment in the cosmetic and pharmaceutical industry as formulation emollients, active ingredients as well as functional products.

Unlike essential oils, fixed oils can be applied directly to the skin without any carriers

(Roy Gardner: 2010).

2.4.14 Trichilia emetica

English: Natal Mahogany, Banket mahogany

Shona: Mutsikiri, Muchichiri

One indigenous plant that has been extensively used in this country as a botanical panacea is the Natal Mahogany. Trichilia emetica belongs to the family Meliaceae. The trees proliferate in riverine settings and open woodlands throughout Zimbabwe. The tree density per square kilometer is however more concentrated in Manicaland and the

Zambezi valley occurring at medium to low altitudes (Van Wyk B.E et al: 2009). Its appearance in clusters of large thriving trees in selected built urban sections like the

Harare Institute of Technology car park, Parirenyatwa group of hospitals grounds and the Hwange town centre is testimony that the tree can be successfully artificially propagated in Zimbabwe.

Figure 2-14: Artificially propagated Trichilia plant and the leaves and fruits of the plant (Pictures taken by author February 2014)

The Natal Mahogany is an evergreen, handsome medium sized tree which can grow up to 12 m high (Van Wyk B.E et al: 2009). The crown is rounded and dense giving a

beautiful shade and the trunk is woody with a greyish to brown bark. The dark green, glossy leaves which are about 12 cm long are hairy underneath and are divided into pairs. The tree flowers with yellowish green petals which give greenish brown fruit. The fruit which matures from December to March is almost spherical, 2.5-3 cm in diameter, splitting into 2 or three valves with a distinct neck. The seeds are black and are almost completely covered by a red envelope giving the impression of a “doll’s eyes” (fig 2.24 and 2-25) (Van Wyk B.E et al: 2009).

In Zimbabwe, like in any other habitat where it is found, the tree is surrounded by an aura of awe, legend and superstition and the fruits and seeds are never eaten by birds or apes (Diallo D et al 2003). Thonga and Ndau tradition has it on good account that the bark of the tree is soaked in water and used as an enema or emetic and the seeds provide a superior quality oil used to anoint the whole body, the oil is also believed to heal fractured limbs (Diallo D et al: 2003). The leaves are traditionally used to induce sleep when placed in the bed at night. The bitter tasting oil or fat ( since it is solid in cold weather) is used for culinary purposes by the Thonga. With its magnificent self- preserving efficacy the fat with slight processing can remain unspoiled for up to two years and is usually traditionally stored as a food reserve. The Ndau and the Ronga use this oil extensively as a cure for rheumatism and as a remedy for leprosy (Germano

M P et al: 2006).

Nonetheless, myth or reality, it is however the cosmeceutical attributes of this mysterious tree that have prompted use in this research study. The women of Gazaland traditionally prepare cosmetic oils from the seed fat. The Thonga apply the oil to bruises as a soothing lotion (Germano M. P et al: 2006). The fruit and seed extracts are 70

applied locally to treat eczema and the seed oil is used extensively to relieve itch and skin dryness (Van Wyk B.E et al: 2009). The oil extract has also been used to make good soap. Perhaps what draws the biggest source of curiosity, interest and attention for every cosmeceutic scientist is the seed kernel. For centuries people in arid areas like the Middle East have used natural oils rich in oleic acid, to combat skin dehydration and reduce hair damage caused by arid conditions. Their intuition told them this worked and it has now been proven scientifically that skin cells minimize the accumulation of polyunsaturated fatty acids to protect themselves against free radical formation and thus cellular damage leading to actinic aging (DermiIS: 2009). In cosmeceutical formulation, this natural affinity allows for easy absorbance without the greasy feeling typical of some oils. The Mutsikiri kernel is believed to have at least 65 % oils from at least 3 different free fatty acids (Germano M P et al: 2006). In cosmeceutics, the availability of stable free fatty acids in the Mutsikiri therefore imparts different spreading rates on skin application leading to aesthetic elegance during the cosmeceutic rub in process

(Germano M P et al: 2006). The elegance from such oils does not quickly disappear nor give a rough tactile perception, attributes to be steered clear of in all day wear cosmeceutic formulations (Gardner R: 2010). Traditionally, in Zimbabwe, Mutsikiri oils or fats are obtained by skimming the surface of water in which the seeds have been boiled after being ground and pounded (Gundidza interview: 2014). The oil is also traditionally rubbed onto the skin to relieve pains and aches and rheumatism, but most critically it is used by locals as a cosmetic emollient (Gundidza et al: 2014). Apart from the fixed oils, Trichilia emetica oil is also rich in Trichlin A, an alkaloid with various medicinal properties. 71

Figure 2-15: Trichilia emertica seeds and the trichlin A chemical structure (source: Pooley E :1993)

2.4.15 Aloe excelsa

English: Tree Aloe, Zimbabwean Aloe

Shona : Gavakava

Figure 2-16: The author illustrating the unique height of naturally growing Aloe excelsa at Khami ruins: 2014

Aloe excelsa is known as the Zimbabwean aloe due to the abundant specimens of the species around the Great Zimbabwe area of Southern Zimbabwe. This unique single 72

stemmed aloe can reach heights of over 5 metres (Figure 2-26). The curved rosette at the apex can be up to 1 metre long (Coates P K: 2002). The leaves are thick and succulent with thicknesses of over 3 cm recorded in some specimens. To protect the plant from animals, the leaves often do have reddish thorny spines in younger shorter plants, which disappear as the plant becomes taller and out of reach for most browsers

(Coates P K: 2002). The scarlet racemes flowers of aloe excelsa are differentiated from other species by their shortness and exhibition of a slight curvature (Figure 2-27). The entire length of the stem is almost always covered in dead leaves (Coates P K: 2002).

Figure 2-17: Picture showing the Inflorescence and short curved aloe excelsa raceme (picture by author: 2014)

Extensive studies by Gundidza et al: 2005, on the plant show that it has widely been used by the native people as a treatment for various skin ailments including burns and wounds. The plant extract contains aloin and various extracts which have spectrophotometric peaks in the UVR ranges of cosmeceutic concern and should therefore act as viable sunscreens (Gundidza et al: 2005). Traditionally, the tree aloe

has also been used as a treatment for sunburns, sores as well as a systemic remedy

(Coopoosamy R.M: 2010)

2.4.16 Myrothamnus Flabellifolia

English: Resurrection bush

Shona : Mufandichimuka / Rufandichimuka

Figure 2-18: Myrothamnus Flabellifolia in (left) the hydrated state and (right)) the dehydrated state ( pictures by author: 2014)

The resurrection bush is a large woody regenerative bush growing up to 1.5 m in height in colonies on rocky inselbergs, in most parts of Southern Africa. The plant has the unique ability to revive itself miraculously when a “dead” plant is exposed to water

(Figure 2-28). In dry conditions, the entire bush has the capacity to dehydrate itself to a complete air-dry state. In this “dead”, state the leaves, twigs shrink, and the plant color changes from a vibrant green to an unflattering brown. When water is supplied to the roots of the plant in this desiccated state. The bush resurrects and transforms back to

its vibrant color, shape and structure within minutes, Tribal names of the plant all seem to be related to this resurrection ability.

M. Flabellifolia is a medicinal plant of note to natives in areas where the plant originates.

Psychologically, its resurrection ability is viewed as a testimony of hope and resilience in African culture. The wide medical uses include inhalation of smoke for depression, and chest pains. The essential oil is used for wound sterilization, as an herbal tea and as a cough remedy. Influenza, hemorrhoids and stomach ailments have been reported to be treated traditionally by the plant. The essential oil has been demonstrated to be effective against a wide spectrum of microbial pathogens. The characterised constituents of the plant correspond with the various traditional medical claims.

2.5 FORMULATION OF DERMATOLOGICAL AND

COSMECEUTICAL PREPARATIONS

Cosmeceutical treatments always have other functions and functional materials other than the active ingredients. Due to the self-administration of most dermatological treatments, formulations must seek the safest, most effective and most convenient way of delivery of cosmeceutical active ingredients (Fishburn: 1965:1).

In cosmeceuticals formulation, the formulator must consider the physical, chemical and biological characteristics of all the active, functional and structural ingredients that are incorporated. The ingredients must all be CIR approved and must be compatible with

each other to ensure product stability, safety and efficacy. The release profiles of the active from the formulation matrix must also be clearly understood (Fishburn: 1965:8).

The formulation is designed to deliver the active pharmaceutical ingredient (API) to the appropriate cutes level. Formulators usually consider the following guidelines and assumptions (Burton J L: 1985.249).

 Skin periphery: no penetration is required, formulations in this category include

physical sunscreens, insect repellants and color cosmetics.

 Stratum corneum: formulations here include anti-fungal preparations.

 Epidermis and dermis: limited permeation is required here, formulations include

topical steroids for eczema and psoriasis.

 Sweat and Sebaceous gland: penetration is required here, examples include

topical applications for acne and anti-precipitants.

 Subcutaneous delivery: absorption is required here for example in the

treatment of angina using glyceryl trinitrate.

Quality control measures are also put in place during manufacture to maintain product stability. Congruent to this, the packaging should be such that the stability is maintained during the product’s shelf life (Gibson: 2004:301).

2.5.1 Regulations that affect cosmeceutical formulation development

The FDA does not legislate any pre-market approval for cosmeceutical products

(www.cfsan.fda.gov). In the EU however, cosmeceutic ingredients and the final products are subject to detailed regulatory control by the EEC council directive 76/7668/EEC and

its various amendments, before they are introduced onto the market (Iwobi M.U et al

2010). FDA, EU and SABS (South African Bureau of Standards) regulate that cosmeceutical products must list the ingredients contained in a product in descending order of prevalence on the labels. The FDA has also published a monograph with a list of approved sunscreens that can be used in formulations and the maximum concentration limits allowed (Gasparo F P et al: 1998).

2.5.2 Formulation of emulsion sunscreen dosage forms.

Formulating sunscreen products is much more complicated than formulating just basic cosmetics or dermatological creams with drug actives. Formulation development and aesthetic science expertise is both an art and a science. Developing a sunscreen emulsion, incorporating botanical extracts and drug actives for albinistic skin will unavoidably present a myriad of challenges to even the most experienced formulator.

The efficacy of the sunscreen or treatment is wholly depended on the formulation

(Carswell M: 2000). The ability of the treatment to protect the skin against UVR induced actinic damage is defined as the sunburn protecting factor (SPF). This dimensionless factor which is a ratio of how long you can stay in the sun after applying the sunscreen without developing barely perceptible sunburn compared to how long you can stay in the sun before developing sunburn without any sunscreen is depended on the formulation. It is influenced by the choice of sunscreen, emulsifiers used in the cream, emollients, the choice of other functional ingredients and the patient adherence to application recommendations (Gasparo FP et al: 1998).

The task at hand for the cosmeceutic formulator is to create a product with the optimum

SPF at the lowest cost and with the best aesthetic properties. This is by far no mean feat, for the formulation is composed of ingredients that are naturally immiscible

(Carswell M: 2000).

2.5.3 Emulsion technology

IUPAC nomenclature describes emulsions as fluid systems in which immiscible droplets are dispersed in a continuous phase (IUPAC: 1997). However, a more practical definition is perhaps, ‘a mixture of immiscible or normally un-blendable liquids’. One liquid is the dispersed phase and the other is the continuous or extended phase.

Emulsions are two phase systems belonging to a category referred to as colloids.

Emulsion technology therefore allows us to blend polar and non-polar ingredients, which is the cornerstone of cosmeceutical science (Aulton, Michael E, ed: (2007). In cosmeceuticals, oil and water based ingredients must be blended and oil and water can only be stably mixed through emulsion technology. A mixture of oil and water may either be an oil in water emulsion (O/W) or a water in oil emulsion (W/O) or even a multiple emulsion which could be an O/W/O or a W/O/W emulsion. Being liquids, emulsion dispersions are presumed to be statistically distributed with no internal structure (Aulton,

Michael E. Ed: 2007).

2.5.4 Emulsifiers used in Cosmeceuticals.

2.5.4.1 Stearic acid (CAS number 57-11-4)

The IUPAC name for stearic acid is octadecanoic acid (IUPAC.1997). A waxy, white, solid, saturated fatty acid constituted by an 18-carbon chain. After palmitic acid, stearic acid is the second most common fatty acid found in nature. It has a mean molecular weight of 284 gmol-1, a specific gravity of 0.94 and a melting point of 69.4 oC (Lide,

David R: 2009). Stearic acid is the primary emulsifier used in cosmetic emulsions like creams and lotions. The esters of stearic acid may be incorporated as pearlising agents in shampoos, soaps and various other cosmetics (David J et al: 2006). Stearic acid is largely the cornerstone of cosmeceutical emulsions, mostly because it is relatively inexpensive, widely available and chemically benign (Susan Budavari, ed: 1989).

2.5.4.1.1 Cetyl alcohol (CAS number 36653-82-4)

The IUPAC name for cetyl alcohol is Hexadecan-1-ol (IUPAC: 1997). Also known as palmityl alcohol, it is a white, flaky, waxy solid at room temperature. The chemical has a mean molecular weight of 244 gmol-1 and a specific gravity of 0.811. Cetyl alcohol has a melting point of 49 oC and is practically insoluble in water (Lide, David R: 2009).

It is a secondary emulsifier in cream and lotion formulation and a primary thickening agent, opacifier and emollient in the manufacture of skin care products (Smolinske,

Susan C: 1992). People living with eczema have been found to be sensitive to cetyl alcohol and its impurities, ironically though, it is used in most skin care formulations

(Gaul LE: 1969) (Soga F: 2004).

2.5.4.1.2 Glycerol Mono stearate (GMS) (CAS Number 123-94-4)

The IUPAC name for GMS is 2, 3-Dihydroxypropyl octadecanoate (IUPAC: 1997). It has a mean molecular mass of 358 gmol-1.It has a specific gravity of 0.97 and a melting point of 59 oC (Lide, David R: 2009). GMS is a hygroscopic, flaky powder, it is an odourless and sweet tasting organic molecule. One of the many glycerol esters of stearic acid, this compound exists in the body after the breakdown of fats (Jens Birk

Lauridsen: 1976). GMS is used mainly as a secondary emulsifier in cosmetic emulsions.

Just like cetyl alcohol, it has thickening effects on emulsions. Apart from emulsification, it has been shown to exhibit controlled release properties in pharmaceuticals as well as preservative properties (The British Pharmacopoeia Secretariat: 2009).

2.5.5 Emollients used in cosmeceuticals

Emollients are ingredients that are added to cosmeceuticals to impart either moisturizing effects or humectancy. The dermis secretes a natural oil called sebum through sebaceous glands; this oil is responsible for lubricating the skin as well as regulating the natural moisturizing factor (NMF) through occlusivity. There are two routes to moisturizing the skin. The first is to form an occlusive layer on the skin surface to replace lost sebum that retards any water loss through the skin. Oils and fats play this role extremely well. The second is to absorb the atmospheric humidity or moisture onto the skin through humectancy (Christoph et al: 2006).

2.5.5.1.1 Glycerol (CAS number 56-81-5)

The IUPAC name for Glycerol (also industrially known as Glycerin or Glycerine) is propane-1, 2, 3-triol (IUPAC: 1997). This heavy liquid has a specific gravity of 1.26, a mean molecular weight of 96 gmol-1 and a boiling point of 290 oC (Lide, David R: 2009).

This simple polyol is highly viscous, odourless, colorless and sweet to the palate. Its non-toxicity, hygroscopicity, humectancy and solubility in water have found the most widespread uses in pharmaceutical and cosmetic formulations than any other chemical

(Segur J.B: 1951). The glycerin structure is the backbone and foundation of all triglyceride fats and oils. Natural glycerine is obtained from the hydrolysis of fatty triglycerides to fatty acids as well as in the production of biodiesel (Nilles: 2005).

Synthetic glycerin is however also widely produced from propylene. Glycerin has found cardinal uses in personal care formulations as an emollient, humectant, and to provide smoothness and lubrication (Christoph et al: 2006).

2.5.5.1.2 Cocoa Butter

Also known as Theobroma oil, cocoa butter is composed of about 60% saturated fats

(mostly stearic and palmitic acid) and unsaturated fats. Cocoa butter has a melting point of 34.1 oC an iodine value of 32 and an acid value of 1.68 (Alfred T: 2002).This is a yellowish, waxy vegetable fat obtained from cocoa beans. Pharmaceutical formulations use it mostly as a base for medicinal suppositories. Being one of the most stable fats known, cosmeceutical preparations employ the velvety feel, aroma and emollient properties for skin care. Its remarkable moisturising capabilities are widely believed to

prevent stretch marks, treatment of chapped skin and to sooth itchy skin (Britannica

Online Encyclopedia: 2014). Cocoa butter is rich in natural anti-oxidants which prevent its own rancidity for up to 5 years (Liendo: 1997).

2.5.5.1.3 Lanolin

Lanolin is also referred to as wool wax or wool grease. This wax is obtained from sebaceous secretions of wool producing domesticated and wild animals. Since lanolin lacks triglycerides but has up to 97% sterol esters as constituents, it is therefore not a true fat even though the pharmacopoeia refers to it as wool fat (Barnett G: 1986).

Due to its waterproofing effect lanolin and its many derivatives are used in skin care as emollients (Alfred Thomas: 2002).

2.5.5.1.4 Vitamin E

The generic term ‘vitamin E’ refers to ten (10) lipid soluble compounds that both include tocopherols and tocotrienols. These are all anti-oxidants that stop the formation of reactive oxygen species (R.O.S) when fat is oxidized. In North America, the most common type of vitamin E is Ỿ tocopherol found in corn oil and soybean oil. The most biologically active variant type which is most common in all other areas is α tocopherol which is abundant in wheat germ oil and sunflower oil ( Brigelius-Flohé R: 1999).

The ten forms of Vitamin E belong to either of two groups, which consist of five tocopherols and five tocotrienols each. The variants are differentiated by Greek prefixes alpha, beta, gamma, delta and epsilon (α, β, Ỿ, ȣ and ɛ) respectively. The natural derivatives of tocopherols are only found in RRR configuration. Synthetic forms of 82

tocopherols are also commercially available as eight different stereoisomers of α tocopherol (Brigelius-Flohé R: 1999). Tocotrienols have no commercially available synthetic analogues mostly because they have been sparsely researched on and only

1% of published medical papers on vitamin E are on tocotrienols (Traber M.G: 1998).

As antioxidants, vitamin E derivatives function as peroxyl radical scavenging molecules, which inhibit the propagation of free radicals in the body through reactions that yield tocopherol radicals, which in the presence of hydrogen donors will be reduced back to a non-reactive state. Vitamin E derivatives are fat-soluble and are therefore incorporated in cell membranes protecting the cells from oxidative damage (Traber M.G: 1998).

2.5.6 Functional ingredients

Functional ingredients are structural raw materials that impart usability and stability properties to cosmeceuticals. They may impart this through adjusting rheology, pH, product preservation as well as spreadability.

2.5.6.1 Carbopol 940®

Carbopol 940® is a carbomer used as a gellant or thickener in most pharmaceutical and cosmetic formulations. Carbomers are water absorbing expanded molecules created by inserting a C2 unit in a given molecule; the molecule will continue to expand into a

‘polymer’ as more and more units are added. Carbomers are also called carbonn molecules. Where “n” is the number of allene groups in an expanded unit. The

technology of creating gellants out of ordinary molecules was developed by Remi

Chauvin (Remi Chauvin: 1995).

2.5.6.2 Triethanolamine (CAS number 102-71-6)

The designated IUPAC name for Triethanolamine (TEA) is Tris (2-hydroxyethyl) amine

(IUPAC: 1997). TEA has a mean molecular mass of 149 gmol-1, a boiling point of 335 oC and a specific gravity of 1.12. This highly viscous colorless to yellow organic chemical has the unique combination of being both a tertiary amine as well as a Triol (a molecule with 3 alcohol groups). By virtue of being an amine, TEA is a weak base. In large quantities, TEA is used as an emulsifier and surfactant. However, in small quantities this organic compound is the most common buffering agent used in the manufacture of emulsions (Simond M. R: 2012).

2.5.6.3 Parabens

‘Paraben’, is a generic term used for a series of parahydroxybenzoates (4- hydroxybenzoic acid). The pharmaceutical and cosmetic industry widely employs parabens, as preservatives. They possess excellent bactericidal and antifungal activity.

Parabens are the most common preservatives used due to their low cost, low toxicity, efficacy and reliability as compared to other preservative alternatives (Golden R: 2005).

They have been linked to breast cancer incidences, even though no direct relationship has been established by scientific research yet (Harvey P.W: 2004).

Parabens as mentioned above are esters of parahydroxybenzoic acid and the subsequent name is derived from the alkyl group esterified. The most common parabens are methyl paraben, ethyl paraben, propyl paraben, butyl paraben and hepta paraben. There are other less common parabens used sparsely that includes isopropyl paraben, isobutyl paraben and benzyl paraben (Soni M.G: 2005).

Although most parabens are found naturally in plant sources like blueberries, most commercially available parabens are synthetic analogues produced by a modified Kolbe

Schmitt® reaction, which utilizes potassium phenoxide and carbon dioxide. For most normal skin types, parabens are non-toxic, non-irritating and non-sensitizing (Soni M.G:

2005).

2.5.7 Aesthetic ingredients

Cosmeceuticals have a dual role as both drugs and cosmetics. Unlike general drugs, which are administered by professionals mostly through prescriptions and do not necessarily have to be appealing to patients. Cosmeceuticals compete for patients in an open market, they have to be aesthetically appealing so as to draw the attention of consumers, they should therefore look good, smell good as well as make the user enjoy the in use experience.

2.5.7.1 Cosmeceutical colorants

Colorants are surprisingly the most stringent category of cosmeceutical ingredients.

Colorants have basically three functions in cosmeceutical products, the first and obvious

one is the product aesthetic appeal, just to make a product look good and to reinforce product imagery. The second one is as a functional ingredient to protect the formulation photosensitive ingredients from UV radiation. The third instance is when colorants are used as the active ingredients in color cosmetics like facial make up and tattoos which may be permanent or temporary (Gardner R: 2009). Whatever the reasons, the formulator has for including colorants to the formulation; the critical aspect is that colorants are controlled cosmetic ingredients. Due to their diversity and variation of purity and sources, robust legislation exists worldwide that govern the use of colorants in cosmeceuticals (Iwobi M.U: 2010).

If a product for topical application incorporates a color additive in the formulation, it is mandatory for it to adhere to the following requirements (www.cfsan.fda.gov).

 Approval: All color additives must be approved by the FDA and the EU, EEC

council.

 Identification: An index exists for all approved color additives. The code of

Federal Regulations issues specifications for the identity of colorants, which must

be met.

 Certification: All approved dyes must be certified by the FDA prior to use.

 Use and restrictions: Color additives must be for the intended use stated in the

regulations. The regulations may also state the maximum permissible

concentration in the formula.

Color additives are further categorized into those subject to certification and those that are exempt from certification. Colors that are subject to certification include all synthetic organic dyes that are derived from petroleum products. The majority of cosmeceutical 86

colorants are in this category. Those that are not certifiable include all colorants that are obtained from natural inorganic, plant or animal sources. Though they are exempt from certification, they are still liable to specifications for identity and use regulations

(www.cfsan.fda.gov).

2.5.7.2 Fragrances

Fragrances are ingredients that are incorporated in cosmeceutical products for both aesthetic and functional reasons. First, they are added in, to mask the base odors of other ingredients, which may be unpleasant. They are mostly used to draw the attention of consumers to specific ingredients. Fragrances also reinforce product imagery

(Klaassen et al: 1991). The emergence of alternative medicines like aromatherapy reinforces the concept that the role of fragrances is not only subtle but also real. They do indeed have a psychodynamic effect, especially those drawn from natural essential oils (Gilman: 1990).

2.6 EFFICACY AND STABILITY TESTING OF

SUNSCREEN EMULSIONS

2.6.1 Sunburn Protection Factor (S.P.F) testing

The FDA approved a standardized SPF testing method in its 1993 tentative final monograph (TFM) currently being used by all researchers involved in SPF testing.

Before that, researchers used the FDA 1978 proposed monograph test method (Agin

P.P: 2002). In this 'one and only' method to determine the SPF of a sunscreen, the prescribed procedure is to determine the Minimum Erythermal Dose (MED) of a sunscreen first on between 20 and 25 subjects, not more and not less. The MED is defined as the amount of radiation required to produce barely perceptible erythema at between 22 to 24 hours after exposure to the irradiation.

The MED is determined by exposing the unprotected skin to a series of five incidences of geometrically increasing UVR. The radiation must be from a solar simulator xenon lamp, which emits radiation between 290 nm and 400 nm. The lamp must have a spectrum similar to the 10o solar zenith angle at sea level. The simulator should emit radiation simulating both UVA and UVB at sea level. The exposures exponentially increase at a rate of 25% of the previous exposure. After 22-24 hours, a trained practitioner appraises the exposed site for erythema (Agin P.P: 2002). The MED obtained for the unprotected skin here is referred to as the MEDUS.

The second stage is now to repeat the exposures, but this time with sunscreen- protected skin. The sunscreen is applied to the patient’s back and given 15 minutes to dry. The site is then exposed to seven, (not five as in MEDUS) geometrically increasing

UV irradiation. The geometric progressions are based on the expected SPF from the product. The MEDPS determined is the lowest dose that produces barely perceptible

sunburn between 22-24 hours after the incidence. The product SPF value is therefore the ratio between the MEDUS/MEDPS (Agin P.P: 2002). Based on the determined SPF, the product is categorized for commercial identification in three product category designations (PCD) as follows in Table 2-3.

Table 2-2: Product category designations of sunscreens

SPF PCD Less than 2 Not labeled as a sunscreen 2-12 minimal 12-30 moderate 30+ high

2.6.2 Franz diffusion studies

Skin diffusion is the random transfer of permeant molecules through a membrane driven by a concentration gradient. The movement is from a phase of high concentration to a phase of low concentration. In-vitro Franz diffusion is therefore the movement of a permeant from a vehicle in a donor chamber through a biological membrane mounted on Franz diffusion cells into a receptor fluid contained in a receptor chamber, This is a passive diffusion process characterized by the following terms (William AC: 2003):

 Permeation is the movement of the permeant molecules into or through the

biological membrane. The permeant may diffuse into the membrane and not

necessarily pass through it.

 Flux is the quantity of permeant molecules crossing the biological membrane into

the receptor chamber fluid. Flux is expressed as dimensions of mass/area/time.

Accumulation therefore becomes the amount of flux within a certain time

expressed in units of mass/time. 89

 Diffusivity is a function of the permeant and is a measure of how easily it

penetrates the membrane. It is designated in units of area/time.

 The permeability coefficient (Kp) is the rate of molecules penetrating the

membrane per unit concentration expressed as distance/time.

 The lag time is a period of increasing diffusion before stabilizing into the steady

diffusion state.

 The lipid permeability barrier depends on the tissue type with regards to lipids

abundance and arrangement. Permeability is not directly related to tissue

thickness.

2.6.3 Franz diffusion experimental design and equipment

Franz diffusion comes in a choice of either static or continuous flow diffusion cells.

Static vertical cells have a fixed volume receptor chamber and controlled temperature for the receptor phase. The cells should have a facility for stirring both the receptor and the donor phases (Broughna L and Maibach HI: 2005).

Franz diffusion cells studies are used to evaluate molecule uptake into or through a bio- membrane. They are used to study the steady state flux of compounds in formulations or even on their own. They can be used to study the finite dose permeation through selected membranes (Broughna L and Maibach HI: 2005).

In static diffusion permeability of membrane and the permeability of compound controls the flux, which ultimately determines the concentration in the receptor phase.

2.6.4 Tape stripping technique for dermato-pharmacokinetics

Wolf and Keddie (Wolf J: 1939) are widely regarded as the originators of the tape stripping techniques for evaluating skin penetration. The methodology was further developed at the Unilever Laboratory in Sharnbrook Bedford by Jenkins and Tresise in the late 1960s (Jenkins HL, Tresise HA: 1968). Human stratum corneum tape stripping is still a versatile, simple and efficient method used broadly for investigating topical and transdermal drug kinetics and penetration depth (Escobar-Chávez JJ: 2007). In tape stripping, a dermatological formulation is evaluated by successful removal of the stratum corneum cell layers using adhesive tape. The tape strips contain corneocytes and penetrating application material, which is subsequently evaluated by instrumental techniques. In its basic form, the tape stripping method is best used to determine the dermato-pharmacokinetics of formulations. The method is also used to study the homogeneous distribution of formulation in the skin stratum corneum. This minimally invasive technique has found application in in-vivo and in-vitro testing in both humans and animals including pigs, rats and rabbits (Escobar-Chávez JJ: 2007). The amount of cell layers removed is depended on a number of both intrinsic and extrinsic factors including anatomical site of excision, the animal age as well as the season.

Dermatological parameters such as Trans Epidermal Water Loss (TEWL), pH, and the

Natural Moisturizing Factor (NMF) are controlled by age, race, season and the skin type of the sample source (Laderman J et al: 2008).

External factors including removal force, the duration of the tape on the skin as well as the formulation characteristics also affect the amount of corneocytes removed

(Laderman J et al: 2008).

2.6.5 Draize Skin sensitivity and ocular testing

This acute toxicity set of tests developed by the famous FDA toxicologists J H Draize and his partner J M Spines in 1944 to ascertain the sensitivity profiles of cosmetics after incidences of blindness caused by mascara is controversial in many ways, though it is still widely in use (Draize J H et al: 1944). The controversy comes from the procedure, which involves the application of 5 ml or 5 g of the test material to the eye or skin of a restrained live animal and evaluation of the potential damage caused before rinsing it out. The infamous tests are carried out for 14 days with the eye or sensitized skin area being observed for oedema and erythema in the skin test. The notorious eye test will test for redness, cloudiness, blindness, discharge or ulceration of the tested eye. The test animal of choice is usually a rabbit. In the case of acute irreversible damage, the animals are euthanized, they are reused if the damage to the skin or eye is reversible

(Draize J H et al: 1944).

2.6.6 Anti Draize tests arguments

Animal rights groups complain that during the tests, the applied chemicals cause intense pain and burning to a very sensitive body part, the rabbit eyes are also kept open by clips for several days. The animals are restrained while they bleed and ulcer for

days with some even going blind. In the analogous skin irritancy test, the chemicals are applied to abraded skin leaving permanent damage (Huxley A: 2006).

2.6.7 Pro Draize tests proponents

Proponents argue that the Draize test is a mild one because the chemicals are washed out as soon as perceptible irritation is observed. The tests are mostly for cosmetics and substances expected to be very painful are not tested with this method. Eye tests are only conducted after evidence of non-sensitivity to the skin. In-vitro pre-screening tests are done before the actual Draize tests (Huxley A: 2006).

2.6.8 Stability testing

The rationale behind stability tests for Cosmeceuticals is to guarantee that the new formulation conforms to specifications for physical, chemical and shelf life standards.

The other objective is to ensure that products maintain their functionality and product aesthetics during storage (Hall N: 1999).

The huge technical differences in product specifications imply that stability tests cannot be standardized or prescribed. Testing protocols must be flexible enough to be modified to maintain a scientific basis for assessment. Specific methods can therefore be development to suit new and unorthodox technologies. Stability tests can either be conducted under accelerated conditions or in real time, under actual appropriate storage and transport conditions (Hall N: 1999).

2.6.9 Stability tests types

 Physical and chemical stability tests are designed to assess the product

aesthetics and functionality through checking for consistencies in pH, color,

viscosity, rheology and emulsion cracking.

 Packaging stability tests asses the compatibility and long-term effects of

packaging on the packaged product.

 Microbial challenge tests evaluate the resistance to contamination from fungal or

bacterial agents. Contamination may occur during manufacturing, during storage

or during the use of the product (Hall N: 1999).

2.6.10 Physico-chemical stability tests

Physical and chemical tests are stress tests to confirm that a product is not vulnerable to extreme conditions such as temperature and light. Manufactures should determine specialized stability testing for every product, which should consider the anticipated storage, transportation, and in use conditions. For all the tests done vital parameters should be monitored for stability. Common tests according to COLIPA (COLIPA: 2007) include:

2.6.10.1 Emulsion stability tests

Emulsion failure and separation into its phases of oil and water is referred to as cracking. Four factors destabilize emulsions and affect product shelf life, and these include coalescence, creaming, phase inversion and Ostwald ripening. A product’s 94

propensity to crack must be evaluated by various methods, which include the following

(Hall N: 1999):

2.6.10.2 Centrifugation

This is one way of evaluating the oil phase tendency to separate and rise to form a layer at the top, a phenomenon known as creaming. The test is carried out by heating the emulsion to 50 oC and then centrifuging it at 3000 rpm. The product is removed and checked for advent of creaming signs.

2.6.10.3 Photo stability

The formulation and the packaging must be evaluated for its stability to light exposure.

The product is placed in a glass as well as the commercial packaging on a window ledge. Another aluminum foil covered glass is placed in the same window as a control.

The product is then checked for discoloration periodically (Hall N: 1999).

2.6.10.4 Shock tests for transit stability

Transit tests are conducted on products to determine whether transportation may damage the product package and its contents. Vibrating tests on a pallet shaker will predict de-mixing of products in transit. The product may also be subjected to actual transit tests by placing it in a distribution or shipping environment for the duration of an actual distribution run (Hall N: 1999).

2.6.10.5 Microbiological challenge tests

There are four routes to microbiological contamination. These are equipment, raw material, post manufacture and in use contamination. Microbial preservation is important for the product functionality and quality as well as the consumer safety.

Various pathogens including candida, traces of pseudomonas aeruginosa and common staphylococcus aureus are notorious for invading poorly preserved cosmeceutic products. It is therefore imperative that stability testing include microbiological challenge tests, which incorporate the following (Hall N: 1999):

 Screening tests are done by easy test kits, which include dip slides or plate

counts. These quick methods are semi quantitative procedures that predict

contamination. Sampling techniques and the subsequent evaluation are quick

and easy and can be done by untrained personnel.

 Quantitative tests are analytical tests that actually determine the count level of

microbiological contamination in a product. These tests require expertise and

should be done by trained personnel. Methods for microbial isolation include

culturing and colony counts.

2.6.11 Instrumental and Classical product Analysis

Assaying of the active constituents of Cosmeceutical products require instrumental and classical analytical techniques. Active Pharmaceutical Ingredients may be analyzed either by chromatographic or spectrophotometric methods.

2.6.11.1 Atomic Absorption spectroscopy (AAS)

AAS is a spectrophotometric technique for analytical, quantitative and qualitative determination of elements based on light (optical radiation) absorption. AAS is sensitive for determining concentrations of elements up to parts per billion (ppb) in samples (Welz

B, Sperling M: 1999). This is a robust pharmaceutical analysis technique developed by

Professor Robert William Bunsen and co-researchers from the German University of

Heidelberg in the late 19th century (Welz B, Sperling M: 1999). AAS has found numerous uses in chemical analysis including:

 Clinical, qualitative and quantitative determination of metals in body fluids and

tissues.

 Quality assurance analysis in pharmaceutical manufacturing processes.

 Analysis of metals and inorganic ions in water samples.

2.6.11.1.1 Principles of AAS

AAS principles are based on the Beer-Lambert Law. It employs techniques of absorption spectrometry to determine concentrations of metallic analytes in samples by comparing measured absorbance of the sample analyte to known concentration standard analyte absorption (A. Walsh: 1955).

The elemental selectivity of AAS comes from the fact that each element has a particular unique radiation flux fingerprint. After absorbing a defined energy quantum, atoms can be promoted to a higher state (orbitals) for just a few nanoseconds. The wavelength of absorbed energy is particular to specific element electron transition. Therefore, specific

wavelengths belong to one element. The radiation flux of the sample analyte and that of the blank sample are measured by the instrument detector. The subsequent ratio of the two absorbances is related to the analyte concentration using the Beer-Lambert Law

(Welz B, Sperling M: 1999).

2.6.11.1.2 AAS Instrumentation

The analyte is atomized by either a flame or an electro-thermal atomizer first and the atoms are irradiated by optical radiation. The radiated atoms then go through the monochromator, which selects the specific element radiation by separating it from the radiation from other sources. This radiation flux is then finally measured by the detector

(Skoog, Douglas: 2007).

 The frequently used atomizers for practical applications are flame atomizers and

electro thermal atomizers. Specialized types are also available for specific

purposes including hydride, cold vapor and glow-discharge atomization.

 Air acetylene flame ionization is the oldest and commonest types used in AAS

and it operates at a temperature of 2300o C. The process includes de-solvation,

whereby the solvent is evaporated from the sample leaving solid particles which

are vapourized before they are atomized or dissociated into free atoms .The free

atoms are then subsequently ionized (Skoog, Douglas: 2007).

2.6.11.2 Inductively Coupled Plasma Atomic Emission

Spectroscopy (ICP-AES)

ICP-AES also known as ICP-OES( ICP-optical emission spectrometry) is a type of emission spectroscopy used for the detection of trace metals in samples. The analytical technique has exceptionally high selectivity and can detect analytes at concentrations down to parts per trillion (ppt). It is adept for detection of metallic atoms and other non- metals at such low concentration making it highly suitable for pharmaceutical trace analysis. This technique involves ionization of analyte with ICP and then employing the

AES to quantify the ions. As compared to other spectrometric methods like AAS, ICP-

AES is a robust, faster, more sensitive and precise technique. Unfortunately, the high precision comes at a cost by making the method more prone to trace contamination from utensils and reagents (Shane Elliott et al: 2004).

2.6.11.2.1 ICP-AES principles

The ICP plasma is obtained by inductively energizing the gas with electromagnetic coils.

This plasma contains enough ions and electrons to be conductive. The plasmas in instrumental analysis are neutral, with an equal amount of ions and electrons. The ICP is generated in a torch consisting of quartz concentric tubes. The temperature of the plasma is extremely high as compared to AAS (Shane Elliott et al: 2004).

By coupling to AES, the ions from ICP are channeled into an AES quadruple by a number of cones. The ions are subsequently separated in accordance to their electromagnetic emission spectrums. The detector then receives the ion signal that is

proportionate to analyte concentration. The concentration of the sample is therefore evaluated by calibrating with certified reference standards (Shane Elliott et al: 2004).

2.6.11.2.2 Applications of ICP-AES

Due to its high precision and sensitivity, this method is used for medical and forensic trace metal detection most specifically for toxicology. Environmental analysis for trace elements in large samples like water bodies and soil samples is also a primary use of

ICP-AES, due to its high sensitivity (Yip Y: 2007). Industrial and biological monitoring of heavy metals contamination also calls for metal analysis courtesy of ICP-AES. The technique has also found use in radiometric dating to analyze comparative abundance of different isotopes (Yip Y: 2007).

Recently, methods for speciation analysis to quantify proteins and biomolecules using

ICP-AES have been developed. By using metal coded affinity tags, it is now possible to track the pharmacokinetics of proteins and peptides in biological tissue and fluids. ICP-

AES is used for the detection of all metal elements with masses from 7-250 (Lithium to

Uranium) except for Argon. Unlike AAS, ICP can simultaneously analyze for more than one element from one sample at any given time, this reduces analytical time for forensic analysis considerably (Yip Y: 2007).

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CHAPTER 3

3. MATERIALS AND METHODS

“Art and science have their meeting point in method”. Edward G. Bulwer-Lytton (1803-1873)

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3.1 RESEARCH FACILITIES, ETHICS AND ANIMAL

TESTING APPROVALS

i. Approval to use laboratory facilities was obtained from the University of Zimbabwe, School of Pharmacy. ii. Approval to use laboratory rabbits for dermatological research was obtained from the Department of veterinary services , Ministry of Agriculture, Zimbabwe under licence L610 iii. Ethical approval to carry out the medical research obtained for the Joint Parirenyatwa research and ethics committee JREC

3.2 NANO-MATERIALS TOXICITY AND SAFETY

INVESTIGATIONS FOR POTENTIAL USE ON

ALBINISTIC SKIN

3.2.1 Ex-Vivo safety investigation of nanometric TiO2 and ZnO

permeation through porcine skin, using vertical, static, Franz

diffusion cells

The following methods (3.1.1) are reproduced from a publication in a refereed journal by the researcher in fulfilment of the DPhil (medicine) degree requirements in a research article referenced as: Chifamba J et al: ex vivo penetration of TiO2 and ZnO across actinically damaged porcine skin: development of an albinistic skin protection treatment. Int J Pharm Sci Res 2015; 6(6): 2339-48.doi:10.13040/IJPSR.0975-8232.6 (6)

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3.2.1.1 Investigation guidelines

The laboratory experiments documented under this ex-vivo study were conducted with reference to related studies by Diembeck et al: 1999, Gamer A. O et al: 2006, relevant excerpts from the OECD guideline number 428 (2004a) and the subsequent guideline document 28 (2004b) as well as the EU Scientific committee opinion SCCNFP O750/03:

2003).

3.2.1.2 Study principles and objectives

The ex-vivo dermato-pharmacokinetics and safety investigation of nanomaterials on actinically damaged porcine skin, aimed at determining whether nanoparticles of TiO2

(CAS number 13463-67-7) and ZnO (CAS number 1314-13-2) can penetrate damaged and compromised skin when formulated in actinic damage retardation treatments for use by albinistic persons leading to systemic exposure. A topical cream was therefore formulated incorporating a wide range of CIR approved ingredients as starting materials including the nanometric metallic oxides (Table 3-1). Vertical static Franz diffusion cells using 15 different porcine skin dermatomes were utilised (Franz T: 1975). The effects of formulation concentration and extent of permeation of trans-epidermal nanometric Ti and Zn was therefore determined by analysing for Ti and Zn in the receptor, donor phases and the skin dermatomes by ICP-AES and AAS respectively.

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Table 3-1: Reagents and starting materials for porcine permeation studies by Ti and Zn nanoparticles.

MATERIAL manufacturer Nanometric Titanium IV oxide Sigma Aldrich Germany (Rutile/anatase) 43-47% w/w dispersion lot MKBN8669V Nanometric Zinc Oxide, 50% Sigma Aldrich Germany w/w dispersion lot MKBQ0692V Cetyl alcohol Savannah South Africa Stearic acid Savannah South Africa GMS Savannah South Africa Liquid paraffin Engen South Africa Petrolatum Engen South Africa Tween 20 Merck South Africa Triethanolamine Merck South Africa Potassium dihydrogen Merck South Africa orthophosphate Ethanol Astra Zimbabwe Sodium PCA Cornelder Zimbabwe EDTA Cornelder Zimbabwe Carbopol 940® Cornelder Zimbabwe Methyl paraben Cornelder Zimbabwe Propyl paraben Cornelder Zimbabwe Cocoa butter Cornelder Zimbabwe lanolin Cornelder Zimbabwe Sodium hydroxide Cornelder Zimbabwe Deionized water UZ -SOP

 All materials were stored at room temperature

3.2.1.3 Investigation Substrates

Full thickness, visually intact porcine skin samples were excised from the abdomen of three, 6 months old, domestic landrace porkers obtained from a commercial abattoir, i.e.

Barnstone enterprises (Domboshava, Zimbabwe). These were dermatomed and used in the study through facilitation by the University Of Zimbabwe Veterinary Science

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Department. Animal ethics approval was granted through license L610 by the

Department of Veterinary Services.

3.2.1.4 Investigation equipment

1. Inductively coupled plasma - atomic emission spectroscopy (ICP-AES Varian;

Wavelength 336nm, external calibration, linear range 0.05-10 mg/l).

2. Flame atomic absorption spectrometry (Varian AAS, Spektr AA220; wavelength

220.5 nm, linear range 0.02 -1mgZN/l).

3.2.1.5 Formulation of cream oil in water (O/W) emulsion dosage

form

All oil soluble starting materials (stearic acid, ceto stearyl; alcohol, GMS, cocoa butter, liquid paraffin, petrolatum, fixed oil extractions, castor oil, propyl paraben) were weighed into a 10 litre thermal jacketed stainless steel vessel and heated to 90 oC. This was referred to as the oil phase. All water soluble ingredients (deionized water, caustic potash, EDTA, carbopol 940®, colorant, methyl paraben) formed the water phase and were weighed into a separate but similar vessel and heated to 90oC. After heating for 5 minutes at the prescribed temperatures, the oil phase ingredients were slowly added to the water phase ingredients while continuously agitating using a variable speed emulsifying mixer at 2200 rpm. Nanoparticles of TiO2 and ZnO were added in at this stage. After the emulsion had formed, triethanolamine was added to buffer the pH and

105

facilitate optimum gelling of carbopol 940® and the resultant emulsion was allowed to naturally cool down by removal of the source of heat.

Oil in water cream emulsions with 5% nanometric TiO2 and 5% nanometric ZnO were formulated and optimized. Standard opaque creams containing 5% normal TiO2 and 5% normal ZnO were also formulated. 18 formulation samples were thus used in the study:

6 with nanomaterials; 6 without any metallic oxides and 6 with normal TiO2 and ZnO. All formulations and starting materials for all three types of creams (with nanomaterials, with normal metallic oxides and without any metallic oxides) were identical.

3.2.1.5.1 Preparation of phosphate buffer solution at pH 7.4

In the study, phosphate buffer saline (PBS) solution was used in the receptor phase of the Franz diffusion cells. PBS solution was prepared by adding 27.8g KH2PO4 together with 6.30g NaOH. The NaOH was diluted to 1560ml with deionized water and the

KH2PO4 was diluted to 1000ml with deionized water. 156.6g of NaCl were then added to the diluted solution. The two solutions were mixed together to make the phosphate buffer solution and the pH was measured with a Shimadzu pH meter. A 10% orthophosphoric acid and 10% NaOH mixture was used to adjust the solution pH to 7.4

(Gamer A. O: 2006). The solution was used in this study for the aqueous solubility determination, standard preparations and as the receptor phase during the Franz cell diffusion studies.

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3.2.1.5.2 Aqueous solubility determination of nanometric TiO2 and ZnO

An excess of TiO2 and ZnO was added to 50 ml of the PBS solution (pH 7.4). The temperature was maintained at 32 °C and the super-saturated solution was constantly stirred by a magnetic stirrer using a water-bath. After 24 hours, the supersaturated sample was filtered, and analysed by atomic absorption spectroscopy. This was conducted in duplicate.

3.2.1.6 Preparation of skin for the skin penetration experiments

The ex-vivo skin diffusion studies made use of abdominal porcine skin obtained from three, 6 months old porkers. Landrace breed porkers weighing between 30 and 40 kg which were being commercially slaughtered were used. The skin was excised from the pig abdomen 48 hours after inducing actinic damage on the area to be excised (using the method outlined below) and within minutes of slaughter. The epidermis was prepared surgically by the heat separation technique, which involved soaking the entire abdominal skin in water at 60°C for 45 seconds, followed by careful removal of the epidermis (Diembeck et al: 1999). The skin was prepared and then frozen within 24 hours of the slaughter. The skin samples were prepared using a surgical blade at a thickness of 1000μm. This thickness included the epidermis and part of the dermis. The dermatomed skin was placed on top of Whatman® filter paper and circles with a diameter of approximately 50mm were punched into the skin. It was ensured that each circle of skin on the filter paper was big enough to cover the Franz cells diffusion area

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(Diembeck et al: 1999). The skin circles were wrapped in foil and quick frozen until needed.

3.2.1.6.1 Simulation of actinic damage in porcine skin

A 49 cm2 (7cm x 7cm) area of the pig’s abdominal section was chosen, the area was cleanly shaven through depilatories. 20ml of a 10% sodium hydroxide solution was applied onto 2g hospital grade cotton wool, the drenched cotton wool was evenly spread with tongs over the selected area demarcated by a stencil and bandaged over by sticky bandages for 30 minutes while the pig was sedated. The high density poly ethylene (HDPE) stencil was left secured over the affected area for the entire 30 minutes. The affected area was then exposed to a xenon arc lamp (solar simulator) for

30 minutes at maximum intensity while the pig was still sedated.

3.2.1.7 In vitro skin permeation studies in Franz diffusion cells

The in vitro skin permeation studies were carried out using static vertical Franz diffusion cells with a diffusional area of 3.2 cm2 (Franz T: 1975). Porcine abdominal skin was mounted between the compartments of the diffusion cell with stratum corneum facing the donor compartment. The receiver phase was PBS: pH 7.4, stirred at 300 rpm by a magnetic stirrer. Six Franz cells were used for each experiment, 4 with the test formulation and two control cells. The buffer (pH 7.4) was pre-warmed to 37oC in a water bath an hour before the experiment commenced. The donor phase formulation was placed in at 32oC. The donor and receptor compartments of each Franz cell were greased with commercial grade vacuum grease. The two compartments of the Franz 108

cell were placed together, sealed with vacuum grease to prevent leakage and secured together with a horseshoe clamp. 50ml of the prepared PBS solution pH 7.4 was added to the receptor compartment and 4g of the cream formulation under investigation was placed into the donor phase compartment. The donor compartment was covered to avoid the loss of constituents by evaporation. The assembled Franz cells were placed on a Franz cell stand and placed in a water bath with a magnetic stirrer ensuring that only the receptor compartment of the Franz cell was immersed (Franz T: 1975). This was recorded as time 0.0h. The entire receptor buffer phase was removed at predetermined time intervals and replaced with fresh buffer to maintain sink conditions

(Gamer A. O: 2006). The extracted samples were analysed for Zn and Ti by ICP and

AAS. Samples were analysed for Ti by inductively coupled plasma - atomic emission spectroscopy (ICP-AES Varian; Wavelength 336nm, external calibration, linear range

0.05-10 mg/l). Flame atomic absorption spectrometry (Varian AAS, Spektr AA220) wavelength 220.5 nm, linear range 0.02 -1mgZN/l) was used for Zn analysis. Twelve hour skin diffusion studies were done and the amount of TiO2 and ZnO that diffused through the skin ex-vivo was determined. It was observed during other studies that very low amounts of TiO2 and ZnO permeate through the skin for the greater duration of the experiments, leading to problems with accurate detection and quantification (Gamer A

O: 2006). So, it was decided to do a single extraction after 12 hours, thus analysing only the total amount of nanomaterials that diffused through the porcine skin to the receptor phase. The unabsorbed cream base remaining in the donor compartment was also analysed to quantify the unabsorbed metallic oxides. After the diffusion process the

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mounted skin membrane was dismounted and crushed. The crushed material was acid treated and subsequently analysed to quantify epidermal Ti and Zn.

3.2.1.8 Statistical Techniques

Statistical analysis was done on the results of the permeation test but the results may be limited because there were only three observations per group. Descriptive statistics for each group were obtained; these include the mean for normal data, median for non- normal data and standard deviation” (Chifamba J et al: 2015).

3.2.2 In-vivo Ti dermato-pharmacokinetics across albinistic skin

investigation by sequential adhesive tape stripping

3.2.2.1 Guidelines and study objectives

The in-vivo investigations documented under this study were guided by relevant excerpts from the OECD guideline number 428 (2004a) as well as the EU SCCNFP opinion number SCCNFP O750/03 (2003). They were also conducted albeit with modifications with reference to related work done by Tokumura F et al: 2006 and Loffler et al: 2004

The objective of the study was to determine the extent of penetration and skin reservoir distribution of nanometric TiO2 (CAS number 13463-67-7) in oculocutaneous albinistic skin typically found in the tropics when formulated in actinic damage retarding treatments. The study also investigated the influence of regional anatomical differences in skin exposure sites on dermato-pharmacokinetics. Only Ti was used and investigated

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for its dermato-pharmacokinetics and potential for systemic exposure across genetically compromised albinistic skin due to the fact that the human skin already has Zn ions and it would not be possible to separate solution Zn already in the body and formulation zinc using the experimental instrumentation and study protocol

3.2.2.2 Human subjects

All test procedures under this section and the number of subjects used were dictated by guidelines from the SCCP and the OECD under Opinion concerning guidelines on the use of human volunteers in compatibility testing of finished cosmetic products - adopted by the Scientific Committee on Cosmetics and Non-food Products intended for

Consumers during the plenary session of 23 June 2009 (Opinion EU SCCNFP 23/2009

Three OCA volunteers participated in the study (2 female, 1 male: aged 23-30 years, mean 26 ± 4). Ethical approval was obtained from the University of Zimbabwe, joint research and ethics committee (JREC) under Parirenyatwa group of hospitals and informed consent was obtained from all subjects prior to the study. A practising dermatologist certified that volunteers exhibited no other significant skin disease except for characteristics of actinic exposure prevalent in tropical albinistic persons including freckles, patches, macules, wrinkles, accentuated expression lines (premature aging) and hyper-keratinization on the facial and neck area. The outer arms were heavily keratinized but free from any primary or secondary skin lesions in all participants, the inner forearms were not hyper-keratinized and were also free from both primary and secondary skin lesions. The general phenotype of all three participants was confirmed to be OCA2 albinism by the dermatologist. For each anatomical site investigated, 111

experiments were conducted on the same subject, and all subjects had all three anatomical sites investigated. Subjects were instructed not to use any other cosmetic cream or treatment for 3 days prior to the experiments except for a specially designed base cream. The studies were carried out in Harare, Zimbabwe (altitude 1490m) in

November 2014, (mean temperature 29.5oC, mean relative humidity 51.5)

3.2.2.3 Investigation substrates

Transpore® polypropylene adhesive tape, 50mm width, with acrylate adhesive obtained from 3M, South Africa was used for stripping.

3.2.2.4 Study methods

3.2.2.4.1 Formulation of base cream O/W emulsion dosage form

The actinic damage retarding cream was formulated from the complete list of starting materials in table 3-2, as follows: a water phase containing deionized water, glycerin,

MPG, colorant caustic potash, methyl paraben, Carbopol® and EDTA was heated to

90oC in a thermal jacketed heating vessel. In a separate vessel, the oil phase was prepared by adding all the oil miscible ingredients and heating them to 90oC. These included lanolin, Trichilia emetica, cocoa butter, ethylene glycerol mono stearate, liquid paraffin, and castor oil, stearic acid, cetyl alcohol and propyl paraben. After maintaining the stated temperatures for 5 minutes, the oil phase was slowly added to the water phase while vigorously agitating with an emulsifying mixer at 2200 rpm. Triethanolamine was then added to buffer the emulsion pH and facilitate viscosity adjustment by

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Carbopol 940 ®. The O/W emulsion cream was then cooled down naturally to 50oC.

The Aloe excelsa and fragrance were incorporated into this cooled state.

The required TiO2 was inputted into the base cream at the expense of an equivalent amount of water to create the treatment cream.

Two different cream formulations were therefore prepared, a standard base cream with all materials except for the titanium IV oxide and the treatment cream containing 5% w/w titanium IV oxide. The Ti concentration in the treatment cream was determined by

ICP-AES analysis before the albinistic skin absorption investigations.

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Table 3-2: Investigation Materials for patch testing

MATERIAL MATERIAL STORAGE Manufacturer FUNCTION Nanometric Titanium IV Sunblock Room temperature Sigma Aldrich oxide (Rutile/anatase) Germany 43-47% w/w dispersion lot MKBN8669V PEG 400 emulsifier Room temperature Savannah South Africa Cetyl alcohol emulsifier Room temperature Savannah South Africa Stearic acid emulsifier Room temperature Savannah South Africa EGMS emulsifier Room temperature Savannah South Africa Ceto stearyl alcohol emulsifier Room temperature Engen South Africa Pharma-oil heavy emollient Room temperature Engen South Africa White Petrolatum emollient Room temperature Engen South Africa Triethanolamine Buffering Room temperature Merck chemicals SA agent Trichilia emertica Emollient/ Room temperature Prepared by active Researcher ingredient Aloe excelsa Active 5-10 oC Prepared by ingredient Researcher EDTA Preservative Room temperature Cornelder Zimbabwe enhancer Carbopol 940® Gellant/ Room temperature Cornelder Zimbabwe thickening polymer Methyl paraben preservative Room temperature Cornelder Zimbabwe Propyl paraben preservative Room temperature Cornelder Zimbabwe Cocoa butter emollient Room temperature Cornelder Zimbabwe lanolin emollient Room temperature Cornelder Zimbabwe Deionized water vehicle Room temperature UZ-SOP

3.2.2.4.2 Determination of cream pH

The pH was determined by dissolving 5 g of the 5% TiO2 cream in 50 ml deionized water in a 100 ml beaker. A Jenway 3510 pH meter was used to analyse for the pH. 114

The equipment was pre-calibrated using buffer pH 7 and buffer pH 4. Determinations were performed at room temperature in triplicate.

3.2.2.5 Skin application of formulations and sample collection

Three application sites were selected on each volunteer (Figure 3-1). The areas were naturally free from terminal hair; vellus hairs present on sites were removed by special clippers 30 minutes before the application of the test material. The selected sites were cleaned by wiping with de-ionized water moistened cotton swabs and dried with a heat blower air stream for 30 seconds.

a b c

Figure 3-1: Selected anatomical sites for sequential tape stripping: (a) the inner forearm, (b) outer forearm of the same arm and (c) the forehead of an albinistic volunteer showing the anatomical site based variations in actinic damage on the same individual

On each of the three test sites, a 50mm width Transpore® adhesive tape was used to demarcate a template study area measuring 30mm x 90mm before stripping. The test area was therefore the stencilled area left by the gaping hole within the adhesive tape perimeter (Figure 3-2a) below. The template was fastened to guarantee reproducibility of sequential stripping from the site. A 0.30 ml (0.27 g) aliquot of the treatment cream was applied onto the selected area using a syringe. The treatment was homogeneously

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distributed over the selected area and left for 15 minutes. Throughout the study, due care was taken not to touch or disturb the test site. After 15 minutes the test sites still with the template stencil was repeatedly wiped clean using a dry cotton swab to remove unabsorbed material. The cotton swabs were placed in a glass test tube for extraction and analysis of recovered Ti. The demarcated area was marked with a felt-tip marker and the template adhesive tape was removed from the site. The 50 mm width stripping adhesive tape was cut into 12 cm strips and the strips were subsequently applied over the demarcated area. The area extracted from was therefore 60cm2 and abundantly exceeded the 27cm2 test area. The stripping adhesive tape was applied successively from tape 1, up to tape 10 on the site and pressed over using a glass rod roller to smooth out all creases and remove all air pockets. A clean sheet of paper was placed between the tape and the roller so as to avoid any transfer of material to the back of the tape. The roller was rolled over back and forth ten times so as to maintain reproducible uniform pressure and achieve optimal bonding between the adhesive tape and the stratum corneum. Using tongs the adhesive strips were gradually removed from the skin in one draw lasting at most 5 seconds. The first strip was placed in the glass test tube containing the cotton swab used to remove excess material. Strips 2-5, 6-8 and 9-10 were put in 3 separate glass test tubes respectively (Figure 3-2).

To all the test tubes containing tape strips including the first one combined with the cotton swab, 5 ml of nitric acid was added and shaken by a rotary shaker for 15 minutes. The resultant acid extract was diluted to 10 ml with deionized water and subsequently analysed by inductively coupled plasma - atomic emission spectrum (ICP-

AES Varian; Wavelength 336nm, external calibration, linear range 0.05-10 mg/l). 116

a b c

d e f

Figure 3-2: Adhesive tape stripping methodology on an albinistic forearm: (a) demarcation of the selected site with an adhesive tape template (b) application of test material on site (c) spreading the cream (d) removal of surface formulation after 15 minutes with cotton swab (e) pressing of adhesive tape over selected site with glass rod and a paper shield (f) removal of the adhesive tape using tongs

Negative controls for Ti content were carried out on the standard base cream without Ti, and on all reagents including deionized water and the acid, on control skin strips taken from non-exposed sites and the tape before any stripping using ICP-AES at a detection level of 50 ppb.

3.2.2.6 Study Statistical Techniques

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3.3 BOTANICAL ACTIVES PREPARATION FOR THE

ALBINISTIC TREATMENT DEVELOPMENT

3.3.1 Fixed oil extraction from Trichilia emetica (Banket mahogany)

seed kernels

3.3.1.1 Guidelines objective

Trichila emetica fixed oil was extracted and stabilised so as to be incorporated in the albinistic treatment formulation. Cold expeller methods were used to extract the bulk of the oil and the little oil that remained in the seed meal was scavenged for, by solvent extraction. The extraction was guided by commercial methods used by TUV organic certified natural products processors including Phytotrade Africa and Bio-Innovation

Zimbabwe.

3.3.1.2 Extraction materials and methods

Trichilia emetica seeds were collected from the Harare Institute of Technology (HIT) in,

Belvedere (17° 49′ 39″ S, 31° 0′ 43″ E) Harare, Zimbabwe (Figure 3-3) where the plants are found in abundance. The red seeds with black dots resembling a doll’s eyes were authenticated by the botanical gardens research officers to belong to the species. The fallen seeds were picked in February 2014 from the numerous trees which have been artificially propagated for shade around the HIT car park.

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The collected seeds were cleaned and the scarlet shells were removed by rubbing the seeds in a trough. The de-hulled seeds were further cleaned with cold water and left to dry naturally.

Figure 3-3: Belvedere area where Trichilia emetica was collected (adapted from www.geohack.com)

The dried seeds were ground into a powder using a manual pulveriser and mill. A portion of the resultant meal (5kg) was weighed into the inlet receptacle of a screw press, which was coupled to an electric motor. The screw press was powered and commenced extracting the oil from the coarse meal without any heating. The expressed oil was collected from a nozzle at the bottom of the expresser and weighed. The remaining meal cake after extraction was recovered from the barrel of the expresser and weighed.

The recovered meal cake was further subjected to solvent extraction for any remaining oil by adding it to 8 litres petroleum ether and, leaving it to extract over 48 hours. After

48 hours the petroleum ether seed cake mix was filtered by cotton wool and the 119

recovered solvent oil mixture was left standing for 2 hours and then transferred to a rotor vapour at 55oC where all the hexane was evaporated off and condensed back to liquid in a different container. The remaining purified oil was weighed and mixed with the rest of the expressed oil from the screw press and weighed. The solvent was recovered and used again for other extractions.

To prevent aerial oxidation, 0.5 % tocopherol acetate and 0.2% BHT were added to the recovered oil. The percentage yield of the extracted oil was calculated using the following equation:

푂푃(푔) + 푂푆(푔) % 푓𝑖푥푒푑 표𝑖푙 푦𝑖푒푙푑 = 푥 100 푆푀(푔)

Where:

OP= the total oil recovered from the seed meal by the screw press

OS= the total oil recovered by the petroleum ether

SM= the total seed meal fed into the screw press.

The resultant oil extract was sent for physical and chemical characterisation by

Chemnet Organics (Pvt) Ltd and a certificate of analysis was issued.

3.3.2 Extraction of Aloe excelsa gel matrix

Aloe excelsa gel was extracted and stabilised for incorporation into the albinistic treatment. There are no documented best proven practice standard guidelines or protocols for extraction of the Zimbabwean aloe in literature. The following method was therefore specifically developed for this study.

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3.3.2.1 Materials and methods

Fresh rosettes of Aloe excelsa leaves, cut off from the stems were collected from the rocky outcrops close to the Great Zimbabwe ancient city ruins (20° 16′ 0″ S, 30° 56′ 0″ E) in Southern Zimbabwe approximately 32 km from the city of Masvingo.(Figure 3-4). The plant material was authenticated taxonomically by the national herbarium in Harare,

Zimbabwe.

The fleshy leaves were cut off transversely from the base of the rosette. The skin was scrapped off by a scalpel and aloe juice was obtained by extracting the gel matrix sap from the leaves and squeezing it through filters to remove solids to remain with a clear juice. The juice was instantly preserved by adding 0.1% citric acid, 0.1% BHT and 0.1% sodium benzoate. The resultant extract was sent for physical and chemical characterisation by a third part, Chemnet Organics (pvt) ltd and a certificate of analysis was issued.

The obtained Aloe excelsa gel matrix yield was calculated by the following equation:

푚푎푠푠 표푓표푏푡푎𝑖푛푒푑 푓𝑖푙푡푒푟푒푑 푒푥푡푟푎푐푡(푔) % 푔푒푙 푚푎푡푟𝑖푥 푦𝑖푒푙푑 = 푥 100 푚푎푠푠 표푓 푝푙푎푛푡 푚푎푡푒푟𝑖푎푙 푢푠푒푑(푔)

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Figure 3-4: Lake Mutirikwi and Great Zimbabwe area (adapted from www.geohack.com)

3.3.2.2 In-vitro Sunburn Protection Factor (SPF) determination of

5% Aloe excelsa in a cosmetic lotion

3.3.2.2.1 Study Guidelines and objectives

This determination was done according to the mandated US-FDA method for determining SPF of cosmeceuticals using Optometrics LLC, 290S Spectrophotometer.

Subsequent calculations of the SPF from the MPF were done according to the FDA and

COLIPA guidelines.

The objective of this particular study was to scientifically determine and confirm whether

Aloe excelsa gel extracts possess sunscreen activity and can potentially be incorporated into actinic damage treatments for albinistic people living within the tropics.

A sunscreen cream base was therefore prepared incorporating all typical cosmetics ingredients shown in Table 3-5.

Table 3-3: In-vitro SPF testing starting materials

MATERIAL SUPPLIER Medilan® Croda South Africa Isopropyl myristate Crest chemicals SA PEG 400 Savannah South Africa Cetyl alcohol Savannah South Africa Stearic acid Savannah South Africa GMS Savannah South Africa Caustic potash Engen South Africa Pharma-oil heavy Engen South Africa White Petrolatum Engen South Africa Triethanolamine Merck chemicals SA Butylated Hydroxy Toluene Silcherm Zimbabwe Carbopol 940® Cornelder Zimbabwe Methyl paraben Cornelder Zimbabwe

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Propyl paraben Cornelder Zimbabwe Castor oil Cornelder Zimbabwe Deionized water UZ-SOP

 All materials listed above were stored at room temperature

3.3.2.2.2 Method for preparing the cosmeceutical cream

Step 1: A water phase was homogenized in a stainless steel thermal jacketed heating vessel, It contained 80% of the required amount of deionized water, caustic potash,

EDTA ,glycerin, carbopol 940® MPG, BHT and methyl paraben. The materials were homogenized by a Silverson mixer and heated to 85oC for 5 minutes.

Step 2: In a separate vessel, the oil phase was prepared simultaneously by adding all the required amounts of the following materials and heating them to 90oC for 5 minutes.

The materials included propyl paraben, liquid paraffin, cocoa butter, GMS, cetyl alcohol, castor oil and stearic acid.

Step 3: The clear, hot oil phase was added into the water phase vessel while consistently agitating with an emulsifying mixer at 2200rpm. Triethanolamine was then added to the newly formed emulsion.

Step 4: The buffered emulsion was then cooled down naturally by removing sources of heat to 50oC. The Aloe excelsa extract was then incorporated at this point. The remaining 20% of the deionized water was also added in to make up the volume.

3.3.2.2.3 Determination of lotion pH

The cosmeceutical lotion pH was evaluated at room temperature by dissolving 20g of the cream in 200 ml deionized water in a glass beaker. The pH was then read from a

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Jenway 3510 pH meter which had been pre-calibrated using known standard buffers of pH 4 and 7. The evaluations were done in triplicate.

3.3.2.2.4 SPF determination materials and instrumentation

 5 samples of the 5% aloe excelsa w/w lotion, prepared specifically to retard

actinic damage in albinistic persons.

 A commercial SPF 30 lotion used as a quality assurance sample

 Optometrics SPF-290S analyser, with xenon arc lamp, solar simulator.

3.3.2.2.5 Invitro SPF determination protocol

Two layers of the 3M Transpore® tape were placed back to back to make a double layer of tape. Using a 1ml fine syringe, 96mg of the sample material was transferred to a 50cm2 area of the tape giving an application rate of 2μl/cm2 being applied to one of the fronts of the Transpore tape®. The material was spotted in tiny droplets at various locations on the Transpore tape® and spread over into a uniform layer by a finger stall as required by the FDA. The treated tape was left to dry for 15 minutes before commencement of measurements. Meanwhile a reference sample of 3M Transpore® tape without any sunscreen was also prepared. The reference sample and the treated tape were placed sequentially in the transmittance port of the Optometrics 290S instrument and exposure to UV radiation with wavelength that ranged between 280-

400nm was initiated. The transmittance was determined for both the blank reference tape sample and the treated tape. Transmittance was measured at 5 random spots with

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each spot being scanned repeatedly twice. The scan reading was taken and the SPF was calculated by built in software according the following equation.

400 400 퐸휆퐵휆 푆푃퐹푠푐푎푛 = ∑ EλBλ / ∑ 푀푃퐹휆 290 200

Where:

MPFλ = Scan MPF value

Eλ = spectral irradiance of terrestrial sunlight under controlled conditions

Bλ = erythermal effectiveness

The whole experiment procedure was repeated with a COLIPA SPF 15 standard, as an assaying guide.

3.3.3 Extraction of Myrothamnus flabellifolia by steam distillation

M. flabellifolia essential oils were extracted for incorporation into the albinistic treatment using steam distillation. Documented guidelines and similar extractions by Gundidza et al (2004) using Clavenger type apparatus and methods described by Marcello N: (2012) were used as references to the extraction.

The resultant extract was sent for physical and chemical characterisation by a third part laboratory, Chemnet Organics (pvt) ltd and a certificate of analysis was issued.

3.3.3.1 Steam distillation materials, and methods

M. flabellifolia Plants were obtained from the rocky inselbergs bordering the shores of

Lake Mutirikwi (20° 14′ 56.12″ S, 31° 1′ 59.74″ E) approximately 40km due South East from

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the town of Masvingo, (Figure 3-4) in Masvingo province of southern Zimbabwe. The plant material, which was in the desiccated “dead” phase of the resurrection bush status was authenticated taxonomically by the national herbarium and was extracted for the essential oil within 48 hours post- harvest. Using scalpel and a manual chopper, the whole plant including roots, stems, branches and leaves were cut into small pieces and

1.560 kg of the plant material was loaded into the material holding section of the steam distillation unit. Clavenger type apparatus (shown in Figure 3-5) was used for the steam distillation process modelled in line with the method described by Marcello N (2012).

Two and a half (2.5) litres of distilled water were used to generate the steam.

Condensed essential oils and water were collected in the condenser at the end of the column. The essential oil which separated from the water and settled at the top in the condenser was obtained after careful decanting off, of the water from the condenser.

Any remaining water was scavenged for by the addition of 5% anhydrous sodium sulphate to the extract. The yield percentage of the essential oil was calculated by the following equation.

푚푎푠푠 표푓표푏푡푎𝑖푛푒푑 표𝑖푙 푒푥푡푟푎푐푡(푔) % 표𝑖푙 푦𝑖푒푙푑 = 푥 100 푚푎푠푠 표푓 푝푙푎푛푡 푚푎푡푒푟𝑖푎푙 푢푠푒푑(푔)

The obtained extract was stored in a refrigerator between 5-10oC.

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Figure 3-5: Extraction of M. Flabellifolia using Clavenger type apparatus in (UZ School of Pharmacy laboratory)

3.4 ALBINISTIC ACTINIC DAMAGE RETARDING

TREATMENT EMULSION DEVELOPMENT

3.4.1 Principles and guidelines

The ternary phase approach was employed for emulsion development. The formulation process was done under guidelines from the international CTFA, the European COLIPA and South African CTFA mandates. Guidelines from Cancer South Africa (CANSA) and

South African Bureau of Standards (SABS) were adhered to throughout the whole process. Only CIR approved ingredients were used and all non-standard ingredients like nanomaterials and botanical extracts were screened for safety. Various formulations were developed and optimised from a standard set of CIR approved ingredients (Table

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3-4) to come up with the safest, most efficacious, most stable and most aesthetic product.

Table 3-4: Actinic damage treatment Starting Materials

MATERIAL STORAGE manufacturer Nanometric Titanium IV oxide Room temperature Sigma Aldrich Germany (Rutile/anatase) 43-47% w/w dispersion lot MKBN8669V Nanometric Zinc Oxide, 50% Room temperature Sigma Aldrich Germany w/w dispersion lot MKBQ0692V Solaveil XT 300 Room temperature Croda South Africa Solaveil XT 100 Room temperature Croda South Africa Medilan ® Room temperature Croda South Africa Aloe excelsa extract 5oC Prepared by researcher Myrothamnus flabellifolia 5oC Prepared by researcher extract Adansonia digitata extract Room temperature Prepared by researcher Dicerocaryum zanguebarium 5oC Prepared by researcher extract Plumbago Zeylanica extract 5oC Prepared by researcher Aloe Zebrina extract 5oC Prepared by researcher Cetyl alcohol Room temperature Savanna South Africa Stearic acid Room temperature Savanna South Africa GMS Room temperature Savanna South Africa Liquid paraffin Room temperature Engen South Africa Petrolatum Room temperature Engen South Africa 128

Tween 20 Room temperature Merck chemicals SA Triethanolamine Room temperature Merck chemicals SA Caustic potash Room temperature M & M chemicals Zimbabwe Pin Lavender Room temperature M & M chemicals Zimbabwe Sodium PCA Room temperature Cornelder Zimbabwe EDTA Room temperature Cornelder Zimbabwe Carbopol 940® Room temperature Cornelder Zimbabwe Methyl paraben Room temperature Cornelder Zimbabwe Propyl paraben Room temperature Cornelder Zimbabwe Cocoa butter Room temperature Cornelder Zimbabwe lanolin Room temperature Cornelder Zimbabwe Sodium hydroxide Room temperature Cornelder Zimbabwe Deionized water Room temperature UZ-SOP Isopropyl myristate Room temperature Cornelder Zimbabwe BHT Room temperature Cornelder Zimbabwe

3.4.2 Formulation protocol

The ingredients were grouped into two lots, the first was for all oil soluble ingredients and the second was for all water soluble ingredients. In varying lots, quantities and in different batches oil soluble starting materials including stearic acid, ceto stearyl; alcohol, GMS, cocoa butter, liquid paraffin, petrolatum, fixed oil herbs, castor oil, and propyl paraben were weighed into a thermal jacketed stainless steel vessel and heated to 90oC. This was referred to as the oil phase and the ratio between the total amount of emulsifiers (stearic acid, cetyl alcohol, ceto stearyl alcohol and GMS) and the other emollients in the oil phase was kept at 1:2.5. The water soluble ingredients including deionized water, caustic potash, EDTA, carbopol 940®, colorant, methyl paraben formed the water phase and were weighed into a separate but similar vessel and heated to

95oC. The oil phase ingredients were slowly added to the water phase ingredients while continuously agitating using a variable speed emulsifying mixer at 2200rpm.

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Nanoparticles of TiO2 and ZnO were added in at this stage. After the emulsion had formed, triethanolamine was added to buffer the pH and facilitate optimum thickening for carbopol 940® and the resultant emulsion was allowed to naturally cool down by removal of heating sources. Once the cream had cooled to 50oC, the volatile components including essential oils (Myrothamnus flabellifolia) and fragrances were added. Degradable herbal extracts (Aloe excelsa) were also added to the cream at this stage.

Various formulations for oil in water cream emulsions with 5% TiO2 and 5% ZnO were formulated and optimized with regards to emulsion stability. Standard opaque creams containing 5% normal TiO2 and 5% ZnO were also formulated for comparison purposes.

At least 18 formulation samples were thus used in the study: 6 with nanomaterials; 6 without any metallic oxides and 6 with normal TiO2 and ZnO. The other starting materials and all formulations with the nanomaterials, without any metallic oxides and those with the normal metallic oxides were identical. For the numerous formulations done during the study, the procedure can be summarised as below.

Step 1: The water phase was homogenized in a stainless steel thermal jacketed

heating vessel, It contained 75% the required amount of deionized water,

humectants and the following in different levels as per formulation, glycerin

caustic potash, EDTA, carbopol 940® and methyl paraben. The materials were

homogenized by a Silverson mixer and heated to 95oC for 5 minutes. The

required amounts of nanomaterials were dispersed into this water phase.

Step 2: In a separate vessel, the oil phase was prepared simultaneously by adding all

the required amounts of the following materials and heating them to 90oC for 5 130

minutes. The materials included propyl paraben, GMS, cetyl alcohol, stearic acid,

ceto stearyl alcohol as standard and varying levels of all other oil soluble

emollients as required by the specific formulation.

Step 3: The clear hot oil phase was added into the water phase vessel while

consistently agitating with an emulsifying mixer at 2200rpm. Triethanolamine was

then added to buffer the newly formed emulsion in all cases.

Step 4: The buffered emulsion was then cooled down naturally by removing sources of

heat to 50oC. The Aloe excelsa, Myrothamnus flabellifolia extract and all other

heat sensitive ingredients were then incorporated at this point. The remaining

25% of the warm deionized water was also added in to make up the volume.

3.4.3 Treatment physico-chemical and stability tests

3.4.3.1 Emulsion pH

The cosmeceutical cream pH was evaluated at room temperature by dissolving 20g of the cream in 200 ml deionized water in a glass beaker. The pH was then read from a

Jenway 3510 pH meter which had been pre-calibrated using known standard buffers of pH 4 and 7. The evaluations were done in triplicate.

3.4.3.2 Specific gravity

A 50ml sample of the emulsion was placed in a calibrated Pyknometer at 25oC and the specific gravity evaluated at room temperature. The evaluations were done in triplicate.

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3.4.3.3 Freeze thaw cycle test

The product was packed in 6 x 100ml HDPE jars and subjected to freeze thaw cycle tests. The 6 samples were frozen at -10oC for 14 days. The 6 jars were removed from the blast freezer and subsequently thawed naturally. Vital emulsion parameters of pH, odour, specific gravity, colour and general appearance were checked against a quality control sample.

3.4.3.4 High temperature stress testing

The emulsion was packed in 6 x 100ml HDPE jars and subjected to temperature stress tests. The jars were placed in a controlled oven at 45oC for 30 days. The samples were removed from the laboratory oven and allowed to cool naturally before vital emulsion parameters of pH, specific gravity, odour, colour and general appearance were checked against a quality control sample.

3.4.3.5 Window ledge tests

The emulsion was packed in 6 x 100ml HDPE jars and subjected to tropical window ledge stress tests. The packed jars were placed on the inside window ledge of a glass window facing to the north for 90 days from January to March 2015. After the period, the samples were withdrawn from the window and vital emulsion parameters of parameters of pH, specific gravity, odour, colour and general appearance were checked against a quality control sample. A separate sample in a clear glass jar was also concurrently subjected to the same window ledge test specifically to evaluate photo stability. 132

3.4.3.6 Stress cycle tests

The finished product in 6 x 100ml HDPE jars was placed at -10oC for 24 hours in a blast freezer and upon removal it was immediately placed at 45oC in a laboratory oven for 24 hours. This process completed one cycle and the product was then subjected to two further cycles of the same test. After completion of 3 cycles vital emulsion parameters of parameters of pH, specific gravity, odour, colour and general appearance were checked against a quality control sample.

3.4.3.7 Centrifugation

A 1200g sample of the final emulsion was heated to 50oC for 10 minutes. Samples were withdrawn and centrifuged at 3000 rpm by a Hettich Universal II centrifuge instrument.

After the process the emulsion was checked for creaming and separation.

3.4.3.8 Transit vibration tests

So as to mimic a worst case scenario for conditions encountered in transportation. The product was packed in 6 x 100ml jars and subjected to transit vibration tests. The packed product was placed in a sealed box and placed under the rear back seat of a commuter omnibus and subjected to 10 return transit trips between two cities 440 km apart over a 16 day period. After the transit trips the product was checked for signs of creaming against a quality control sample.

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3.4.3.9 Preservative challenge tests

Preservative challenge tests were conducted according to the USP<51> test method to challenge the products with the help of the Harare institute of technology department of pharmaceutical technology. Five microorganisms which included 3 bacteria and 2 fungi were employed to challenge the actinic damage cream.

The 3 bacteria strains were pooled together and the 2 fungi were also pooled together before being added to the product in 2 separate containers. After a 7 day contact time the containers were tested for any remaining bacteria and fungi. The preservative system was passed as effective if there were no bacteria remaining in the creams after the contact period.

3.4.3.10 Spreadability tests

A Brookfield CT3 instrument was used to evaluate the spreadability of the cream.

3.4.3.10.1 Equipment and materials

 Brookfield CT3

 Matched Perspex male and female cones set

 Software for spreadability graphs

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3.4.3.10.2 Sample preparation

The female cone was filled with the sample using a laboratory spatula and all air pockets were driven out. The surface of the cone, after filling was levelled using the back of the spatula (Figure 3-6)

3.4.3.10.3 Procedure

The filled female cone was inserted into the female cone holder; the male cone was then attached to the load cell and aligned directly above the female cone. Once alignment had been achieved the screws on the base table were tightened to avoid movement which may lead to loss of alignment. The test was then commenced with the male cone being lowered into the female cone while the software recorded the spreadability curve and work hardness.

3.5 PRODUCT SENSITIVITY TESTING

3.5.1 In-vivo sensitivity testing: Draize skin sensitivity test

3.5.1.1 Materials and methods

The Draize skin sensitivity test was carried out on the final formulation using 3 adult male New England breed white laboratory rabbits, weighing between 1.3-1.8 kg. The rabbits were checked for their suitability for the study over a 7 day acclimatisation period under the guidance of a qualified veterinary doctor (Dr S Chinyoka, University of

Zimbabwe Veterinary School).

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The rabbits were kept in a rodent facility within the University of Zimbabwe veterinary sciences department in a limited access facility. They fed on a typical commercial rabbit diet sourced from Agrifoods Zimbabwe (pvt) ltd and had unlimited access to drinking water. Animal ethics approval was obtained before commencement of the protocol.

Prior to the test, the backs of the rabbits were shaved by depilatories and the shaved area was divided into two marked parts measuring 25cm2 each. The first marked area was used for the application of the actinic damage treatment and the second demarcated area was used as the control for testing the irritation as per the method by

Draize.

3.5.1.2 Cream application

To the test area, 5ml of the test cream was applied by a syringe and spread evenly over the 25cm2 demarcated shaved area of each animal. The application site was covered by gauze and the area was lightly covered by non-sticky bandages. The treated rabbits were then returned to their respective cages and observations were made to the sites at

24, 48 and 72 hours. Any sensitivity or reactions to the treatment was evaluated by the following criteria as per Draize documented method (Table 3-5).

3.5.1.3 Score of primary irritation

Table 3-5: Draize irritation classification protocol

Reaction Reaction Score Erytherma No erytherma 0 Very slight erytherma 1 Well defined erytherma 2 Moderate to severe erytherma 3 136

Severe erytherma to eschar formation 4 Oedema No oedema 0 Very slight oedema 1 Well defined oedema 2 Moderate oedema (raising 1mm) 3 Severe oedema (raised more than 1 mm and 4 extending beyond area of exposure Total possible score for primary irritation 8

The marked control site was treated in the same way with the base cream without any nanomaterials and herbal extracts. Observations were made in the same manner as the treated sites.

The Score of Primary Irritation (SPI) was calculated by the following equation for both the treated and the control sites.

푒푟푦푡ℎ푒푟푚푎 푎푛푑 표푒푑푒푚푎 푔푟푎푑푒 푎푡 24,48,72ℎ푟푠 푆푃퐼 = ∑ 푛푢푚푏푒푟 표푓 표푏푠푒푟푣푎푡𝑖표푛푠

3.5.1.4 Primary irritation index (PII)

The Primary Irritation Index (PII) was derived from the differences between the summed SPI scores for the treated site and the control sites. The PII was calculated by the following equation:

∑ 푆푃퐼(푡푒푠푡) − ∑ 푆푃퐼 (푏푎푠푒) 푃퐼퐼 = 푛푢푚푏푒푟 표푓 푎푛𝑖푚푎푙푠

The degree of irritation was then categorised according to the Draize irritation response categories in Table 3-6.

Table 3-6: Draize Irritation response categories

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Category Primary Irritation Index (PII) Negligible irritation 0-0.4 Slight irritation 0.5-1.9 Moderate irritation 2-4.9 Severe Irritation 5-8

1.0.1 Product ocular sensitivity test: Low volume eye test (LVET)

The low volume eye test was opted for ahead of the Draize ocular sensitivity, and was only done after the skin sensitivity results had been evaluated. The LVET was carried out on the final formulation using 3 adult male New England breed white laboratory rabbits, weighing between 1.3-1.8 kg. The rabbits were checked for their suitability for the study over a 7 day acclimatisation period under the guidance of a qualified veterinary doctor (Dr S Chinyoka) The rabbits were kept in a rodent facility within the University of Zimbabwe veterinary sciences department in a limited access facility. The facilities were designed so as to exclude any materials that may induce eye irritation or cause eye injury including saw dust and wood chips as per the test requirements. They were fed on a typical commercial rabbit diet sourced from Agrifoods Zimbabwe (pvt) ltd and had unlimited access to drinking water. Animal ethics approval was obtained before commencement of the protocol.

3.5.1.5 LVET Procedure

The animals were held firmly but gently while directly to the cornea of the animals, 0.01g of the treatment cream was applied to the right eye of test animals. The left eye was left untreated as a standard control. Both eyes were observed after 1 hour, 24 hours and 72 hours after treatment for adverse reactions. A qualified veterinary surgeon (Dr S Chinyoka) guided the eye observations and interpretation of results. Observations were done using both gross external observation and a slit lamp bio-microscope with a slit width of 5mm and a magnification of 10xs to determine both minor irritations and long lasting damage to the

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eye. If no adverse reactions were observed the rabbit eyes were washed with plenty water and the test was concluded at 72 hours If adverse effects were observed, the experiments were continued up to 21 days or until the damage had reversed to normalcy. The total irritancy of the cream was ranked on a score of 1-10 as per the Draize eye test grading system. The test score was the aggregate of the individual scores of irritancy for the rabbit cornea, conjunctiva and the iris each after grossing up by a given factor.

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Total eye irritancy grading

Table 3-7: Scale of weighted scores for grading the severity of ocular lesions (Draize et al 1944) Reaction Reaction Score Cornea opacity (A) Scattered or diffuse area-iris clearly visible 1 Easily discernible translucent 2 Opalescent areas, no details of iris visible pupil 3 barely discernible Opaque iris invisible 4 Area on cornea One quarter or less, but not zero 1 involved (B) Greater than one quarter but less than half 2 Greater than half but less than three quarters 3 Greater than three quarters 4 Score equals A x B x 5 total maximum score is 80 Iris Values (A) Folds above normal, congestion, swelling, iris still 1 responding to light No reaction to light, hemorrhage, gross 2 destruction or any one of these Score equals A x 5 total maximum score is 10 Conjunctivae Vessels injected above normal 1 Redness (A) Vessels more diffuse, deeper crimson red 2 Beefy red 1 Chemosis (B) Any swelling above normal 1 Obvious swelling with partial eversion of lids 2 Swelling with lids half closed 3 Swelling with lids half to completely closed 4 Discharge Any amount different to normal 1 Discharge with moistening of lids and hairs 2 adjacent to lids Discharge with moistening of lids and areas 1 surrounding eye Score = (A+B+C) x 2, Total maximum is 20

Total maximum score is the sum of all scores form cornea , iris and conjunctivae

A rabbit was considered to have exhibited positive ocular irritation if the cream produced any minimum readings of cornea ulceration, opacity of the cornea or inflammation of the iris, swelling of the conjunctivae and partial to full closing of the lids.

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3.5.2 In-vivo Human sensitivity tests: Patch testing

After animal sensitivity tests, the prepared formulations were tested for safety on human volunteers by patch testing. The tests were conducted as per the guidelines of the

European Scientific Committee on Cosmetics and Non-food Products intended for

Consumers opinions (SCCNFP O750/03). The guidelines determine the number of participants to be used and the test procedures.

3.5.2.1 Human subjects

Eight human volunteers participated in the study. The participants included 5 albinistic volunteers (4 females and 1 male) and 3 normal Negroid skin types (2 female, 1 male) aged 23-30 years, mean 26 ± 4). Ethical approval was obtained from the University of

Zimbabwe, Joint Research and Ethics Committee (JREC) under Parirenyatwa group of hospitals and informed consent was obtained from all subjects prior to the patch testing.

The volunteers filled in medical questionnaire which confirmed them to be healthy.

Subjects were instructed not to use any other cosmetic cream on the selected areas to be patch tested for 3 days prior to the experiments except for a specially designed base cream. The studies were carried out in Harare, Zimbabwe (altitude 1200m) in October

2014, (mean temperatures 28.8oC, mean relative humidity 30%).

3.5.2.2 Patch testing procedure

The dorsal skin was cleaned with plenty of warm water without any soap and air dried before commencement of the patch tests. On both, the right and left forearms, 25cm2 141

areas were demarcated for the tests. On the right forearm 2g of the cream was applied and uniformly spread over the stencilled area on all subjects. The patches were covered with surgical dressing for 48hours. On the left forearm, the base cream without nanomaterials and herbal extracts was applied at the same concentration and protocol as on the right forearm. After removal of the patches the forearms were washed with physiological saline solution and the forearms were then observed for erytherma, oedema, urticaria and irritation at point of removal, 15 minutes, 1 hour and 24 hours after removal of the patches.

3.5.2.3 Albinistic consumer panel tests

The consumer panel tests documented under this section were guided by relevant excerpts from the OECD guideline number 428 (2004a) as well as the EU SCCNFP opinion number SCCNFP O750/03 (2003).

Five albinistic human volunteers participated in the study. The participants included 4 females and 1 male (aged 22-27 years, mean 23 ± 3). Ethical approval was obtained from the University of Zimbabwe, JREC under Parirenyatwa group of hospitals and informed consent was obtained from all subjects prior to the patch testing. The volunteers filled in medical questionnaire which confirmed them to be healthy. Subjects were allocated with 2 x 200ml tubes of the cream each and instructed not to use any other cosmetic cream but the actinic damage retarding cream for a minimum of 14 days.

They were then required to fill in a prepared questionnaire (Table 3-7) on the aesthetics

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and functionality parameters of the cream and rate the cream compared to other commercial sunscreens they had used before.

Table 3-8: Treatment panel test score sheet

parameter observation Score Product aesthetics poor 0 colour, fragrance tolerable 1 and feel good 2 Very good 3 excellent 4 Moisturisation and poor 0 emolliency Slight moisturisation 1 Moderate moisturisation 2 High moisturisation 3 Exceptional moisturisation 4 Application poor 0 functionality, acceptable 1 good 2 spreadability, in use Very good 3 and skin after feel exceptional 4 Comparison with poor 0 current donor The same 1 product Slightly better 2 Much better 3 exceptional 4 Packaging, pack poor 0 size ,labeling and acceptable 1 general presentation good 2 Very good 3 exceptional 4 Total possible panel points 20

The scored points from each individual panellist for each parameter were averaged out for the 5 participants. The average scores for each parameter were then added up to give the final score which was expressed as a percentage.

∑ 푎푣푒푟푎푔푒 푡푒푠푡 푠푐표푟푒 푓표푟 푒푎푐ℎ 푝푎푟푎푚푒푡푒푟 % 표푣푒푟푎푙 푝푎푛푒푙 푠푐표푟푒 = 100 푥 20

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A commercial leading sunscreen product was removed from its original packaging and packed similarly to the product being reviewed. Using the same criteria described above, the participants also evaluated the product consumer attributes. The results for both products were then compared to each other.

3.5.3 Ti and Zn assay in finished product

The total Ti and Zn in the finished product was assayed simultaneously for all samples including those used for stability tests by ICP-AES. The method involved adding 1g of the cream sample to a test tube. To the test tubes containing the cream sample, 5 ml of nitric acid was added and shaken by a rotary shaker for 15 minutes. The resultant acid extract was diluted to 10 ml with deionized water and subsequently analysed by

Inductively Coupled Plasma - Atomic Emission Spectrum (ICP-AES Varian; Wavelength

336nm, external calibration, linear range 0.05-10 mg/l) to ascertain the amount w/w of Ti and Zn in the cream. Standard nanometric TiO2 47% w/w dispersion from Sigma Aldrich was used as a quality control standard for Ti and standard nanometric ZnO, 50% dispersion also from Sigma Aldrich was used as the assaying quality control standard for Zn in the cream.

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3.5.4 In-Vitro SPF determination of final albinistic treatment

3.5.4.1 Study Protocol

Five aliquots of 80 mg of the final actinic damage retarding treatment samples was spread over a 40cm2 area of Transpore® surgical tape in this determination and a

Colipa SPF 30 sunscreen standard was used as an assaying guide. The instrumentation, substrates and the methods were identical to the protocol for determining the SPF of the 5% Aloe excelsa lotion described in section 3.3.2.2 above.

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CHAPTER 4

4. RESULTS

“He who knows all the answers has not been asked all the questions.” Confucius (Died 479BC)

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4.1 EXPERIMENTAL STUDIES RESULTS

4.1.1 Ex-Vivo safety investigation of nanometric TiO2 and ZnO

permeation through porcine skin, using vertical, static, Franz

diffusion cells

These results (Section 4.1.1) are reproduced from a publication in a refereed journal by the researcher in fulfilment of the DPhil (medicine) degree requirements in a research article referenced as: Chifamba J et al: ex- vivo penetration of TiO2 and ZnO across actinically damaged porcine skin: development of an albinistic skin protection treatment. Int J Pharm Sci Res 2015; 6(6): 2339-48.doi:10.13040/IJPSR.0975-8232.6 (6).

4.1.1.1 Cosmeceutical cream formulation

“The following formulation (Table 4-1) was developed as the base vehicle cream for the permeation of nanomaterials studies.

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Table 4-1: Optimized O/W emulsion containing nanometric TiO2 and ZnO

Material Usage % Stearic acid 4 Cetyl alcohol 3 Glycerol monostearate 2 Peg 400 1 Cocoa butter 1 Castor oil 2 Liquid paraffin (heavy) 1 Petrolatum 1 Glycerin 3 Monopropylene glycol 2 Sodium PCA 1.5 Carbopol 940 0.07 Methyl hydroxybenzoate 0.2 Propyl hydroxybenzoate 0.2 Caustic Potash 0.4 Triethanolamine 0.4 Nanometric TiO2 5 Nanometric ZnO 5 Watermelon fragrance 0.03 EDTA 0.4 Distilled Water qs

4.1.1.2 Nanomaterials aqueous solubility tests

The aqueous solubility of ZnO was found to be 1.6 mg/ml. TiO2 was totally insoluble in water. The results agree with literature.

4.1.1.3 Porcine skin preparation and induction of actinic damage

Skin irritations were successfully induced on three animals by caustic soda and the solar simulator to replicate actinic damage on porcine skin (Figures 4-1 to 4-3). The actinic damage induced compared very well with that which is observed in albinistic people living within the tropics. Tropical albinistic skin is characterised by inflammation,

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erytherma as well as various primary lesions which include papules, macules, patches, nodules wrinkles and fissures.

a b

Figure 4-1: Porcine skin 1 before (a) and after (b) simulated actinic damage

a b

Figure 4-2: Porcine skin 2 before (a) and after (b) simulated actinic damage

a b

Figure 4-3: Porcine skin 3 before (a) and after (b) simulated actinic damage

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4.1.1.4 Skin penetration studies

The total Ti recoveries from the dermatomes were in the range 97.83- 99.49%, the mean was 98.90 ± 0.51% for the samples analysed. Almost all the Ti applied was recovered from the skin surface in both sets of skin types. A small quantity of Ti was found in the upper layers of the skin epidermis. The average Ti recovered from the skin membranes ranged from 0.02 to 5.18%. The mean was 1.91±1.91%. Epidermal recoveries were marginally higher in actinically damaged skin than intact skin. No Ti was detected in the receptor phase (Tables 4-2, 3, 4,). There was no significant difference in the total recovered Ti from nanomaterials and that from macromolecules of

Ti for both damaged and intact skin. The significant difference noted was in the distribution of total recovered Ti between the epidermal extracts and the unabsorbed base, (Figure 4-4 and 4-5).

In the permeation experiments, the total recoveries for the Zn ions ranged between

101.35 and 103.20%. The average recovery was 102.23± 0.54% (Tables 4-5, 6). It was noted that in either intact skin or actinically damaged skin, virtually, all the total applied

Zn was recovered in the donor compartment. Zn was detected in the skin membranes and the receptor phase which was comparable to untreated vehicle used as standard.

The total recovered Ti and Zn from all treated samples showed corresponding dependence on skin integrity (Figure 4-6)” (Chifamba J et al: 2015).

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Table 4-2: Nanometric Ti recovery from damaged and non-damaged porcine skin

Formulation 5%TiO2 cream (Nano) 5% TiO2 cream (Nano)

Skin Type induced actinic damage No actinic damage animal animal animal animal animal animal Animal Average StDev.S Average StDev.S 1 2 3 1 2 3 Total recovery % 97.83 98.39 98.96 98.39 0.57 98.86 99.23 99.02 99.04 0.19

Unabsorbed base % 94.54 93.21 94.32 94.02 0.71 98.2 98.45 98.78 98.48 0.29

Epidermal extraction % 3.29 5.18 4.64 4.37 0.97 0.66 0.78 0.24 0.56 0.28

Receptor fluid % 0 0 0 0 0 0 0 0 0 0 Table 4-3: Normal Ti recovery from damaged and non- damaged porcine skin

Formulation 5% TiO2 cream, (normal) 5% TiO2 cream (Normal)

Skin Type Induced actinic damage No actinic damage

animal animal animal animal animal animal Animal Average StDev.S Average StDev.S 1 2 3 1 2 3 Total recovery % 99.15 99.49 98.27 98.97 0.63 99.33 99.48 98.9 99.24 0.3

unabsorbed base % 97.52 95.63 95.82 96.32 1.04 99.27 99.36 98.88 99.17 0.26 Epidermal extraction 1.63 3.86 2.45 2.65 1.13 0.06 0.12 0.02 0.07 0.05 % Receptor fluid % 0 0 0 0 0 0 0 0 0 0

Table 4-4: Average Ti recovery from 12 porcine permeation studies

5 % TiO2 Nano 5 % TiO2 nano 5% TiO2 Normal 5% TiO2 Normal Formulation Cream cream Cream cream Skin Type Actinic damage No Damage Actinic Damage No Actinic damage

Code Actinic Damage Intact skin Actinic Damage intact skin

Animals 1,2,3 - % Average Ti Recovery from Table 4 Average StDev.S Total recovery % 98.73 99.1 98.97 99.24 99.01 0.19 unabsorbed base % 94.02 98.48 96.32 99.17 97 2.02 epidermal extraction % 4.7 0.63 2.65 0.07 2.01 1.82 Receptor fluid % 0 0 0 0 0 0

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Aggregate Ti recoveries 100.00 99.00 98.00 recovered from epidermal extract 97.00 96.00

95.00 recovered from 94.00 unabsorbed base

93.00 percentage Ti recoveries 92.00

91.00

Actinic

Damage

NanoTi-

Actinic

Damage

Normal Ti-

Damage

NanoTi- No Normal Ti-No

Figure 4-4: Total Ti recoveries and distribution in dermatomes by ICP-AES

6 Nano epidermal Ti % 5 in damaged porcine skin 4 Nano epidermal Ti % in normal porcine 3 skin Normal epidermal Ti 2 % in damaged porcine skin 1 Normal epidermal Ti % in normal porcine 0 skin percentageepidermal Titanium 1 2 3 Study animal

Figure 4-5: Percentage nanometric and normal Ti recovered from epidermal tissue of 12 porcine dermatomes by ICP-AES

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Table 4-5: Nanometric Zn recovery from damaged and non-damaged porcine skin

Formulation 5% ZnO cream (Nano) 5% ZnO cream (Nano) Skin Type induced actinic damage No actinic damage Animal animal 1 animal 2 animal 3 Average StDev.S animal 1 animal 2 animal 3 Average StDev.S

Total recovery % 101.75 101.95 101.96 101.89 0.12 101.35 102.3 102.04 101.90 0.49

Table 4-6: Normal Zn recovery from damaged and non- damaged porcine skin

Formulation 5% ZnO cream (normal) 5% ZnO cream (Normal) Skin Type Induced actinic damage No actinic damage Animal animal 1 animal 2 animal 3 Average StDev.S animal 1 animal 2 animal 3 Average StDev.S

Total recovery % 103.2 101.35 103.2 101.88 1.07 102.4 102.86 101.96 102.41 0.45

104 Total nanomterials recoveries from dermatomes

103 % Total Ti 102 recoveries from the 12 101 Porcine 100 dermatomes 99 98 % Zn Recoveries

97 from 12 Totalpercentage recovery 96 porcine dermatomes 95 1 2 3 4 5 6 7 8 9 10 11 12 porcine dermatome

Figure 4-6: Total Percentage Ti recovered from 12 porcine dermatomes by ICP-AES analysis and Zn recoveries from 12 porcine dermatomes by Flame AAS analysis.

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4.1.2 In-vivo TiO2 dermato-pharmacokinetics investigations across

albinistic skin by sequential adhesive tape stripping

4.1.2.1 Cream formulation

Table 4-7: Optimized O/W emulsion cream formulation for dermato-pharmacokinetics studies

Material Usage % Stearic acid 4 Cetyl alcohol 3 Glycerol monostearate 2 Cocoa butter 1 Trichilia emetica 2 Liquid paraffin (heavy) 1 Petrolatum 1 Glycerin 3 Monopropylene glycol 2 Carbopol 940® 0.07 Methyl hydroxybenzoate 0.2 Propyl hydroxybenzoate 0.2 Caustic Potash 0.4 Triethanolamine 0.4 Nanometric TiO2 5 Watermelon fragrance 0.03 Approved FDC Colorant 0.0055 Trace Trichilia emetica 0.5 Aloe Excelsa extract 2 EDTA 0.4 Distilled Water qs

The base cream showed good consistency and aesthetics. Table 4-8 shows the treatment cream analytical report.

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Table 4-8: Characteristics of the actinic damage retarding cream

Parameter Result Description Off-white smooth viscous cream and aesthetically pleasing slippery feel when rubbed between two fingers. pH 5.9 Ti content by ICP 3.08 ± 0.3%

4.1.2.2 Skin stripping tests

The total Ti recovered from all volunteers and all sites was in the range 97.07 to

99.76%, the mean was 98.50±0.83% (Figure 4-7). There was no significant difference in recoveries between the male and the female volunteers. However, distribution of Ti in the skin profile was site depended (Figure 4-7). From the surface 67.52 ± 0.95% was recovered from the inner forearm compared to 61.86 ± 0.76% and 54.94 ± 0.34% from outer forearm and forehead respectively. The mean recoveries for upper stratum corneum strippings (strips 2-5) were 29.41±0.60%, 34.29±0.61 and 40.55±1.79% for inner forearm, outer forearm and thee forehead respectively. No Ti was recovered from strippings 9 and 10 for all sites (Table 4-13 to 4-15) (figure 4-8). Results from forehead strips showed the widest standard deviations compared to other sites (Table 4-16).

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120 Total recoveries from all sites Strips 9-10 100 recoveries

80 Strips 6-8 recoveries 60 strips 2-5

40 recoveries Percentage recovery 20 Cotton swab and strip 1 recovery 0 IF1 IF2 IF3 UF1 UF2 UF3 FH1 FH2 FH3 Anatomical site

Key IF-Inner forearm, UF-Outer forearm, FH- Forehead

Figure 4-7: Percentage Ti recoveries from all sites and skin profiles following application of 3.08% Ti cream

Table 4-9: Ti recoveries from inner volar forearm adhesive strippings in the three OCA human volunteers

Formulation 3.08% w/wTiO2 cream (Nano) Anatomical Region Inner forearm Volunteer 1 2 3 Average StDev.S

Total recovery % 98.76 98.23 97.07 98.02 0.86 Cotton Swab and strip 1 67.58 68.44 66.55 67.52 0.95 strips 2-5 29.94 28.76 29.54 29.41 0.60 strips 6-8 1.24 1.03 0.98 1.08 0.14 Strips 9-10 0.00 0.00 0.00 0.00 0.00 Table 4-10: Ti recoveries from upper forearm adhesive strippings in the three OCA human volunteers

Formulation 3.08% w/w TiO2 cream (Nano) Anatomical Region Upper forearm Volunteer 1 2 3 Average StDev.S

Total recovery % 98.38 98.59 97.71 98.23 0.46 Cotton Swab and strip 1 61.8 62.65 61.14 61.86 0.76 strips 2-5 34.8 33.62 34.45 34.29 0.61 strips 6-8 1.78 2.32 2.12 2.07 0.27 Strips 9-10 0.00 0.00 0.00 0.00 0.00

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Table 4-11: Ti recoveries from volar forehead adhesive strippings in the three OCA human volunteers

Formulation 3.08% w/w TiO2 cream (Nano) Anatomical region Forehead Volunteer 1 2 3 Average StDev.S

Total recovery % 98.45 99.54 99.76 99.25 0.70 Cotton Swab and strip 1 54.6 55.28 54.94 54.94 0.34 strips 2-5 38.65 42.2 40.8 40.55 1.79 strips 6-8 5.2 2.06 4.02 3.76 1.59 Strips 9-10 0.00 0.00 0.00 0.00 0.00

Table 4-12: Average Ti recoveries from all three sites from all participants

Formulation 3.08 % w/w TiO2 cream (Nano) Anatomical region Average Volunteer inner forearm upper forearm forehead Average StDev.S

Total recovery % 98.02 98.23 99.25 98.50 0.66 Cotton Swab and strip 1 67.52 61.86 54.94 61.44 6.30 strips 2-5 29.41 34.29 40.55 34.75 5.58 strips 6-8 1.08 2.07 3.76 2.31 1.35 Strips 9-10 0.00 0.00 0.00 0.00 0.00

80 Average site recoveries of Ti 70

60 Surface recoveries AVG 50

40 Strips 2-5 AVG 30

Average Average percentrecovery Strips 6-8 AVG 10

0 inner forearm upper forearm forehead Anatomical site

Figure 4-8: Average percentage Ti recoveries from all three sites from all participants according to number of tape strips.\

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4.2 PRODUCT SENSITIVITY TESTING

4.2.1 In-vivo sensitivity testing: Draize skin sensitivity test

The skin irritation and sensitivity test on rabbits for the treatment cream and the control base cream was evaluated according to Draize irritation classification protocol (Table 4-

8).

4.2.1.1 Erytherma

After 24 hours, the skin irritation score for erytherma and oedema in all 3 rabbits ranged from 0-1 for both the treatment cream and the control base cream, (table 4-13). The results were identical for both the control and the test creams. No erythermal or oedema values above 1 were observed on any rabbit in all the studies carried out (table 4-13).

The limited sites which recorded very minimal erythermal scores of 1 after 24 hours, recorded scores of 0 after 72 hours, showing disappearance of the slight erytherma in limited time.

4.2.1.2 Oedema

For the oedema, the results for the treatment and the control base cream were also identical. No animal exhibited any signs of oedema formation after application of both creams. After the 72 hours, time frame, no rabbit was exhibiting any form of erytherma or oedema and the experiments were concluded as per the guidelines.

Table 4-13: Irritation, erytherma mad oedema scores for rabbit skin sensitivity

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Treatment Control base cream cream Rabbit sensitivity 24hrs 48hrs 72hrs 24hrs 48hrs 72hrs reactions 1 Erytherma 0 0 0 0 0 0 oedema 0 0 0 0 0 0 2 Erytherma 1 0 0 1 0 0 oedema 0 0 0 0 0 0 3 Erytherma 1 1 0 1 0 0 oedema 0 0 0 0 0 0

4.2.1.3 Primary irritation index

For both creams, the Primary irritation index, for all animals, were found to be between

0.04 and 0.09. According to the scoring criteria, this classifies the cream as a “negligible irritant”.

4.2.2 LVET ocular evaluations

No adverse reactions were observed after 24 hours to any of the rabbit eyes.

4.2.3 In-vivo Human sensitivity tests: Patch testing

The patch tests on humans show that the actinic damage treatment poses no irritation and sensitization potential for both albinistic (table 4-14) and normal skin type (table 4-

15) volunteers. The tests were conducted as per the guidelines of the European

Scientific Committee on Cosmetics and Non-food Products intended for Consumers

(SCCNFP opinions 1997-2004).

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Table 4-14: Cutaneous patch reaction after application of treatment on albinistic skin

Treatment cream Control base cream sensitivity Volunteers reactions 15 mins 1 hour 24 hours 15 mins 1 hour 24 hours Pruritus Nil Nil Nil Nil Nil Nil Albinistic female 1 Erytherma Nil Nil Nil Nil Nil Nil urticaria Nil Nil Nil Nil Nil Nil allergy Nil Nil Nil Nil Nil Nil irritation Nil Nil Nil Nil Nil Nil oedema Nil Nil Nil Nil Nil Nil Albinistic female 2 Pruritus Nil Nil Nil Nil Nil Nil Erytherma Nil Nil Nil Nil Nil Nil urticaria Nil Nil Nil Nil Nil Nil allergy Nil Nil Nil Nil Nil Nil irritation Nil Nil Nil Nil Nil Nil oedema Nil Nil Nil Nil Nil Nil Albinistic female 3 Pruritus Nil Nil Nil Nil Nil Nil Erytherma Nil Nil Nil Nil Nil Nil urticaria Nil Nil Nil Nil Nil Nil allergy Nil Nil Nil Nil Nil Nil irritation Nil Nil Nil Nil Nil Nil oedema Nil Nil Nil Nil Nil Nil Albinistic female 4 Pruritus Nil Nil Nil Nil Nil Nil Erytherma Nil Nil Nil Nil Nil Nil urticaria Nil Nil Nil Nil Nil Nil allergy Nil Nil Nil Nil Nil Nil irritation Nil Nil Nil Nil Nil Nil oedema Nil Nil Nil Nil Nil Nil Albinistic male 1 Pruritus Nil Nil Nil Nil Nil Nil Erytherma Nil Nil Nil Nil Nil Nil urticaria Nil Nil Nil Nil Nil Nil allergy Nil Nil Nil Nil Nil Nil irritation Nil Nil Nil Nil Nil Nil oedema Nil Nil Nil Nil Nil Nil

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Table 4-15: Cutaneous patch reaction after application of treatment to normal skin types

Treatment cream Control base cream sensitivity 15 Volunteers reactions 15 mins 1 hour 24 hours mins 1 hour 24 hours Normal skin female 1 Pruritus Nil Nil Nil Nil Nil Nil Erytherma Nil Nil Nil Nil Nil Nil urticaria Nil Nil Nil Nil Nil Nil allergy Nil Nil Nil Nil Nil Nil irritation Nil Nil Nil Nil Nil Nil oedema Nil Nil Nil Nil Nil Nil Normal skin Female 1 Pruritus Nil Nil Nil Nil Nil Nil Erytherma Nil Nil Nil Nil Nil Nil urticaria Nil Nil Nil Nil Nil Nil allergy Nil Nil Nil Nil Nil Nil irritation Nil Nil Nil Nil Nil Nil oedema Nil Nil Nil Nil Nil Nil Normal skin male Pruritus Nil Nil Nil Nil Nil Nil Erytherma Nil Nil Nil Nil Nil Nil urticaria Nil Nil Nil Nil Nil Nil allergy Nil Nil Nil Nil Nil Nil irritation Nil Nil Nil Nil Nil Nil oedema Nil Nil Nil Nil Nil Nil

4.2.4 Formulation development of the albinistic treatment

4.2.4.1 Extraction of Trichilia emetica (Banket mahogany)

The following results in table 4-16 and 4-17 were obtained after extraction of the seed oil; the results are a summary from work done by the researcher and a third part analytical laboratory where the sample was sent for instrumental analysis and characterisation.

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Table 4-16: Physico-chemical analysis of Trichilia emetica

PARAMETER RESULTS EXPECTED RESULTS Extraction yield 26% No available literature turbid liquid , which quickly No available reference data Appearance solidifies at lower temperatures Color Yellow golden brown No available reference data Odour Characteristic dull fatty scent Rel. Density 0.91 0.90-0.93 (ISO279) Ref Index (ISO 280) 1.45 1.3-1.8 Acid value 3.45 No available reference data Iodine value 74 No available reference data Peroxide value 11.85 No available reference data Saponification 178 130-180 value *analysis in shaded areas were done by a third part analytical laboratory

Table 4-17: Fatty acid (%) composition of Trichilia emetica

FATTY ACID COMPOSITION % RESULTS Myristic Not found Saturated Palmitic 45 stearic 4.2 Oleic 51 linoleic 16 α linoleic Not found Unsaturated linolenic 16 erucic Not found Arachdonic Not found

 Only the fatty acids in the shaded regions were found in significant quantities

4.2.5 Extraction of Aloe excelsa gel matrix

The following results (table 4-18 and 4-19) were obtained after extraction of the plant material; the results are from work done by the researcher and a third part analytical laboratory where the sample was sent for instrumental analysis.

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4.2.5.1 Aloe excelsa physico-chemical analysis

Table 4-18: Physico-chemical analysis of Aloe excelsa

PARAMETER RESULTS EXPECTED RESULTS Extraction yield 11% No available reference data Appearance Frothy gel paste No available reference data Color Off white-pale greenish No available reference data Odour Non characteristic No available reference data Rel. Density 0.98 No available reference data (ISO279) Ref Index (ISO 280) 1.85 No available reference data

Table 4-19: Aloe excelsa leaf crude composition

Compounds % availability Crude protein 7 Carbohydrates 28 Crude fiber 50 Crude fat 1.8 Ash 12

4.2.5.2 In-vitro Sunburn Protection Factor (SPF) determination of

5% Aloe excelsa in a cosmetic lotion

After various formulations were tried, the optimum stable formulation incorporating Aloe excelsa had the following composition.

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4.2.5.2.1 Formulation

Table 4-20: optimized aloe excelsa base cream formulation

Material % incorporation Stearic acid 5 Cetyl alcohol 2 Ceto stearyl alcohol 2 Glycerol monostearate 2 Cocoa butter 1 Castor oil 1 Medilan 0.5 Liquid paraffin (heavy) 1 Petrolatum 2 Glycerin 4 Monopropylene glycol 1 Carbopol 940® 0.07 Methyl hydroxybenzoate 0.2 Propyl hydroxybenzoate 0.2 Caustic Potash 0.3 Triethanolamine 0.6 Aloe Excelsa extract 5 EDTA 0.3 Distilled Water qs

4.2.5.2.2 Analytical report

The physico-chemical analysis done for the cream yielded the following results.

Table 4-21: Aloe excelsa cream Analytical Report

Parameter Result Description Off white, smooth, viscous cream that is esthetically pleasing slippery, feel when rubbed between two fingers. pH 5.80 Odour Characteristic of un-fragranced base cream

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4.2.5.3 SPF Determinations of 5% Aloe excelsa cream

The SPF for the 5% Aloe excelsa cream was determined to be 1.85 by Optometrics® spectrophotometric 290s instrumentation. The instrument plotted graph is shown in

Figure 4-9. The graphs are difficult to discern due to the standard instrumentation plot calibration which shows the chart plot area from 0. An excel spread sheet close up was therefore done in Figure 4-10, to help with graph resolution.

2 1.8 1.6 1.4 SCAN 1 1.2 SCAN2 1 MPF SCAN 3 0.8 0.6 SCAN 4 0.4 SCAN 5 0.2 AVG MPF

284 292 300 308 316 324 332 340 348 356 364 372 380 388 396 WAVELENGTH

Figure 4-9 Instrument MPF scans for Aloe excelsa cream

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1.8 1.75

1.7 SCAN 1 1.65 SCAN 2

MPF 1.6 SCAN 3 1.55 SCAN 4 1.5 SCAN 5 AVG MPF

1.45

284 292 300 308 316 324 332 340 348 356 364 372 380 388 396 WAVELENGTH

Figure 4-10: Excel spread sheet close up of instrument MPF scans for aloe excelsa cream

4.2.6 Extraction of Myrothamnus flabellifolia by steam distillation

The following results were obtained after extraction of the plant; the results are from work done by the researcher and a third part analytical laboratory where the sample was sent for instrumental analysis.

4.2.6.1 M. flabellifolia physico-chemical analysis

The physico chemical analysis from wet techniques and instrumental are shown in table 4-22

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Table 4-22: Physico-chemical analysis of the Myrothamnus flabellifolia extract

PARAMETER RESULTS EXPECTED RESULTS Extraction yield 2.85±0.14 % No available literature Clearly liquid above 25o C , No available reference data Appearance becomes turbid at lower temperatures Color Pale yellow No available reference data Characteristic terpinene smell Characteristic terpinene Odour smell Rel. Density 0.92 Below 1 (ISO279) Ref Index (ISO 280) 1.505 1.3-1.8 Op Rotation (ISO +7 +5-+15 592) Gas Chromatography analysis after desiccation 1-8-cineole 2.2% No available reference data Terpinene -4-0l 8% No available reference data Sucrose 22% No available reference data Arbutin 38% No available reference data 4.3 ALBINISTIC ACTINIC DAMAGE RETARDING

TREATMENT EMULSION DEVELOPMENT

4.3.1 Treatment formulation protocol

Out of the 18 potential pilot formulations done, which all successfully incorporated the nanomaterials and the herbal extracts. All the formulations with nanomaterials were translucent compared to the opaque macromolecular formulations (Figure4-11). Nine formulations met the required pH specification which was used as the basis for an initial screening. The selected nine formulations were subjected to various physical and stability tests as well as microbial challenge tests to screen for the best treatment (Table

4-23). Treatments SG3 (Solar-Guard 3), SG7 and SG8 passed all stringent physical stability testing and challenge tests with shelf lives far exceeding the minimum required 167

18 months. The 3 alternative treatments with formulations shown in table 4-24 passed all tests,

The test results were too close to call any one of the formulations “the best among the

3”. SG3 was however chosen as the optimum treatment after costing formulations (table

4-24) showed that the product was 4% lower in total supply cost than the SG7 and SG8 as shown in table 4-24.

Figure 4-11: Application of macromolecular cream (L) and the nanometric cream (R) on a research participant

4.3.1.1 Stability tests results for the 9 selected formulations (SG1-

SG9)

Table 4-23: Overall stability test results for the final 9 formulations

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Treatment formulation

Specifications SG1 SG2 SG3 SG4 SG5 SG6 SG7 SG8 SG9

Emulsion pH 5.3-6.0 6.2 5.3 5.9 5.8 5.4 5.8 5.8 5.9 5.9

Specific gravity 0.91-0.95 0.94 0.92 0.94 0.95 0.93 0.96 0.94 0.93 0.92

Freeze thaw cycle test pass pass pass pass pass pass pass pass pass pass

High temp stress testing pass pass pass pass pass pass pass pass pass pass

Window ledge tests pass colour colour pass pass colour pass pass pass pass loss loss loss

Stress cycle tests pass pass pass pass pass pass pass pass pass pass

Centrifugation pass pass pass pass pass pass fail pass pass pass

Transit vibration tests pass pass pass pass pass pass fail pass pass pass

Spreadability tests pass pass pass pass pass pass pass pass pass pass

Preservative challenge tests 5 out of 5 ⅗ pass pass ⅖ pass pass pass pass ⅖

Minimum Shelf life 18 months 18+ 18+ 18+ 18+ 18+ 18+ 18+ 18+ 18+

Formulation pass/fail fail fail pass fail fail fail pass pass fail

SG –Solar guard is the code for the actinic damage retarding treatments

4.3.1.2 Batch and costing formulations for the final optimized

treatments (SG3, SG7, SG8)

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Table 4-24: Costing formulations for the final 3 treatments

FORMULATIONS 100kg BATCH COSTING

Starting material Cost SG3 SG 7 SG8 SG3 SG7 SG8 price/Kg % input % input % input Cost/100kg Cost/100kg Cost/100kg) Stearic Acid T/P F $2.50 5.0 4.0 5.0 $12.50 $10.00 $12.50 Cetyl Alcohol $6.00 2.5 2.0 2.5 $15.00 $12.00 $15.00 Cetyl Stearyl Alcohol $6.00 1.0 2.0 $6.00 $12.00 $0.00 EGMS $12.00 2.0 $0.00 $0.00 $24.00 PEG 400 $7.00 $0.00 $0.00 $0.00 GMS S/E $8.00 1.0 1.0 1.0 $8.00 $8.00 $8.00 White Petroleum Jelly $2.00 1.0 2.0 3.0 $2.00 $4.00 $6.00 Mineral Oil 68 (Ondina) $3.00 1.0 2.0 1.0 $3.00 $6.00 $3.00 Castor oil $7.00 $0.00 $0.00 $0.00 Trichilia emetica $15.00 2.0 2.0 2.0 $30.00 $30.00 $30.00 Lanolin BP ANHY $20.00 0.3 1.0 1.0 $6.00 $20.00 $20.00 Myrothamnus Flabellifolia $50.00 1.5 1.5 1.5 $75.00 $75.00 $75.00 Aloe excelsa $50.00 3.0 3.0 3.0 $150.00 $150.00 $150.00 Urea $30.00 0.0 1.0 0.0 $0.00 $30.00 $0.00 Glycerin $3.00 3.0 3.0 5.0 $9.00 $9.00 $15.00 Carbopol 940 $40.00 0.1 0.1 0.1 $2.80 $2.80 $2.80 Mono Propylene Glycol $5.50 1.0 1.0 2.0 $5.50 $5.50 $11.00 EDTA Tetra sodium $16.00 0.1 0.1 0.1 $1.28 $1.28 $1.28 Triethanolamine 85% $12.00 0.6 0.6 0.6 $7.20 $7.20 $7.20 Caustic Potash $4.50 0.4 0.0 0.2 $1.80 $0.00 $0.90 lavender perfume $36.00 0.2 0.2 0.2 $7.20 $7.20 $7.20 Methyl paraben $9.00 0.2 0.2 0.2 $1.80 $1.80 $1.80 Propyl paraben $9.00 0.2 0.2 0.2 $1.80 $1.80 $1.80 Sola-guard XT 100* $100.00 2.5 2.5 2.5 $250.00 $250.00 $250.00 Sola-guard XT 300* $100.00 3.0 3.0 3.0 $300.00 $300.00 $300.00 Water 70.45 67.65 65.45

Total Batch 100.000 100.000 101.500 $895.88 $943.58 $942.48

Cost /200ml $1.79 $1.89 $1.88 Packaging cost $0.60 $0.60 $0.60 Total product cost $2.39 $2.49 $2.48 Manufacturing Direct expenses (Utilities, labour and statutory expenses) $0.36 $0.37 $0.37 Indirect expenses (Overheads and distribution costs) $0.48 $0.50 $0.50 Expected total treatment Supply Cost $2.75 $2.86 $2.86

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*Sola-guard 100XT- The nanometric titanium dioxide based dispersion developed by the researcher during the study and awaiting patenting

*Sola-guard 300XT- The nanometric zinc oxide based dispersion developed by the researcher during the study and awaiting patenting

4.3.1.3 Panel test comparison of SG3 and Nivea SPF30 sunscreen

Figure 4-12: SG3 lotion in its final packaging as compared to Nivea SPF 30 lotion The panel results from a comparison between SG3 and commercial Nivea SPF 30 are shown in table 4-25. The general comments from the panellists are shown in table 4-26

Table 4-25: Comparative consumer panel test scores for SG3 and a commercial SPF 30 product

Parameter SG3, SPF 15 Nivea SPF30

Product aesthetics 3.7 3.3 Functionality and emolliency 4 3.8 Spreadability and after feel 3.2 3.1 Comparison with current donor product 3.8 3.1 Packaging size and general presentation 3 3 Total panel scores 17.7 16.3

Percentage consumer panel score 88.50% 81.50%

Table 4-26: General comments from panellists on panel test product use scoring 171

Panelist Comment “Both test products are way better than the donor Female panelist 1 sunscreen products, they moisturize and are real cosmetics” “These products are very good, but the commercial one Female panelist 2 dries my skin” “The donor products are too liquid, product SG3 is very Female panelist 3 thick and stays on skin for a long time” Female panelist 4 “Product SG3 is very moisturizing but a bit greasy “SG3 is very thick, you can even use it on the lips and it Male panelist 1 stays there”

4.3.1.4 Pricing comparison

SG3 lotion 200ml, expected selling price is below $6.00 compared to $21.05 for Nivea SPF 30 lotion.

4.3.2 In-Vitro SPF determination of SG3 albinistic treatment

4.3.2.1 SPF determination

The SPF of the SG3 treatment was determined to be 16.64. Figure 4-12 shows the Optometrics 290S graph for the combined 5 instrument scans. The data was transferred to excel spread sheets, to show a higher resolution of the graphs (Figure 4-13).

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18 16 14 12 SCAN 1 10 SCAN2

MPF 8 SCAN 3 6 SCAN 4 4 SCAN 5 2 AVG MPF

284 292 300 308 316 324 332 340 348 356 364 372 380 388 396 WAVELENGTH

Figure 4-13: Optometrics 290S instrument scans for the SG3 actinic damage treatment

16.8

16.75

16.7 SCAN 1 16.65 SCAN 2

MPF 16.6 SCAN 3 SCAN 4 16.55 SCAN 5 16.5 AVG MPF

16.45

284 292 300 308 316 324 332 340 348 356 364 372 380 388 396 WAVELENGTH

Figure 4-14: Amplified excel presentation of the actinic damage treatment MPF scans

4.3.2.2 Active ingredient assay

The final product analysis for the content of Ti and Zn for SG3, 7 and 8 is shown in table 4-27. 173

Table 4-27: Active ingredient assay for Ti and Zn by ICP-AES

Ingredient Specification SG3 SG7 SG8 Mean

Titanium assay by ICP-AES 3% 3.08% 3.11% 3.03% 3.07% Zinc assay by ICP-AES 4% 3.75% 3.64% 3.79% 3.73%

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CHAPTER 5

5. DISCUSSION

“The essence of knowledge, is having it, to apply it” Confucius (Died 479BC)

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5.1 MATERIAL SAFETY INVESTIGATION

5.1.1 Ex-Vivo safety investigation of nanometric TiO2 and ZnO

permeation through porcine skin, using vertical, static, Franz

diffusion cells

The following discussions (section 5.1.1) are reproduced from a publication in a refereed journal by the researcher in fulfilment of the DPhil (medicine) degree requirements in a research article referenced as: Chifamba J et al: ex vivo penetration of TiO2 and ZnO across actinically damaged porcine skin: development of an albinistic skin protection treatment. Int J Pharm Sci Res 2015; 6(6): 2339- 48.doi:10.13040/IJPSR.0975-8232.6 (6).

Naik et al: 2009, demonstrated that for skin penetration to occur, an active ingredient

(API) should have an aqueous solubility of at least 1 mg/ml; a molecular weight below

500 Da; a melting point below 200 °C and a partition coefficient (Log P) between 1 and

3.9 (Naik et al: 2009). Such parameters and other physical properties of normal macromolecular Ti and Zn do not lie within the required range to facilitate skin penetration and therefore do not pose any threat to systemic exposure when used in sunscreens. Normal Zn and Ti are therefore excellent safe, broad spectrum sun blocks that remain on the skin periphery with no risk of systemic exposure. The drawback however is the opaqueness in formulations which make preparations unsuitable for all day wear. This problem has recently been circumvented by introduction of nanometric

TiO2 and ZnO, which can be incorporated in clear aesthetic sunscreens (Gamer A.O et al: 2006). However, there is debate on whether these safe physico-chemical properties

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of the normal molecules are affected or changed in nano-molecular analogues (Gamer

A.O et al: 2006). For practical product development purposes, it is not fully possible to predict skin diffusion by the use of a few physico-chemical properties from literature, especially when considering that albinistic skin in the tropics is almost always actinically damaged. Dermatological studies only tend to focus on drug permeation through normal healthy skin. Various researchers have carried out comparative studies on the diffusion of nanomaterials across human and porcine skin, though, using healthy uncompromised skin and its equivalents (Marisa D Newman et al: 2009). All these studies done before are therefore not relevant to commercial actinic damage retarding treatments product development for PLWA and living in the tropics.

This study therefore carried out tests on simulated actinic damage induced porcine skin that exhibited most symptoms afflicting PLWA except for open wounds which would obviously compromise accuracy of results. Literature on hand tentatively predicts that nanomaterials might not permeate normal healthy skin that is not compromised in any way (Newman M D et al: 2009). To the best of my knowledge, no published studies are available on investigations using damaged skin exhibiting symptoms prevalent in oculocutaneous albinism. This experimental study using damaged porcine skin contributes to the debate on safety of nanomaterials in practical commercial treatments by focusing on actinically damaged skin in tropical conditions and set ups.

Diffusion through the (non-living) stratum corneum peripheral layer of skin is the rate limiting step for percutaneous absorption (Dugard P H: 1984). The permeability properties of the stratum corneum remain unchanged with removal from the body and therefore ex-vivo experiments like this one, are appropriate and offer significant 177

advantages over whole animal or human volunteer experiments (Van Ravenzwaay et al:

2004). The study investigates the health risks posed by nanometric titanium and zinc oxides in compromised skin, through absorption across excised porcine skin mounted on static vertical Franz diffusion cells. Porcine skin was an excellent choice for this study because various studies have validated in numerous investigations that it is similar in structure to that of human skin (Kaidi Z, Jagdish S: 1999).

More so, the studies were practical in that they determined the dermato- pharmacokinetics of the nanomaterials in actual commercial grade aesthetic formulations which did not exclude the possible effects of other typical cosmeceutical starting materials. The formulation contained no trans-epidermal vehicle with a molecular weight below 500 Da and no parameter that could promote skin absorption.

The induced actinic damage was certified by a practicing dermatologist as a comparable replicate of actinic damage characteristics in oculocutaneous albinism afflicted people living within the tropics.

Employing the various techniques and methods mentioned above, the investigations showed that irrespective of typical albinistic actinic damage characteristics, there is no evidence of penetration through the skin of Zn and Ti particles regardless of the shape and particle size of the metallic oxides. The nanomaterials and the normal metallic oxides were mostly deposited on the stratum corneum periphery; the small quantities recovered from the epidermal extractions were most likely to be from epidermal furrows wrinkles and hair ducts as evidenced by the wide standard deviations of epidermal recovered materials from the mean in actinically damaged porcine skins as compared to intact skin. There are no marginal differences in distribution of the nanomaterials in both 178

intact and damaged skin from that of the normal molecules. The slight discrepancies observed in the results are most likely to be from the higher probability of nanosized materials being trapped in hair ducts, skin furrows and ridges than any significant active penetration.

In all the 4 study categories documented above involving 12 animal dermatomes, for skin penetration of both nanometric and normal Ti, the average total recoveries for each category were very closely related and ranged from 98.73 to 99.24% with a standard deviation of 0.19. Virtually no Ti was found in the receptor phase for all investigations.

What differed was the distribution of the recovered materials. On average 98.83% Ti was recovered from the unabsorbed base in the 2 categories with intact skin compared to an average 95.17% recovery from the 2 investigations with actinically induced damage. Damaged skin retained an average of 3.70% of both normal and nanometric Ti compared to 0.35% for normal intact skin. This was most likely due to accentuation of wrinkles and furrows as a result of actinic damage to the upper layers of the stratum corneum rather than any technical penetration. The standard deviation of epidermal recoveries was very wide at 1.82 implying that the nature and extend of actinic damage and alteration of skin profiles is related to extend of penetration.

In the experiments with zinc oxide, recoveries of more than 100% of applied zinc were observed. It was noted that the untreated skin samples and those treated with a base without the sunscreens already had up to 3 µg of intrinsic zinc which corresponds to at least 2 % concentration of the applied dose in the donor compartment.

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The amounts of recovered zinc from the skin samples epidermal extractions and receptor phase were comparable for all skin samples treated and untreated, implying that there were no significant contributions from donor phase formulation zinc.

The virtual recovery of Zn from untreated samples also indicates that the instrumental techniques and analytical methods were adequate for the purposes of this study. The instrument sensitivity of the analytical tests was also sufficiently high” (Chifamba J et al:

2015).

5.1.2 In-vivo TiO2 dermato-pharmacokinetics investigations across

albinistic skin by sequential adhesive tape stripping

For tape stripping studies to produce reproducible results, standardization of procedures and influencing conditions is paramount. The type of tape used, the application pressure and the velocity of tape removal are all investigator depended factors that can influence results (Loffler H et al: 2007). Seasonal conditions like temperature and humidity have well been demonstrated to influence trans-epidermal water loss (TEWL) and consequently dermato-pharmacokinetics (Tokumura F et al: 2006). Differences in spontaneous desquamation are depended on underlying corneocytes structures which are in turn, site depended. Some anatomical sites especially those adjoining joints areas are also prone to muscular movements that can affect the results. Earlier studies have confirmed that many factors mentioned above significantly affect outcomes of stripping studies (Loffler H et al: 2004). In this investigation therefore, an attempt to

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carefully and adequately define influencing parameters and procedures was done so as to minimize results variability.

It has been demonstrated that even after 50 strippings, complete removal of the stratum corneum is not possible (Laderman J et al: 2008), therefore, tape stripping as a non- invasive technique is a very appropriate tool for this study. The absorption of Ti in a commercial albinistic treatment was studied because it is recognized that percutaneous absorption is vehicle depended (Hostynek JJ et al: 2002) and analysis is best carried out with formulations as they would be used by patients. The choice of the three anatomical sites used was motivated by visual appraisal of tropical albinistic skin, the facial and neck skin is frequently the most actinically damaged. The outer (back) of the volar forearm is heavily keratinized and the inner forearm is usually the least actinically damaged of all exposed tropical albinistic skin.

The experiments were essentially carried out in summer at a subtropical location.

Seasonal variations in skin TEWL, electrical conductance and susceptibility to dermal irritations are all higher in summer and all have effects on dermato-pharmacokinetics and skin permeability (Tokumura F et al: 1999). It is therefore expected that the results represent a worst case scenario for dermal absorption. The studies assumed actinic damage in albinistic persons living within the tropics to be at its worst during this season. The application rate of 10 mg/cm2 used in the study is also 5 times higher than the average expected sunscreen application rate and the FDA recommendation of 2 mg/cm2.

In the studies all the Ti was recovered from the first ten strips which represent the surface and the upper layers of the stratum corneum. This is in line with the results from 181

the previous ex-vivo studies on the absorption of Ti through damaged porcine skin.

Total Ti recovered from all sites was comparable for all sites. All Ti was confined to the surface, upper and lower stratum corneum and no Ti was detected beyond the stratum corneum. Skin permeation across the stratum corneum was not expected because for permeation to occur the active material must have a partition co-efficient between 1 and

3.9, reasonable aqueous solubility and a molecular weight below 500Da (Naik et al:

2006) which is not the case with Ti. The formulation was based on typical CIR approved ingredients. The ingredients did not include any material that could facilitate transdermal mobility or promote skin absorption. The formulation as an actinic damage treatment was expected to stay on the skin periphery where Ti would refract UVR. The pH was adjusted to 5.9 so as to minimize the potential for skin irritation due to wide discrepancies between the treatment and albinistic skin pH which from this study was found to be between 5.8 and 6. Albinistic skin maintained its skin integrity against percutaneous nanometric Ti absorption incorporated in the treatment formulation.

However the distribution of the nanomaterials in the stratum corneum was influenced greatly by anatomical site. The forehead area which exhibited greater actinic damage had much more material recovered by tape strippings 2-5 than any other site. The facial skin had marginally more wrinkles and keratinized skin than all the other sites, the material was therefore most likely logged in skin folds and crevices and could not be recovered by the cotton swab and the first strip. It was also noted that all heavily keratinized sites of the face and neck exhibited sparse terminal hair follicles not typically observed on non-hyper-keratinized areas and in normal female skin types. Sparse vellus hairs appear to be hyper-keratinized to terminal hair in all areas exhibiting rough 182

skin. The differences between inner and outer forearms could be explained by the higher keratinisation and higher volumes of vellus and terminal hair follicles in the outer volar forearm. The accentuated roughened skin furrows and the hair ducts could have trapped the material and prevented it from being recovered from the surface. It is also recognized that there is a difference in the number of cell layers and thickness of the stratum corneum between the forehead and the forearm (Laderman J et al: 2008). The fewer layers on the forehead could have facilitated a faster movement of Ti from the surface to lower stratum corneum levels.

The tape application pressure which affects corneocytes removal rate (Loffler H et al:

2004) could have been depended on underlying structures and biomechanical properties. The bone structure immediately found under the skin of the forehead could possibly have increased the tape pressure compared to forearm skin which is not immediately supported by underlying bone structures. Apart from the absence of internal exposure, the investigation also demonstrated that the results obtained from skin stripping are depended on the procedure, site and general skin condition. The differences in site distribution of Ti in the stratum corneum appear to be more related to extend of actinic damage rather than a direct implication that the skin on the inner forearm is less penetrable by Ti than the outer forearm and the facial area.

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5.2 PRODUCT SENSITIVITY TESTING

5.2.1 Draize skin sensitivity

The skin is always exposed to potential irritants, both intentionally and accidentally during the use of topical treatments. It is therefore imperative that the potential for any topical application to cause adverse skin effects is evaluated.

Adverse effects of formulations on skin which are generally referred to as skin irritations are due to various factors which include the concentration of the irritants in the product, the duration of the contact time, the body site that has been exposed to the potential irritant, the skin permeability and toxicity profile of the potential irritant. The determination of the potential to cause irritancy by any skin care product is therefore necessary and mandated. The OECD guideline 404, COLIPA as well as the FDA recommend the rabbit skin irritancy evaluation based on the methods document by

Draize (Draize H: 1944) done in this study.

For ethical reasons, OECD guideline 404 recommends a decision making protocol prior to any in-vivo animal test for skin irritation and corrosivity based on the product analytical report evidence and product pH (OECD TG 404: 2002) which was adhered to in this study. Examples of the decision making considerations done include;

 Substances with pH below 2 or with pH above 11.5 should never be tested on

animals under the Draize tests due to the obvious adverse effects on animals,

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 Any substances previously found to be corrosive in any other sensitivity tests

documented under Annex V of Dir. 67/548/EEC should never be included in the

Draize tests (OECD TG 404: 2002).

In this study, the product analytical report and pH justified the use of the Draize tests.

Another point to note is that despite the ethical concerns, to date, there is no validated alternative method for evaluating skin irritancy and corrosivity to the Draize tests (Zuang et al: 2002).

The standard rabbit skin test is an adequate appraisal method for skin sensitivity. The results are acceptably indicative of the safety of the treatment on humans even though it is known that rabbit skin is physiologically different to human skin and the response to environmental and chemical agents is expected to differ. However, after considerable tests, Professor Wilhelmus concluded that even though the rabbit skin was anatomically different from the human skin, it was more sensitive to chemicals making it a conservative and acceptable model of the human skin. He endorsed the Draize tests and concluded that they had assuredly saved man from harm (Wilhelmus K R: 2001). A lot of other scientists have endorsed the use of rabbit skin in sensitivity testing and regulatory agents still maintain it, as a credible assessment of irritation potential (More B

H et al: 2013).

In these studies, no significant oedema and erytherma was observed on any of the test animals within the specified test period. The PII for all animals, which ranged between

0.04 and 0.09, is negligible irritation potential. Skin irritancy, was not expected from the formulation since only CIR approved ingredients, had been used in the product and the

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previous test on damaged porcine skin using Zn and Ti documented above had showed no adverse effects on skin.

5.2.2 Low volume eye test (LVET) irritation tests

Even though, industry and regulatory bodies are actively looking for alternatives to supplant the Draize tests. The OECD was yet to validate any alternative method for assessing eye and skin irritation potential by 2009 (OECD: 2009). However, in 2009 the

OECD validated two alternative methods, which included an Isolated Chicken Eye test

(ICE), and the Bovine Cornea Opacity Test (BCOP) (reference?). However, a previous study conducted in 2005 had however shown that none of these methods was as reliable as the Draize test (Doucet O et al: 2006). A tiered system has therefore been adopted by most regulatory bodies and researchers in using alternatives to the Draize test when there is severe envisaged potential damage to the eye and skin (Doucet O et al :2006). In this study, the LVET was conducted after ethical considerations in using

Draize eye tests. The LVET was also opted for on the basis that (Weil and Scala: 1971) proved that the Draize tests were over predictive of irritation effects of agents on humans due to the differences in rabbit and human anatomy. Nixon et al: 1975 also recognized that the design of Draize ocular tests and the visual grading of effects are highly subjective and might have very little relevance to humans.

The treatment under study is not intended for eye use, exposure to the eyes is therefore only expected to be accidental. Low volumes (LVET) were used after considering the size of the rabbit eye and the probability of potential exposure to the eyes during use. 186

As expected, however, no adverse ocular effect was observed during the studies.

Studies by the FDA have shown that substances that are irritants to the skin are also potential irritants to the eyes (FDA: 1965). The ocular tests were only carried out after the skin sensitivity tests had rated the product as a negligible irritant and therefore the results for LVET were predictable and came out as expected.

5.2.3 In-vivo Human sensitivity tests: Patch testing

The majority of contact dermatitis cases at referral health clinics are a result of adverse reactions to cosmetics and topical preparations (Nigam P.K: 2009). However, most of these reactions are mild and irritant in nature rather than allergic. Acute adverse reactions may occur in various forms and formats including percutaneous absorption, acute and sub-acute toxicity, skin and eye irritations and sensitizations, photo toxicity as well as photo irritations. Product safety assessment through patch testing is therefore mandatory for new products and primarily depends on how and where the product is applied. Before commercialization, topical treatments are mandatorily tested on a small controlled human population to confirm compatibility and acceptability as safe and non- irritant topical treatments (Nigam P K: 2009).

The studies were guided by mandatory Technical guidelines from the OECD and the

Opinion concerning guidelines on the use of human volunteers in compatibility testing of finished cosmetic products. This opinion was adopted by the Scientific Committee on

Cosmetics and Non-food Products intended for Consumers during the plenary session of 23 June 2009. The opinion limits the number of human subjects to be used in compatibility tests for cosmetics as between 5 and 8 and goes on to recommend the 187

least possible number of subjects (Council Directive (76/768/EEC). The EEC is mindful of an environment where consumers are exposed to numerous harmful treatments due to the uncontrolled nature and proprietary nature of “gimmick” cosmetic active ingredients. The final decision in selecting a cosmetic product is also based on aesthetic tastes and non-technical which are very subjective and consumer depended (Council

Directive (76/768/EEC).. The population size of consumers in panel tests is not related to commercial success neither does it guarantee product acceptability to an extent.

In these studies, even though the treatment is expected to be used on the open skin, closed patch testing was conducted because it represents the worst case scenario in product use since occlusion is believed to worsen sensitization by skin irritants (Nigam

P K: 2009). The amount of product used in the study was also higher than recommended application of sunscreens at 2 mg/cm2 .Although this could have given false positive reactions due to increased concentration of potential irritants per cm2. The justification for using higher doses in these studies was due to the fact that, cosmeceuticals are ‘leave in’ treatments, rates of applications and replenishment intervals by users are very subjective and varies from patient to patient. It was therefore decided to use worst-case scenarios of application concentrations, occlusions and treatment residence time before washing off. Adverse skin reactions to actinic damage treatments including photo toxicity and photo allergic reactions are usually from chemical sunscreens like benzophenones and salicylates. Additives such as fragrances and preservatives also contribute to skin irritations (Linda B: 2005). In this treatment, no chemical sunscreen with a potential to penetrate the skin was used. TiO2 and ZnO are 188

non-irritants and therefore no adverse reactions were expected from these sunscreens as proved by the foregoing experimental work in this research. No artificial fragrance and colorant was used in the product. This also eliminated the other sources of potential irritants. The rest of the ingredients used in the treatment formulation are CIR approved non-irritants. It was therefore expected that the product formulation will not induce adverse reactions to users.

5.3 FORMULATION DEVELOPMENT OF THE

ALBINISTIC TREATMENT

5.3.1 Extraction of Trichilia emetica fixed oil

The Trichilia emetica extractions employed both physical and chemical methods. The scarlet envelopes of the seeds were removed before cold expeller oil extraction because the colour of the envelopes would be imparted to the oil. Cold extraction methods are highly recommended for fixed oils with potential use in pharmaceuticals due to the need to preserve heat sensitive active constituents and also to avoid contamination by toxic organic solvents. The majority of the oil was extracted through cold expression. However, the method has low yields and substantial oil remains in the resultant cake. The cake was therefore subsequently further extracted by solvents to scavenge for the remaining oil. Fixed oils are notorious for aerial oxidation leading to quick discoloration and rancidity. Anti-oxidants including tocopherol acetate and BHT were therefore added to control aerial oxidation.

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The overall oil extraction yields are high and commercially viable and at 26%, are comparable to other seed oil extractions. The turbid yellow liquid quickly solidified at lower temperatures, most likely as a result of the high levels of saturated palmitic acid as well as unsaturated, oleic, linoleic and linolenic fatty acids.

Palmitic and oleic acids are excellent permeation enhancers (Kim et al: 2008) and it is prudent to assume that the oil has immense potential use in cosmeceuticals. Other research has shown that materials with high oleic acid levels are natural anti-oxidants, it is therefore reasonable to predict again that T. emetica oil should exhibit anti-oxidancy and consequent skin protection in formulations (Talcott et al-2005). The iodine value at

74 points to a higher degree of saturation compared to lighter oils like sunflower and soybean oils with Iodine values above 120. This essentially makes Trichilia emetica oil a semi “hard oil” with characteristics comparable to other kernel oils like palm stearin though not as “hard” as coconut oil. The iodine value and the saponification value at

178 points to potential use of the oil in soap making and as an emollient in cosmeceutic formulations. There is not much reference data available on Trichilia emetica oil to make comparisons with, and no published information on product development using the oil in formulations is available. The presence of limonoids like Trichlin A in the oil suggests that Trichilia emetica oil must have antimicrobial and anti-inflammatory effects.

5.3.2 Extraction of Myrothamnus flabellifolia essential oil

Steam distillation was the extraction method of choice for essential oils of Myrothamnus flabellifolia. The 2.85% extraction yield is comparable to other essential oil extractions like tea tree oil and eucalyptus. The odour is characteristic of terpinene containing 190

essential oils and is due to the 8% terpinene-4-ol found in the extract by gas chromatography. The anti-fungal and anti-septic value of the essential oil is most likely from the abundance of cineole’s and terpinene in the oil as analyzed.

The availability of arbutin (38%) in the extract and the unusually high levels of sucrose

(22%) in the desiccated leaves are probably responsible for the purported wide medicinal properties and the extreme resistance of M. flabellifolia to stress. It has been demonstrated that most plants that have arbutin and sucrose in high quantities are resistant to environmental stress (Suau R et al: 1991).

5.3.3 Extraction of Aloe excelsa gel

The fleshy leaves of aloe excelsa were extracted physically, for the gel matrix after maceration. The gel yield was 11% and in the absence of a standardized extraction method and analytical reference data, the assumption is that the extraction method is commercially viable. The analysis shows a high level of carbohydrates (28%) which are likely responsible for the high resistance to environmental stress and the proliferation of the Zimbabwean aloe in arid conditions. The high sucrose levels, have been correlated to anti-oxidancy in other studies and might be responsible for the anti oxidancy of aloe excelsa (Suau R et al: 1991). There are close similarities in traditional uses between the sparsely researched Aloe excelsa and the more common Aloe ferrox, Aloe barbadiensis and Aloe vera. The existence of aloin A and aloe emodin in all species might suggest that aloe excelsa is also a very good antifungal plant since the compounds are proven, competent antifungals when extracted from other aloe species. Plants with lectins and lectin derivatives are potent in treating severe burns and in injuries that require blood 191

clotting to avoid hemorrhage (Coopoosamy R M: 2010). It has been demonstrated scientifically that lectins will produce agglutination even at very small dilution concentrations. The presence of lectins in aloe excelsa which is well documented

(Coopoosamy R M: 2010) could therefore explain its traditional uses in treating burns and wounds and could justify the use of aloe excelsa in treatments for actinic damage.

Research has also shown that lectins are effective in anti-cancer treatments due to the capability of lectins on tumor cell surfaces for endocytosis of carbohydrate containing exogenous material (Fujita et al: 1978). This therefore points in the direction for potential use of aloe excelsa in treatments for albinistic persons who are prone to squamous and basal cell carcinomas.

The SPF for the 5% Aloe excelsa cream was determined to be 1.85 ± 0.4 by

Optometrics® spectrophotometric 290s instrumentation. Any product with an SPF value above 2 can be sold as a commercial sunscreen and therefore the determination of an

SPF value close to 2 for a lotion with just aloe excelsa and no commercial sunscreens suggest that Aloe excelsa can potentially be used in sunscreen cosmeceuticals. Even though the value is not above 2, further optimisation of aloe excelsa formulations and perhaps an increase in concentration in the final product will definitely yield SPFs above

5.3.4 Formulation protocol

The various formulations developed in this study were oil in water emulsions based on 4 basic emulsifiers that are used most frequently in cosmeceutic preparations and are 192

approved by CIR. Stearic acid was the primary emulsifier and cetyl alcohol; ceto-stearyl alcohol as well as glycerol monostearate were the secondary emulsifiers. In optimizing the emulsion, the idea was not to build nanoemulsions but rather to disperse nanomaterials of titanium dioxide and zinc oxide in a macromolecular emulsion for aesthetic reasons and formulation photostability. Macromolecular emulsions are opaque and more viscous, a quality that was sought for in the formulation process. Opacity tends to protect delicate ingredients like aloe excelsa and Myrothamnus flabellifolia from photo degradation. Nano-emulsions and micro-emulsions, which are special classes of emulsions unlike ordinary macromolecular emulsions, appear translucent because of their small droplet sizes which are below 100 nm. It is understood that light waves will only be scattered by droplets with sizes that exceed a quarter of the incident light wavelength. Given that the visible section of the solar spectrum is between 390 and

750nm, light will therefore pass through an emulsion below 100 nm in droplet size without any light scattering taking place (Mason TG et al: 2006). The emulsion was deliberately macromolecular to promote opacity and also to make sure that droplet size remained optimally large so as to minimize the risk for percutaneous absorption of small nano- molecules. Physical sunscreens must remain on the skin periphery where they will scatter, absorb and reflect photo rays and protect the skin. They must never be absorbed. The numerous interfaces in the emulsions scatter light as it passes through it giving emulsions a turbid, milky appearance. During the formulation trials, dilute emulsions appeared bluish due to the fact that higher frequency light rays were being scattered more than lower frequency light. The best formulated emulsions appeared uniformly white because visible light scattering was uniform throughout the emulsion. 193

This phenomenon is referred to as the Tyndall effect. As the dispersed phase concentration was increased, the emulsion tended to be yellower because of lower frequency light rays that were now being scattered more (Aulton M.E: 2007).

Formulations were chosen as successfully emulsified on this basis during the studies.

This aspect was very critical in controlling emulsion formulation stability and aesthetics.

Formulations with macromolecular metallic oxides of zinc and titanium gave unaesthetic chalky, white and tactile products that were not suitable for all day use. At the same levels of incorporation, formulations with nanomaterials of metallic oxides gave aesthetic products. This was due to the fact that titanium and zinc nanomaterials used in the study do not back scatter visible light since their dimensions are below a quarter of the wavelength of incident light and they therefore do not scatter visible light waves and they remain translucent in formulations.

This manipulation of metallic oxides using nanotechnology to achieve nanomaterials with desirable attributes that are not found in their macromolecular analogues is what made this formulation of an aesthetic actinic damage retarding treatment for use by albinistic persons using previously unsuitable metallic oxides possible. In the formulation process a balance had to be struck, since formulating an aesthetic product using normal TiO2 and ZnO is not possible, manipulated nanosized metallic oxides were used. However the emulsion base had to remain macromolecular so that it would protect the plant extracts and also to provide the emolliency sought in cosmeceutic formulations.

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5.3.5 Emulsion stability

During the formulation development in this study, it was borne in mind that oil and water will never permanently mix; they will eventually separate back into oil and water. This may take anything from a few seconds to several decades. Macro emulsions are generally unstable and do not form spontaneously. The Bancroft rule applies to emulsion formation and determines whether a W/O or an O/W emulsion forms when oil and water are mixed to formulate a cosmeceutical product. To achieve stable emulsification, aspects of Stoke’s equation for predicting potential for emulsion separation were borne in mind (Aulton M E: 2007). Appropriate emulsifiers such as stearic acid and cetyl alcohol used in this study which imparted kinetic stability to the cosmeceutic emulsion so that dispersion droplet size did not change and the emulsion resisted the various forms of emulsion instability which includes creaming, phase inversion, flocculation and coalescence were employed (Aulton M E: 2007). A high shear (2200rpm) emulsifying mixer was used for agitation and mixing the two phases together. Energy was imparted to the system through homogenizing and agitation using an emulsifying mixer to form the stable emulsion with small globule size as required by

Stoke’s law (Mason TG et al: 2006). Carbopol 940®, a matrix building polymer was incorporated into the formulation so as to increase and fix viscosity, since viscosity is inversely proportional to the rate of emulsion cracking as per Stoke’s law. A buffering agent, triethanolamine was used to set the pH at the optimum level for Carbopol 940® to gel and thicken the emulsion. By trial and error the total emollients “oils” were carefully adjusted and incorporated at a final ratio of 1:2.5 with the total emulsifiers so

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as to ensure complete emulsification. The preservative system was based on parabens.

Since, solubility of parabens decreases with molecular weight while potency increases with molecular weight. A combination of coupled methyl and propyl parabens was used to ensure that methyl paraben (water soluble) preserves water phase ingredients and propyl paraben (lipid soluble) preserves oil phase ingredients. The preservative system was enhanced by addition of chelating agents including EDTA tetra sodium.

Formulation humectancy and extra moisturisation was achieved by the addition of glycerin, sodium PCA and Monopropylene glycol. The nanomaterials were dispersed into the hot emulsion during emulsification and agitation was continued until they were fully dispersed. Using these techniques and the ternary phase technical approach, the research work therefore managed to produce stable emulsions which surpassed minimum shelf life of 18 months expected from cosmeceutical formulations through optimization of emulsion formulation protocols.

5.3.6 Treatment SPF

The treatment was formulated to have an optimum SPF of 15. A result of 16.64 ± 0.32 obtained is within specification and the slight increase from expected results could be from the differences in altitude, which are not considered by the mandated test method.

Most people, including cosmeceutic experts seem to misunderstand SPF (Carswell .M

(2000). SPF generally depicts the dimensionless ratio of the time taken for potential development of perceptible sunburn. After application of the recommended dose of a selected sunscreen on a specific user, and the actual time taken for the same user to get sunburn without the sunscreen, on application of controlled UVR which mimics solar 196

irradiance of both UVA and UVB (Carswell .M (2000). For example, if a person receives sunburn in 10 minutes after exposure to the xenon-arc lamp as prescribed by the FDA and the same person were to last for 150 minutes under the same exposure before getting perceptible sunburn after application of a sunscreen, the SPF of the sunscreen will be calculated as 150/10, which gives an SPF of 15. It entails therefore that, the higher the designated SPF figure of a sunscreen, the better it is at protecting the skin

(Carswell .M (2000).

The methods for testing the efficacy of sunscreens through SPFs are standard. They are mandated by the FDA, EU and the OECD (M389/EN) through council directive

76/768/EEC of 27 July 1976. Only 2 methods exist and are recognized by the bodies as suitable methods to approximate the SPF of a sunscreen. One is an in-vivo method based on visually appraising the effects of increased erythermal doses of UVR on human subjects and the second is an in-vitro spectrophotometric method. In this study, the in-vitro testing protocols using Optometrics 290S were employed.

SPF determinations were performed on the developed treatments as mandated by regulatory bodies despite concerns and reservations about envisaged inadequacies of the test methods, the suitability of testing protocols and irrelevance of using SPF measurements as the basis for actinic damage protection in albinistic skin, for the following reasons:

1. SPF is a measurement of sunburn, oedema and urticaria induced by UVB on

skin. It does not measure the effects of UVA. While UVA might not cause much

acute damage in normal skin, it is the precursor of chronic damage in albinistic

skin and all melanin deficient skin types, including various cancers, skin fragility 197

and premature aging. The declaration of product SPFs excluding measurement

of UVA protection is glaringly inadequate, for use as a protection factor for PLWA

treatments.

2. The second major reason that mocks the mandated FDA and EU methods for

determining SPF values, in relation to albinistic persons is that, the related

mandated protocol is the persistent pigmentation darkening method (PPD). This

method observes potential actinic damage protection by the ability of a

sunscreen to protect the skin from persistent darkening on exposure to UVR.

Pigment darkening is a function of melanisation and is a self-protective

mechanism by the body during UVR onslaughts. This mechanism is absent in

albinistic persons, the lack of melanin makes it impossible to react to sunlight in

the way the mandated testing methods evaluate skin damage. There is no way

such a method can be relevant to genetically compromised skin, which does not

tan like albinistic skin.

3. Melanin is part of the wound healing system. The mandated SPF testing method

is based on observing responses to photo onslaughts on normal skin with well-

known UVR damage pathways. Albinistic skin is not normal skin. The pathways

are different, the wound healing system lacks melanin and even benign UVR

damage is not reversible. It is therefore not possible to approximate the sunburn

protection factor of compromised skin using the mandated methods.

4. The SPF measurement method is based on Fitzpatrick skin categories. A set of

skin types that were put into compartments with regards to colour and potential

response to UVR damage. Albinistic skin is not included on the Fitzpatrick 198

classification system on which the mandated test methods are based on. It

therefore flies in the face of logic that such methods should be used on albinistic

skin SPF testing.

5. The mandated test methods seem to ignore the influence of altitude and latitude

on UVR incidences and photo damage. The test methods were developed in

temperate areas. In the FDA TFM (1993) mandated method of SPF testing, the

radiation must be from a xenon solar simulator lamp, which emits radiation between

290nm and 400nm. The lamp must have a spectrum similar to the 10o solar zenith angle

at sea level. The simulator should emit radiation simulating both UVA and UVB at sea

level. Albinism is mostly biased towards people living in tropical Africa. If there is

a difference in water boiling points for sea level and tropical regions, surely, there

must be a difference in the effects of UVR effects for different environments

which must be factored in or corrected in the mandated test methods. According

to this research, the in-effectiveness of most commercial sunscreens on albinistic

humans in Zimbabwe can be attributed to the various discrepancies in climatic

and geographic conditions that are not found in temperate regions where the

products are developed and intended for use.

The observations from this research are negatively, critical of the mandated test methods for SPF by the FDA, the EU, CTFA and the OECD on actinic damage treatments for albinistic persons. In as much as an SPF figure was determined on the researched product for regulatory purposes, its relevance to albinistic skin is questionable.

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Apart from the above concerns with regards to albinistic skin, observations from this research show that the general role of SPF in skin protection may also be unclear even to regulators, users and other formulators. Though high SPF protects the skin better from UVR onslaughts, the user benefits of very high SPF are commercially debatable.

Theoretically, an SPF 30 product will block 96% of the incident UV radiation responsible for actinic damage; an SPF 60 will block 98% of the incident rays, which is practically just 2% more sunscreen effect. High SPF products require higher doses of sunscreens, which is costly and have more skin irritant potentials, at proportions that can hardly justify the little extra protection (Gasparo FP et al: 1998). After the formulation passes the SPF requirements, a formulation must also pass the controversial water or sweat resistance test to ensure that it can stay on the skin even in the event of excessive sweating or exposure to water, this requirement makes formulation of sunblock products much more cumbersome (Carswell M: 2000).

The consensus among most commercial formulators is that, sunblock treatments must meet the requirements of the mandatory SPF and water resistance SPF test methods by regulatory bodies first, then meet the consumer and patient requirements second

(Carswell M: 2000). This is grossly inappropriate in the development of a life-saving product like an actinic damage retarding treatment for albinistic persons.

The complication about developing water proof products comes from the fact that very little emulsifiers must be used in the formulation. This proves difficult to achieve for a product developed for people with compromised skin that needs moisturisation like

PLWA. The FDA mandated requirement for waterproof SPF therefore is not only inappropriate, but may lead to harmful products being developed for albinistic persons. 200

In formulating actinic damage treatments, it should be noted that the thicker the application, the better the spreadability and the efficacy. Creams have higher SPF values than corresponding lotions with the same concentration of sunscreens (Gasparo

FP et al: 1998). Since the major cost driver in sunscreen formulations is the sunscreen active, the formulator must strive to get the highest SPF with the least concentration through optimization of the formula (Carswell M: 2000). The general observation is that the formulation must therefore branch out from accepted mandated protocols if the need is to create a unique product that addresses a unique need like albinism.

5.3.7 Treatment functionality and aesthetics

Albinistic skin is compromised skin; mechanisms that naturally protect and offer skin integrity are not in place. The treatment regimes that work for normal skin are not guaranteed to work here. Most cutaneous conditions can be successfully treated by a small number of specific preparations in normal skin. The skin absorption routes and responses to medications are clearly defined and understood. In albinism however, the skin is typically damaged and traumatized and a careful weighing of the pros and cons of each treatment on broken skin will be required. In most cases, cutaneous conditions will go away in normal skin types and treatments are once off, time framed and finite, which is not the case with albinism. This is a lifelong condition and the treatments have to be practical, affordable as well as easy to adhere to. Most dermatological conditions are self-limiting, they will regress and some of them only affect patients once in their lifetime. In albinistic treatments, due to the longevity of treatment regimens, side effects cannot be tolerated and drug kinetics in compromised skin will have to be understood. 201

5.3.8 Active ingredient assay

The formulations had 5% ZnO and 5% TiO2 incorporated. The assay howver does not test for ZnO or TiO2 but for pure metallic Ti and Zn ions. 5% ZnO corresponds to about

4 % Zn and 5% TiO2 corresponds to about 3% Ti.

5.3.9 Treatment pH, functionality and aesthetics

Skin is referred to as the acid mantle, Schade and Marchionini illustrated in 1929 that skin has to maintain an acidic pH so as to discourage fungal and bacterial growth. They subsequently referred to this acidic protective film as the ‘acid mantle’. Skin pH is homeostatically maintained at 5.5. It is well understood in modern dermatology that chronic alkalization can destabilize this mantle leading to severe dermatitis and adverse skin conditions (Ali S.M et al: 2013). Understanding the factors that destabilize and factors that restore the acid mantle is clinically significant. However, application of this concept in formulation development is lacking. In this research we found the average albinistic skin pH to be slightly higher than normal at almost pH 6, this might be the reason why albinistic persons are afflicted by a myriad of skin conditions which are linked to aberrant skin pH like dermatophytoses, acne, ichthyosis pruritus, bacterial

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infections, candidal intertrigo and others (Ali S.M et al: 2013). The inclusion of topical agents in formulations that preserve the acid mantle has been recommended as helpful in alleviating these conditions (Ali S.M et al: 2013). The treatments developed in this study were all designed to have a pH between 5 and 5.5 so as to restore the acid mantle in albinistic persons. The treatment pH could be the foremost reason why panellists and patients felt immediate relief from discomfort on application of the developed products.

The use of more than 4 different emollients with different molecular weights and degrees of saturation gave the treatment the luxurious feel and after-feel on application as well as the excellent spreadability which was scored higher than commercial alternatives.

By and large, the treatment was rated higher than leading commercial products because it was specifically designed for a unique need and the formulation process considered the effects of altitude and latitude in optimizing actinic damage treatments for efficacy, safety and product functionality.

5.3.10 Nanocosmeceuticals

The manipulation and application of the favorable properties of materials at the nanometric level offers novel breakthrough solutions to age-old cosmeceutic and pharmaceutical problems. Despite the overwhelming benefits of nanotechnology in pharmaceutical products, research work in commercial nanotech consumer products categories like this study must be mindful of the perceived potential risks and

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sentiments of consumer and regulatory bodies, which inevitably impact on the successful commercialization of innovations (Fritz Allhoff et al 2010).

The perceived risks raised with regard to nanotech should be allayed by this and related work.

5.3.11 Perceived health and safety risk of nanomedicine

Nanomaterials have always been present in nature; their presence in consumer products is not in itself a threat. The perceived risk comes from their ‘unknown’ increased mobility and reactivity. The argument is that, nanoparticles are not morphologically similar to their macromolecular analogues and it is therefore not prudent to extrapolate their perceived adverse effects from the known and published toxicity of the macromolecules. This therefore ushers in significant need to address the health impact of nanoparticles independent from the macromolecules (Mehta et al:

2006). What complicates research work further, is that, most nanometric products are never mono disperse, they contain a wide range of particle sizes. Analytical work on the experiments is complicated because larger particles might behave differently from smaller ones. The smaller particles readily tend to aggregate and the resultant aggregates behave differently (Robert A. Freitas Jr. 1999).

Nanotechnology health impacts can either be through the applications of nanomaterials in nano-medical applications or the accidental exposure to nanomaterials in occupational environments. Both have perceived potential nano toxicological impacts, which have to be allayed by research work like this one (Robert A. Freitas Jr. 1999).

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This study has gone to lengths to allay such fears in the exhaustive investigation of nanometric metallic analogues of normally safe metallic oxides in consumer everyday products.

The American Institute for Occupational Safety and Health (NIOSH) has conducted widespread research on exposure to nanoparticles and subsequent interactions by workers. The research has led to a compilation of interim guidelines for handling nanomaterials in the manufacturing environment according to the scientific knowledge available (NIOSH, Bulletin 63: 2007). In Europe, the National Personal Protective

Technology Laboratory (NPPTL) on the penetration of EU marketed respirators by nanomaterials did comparative work (Shaffer RE, Rengasamy S: 2009). In Zimbabwe and much of the developing world, such frameworks and focus do not exist yet. It is imperative therefore that each new nanomaterial is assessed individually and independent research work conducted since occupational standards and toxicology profiles of ordinary molecules cannot be directly applied to nanoparticles( Wagner V, et al: 2006).

5.3.12 Regulation of nanotechnology

Much debate is currently raging, on whether to have a blanket regulation on nanotechnology research and products. Even though the FDA and various European

Commission bodies are looking at the potential risks of nanoparticles based consumer products, there is no regulation regarding manufacturing, processing, handling, packing, labeling and distribution. Material safety data sheets are not required to differentiate between macro and nano sized scale of materials (Bowman D, and Fitzharris, M: 2007). 205

It is argued that comprehensive regulation of nanotechnology is essential to ensure that the application of nanotechnology in consumer products benefits are not overshadowed by the risks associated (Bowman D, and Fitzharris, M: 2007). Despite the numerous potential novel applications of nanomaterials in improving the efficacy of everyday consumer, products and resolving problems found in the developing world. Most of the research-work on application and commercialization of nanotechnology based consumer products is however, confined in the west. Zimbabwe and other developing countries must actively develop frameworks that not only regulate, but also promote similar researches to this one that promote the safe and novel applications of nanotechnology and other emerging technologies for the betterment of the human condition.

5.3.13 Study limitations and recommendations for further studies

The study was severely limited by a lack of previous work done in the research area and an absence of published information for reference purposes. Most of the studies carried out had no appropriate scientific comparison. The number of albinistic volunteers was also very small due to the prevalence of stories regarding their abuse in communities. PLWA appeared to be very scared of possible abuse in researches and were very uncooperative.

The study referred to albinistic skin as if it was one skin type. It should be noted here that just like ordinary skin types, we have variations in PLWA regarding oiliness and dryness of skin leading to distinct albinistic skin types. Skin dermato-pharmacokinetics and skin conditions are depended on such factors. It is imperative therefore that further 206

studies be done to discern the different albinistic skin types and their requirements with a view to developing specific product types.

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CHAPTER 6

6. CONCLUSIONS

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6.1 EXPERIMENTAL STUDY CONCLUSIONS

6.1.1 Nanometric Zn and Ti safety risk

The ex-vivo experimental work using Franz diffusion cells demonstrates, direct evidence that neither Zn nor Ti can penetrate actinic damage induced porcine stratum corneum. It is consequently, extrapolated that, there is no indication that nanoparticles of Ti and Zn can penetrate both intact and actinically damaged skins causing internal exposure. In the experiments done, Ti and Zn were deposited, solely on the surface and upper layers of the epidermis where they are needed to reflect, scatter and absorb UV energy and protect the underlying dermal membranes. Based on this absence of internal exposure, it is concluded that, the use of nanometric metallic oxides of Ti and Zn in actinic damage treatments for OCA afflicted persons living in the tropics does not pose a health risk.

6.1.2 Albinistic anatomical skin profiles and Ti dermato-

pharmacokinetics

The in-vivo sequential adhesive tape stripping studies scientifically confirm for the first time that albinistic dermato-pharmacokinetics is depended on anatomical site and extent of UVR exposure. Physiological and anatomical site differences in albinistic skin response to UVR have an influence on skin reservoir properties and treatment outcomes. Areas exposed to severe actinic damage have more creases and folds that trap treatments and thereby reducing the surface area available for physical sunscreens to refract UVR. The FDA recommendation of a fixed sunblock dose of 2 mg/cm2 will

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probably not have reproducible treatment outcomes on PLWA in the tropics. The investigations also confirm that nanometric Ti in treatments for actinic damage do not penetrate beyond the lower layers of the stratum corneum in OCA persons in tropical conditions thereby causing internal exposure, whether the skin is actinically damaged or non-damaged and regardless of anatomical site.

6.1.3 Skin sensitivity tests

The in-vivo Draize sensitivity tests and the LVET ocular tests with rabbits showed that the treatment incorporating nanomaterials and the herbal concoction exhibit no potential threats that can contribute to skin irritation. This was confirmed by the in-vivo human patch tests, which also showed that the irritation potential of the treatment was negligible on both normal and compromised albinistic skin types when observed for pruritus, erytherma, urticaria, allergy, irritation and oedema in a closed patch test over an extended time period, representing a worst case scenario. It is therefore concluded that nanomaterials of Ti and Zn in actinic damage treatments are negligible skin irritants, and the safety profile is still retained when combined with natural herbal extracts.

6.1.4 Plant extracts

The high extractions yield for T.emetica, A.excelsa and M.flabelifollia demonstrate that these indigenous plants can be commercially used in cosmeceutical treatments sustainably and viably. The phyto-constituents of the respective plants show a

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correlation with the traditional uses of the plants and these compositions confirm the authenticity of Zimbabwean indigenous knowledge systems on plant remedies of medicinal value. It is concluded that these herbs can be incorporated efficaciously, safely and in a cost sustainable manner in everyday consumer treatments.

The research-work also confirmed scientifically for the first time that Aloe excelsa exhibits sunscreen effects and has an SPF value at low extract concentration that approach the levels designated by the FDA as minimum for commercial sunscreen designation. Therefore, it is concluded that aloe excelsa can be commercially developed as a safe and efficacious cosmeceutical sunscreen

6.1.5 Panel evaluations and product costing

The blind panel evaluations of the final treatment formulation by both albinistic and normal skin types users scored 88.5% compared to 81.55% for Nivea® SPF30. This shows that the treatment aesthetics and functionality are far superior to leading existing products and validates the technology employed in this formulation development work as novel. The product has potential to compete favourably with other products currently being retailed in Zimbabwe in both aesthetics and functionality.

The costing price and expected retail price of the treatment is about 20% of the current price of leading sunscreens like Nivea ® SPF 30. This is quite an achievement for a product which is not just a mere sunscreen but a complete actinic damage retardation cosmeceutical and acute solar damage treatment. It is therefore concluded and validated that the technology employed in the development of the treatment, is novel and groundbreaking in topical treatments and cosmeceuticals in particular. 211

6.2 OVERALL CONCLUSION

Based on these foregoing findings, it is concluded that the use of nanometric oxides of

Ti and Zn in actinic damage treatments incorporating of T. emetica, M. flabellifolia and

A. excelsa to retard actinic damage in PLWA living within the tropics is feasible, efficacious, and commercializable and does not pose any health risk.

6.3 SIGNIFICANCE OF THE STUDY

The study lead to the development of a treatment for actinic damage in albinism which will not only alleviate pain and suffering but will offer a rare opportunity for a normal life for PLWA. They will not only be able to live longer lives but to participate in ordinary outdoor activities without any constraints. The study also provided much valuable new knowledge with regards to albinism and albinistic dermato-pharmacokinetics which was non-existent. The study contributed immensely to the on-going debate on the safety of nanomaterials in consumer products. Commercialization of products incorporating indigenous herbs will assist in community development through utilisation of neglected plant species and the inclusion of marginalised groups in main stream commercial sectors.

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7. REFERENCES

Agin P, Edmonds S (2002) Testing high SPF sunscreens ;Photodermatol ,Photo immunol ,Photomed 2002 :18:169-174 ,Blackwell ,Munksgaard

Agren MS (2009). "Percutaneous absorption of zinc from zinc oxide applied topically to intact skin in man". Dermatologica 1: 36–9.

Ainsworth C; Lepage, M (2007). "Evolution's greatest mistakes". The New Scientist 195 (2616): 36–39.

Armstrong A et al (2005). "Nanotubes with the TiO2-B structure". Chemical Communications (19): 2454–6.

Aubrey D et al (2007). "Life Span Extension Research and Public Debate: Societal Considerations" (PDF). Studies in Ethics, Law, and Technology 1 (1). . Article 5.

Aulton E ed. (2007). Aulton's Pharmaceutics: The Design and Manufacture of Medicines (3rd ed.) Churchill ,livingstone . pp. 92–97, 384, 390–405, 566–69, 573–74, 589–96, 609–10, 611

Barnett G (1986). "Lanolin and Derivatives". Cosmetics & Toiletries 101: 21–44.

Baum B D (2006). The Rise and Fall of the Caucasian Race: A Political History of Racial Identity. New York University: 2006.

Beeson S; Mayer, J W. "Discoveries beyond the visible”. Patterns of light: chasing the spectrum from Aristotle to LEDs. New York: Springer. p.149.ISBN 978-0-387-75107-8.

Bhopal R S: 2014 "Migration, Ethnicity, Race, and Health in Multicultural Societies", 2nd edition, Oxford University Press, p. 350.

Bowman D, and Fitzharris, M (2007). "Too Small for Concern? Public Health and Nanotechnology". Australian and New Zealand Journal of Public Health 31 (4): 382– 384.

Bowman D, and Hodge G (2007). "A Small Matter of Regulation: An International Review of Nanotechnology Regulation". Columbia Science and Technology Law Review 8: 1–32.

Brenner M, Hearing V J :2008). The protective role of melanin against UV Damage in human skin. Photochemistry and Photobiology 84 (3): 539–49.

213

Brigelius-Flohé R, Traber M G (1999). "Vitamin E: function and metabolism". FASEB J. 13 (10): 1145–1155

Broughna L et al HI, Eds, Percutaneous absorption, drugs and cosmetics and methodologies 4th edition, Boca Raton FL; 2005

Bruneton J .Pharmacognosy ,Phytochemistry ,Medicinal Plants , Intercept , Hampshire 1995

Budavari S, ed. (1989). Merck Index (11th ed.). Rahway ,New Jersey :Merck and Co,Inc p. 8761.ISBN 978-0-911910-28-5

Burkhart C.N et al ; Tinea corporis in human immunodeficiency virus-positive patients: case report and assessment of oral therapy; The International Society of Dermatology; (2003); 42; 839–843

Burnett M E, Wang S Q (April 2011). "Current sunscreen controversies: a critical review". Photodermatol, Photoimmunol, Photomed27 (2): 58–67.

Burton J L .Essentials of dermatology.churchill livingstone .ISE 1988.ISBN0 443 0408.2

Callen, J (2000). Color atlas of dermatology. Philadelphia: W.B. Saunders. ISBN 0- 7216-8256-1.

Carden S M et al , Albinism, Modern molecular diagnosis, and The British journal of ophthalmology 19198 82-2 89-95

Carswell M (2000). Sunscreen formulation and testing, Chapter 3 in Volume 2, Chemistry and manufacture of cosmetics .MLSchlossman. ed ,3rd edition , Allured Publishing

Castelvecchi D; (2007). Dark Power, Pigment and radiation’. Science News 171 (21): 325..

Chauvin R (1995): Carbomers; a general concept of expanded molecules Tetrahedron letters Volume 36, Issue 3 , 16 January 1995, Pages 397-400

Chifamba J et al: ex vivo penetration of TiO2 and ZnO across actinically damaged porcine skin: development of an albinistic skin protection treatment. Int J Pharm Sci Res 2015; 6(6): 2339-48.doi:10.13040/IJPSR.0975-8232.6 (6)

Christoph, R et al (2006). Glycerol. "Ullmann's Encyclopedia of Industrial Chemistry". Ullmann's Encyclopedia of Industrial Chemistry.

214

Cocoa butter –Britannicca Online. Britannica Encyclopedia article. July 1998. Retrieved March 2014.

COLIPA, The European Cosmetic ,Toiletry and perfumery association,Quality management Guidelines 1997 ;www.colipa.com

Coopoosamy R.M J Med. Plant Res Vol 4(17) pp1738-1742

Craddock, P. T. (2008). Minng and Merttarlurgy ,Chapter 4. The Oxford Handbook of Engineering and Technology in the Classical World. Oxford University Press. pp. 111– 112

Craddock; P. T. et al. (1998). "Zinc in India". 2000 years of zinc and brass. British Museum. p. 27

CTFA, The Cosmetic Toiletries and fragrances Association ,Cosmetics Guidelines, 1993 www.ctfa.org

David J. et al "Fatty Acids" in Ullmann's Encyclopedia of Industrial Chemistry 2006, Wiley-VCH, Weinheim.

David R. 2010. Miocene Hominids and the Origins of the African Apes and Humans. Annual Review of Anthropology, Vol. 39: 67 -84

De Gruijl et al. 2002 Photo carcinogenesis: UVA vs. UVB Radiation, Skin Pharmacol. Applied Skin Physiol 15 (5) .3416-320

De Polo K F: A short Textbook of cosmetology .H.Ziolkowsky GmbH 1998.

DermIS 2005 Dermatology Information system: http://dermis.net ,downloaded March 2013

Detoni C.B et al: Penetration, photo-reactivity and photo-rotective properties of nanosized ZnO, Photochem. Photobiol. Sci.2014, 13, 1253

Diallo D et al 2003 ,The Malian Medicinal Plant , Trichilia emertica . J ethnopharmacol ,84: 279-287

Diembeck et al 1999. Test guidelines for invitro assessment of dermal absorption, percutaneous absorption of cosmetic ingredients. Food chem. Toxicology 37 191-205

Dingler A, Gohla S(2001) Production of Solid Lipid Nanoparticles(SLN) Scaling up feasibilities Microencapsulation 19 ,11-6 2001

215

Double K L (2006). "Functional effects of Neuromelanin and synthetic melanin in model systems". J Neural Transm 113 (6): 751–756.

Doucet O et al. (June 2006). "Reconstituted human corneal epithelium: a new alternative to the Draize eye test for the assessment of the eye irritation potential of chemicals and cosmetic products". Toxicol in Vitro 20 (4): 499–512.

Draize J.H et al. (1944). "Methods for the study of irritation and toxicity of substances applied topically to the skin and mucous membranes". J. Pharmacol. And Exp. Therapeutics 82: 377–390

Dugard, P.H Scott R.C (1984) .Absorption through skin, in: Baden H.p (Ed) Chemotherapy of Psoriasis, Pergamon press Oxford pp 125-144

Escobar-Chávez J J et al, The tape stripping method for skin evaluation :2007

FDA/CFSAN Homepage :2005 www.cfsan.fda.gov , downloaded March 2013

Filipponi L and Sutherland D (2013): Nanotechnologies: Principles, Applications, Implications and Hands-on activities, European Commission Directorate-General for Research and Innovation Industrial technologies (NMP) programme PP 76-78, 103-104

Fitzpatrick K et al (2005). Fitzpatrick's color atlas and synopsis of clinical dermatology. McGraw-Hill Medical Pub. Division. ISBN 0-07-144019-4.

Food Drug and Administration(1965) , Illustrated Guide for grading Eye irritation by hazardous substances. Government Printing Office Washington DC

Franz T. 1975, Percutaneous absorption, on the relevance of invitro data J. Invest, dermatol, 64 .190-195

Frauenkron M ( 2002) “Ethanolamines and Propanolamines” in Ullmann's Encyclopedia of Industrial Chemistry, Wiley-VCH, Weinheim

Fritz A et al , What Is Nanotechnology and Why Does It Matter?: From Science to Ethics (Oxford: Wiley-Blackwell, 2010

Gamer A O et al , the invitro absorption of microfine Zinc oxide and titanium dioxide through porcine skin Toxicology in Vitro 20(2006) 301-307

Gardner R, Society of Cosmetic Chemists, Modules 1-7, 2010

Gardner R: Society of Cosmetic Chemists, Module 9, 2010 11-15

216

Gasparo F P et al (1998). Review of sunscreen safety and efficay. Photochem, Photobiol 68(3) 243-256 (1998)

Gaul L E (1969). "Dermatitis from cetyl and stearyl alcohols". Archives of dermatology 99 (5): 593.

Germano M P et al (2006)Evaluation of the antioxidant properties of Trichilia emertica .J ethnopharmacol 105 -368-373

Gilman A et al (1990). Goodman and Gilman's The pharmacological basis of therapeutics. New York: Pergamon Press.

Glogau R G. (2000). The risk of progression to invasive disease. Journal of the American academy of dermatology, 42(1):s23-s24.

Golden R, et al (2005). "A review of the endocrine activity of parabens and implications for potential risks to human health". Critical Reviews in Toxicology 35 (5): 435–58.

Goresy El et al (2001). "An ultradense polymorph of rutile with seven-coordinated

Greco G et al (April 2011). "Uncovering the structure of human red hair pheomelanin: benzothiazolylthiazinodihydroisoquinolines as key building blocks". Journal of Natural Products 74 (4): 675–82.

Greenwood N et al (1984).Chemistry of the elements Oxford: Pergamon press pp. 1117–19..

Gronskov K, et al (2 November 2007). Oculocutaneous Albinism. Orphanet Journal of Rare Diseases 2: 43.

Gronskov K, et al K (2 November 2007).Oculocutaneous Albinism. Orphanet Journal of Rare Diseases 2: 43.

Gross A et al (1995). "Autosomal-recessive neural crest syndrome with albinism, black lock, cell migration disorder of the neurocytes of the gut, and deafness: ABCD syndrome". Am J Med Genet 56 (3): 322–6.

Gundidza M (Interview on 23 March 2014, Harare, Zimbabwe)

Gundidza M, Zwavig J.H (2005). The chemical composition of the essential leaf oil of Helichrysum odoratissimum from Zimbabwe .J essen oil Res 5:351-343.

Guomin H et al. (2008). "Fabrication of ZnO nanowire arrays by cycle growth in surfactantless aqueous solution and their applications on dye-sensitized solar cells". Materials Letters 62 (25): 4109–4111 217

Gyorgy Scrinis (2007).”Nanotechnology and the environment” Chain Reaction 97: 23– 26.

Harper, Douglas. “oil” .online etymology dictionary 2014

Harvey P W, Everett DJ (Jan 2004). "Significance of the detection of esters of p- hydroxybenzoic acid (parabens) in human breast tumours". Journal of Applied Toxicology 24 (1): 1–4

Hockberger P E. (2002) et al "Toxic effects of ultraviolet radiation on the skin". Toxicology and Applied Pharmacology195 (3): 298–308. http://microvet.arizona.edu/Courses/vsc422/secure/VSC422AppledHistologyLabHandou t.pdf

Hull D, Hall N: Unilever, Internal Research and development updates. R.I.C 12 November 2002

Hung D et al (2006). Engaged learning with emerging technologies. Dordrecht: Springer.

Invernizzi N et al (2008). "Nanotechnology's Controversial Role for the South". Science Technology and Society 13 (1): 123–148.

IUPAC (1997). Compendium of Chemical Terminology (The "Gold Book"). Oxford:

James, W D et al. (2006). Andrews' Diseases of the Skin: Clinical Dermatology. Saunders Elsevier. ISBN 0-7216-2921-0.

Jeffrey H et al . (2006). Lookingbill and Marks' Principles of Dermatology. Saunders.

Jenning V .Gohla S, Encapsulation of retinoid in Solid Lipid Nanoparticles ,(SLN) J Microencapsulation 18 ,149-158 ,2001

Jobling R (2007). "A patient's journey: Psoriasis". Br Med J 334 (7600): 953–4. doi: 10.1136/bmj.39184.615150.802.

John A. et al (1982). "Erythema and melanogenesis action spectra of normal human skin” Photochemistry and Photobiology 36 (2): 187–191.

John Knowlton, Steven Pierce: Handbook Of cosmetic Science and technology, Cotswold Publishing 1997

Kaidi, Z and Jagdish, S (1999). In vitro percutaneous absorption enhancement of propanol hydrochloride through porcine epidermis by terpenes/ethanol. 62: 359-366. 218

Kaldis B (2010). "Converging Technologies". Sage Encyclopedia of Nanotechnology and Society, Thousand Oaks: CA, Sage

Kaplan J et al DM (January 2008).”chediak-Higashi syndromeCurr. Opin. Hematol. 15 (1): 22–9.

Kearnes M et al (2006).” From Bio to Nano. Science as Culture. Science as Culture (Routledge, published December 2006) 15 (4): 291–307

Klaassen C D et al (1991). Casarett and Doull's toxicology: the basic science of poisons. New York: McGraw-Hill

Knowlton J, Pierce S Handbook of cosmetic Science and technology, Cotswold Publishing 1997

Krueger G, Ellis C N (2005). "Psoriasis—recent advances in understanding its pathogenesis and treatment". J. Am. Acad. Dermatol. 53 (1 Suppl 1): S94–100.

Kurzweil R (2005). The Singularity is Near. Penguin Books .ISBN 0-14-303788-9

Laderman J et al: The tape stripping Procedure .Euro.J.Pharma.BioPharm:2008.

Lasic D D: Novel Applications of liposomes, Trends in Biotechnology 16, 7,307-

Lauridsen J K (1976): "Food emulsifiers: Surface activity, edibility, manufacture, composition, and application". Journal of the American Oil Chemists' Society 53 (6): 400–407.

Lewis N, ’Nanocrystalline TiO2” Research. California Institute of Technology. Retrieved 9 October 2009.

Lide D R, (2009). Handbook of chemistry and physics. (90 ed.). Boca Raton, Florida: CRC Press. ISBN 978-1-4200-9084-0.

Lide D R, ed. (1994). CRC Handbook of Data on Organic Compounds (3rd ed.). Boca Raton, FL: CRC Press. p. 4386.

Lieberman L: (2001) .How "Caucasoids" Got Such Big Crania and Why They Shrank:, Current Anthropology, Vol. 42, No. 1, February 2001, pp. 69–95.

Liendo R et al (November 1997). "Characterization of cocoa butter extracted from Criollo cultivars of Theobroma cacao L.". Food Research International 30 (9): 727–731.

Lippens J et al (April 2009). "Cell death in the skin". Apoptosis 14 (4): 549–69

219

Machipisa L: (2004): Rights-Zimbabwe, the last minority group to find a voice. Interpress news Agency.

Machipisa lewis: Rights-Zimbabwe, the last minority group to find a voice. Interpress, news Agency. (February 2004)

Malcolm D et al. "Introduction". Fungal Infection: Diagnosis and Management. John Wiley & Sons, 2012. p. 5

Marchand R et al M. (1980). "A new form of titanium dioxide and the potassium octatitanate K2Ti8O17". Materials Research Bulletin 15 (8): 1129–1133.

Marissa D Newman et al. The safety of nanosized particles in TiO2 and ZnO based sunscreens. Journal of the American Academy of Dermatology 2009, Volume 61, no 4 685-691

Marks J et al (2006). Lookingbill and Marks' Principles of Dermatology (4th ed.). Elsevier Inc. Page 7.

Mason TG et al, "Nanoemulsions: formation, structure, and physical properties", Journal of Physics: Condensed Matter, 2006, 18(41): R635-R666

Matsumu Y, Ananthaswamy H.N. (2004)."Toxic effects of ultraviolet radiation on the skin".Toxicology and Applied Pharmacology195 (3): 298–308.

Mbuya Chiparatani: Registered Traditional Healer (Interview October 2013, Chivhu ,Zimbabwe

McGrath J. et al. (2004). Rook's Textbook of Dermatology (Seventh Edition). Blackwell Publishing. Pages 3.7-3.8.

McGraw Hill dictionary of Modern medicine 2002. McGraw Hill Company Inc.

McHenry H.M (2009). "Human Evolution". In Michael Ruse & Joseph Travis. Evolution: The First Four Billion Years. Cambridge, Massachusetts: The Belknap Press of Harvard University Press

Mehta M et al (2006). Nanotechnology: Risk, Ethics and Law. London:

Miller H et al, (2006). Lookingbill and Marks' Principles of Dermatology. Saunders. ISBN 1-4160-3185-5.

More B H et al ,Evaluation for skin irritancy testing for Butea Monosperma formulations. Int J of Toxicol and Applied Pharmacology: 2013 (1) .10-13

220

Mossie Brues A: 1990 "People and races", , Waveland Press, 1990, notes how the term

Naik A , Kaila N Y ,Guy R H ,2000 , Transdermal drug delivery, overcoming the skin’s barrier function, Pharmaceutical Science and technology today, 3 (9),318-326

Nestle F O et al (2009). "Psoriasis". N. Engl. J. Med. 361 (5): 496–509.

Norval M et al (2009 Oct): "Does chronic sunscreen use reduce vitamin D production to insufficient levels?”? The British journal of dermatology161 (4): 732–6.

OECD (2002) OECD test guideline 429: Testing for acute dermal irritation/corrosion, Paris, France OECD

OECD (2009). OECD test guideline 404. Acute skin irritation and corrosive testing. OECD guidelines for testing of chemicals., Paris. France OECD

OECD 2004b OECD Guideline document for the conduct of skin absorption studies. OECD

OECD, 2004a, OECD Guideline for testing of chemicals, method no 428, Skin absorption in vitro Method, adopted April 13 2004.

Okulicz J F, Schwartz R A (2003). "Hereditary and acquired ichthyosis vulgaris". International Journal of Dermatology 42 (2): 95–8. doi: 10.1046/j.1365- 4362.2003.01308.x. PMID 12708996.

Osgood J et al (1982). "The Sensitization of Near-Ultraviolet Radiation Killing of Mammalian Cells by the Sunscreen Agent Para-amino benzoic Acid". Journal of Investigative Dermatology 79 (6): 354–7. doi:10.1111/1523-1747.ep12529409. PMID 6982950.

Ovaere P et al. (2009). "The emerging roles of serine protease cascades in the epidermis". Trends in Biochemical Sciences 34 (9): 453–463.

Özgür Ü et al (2005). "A comprehensive review of ZnO materials and devices". Journal of Applied Physics 98 (4): 041301.

Palgrave C (2002) .Trees of Southern Africa, Struik Publishers, ISBN 0-620-17697-1.

Pappas P G (2006). "Invasive candidiasis". Infect. Dis. Clin. North Am. 20 (3): 485–506.

Petersen R (2008) Nanocrystals for use in cosmetics formulations ,Abbot GambH and Co US Patent 60/866233

221

Pooley, E. (1993). The Complete Field Guide to Trees of Natal, Zululand and Transkei. ISBN 0-620-17697-0.

Porter,F.(1991). Zinc Handbook: Properties, Processing, and Use in Design. CRC Press.

Prajapati V, Barankin B (May 2008). "Dermacase. Actinic keratosis". Can Fam Physician 54 (5): 691, 699. PMC 2377206. PMID 18474700

Randy S et al: Beginning cosmetic Chemistry, Allured Publishing 2002

Rapini R P et al. (2007). Dermatology: 2-Volume Set. St. Louis: Mosby.

Raymond E et al., at eMedicine, 22 August 2005. Retrieved 31 March 2007

Raysman, R. & Raysman, R. (2002). Emerging technologies and the law: Forms and analysis. Commercial law intellectual property series. New York, N.Y.: Law Journal Press.

Richard D et al, Facts about Albinism, Cosmetics and toiletries 19, 2006 36-39

Saladi R N, Persaud A N (Jan 2005). "The causes of skin cancer: a comprehensive review.” Drugs of today (Barcelona, Spain: 1998) 41 (1): 37–53.

Salamanca B et al ( 2005): Nanotechnology for the developing world .PloS Med, 2(5), and 97

Sánchez J. M. et al 1999, Solvent Extraction and Ion Exchange, 17, 455–474.

SCCNFP O750/03, Basic Criteria for the invitro assessment of dermal absorption of cosmetic ingredients , 0ctober 2003

Scholz, F et al (February 2000). "A new access to Gibbs energies of transfer of ions across liquid interfaces and a new method to study electrochemical processes at well- defined three-phase junctions". Electrochemistry Communications 2 (2): 112–118..

Schueller R, Ramanowski W: Beginning cosmetic Chemistry, Allured Publishing 2002ISBN 0-620-17697-0.

Segur J. B; Oberstar, H. E. (1951). "Viscosity of Glycerol and Its Aqueous Solutions". Industrial & Engineering Chemistry 43 (9): 2117.

Shaffer R E, Rengasamy S (2009). "Respiratory protection against airborne nanoparticles: a review". J Nanopart Res 11: 1661–1672.

222

Shane E et al: A Change in Direction in ICP-MS, published on Mar, 2004 in American Laboratory,

Shaw T, et al (August 2010). "True photoallergy to sunscreens is rare despite popular belief". Dermatitis 21 (4): 185–98.

Sheree E et al. Human skin penetration of sunscreen nanoparticles. Skin Pharmacology and physiology journal 2007, Volume 20 .148-154 series on testing and assessment no 28

Skoog D (2007). Principles of Instrumental Analysis (6th ed.). Canada: Thomson Brooks/Cole. ISBN 0-495-01201-7.

Smijs T G, Pavel S (2011): Titanium dioxide and zinc oxide nanoparticles in sunscreens: focus on their safety and effectiveness, Nanotechnology, Science and Applications 2011:4 95–112

Smolinske S C (1992). Handbook of Food, Drug, and Cosmetic Excipients. CRC Press. pp. 75–76.ISBN 0-8493-3583-X

Soga F et al (2004). "Contact dermatitis due to lanoconazole, cetyl alcohol and diethyl sebacate in lanoconazole cream". Contact dermatitis 50 (1): 49–50.

Sperling-Veitmeier K et al : UV filters for Skin and product protection, Uvinul T150, Perfumerie and Kosmetik, 71, Jahrgan Nr 3, 90 172-190

Svenson S, Tomalia D A (2005) Dendrimers in ,biomedical applications Adv Drug deliv , Rev,57,2106-2129,2005

Tattersall I, Schwartz J (2009). "Evolution of the Genus Homo". Annual Review of Earth and Planetary Sciences 37: 67–92

The British Pharmacopoeia Secretariat (2009). Index.BP2009 Retrieved 18 March 2014.

Thomas A (2002). Fats and Fatty Oils. "Ullman’s Encyclopedia of Industrial Chemistry". Ullman’s Encyclopedia of Industrial Chemistry. Weinheim: Wiley-VCH.

Trabe, M (1998).The biological activity of Vitamin E. The Linus Pauling Institute. Retrieved 6 March 2011.

Van Hal D et al, Vanrensen A, Devringer T, and Diffusion of estradiol from non- ionic surfactant vesicles through human Stratum Corneum, in-vitro, and STP Pharm .Sci 6 72-78, 1996

223

Van Ravenzwaay et al. (2004). The significance of invitro rat skin absorption studies to human risk assessment. Toxicol, in vitro 18. 219-225

Van Wyk B et al , Medicinal plants of South Africa, 2nd edition, Briza Publication 2009 ,pp 296-297 ISBN 078-1-875093-37-3

Wakamatsu K (2003) Quantitative analysis of eumelanin and pheomelanin in humans, mice, and other animals: a comparative review. Pigment Cell Res. 2003 Oct;16(5):523- 31 http://www.nlm.nih.gov

Walsh A (1955), The application of atomic absorption spectra to chemical analysis, Spectrochim. Acta 7: 108–117.

Welz B, Sperling M (1999), Atomic Absorption Spectrometry, Wiley-VCH, Weinheim, Germany, ISBN 3-527-28571-7.

Wilhelmus K R (2001). "The Draize eye test". Surv Ophthalmol 45 (6): 493–515.

William A C: Transdermal and topical drug delivery .London(u.a) ,Pharmaceutical press PhP :2003

Winkler J (2003). Titanium Dioxide. Hannover: Vincentz Network. pp. 115–116.

Wolff K et al. (2008). Fitzpatrick's Dermatology in General Medicine. McGraw-Hill Medical. ISBN 0-07-146690-8.

Zuang L et al: ECVAM pre validation study on invitro tests for acute skin irritation : Atla 30. 109-129 :2002

Zumdahl S. (2009). Chemical Principles 6th Ed. Houghton Mifflin Company. p. A23.

224

8. APPENDICES

Appendix 1 Publication 1

Appendix 2 Publication 2

Appendix 3 Signed consent form

Appendix 4 Newspaper features on research

Appendix 5 Product pictures

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