Pharmaceutical Applications and Toxicity of Extracted Alkenones from Marine

A Dissertation

Entitled:

Pharmaceutical applications and toxicity of extracted alkenones from marine

Isochrysis algae

Kyle McIntosh

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the

Pharmacology and Experimental Therapeutics Degree in College of Pharmacy and

Pharmaceutical Sciences The University of Toledo

______Dr. Amit K. Tiwari, Committee Chair

______Dr. Frederick E. Williams, Committee Member

______Dr. Gabriella Baki, Committee Member

______Dr. Jeffrey Sarver, Committee Member

______Dr. Cyndee Gruden, Interim Dean College of Graduate Studies

The University of Toledo

August 2019

This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of

Pharmaceutical applications and toxicity of extracted alkenones from marine Isochrysis algae

Kyle McIntosh

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Pharmacology and Experimental Therapeutics Degree in Pharmaceutical Sciences

The University of Toledo August 2019

Isochrysis is a commercially available marine algae used for animal feed, human nutrient supplements, and biodiesel. The Isochrysis galbana species is one of four genera of haptophytes that produces unique, long-chain lipids known as alkenones. However, there is a lack of physical characteristics and toxicity data for alkenones in animals, thus, limiting their use in humans. In the first aim of this study, alkenones derived from

Isochrysis sp. were evaluated for their chemical structure characteristics as an alternative for waxes used in personal care products. The melting point of the alkenone was determined (71.1–77.4°C), and their thickening capability in ﬁve emollients was evaluated and compared to microcrystalline wax and ozokerite. Alkenones showed compatibility with three emollients, isopropyl isostearate, C12-C15 alkyl benzoate, and ethylhexyl methyxycinnamate, and they thickened the emollients similar to the other tested waxes. Lipsticks and lip balms were formulated with and without alkenones. All products remained stable at room temperature for 10 weeks. Lipstick formulated with alkenones was the most resistant to high temperature. Finally, alkenones were compared to three cosmetic thickening waxes in creams. Viscosity, rheology and stability of the

iii creams were evaluated. All creams had a gel-like behavior. Overall, the alkenones in these formulas were comparable to the other three waxes. Thus, alkenones can offer a potential green choice as a new personal care structuring compound.

For the second aim of this study, we performed acute oral, acute dermal and repeated 28-day dermal toxicity studies using female SAS (an acronym for the company

SASCO where the colony was bred) Sprague Dawley rats. Our behavioral studies (level of grooming, eye opening, walking, exploring, body posture, breathing, scratching, and nose flattening) indicated that the specific alkenones had no visible behavioral effects at oral doses up to 4000 mg/kg. In addition, there were no significant changes in food consumption or body weight, and there was no significant histopathological changes in the liver, kidneys, spleen, heart or skin compared to animals treated with vehicle. In the acute and chronic dermal toxicity studies, the alkenones produced less irritation and did not significantly damage the skin based on the Draize skin reaction scale and transepidermal water loss readings compared to the positive control, 1% sodium lauryl sulfate. Overall, our results indicated that alkenones are safe in female Sprague Dawley rats, suggesting that they could be used for both oral and dermal formulations, although additional studies would be required before these alkenones could be applied to human personal care products.

iv Acknowledgements

I want to thank my parents, Darryl and Cara, my family, and my friends for their endless support, love, and encouragement. I thank Hope Lutheran Church and its members for their spiritual support. I thank God for paving this journey for me and for placing marvelous individuals in my life.

My biggest thanks go towards Dr. Amit K. Tiwari for being my advisor for these past four years. He has taught me so much when it comes to research; learning and moving past mistakes in experiments, prioritizing daily tasks, and to become passionate in the work you accomplish. He has also taught me valuable life-long skills which I will practice and carry with me throughout my future careers. I want to thank Dr. Baki for her knowledge of testing cosmetic ingredients, for allowing me to run her devices for this project, for the support of her students who assisted me, and her assistance in the planning of work needed with the alkenones. Dr. Sarver for opening his door to me when

I had concerns with software programs to use and for his help with the statistical data. Dr.

Williams for keeping me on track, his feedback and support for not just me, but all graduates in the program.

Lastly, I want to thank my lab colleagues, Angelique Nyinawabera, Noor

Hussein, Haneen Amawi, Saloni Malla, Mariah Pasternak, and Dr. Diwakar Bastihalli

Tukaramrao and past lab colleagues. They have challenged me, helped see the value in myself, and know what I have to offer when working in a team setting. These people and others have made my time at Toledo unique and rich with a cultural experience and have given me memories I will treasure.

v Table of Contents

Abstract ...... iii

Acknowledgements ...... v

Table of Contents ...... vi

List of Tables ...... ix

List of Figures ...... x

List of Abbreviations ...... xii

List of Symbols ...... xv

I. Introduction ...... 1

A. Marine Microalgae ...... 1

B. Extractions Obtained from Marine Microalgae ...... 2

C. Alkenones ...... 3

D. Current Uses of Alkenones ...... 5

a. Paleoclimatology ...... 5

b. Biofuel...... 6

c. Cosmetics ...... 7

d. Medicine ...... 7

II. Materials and Methods ...... 10

A. Test Materials ...... 10

B. Animals ...... 11

C. Chemical Characterization Methods ...... 12

a. Establishment of Solubility ...... 12

b. Determination of Melting Point ...... 12

vi c. Thickening Capability Test ...... 13

d. Developing the Lipstick and Lip Balm Formulation ...... 14

e. Cream Formulation ...... 15

f. Determination of Viscosity, Thixotropy, and Rheology ...... 17

g. Observing the Liquid Crystalline Structure ...... 18

h. Stability Testing ...... 19

D. Animal Study Design ...... 19

E. Suspension Formulation ...... 21

F. Acute Oral Gavage Administration of the Alkenones ...... 22

a. Quantification of White Blood Cells (WBCs), Red Blood Cells

(RBCs), Platelets (PLTs), Monocytes and Hemoglobin (Hb) ...... 23

b. Determination of Blood Parameters...... 23

c. Histology ...... 24

G. Single and Repeated Dermal Application of the Alkenones ...... 25

a. Skin Irritation Index Score ...... 27

b. Determination of Skin Water Loss ...... 27

H. Statistical Analysis ...... 28

III. Results ...... 29

A. The Determination of Solubility and Thickening Capability Test ...... 29

B. Melting Point Determination ...... 30

C. Lipstick and Lip Balm Stability ...... 32

D. Viscosity, Thixotropy, and Rheology ...... 35

E. Liquid Crystalline Structure ...... 37

vii F. Cream Stability ...... 38

G. The Effects of the Acute Oral Administration from Alkenones ...... 39

H. Acute Dermal Treatment ...... 44

I. Repeated Dermal Applications from Alkenones-Containing Paste ...... 47

IV. Discussion ...... 52

A. Physical Characteristics ...... 52

B. Lipstick, Lip Balm, and Cream Stability...... 54

C. Viscosity, Thixotropy, Rheology, and Liquid Crystalline Structure ...... 56

D. Toxicity ...... 58

V. Conclusion ...... 64

A. Highlights ...... 64

References ...... 67

Appendix A List of manuscripts in preparation and conference presentations based on

this dissertation ...... 83

viii List of Tables

Table 1 Lipstick formulas ...... 14

Table 2 Lip balm formulas ...... 15

Table 3 Cream formulas ...... 16

Table 4 Parameters calculated by Formulating for Efficacy™ ...... 30

Table 5 Stability of lipsticks and lip balms ...... 32

Table 6 Viscosity and storage modulus (average ± SD) of the creams at room

temperature...... 35

Table 7 Stability of creams ...... 36

ix List of Figures

Figure 1 Structure of a common alkenone isolated from Isochrysis microalgae. Image

taken of the Alkenones as a dried wax ...... 5

Figure 2a Experimental design showing the duration of administration of alkenones

for three different studies in SAS Sprague Dawley rats...... 19

Figure 2b Experimental design showing the site (6.25 cm2) of acute dermal

applications of different treatments in two stand-alone studies...... 19

Figure 2c Experimental design showing the site (6.25 cm2) of repeat dermal

applications of different treatments in two stand-alone studies ...... 20

Figure 3 Viscosity of wax-emollient mixtures 24 h after formulation (average ± SD). 29

Figure 4 (a) Viscosity-shear rate data and power law fitting for the four creams

(b) Viscosity-shear rate data showing the extent of thixotropy in the four

creams ...... 34

Figure 5 Determination of (a) body weight, and (b) food consumption, following one-

time oral administration of alkenones (4000 mg/kg) compared to vehicle in

female SAS Sprague Dawley rats (n=6)...... 37

Figure 6 Histopathological evaluation of the (a) liver, (b) spleen, (c) heart, (d) kidney

and (e) skin of SAS Sprague Dawley rat after acute oral exposure to vehicle

control and alkenones (4000 mg/kg) after 14 days...... 39

Figure 7 Blood chemistry profile in SAS Sprague Dawley rats after one-time oral

exposure to vehicle control and alkenones (4000 mg/kg) for 14 days...... 40

x Figure 8 Blood chemistry profile to determine organ toxicity in SAS Sprague Dawley

rats after a one-time oral exposure to vehicle control and alkenones (4000

mg/kg) for 14 days...... 41

Figure 9 Determination of (a) body weight, and (b) food consumption, following

acute dermal exposure of Study 1 compared to Study 2 in female SAS

Sprague Dawley rats (n=5) ...... 42

Figure 10 Line graphs of the transepidermal water loss (TEWL) was measured before

and 24 hours after one-time dermal application...... 43

Figure 11 Bar graph of the transepidermal water loss (TEWL) measured before and

after one-time application on dermal skin of SAS Sprague Dawley rats...... 44

Figure 12 The determination of (a) body weight, and (b) food consumption, following

repeated dermal exposure of Study 1 compared to Study 2 in female SAS

Sprague Dawley rats (n=5)...... 45

Figure 13 The transepidermal water loss (TEWL) was measured before and after 6

hours repeated dermal exposure every day for 28 days on skin area exposed

to (a) 1% SLS (positive control), (b) 1000 mg/kg alkenone, (c) 200 mg/kg

alkenone, (d) 50 mg/kg alkenone, (e) normal saline (negative control), and

(f) glycerin...... 46

Figure 14 The transepidermal water loss (TEWL) measurements before and after 6

hours repeated dermal exposure of different treatments each day at (a) Day

1, (b) Day 2, (c) Day 8, (d) Day 14, (e) Day 21, and (f) Day 28...... 47

xi List of Abbreviations

ALP ...... Alkaline phosphatase ALT ...... Alanine aminotransferase AMY ...... Amylase ANOVA ...... Analysis of Variance

BUN ...... Bilirubin urea nitrogen

CA ...... Total calcium CO2 ...... Carbon dioxide COX-2 ...... Cyclooxygenase CRE ...... Creatinine

DHA ...... Docosahexaenoic acid DMEM ...... Dulbecco’s modification of Eagle’s medium DMSO ...... Dimethyl sulphoxide DNA ...... Deoxyribonucleic acid DSC ...... Differential Scanning Calorimetry

EPA ...... Eicosapentaenoic acid

FATPs ...... Fatty acid transport proteins FFE ...... Formulating for Efficacy

GHS...... Globally Harmonized Classification System GLU ...... Glucose

Hb/HGB ...... Hemoglobin HLB...... Hydrophyl lipophyl balance HPMC ...... Hydroxypropylmethylcellulose HSP ...... Hansen Solubility Parameter HTS ...... Hypertrophic scars

IACUC ...... Instituional Animal Care & Use Committe INCI ...... International Nomenclature of Cosmetic Ingredients ISG ...... Ingredient-Skin Gap

xii

K+ ...... Potassium

LogK ...... Octanol/water partition coefficient LVE ...... Linear viscoelastic region

MAAs ...... Mycosporine-like amino acids MVol ...... Molar Volume

Na+ ...... Sodium

OECD ...... Organization for Economic Cooperation & Developement O/W ...... Oil-in-water

PHOS ...... Phosphorus PUFAs ...... Polyunsaturated fatty acids

RNA ...... Ribonucleic acid

SAS ...... Shortened from SASCO, where rats were first bred SD ...... Sprague Dawley SLS ...... Sodium lauryl sulfate SMILES ...... Simplified molecular-input entry systems SolS ...... Solubility in Skin

TA ...... Thermal Advantage TBIL ...... Total bilirubin TEWL ...... Transepithelial water loss

USA...... United States of America USP ...... United States Pharmacopeia UV ...... Ultraviolet

WO ...... Work Order

xiii 5-FU ...... 5-fluorouracil 5-LOX ...... 5-lipoxygenase

xiv List of Symbols

C#...... Carbon(i.e.31,38) ºC ...... Celsius cP...... Centipoise

G’ ...... Storage modulus G” ...... Loss modulus g...... Gram

kg...... Kilogram

LD50...... Lethal Dose 50

mg ...... Milligram min ...... Minute mL ...... Milliter mm ...... Millimeter mMol ...... Millimoles

n...... Shear thinning index

sp...... Species

™ ...... Trademark

% ...... Percent

w/w ...... Weight/weight w/v...... Weight/volume

Uk’37 ...... Unsaturated index formula µL ...... Microliter

Chapter One

Introduction

Marine Microalgae

Algae have many advantages when compared to traditional terrestrial plant agriculture. One significant advantage is the lack of required soil for growth; thus algae do not compete with agricultural products (Trentacoste, Martinez, & Zenk, 2015).

Secondly, they consume less water compared to land crops and yield a high production per acre due to their high growth rate (Michael J Walsh et al., 2016 ). For these reasons, culturing algae over terrestrial plants should prove cost-effective while also avoiding bioenergy-food tradeoffs.

Microalgae species or plankton are a primary producer of organic matter in aquatic environments by photosynthesis and provide a food source for the marine habitat

(Matsunaga, Takeyama, Miyashita, & Yokouchi, 2005). Microalgae can use carbon dioxide, nutrient salts, organic matter, trace elements and convert energy from the sun using photosynthesis (Kirst, 1990; Paerl, Bebout, Joye, & Des Marais, 1993; Sayre, 2010;

Subramanian, Barry, Pieris, & Sayre, 2013). Microalgae are present in either freshwater or seawater environments (Taleb et al., 2016). When exposed to extreme conditions, marine microalgae must adapt to new environments (Duong, Thomas-Hall, & Schenk,

1 2015), where they typically produce diverse secondary, biologically active metabolites that are not present in terrestrial plants (Kim SK, 2010 ). There are an estimated 200,000 to several million species of microalgae (S.-k. Kim, 2015). Only 40,000 species have been identified (Hu et al., 2008), and their unique metabolites could be of importance in the development of personal care products. Currently, marine microalgae derived from harvested seaweed are used as animal feed, wastewater treatment, carbon dioxide sequestration, biofuels, pharmaceuticals, (S.-k. Kim, 2015) and for nutraceutical and personal care applications (Gellenbeck, Salter-Venzon, Lala, & Chavan, 2012).

Extractions Obtained from Marine Microalgae

Bioactive molecules such as carbohydrates, amino acids and proteins, pigments, vitamins, unsaturated lipids and fatty acids, sterols, etc., may be extracted from microalgae (Patras, Moraru, & Socaciu, 2018). The methods of extraction include mechanical and biochemical methods (Patras et al., 2018). Sosa-Hernández et al. (Sosa-

Hernandez, Escobedo-Avellaneda, Iqbal, & Welti-Chanes, 2018) have reviewed recent state-of-the-art extraction techniques such as supercritical-fluid extraction, enzyme- assisted extraction, and microwave-assisted extraction, among others. Some of these compounds could be incorporated into personal care products, (Bhatia et al., 2011;

Hartmann, Becker, Karsten, Remias, & Ganzera, 2015; T. M. Lee & Shiu, 2009)

(Aussant, Guiheneuf, & Stengel, 2018; Berthon et al., 2017; Carneiro et al., 2018;

Gutierrez-Rodriguez, Juarez-Portilla, Olivares-Banuelos, & Zepeda, 2018; Kostetsky,

Chopenko, Barkina, Velansky, & Sanina, 2018; Lin et al., 2018; Martinez-Hernandez,

Castillejo, Carrion-Monteagudo, Artes, & Artes-Hernandez, 2018; Sun, Wei, Zhou,

2 Ashokkumar, & Liu, 2018) including mycosporine-like amino acids (MAAs) for sunscreens, glucosyl glycerols as a humectant, sulfated polysaccharides as thickening compounds, polyphenols and lipids. New potential drugs have emerged from algae- derived compounds (Ercolano, De Cicco, & Ianaro, 2019) as well as shown antiproliferative and apoptotic effects on certain cancer cell lines such as mouse neuroblasts

(Kurt, Ozdal-Kurt, Akcora, Ozkut, & Tuglu, 2018). The focus of this introduction section will be solely on alkenones which are further broken down from lipids.

Alkenones

Currently, only four strains of the phylum Haptophyta, from the order

Isochrysidales (Emiliania huxleyi, Gephyrocapsa oceanica, Isochrysis galbana and

Chrysotila lamellose), are known to produce unique long hydrocarbon chains such as C31,

C33, C37, or C38 alkenes (Nakamura, Sawada, Araie, Suzuki, & Shiraiwa, 2015), whereas

C37-39 are known as alkenones (Richter et al., 2019). The only difference between the

C37/C38 alkenes and alkenones is an unsaturated methyl or ethyl ketone group on the alkenones that are used for mapping past sea surface temperatures (Prahl & Wakeham,

1987). The genus Isochrysis has been selected as a viable candidate for biodiesel development and production due to its different strains containing high amounts of carbon chains as lipids (Feng, Chen, Xue, & Zhang, 2011; Roopnarain, Gray, & Sym,

2014; Song, Pei, Hu, & Ma, 2013). Isochrysis galbana are readily consumed by small

(larval) invertebrates due to its lack of a rigid cell wall and small size (4-7 µm) (Davis,

1953; Jeffrey, Brown, & Volkman, 1994; C.-P. Liu & Lin, 2001; van Bergeijk, Salas-

Leiton, & Cañavate, 2010). This microalga is characterized by a lack of toxins, quick

3 growth rate and salinity tolerance (Jeffrey et al., 1994). The most common cultured version is the Tahitian strain, “T-iso” (Borowitzka, 1997).

The Isochrysis sp. is a non-toxic food source for cultivated marine animals

(Yaakob, Ali, Zainal, Mohamad, & Takriff, 2014) as it produces other essential polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA, 20:5ω3) and docosahexaenoic acid (DHA, 22:6ω3) that are required for the growth and development of fish larvae (Batista, Gouveia, Bandarra, Franco, & Raymundo, 2013; Fidalgo, Cid,

Torres, Sukenik, & Herrero, 1998). Important biomolecules such as pigments, sterols tocopherols (Narcisa M. Bandarra, Pedro A. Pereira, Irineu Batista, & Vilela, 2003) from

Isochrysis galbana make them potential candidates for nutraceutical companies. Both

EPA and DHA decrease pro-inflammatory eicosanoid production by competing with arachindonic acid as a substrate for cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-

LOX) (Komprda, 2012). It has been reported that the administration of Isochrysis galbana by oral gavage daily for eight weeks significantly decreased inflammation in diabetic rats (Nuño et al., 2013). However, a later study reported that the lipid extract of

Isochrysis galbana only inhibited COX-2-mediated inflammation prior to digestion and these compounds were not bio-accessible (Bonfanti et al., 2018).

Isochrysis is only one of a limited number of marine algae grown industrially and harvested for marine culture purposes (Y.-K. Lee, 1997; Priyadarshani & Rath, 2012).

Until recently, only the fatty acid methyl esters (FAMEs) were harvested for biofuel in

Isochrysis sp., until Drs. Chris Reddy and Gregory O’Neil discovered that with more prolonged exposure to gas chromatography, about 14% (w/w) of predominantly C37 and

C38 long-chain alkenones were present (Gregory W. O’Neil et al., 2012). Alone, these

4 alkenones are too large to be used for biofuel, but when broken down by the chemical process of olefin metathesis (Sabatino & Ward, 2019) at its double bonds, the byproducts become small enough to be used in jet fuel (Institution, 2015). Alkenones are an essential component in the stationary phase cultures of Isochrysis sp. (composing of 25.6% of the total lipid) (C.-P. Liu & Lin, 2001). These alkenones have a high melting temperature

(~70 °C), (Gregory W. O’Neil et al., 2012), thereby making them potential compounds for use in pharmaceuticals formulations when developing topically delivered drugs.

Figure 1 shows the common structure of alkenones derived from Isochrysis galbana, as well as the dried off white alkenones.

Figure 1: Structure of a common alkenone, i.e., 37:2 methyl alkenone, isolated from

Isochrysis microalgae. Alkenones contain trans double bonds and a methyl or ethyl ketone. Image taken of the Alkenones as a dried wax.

Current Uses of Alkenones

Paleoclimatology. When determining the changes in the Earth’s climate, monitoring the sea surface temperatures is an important way of ascertaining and tracking of such changes (Henson, Beaulieu, & Lampitt, 2016). Alkenones were first identified in ocean sediments (de Leeuw, v.d. Meer, Rijpstra, & Schenck, 1980). The number of unsaturated bonds in alkenones is known to increase when the microalgae are grown or maintained at low temperatures (Grossi, Raphel, Aubert, & Rontani, 2000). Because of

5 this characteristic, an unsaturated index formula, termed Uk’37, is calculated as a parameter of the ratio of C37:2 to C37:3 alkenones (J. K. Volkman, Barrerr, Blackburn,

& Sikes, 1995; Zheng, Dillon, Zhang, & Huang, 2016). The significant correlation between alkenones abundances and sea surface temperatures make alkenones a widely used biomarker for paleoclimate studies (Pelejero, Grimalt, Sarnthein, Wang, & Flores,

1999; J. Volkman, M. Barrett, I. Blackburn, & Sikes, 1995). It should be noted that a common misconception is that distinct alkenone signatures alone can directly identify specific species of microalgae. This misconception has been disproved by sampling 15 alkenone-containing lake surfaces and showing that DNA fingerprinting is a better approach to identifying species (Theroux, J. D'Andrea, Jaime, Amaral-Zettler, & Huang,

2010).

Biofuel. Due to the high photosynthetic efficiency of algae and less reliance on land usage for growth (Martin & Allen, 2018), they have been suggested to be a promising alternative fuel source over fossil fuels. Diesel fuel derived from algae was shown to be less toxic in seawater when compared to conventional jet and marine diesel fuels (Rosen, Dolecal, Colvin, & George, 2014). Besides having lower amounts of sulfur and particulate matter emissions, biodiesel improves performances in brake thermal efficiency and brake specific energy consumption (Mahla & Dhir, 2019). Furthermore, blending algae biocrude and petrocrude could yield lower sulfur emission (Lavanya et al.,

2016).

The central extraction obtained from biofuel is lipids (Y. Li et al., 2014). There are numerous methods for extracting these lipids, as summarized in Harris et al. (Harris,

Vine, Champagn, & Jessop, 2018). Certain techniques, when growing the microalgae

6 under anaerobic conditions, along with Thermosipho globiformans and

Methanocaldococcus jannaschii (Yamane et al., 2013), or through a ten-step process using commercially available Isochrysis algae, can yield a high amount of n-alkane compounds (O'Neil, Williams, Wilson-Peltier, Knothe, & Reddy, 2016). Being a unique type of lipid, alkenones have been grown and developed for use as biofuels. Although alkenones with long chains of 37-39 carbons alone are not suitable for use as jet fuel, once broken down to 8-13 carbons in length, they can be used for jet fuel (O'Neil G.W et al., 2019).

Cosmetics. Microalgae extracts have been used as protection against extrinsic skin aging from UV radiation as well as act as moisturizing and thickening agents in cosmetic applications (Ariede et al., 2017; Wang, Chen, Huynh, & Chang, 2015). As emulsions, the alkenones were considered to be acceptable based on experiments for cream evaluation. A follow-up study on alkenones derived from Isochrysis sp. used in sunscreen were combined with three “reef-safe” UV filters (Huynh A et al., 2019). Here, alkenones were compared with two other commonly used waxes in sunscreen, beeswax and cetyl alcohol, and the alkenones signiﬁcantly increased the in vitro sun protection factor of the three “reef-safe” UV ﬁlters.

Medicine. There have been no reported medical uses of alkenones. However, certain medications are prepared in creams, gels, or liquid solutions and used topically.

One example of this is topical 5-fluorouracil (5-FU), a standard treatment for actinic keratosis, which is a precursor or form of squamous cell carcinoma (Green & Olsen,

2017; Prince, Cameron, Fathi, & Alkousakis, 2018; Rossi, Mori, & Lotti, 2007). First reported as safe and effective for hypertrophic scarring (HTS) (Fitzpatrick, 1999), 5-FU

7 is well-known for its significant efficacy in treating advanced colorectal cancer (Gamelin et al., 1999; Noordhuis et al., 2004), squamous cell carcinoma of the skin (Nguyen & Ho,

2002; Sadek et al., 1990), non-small cell lung cancer (Kawahara et al., 2001) and breast cancer (Zhang et al., 2012). 5-FU inhibits DNA synthesis and RNA transcription by blocking the methylation of uracil, a precursor of thymidine (L. S. Li et al., 2009;

Noordhuis et al., 2004). 5-FU has also been shown to inhibit fibroblast proliferation

(Ibrahim & Chalhoub, 2018), which may explain its efficacy in reducing post-surgical scarring or HTS. However, allergic contact dermatitis to either 5-FU or to a vehicle component has been reported (Meijer & de Waard-van der Spek, 2007). One study

(Dohil, 2016) showed that changing the novel formulation and reducing the number of applications could yield better patient compliance and treatment outcomes. Other drugs used in early topical stage of actinic keratosis are diclofenac, imiquimod, and ingenol mebutate (de Oliveira et al., 2019). If alkenones derived from Isochrysis sp. are non-toxic and do not produce allergic reactions, these compounds could be used as novel topical compounds for delivering these drugs.

There have been numerous studies showing different extracts from marine algae species that have been incorporated into certain medicines, such as supplements for consumption (Martinez-Hernandez et al., 2018) or topical gels like porphyra-334 derived from Porphyra vietnamensis (Bhatia et al., 2010). Alkenones from algae are gaining attention, but unfortunately there is a paucity of information on the toxicity of these alkenones derived from the Isochrysis sp. The long chain structure of the alkenones limited us from testing it in vitro due to the difficulty in finding a suitable solvent (Di &

Kerns, 2006; Di, Kerns, & Carter, 2009). We conducted experiments to first define the

8 physical properties (melting point, thickening capability, viscosity and rheology) of the alkenones, as well as its stability potential compared to other commonly used solvents in lip balms, lip sticks, and creams. Upon obtaining that information, we conducted experiments to determine if alkenones would be suitably tolerated in animals. We conducted behavioral monitoring and determination of grip strength, blood analysis and histology of major organs, food consumption and body weight as indices of oral toxicity.

We used transepidermal water loss (TEWL) irritation score, behavioral, food consumption and body weight monitoring in the dermal toxicity study using SAS

Sprague Dawley (SD) female rats.

Chapter Two

Materials and Methods

Test Materials

The marine microalgae Isochrysis was purchased from Necton S.A. (Olhão,

Portugal). Alkenones were isolated, purified and chemically characterized from the

Isochrysis biomass as previously described (O'Neil et al. 2016; O'Neil G.W et al. 2019).

The chemical characterization process is described in the patent WO 2017/087539 A1.

Sodium chloride (0.9%) was purchased from BioVision Incorporated (Milpitas,

California, USA). Microcrystalline wax, ozokerite, castor oil, triglyceride, isoeicosane, meadowfoam seed oil, lanolin alcohol, candelilla wax, carnauba wax, Red 7, mica pearl white, tocopherol, petrolatum, jojoba oil, almond oil, avocado butter and xanthan gum were purchased from Making Cosmetics (Snoqualmie, Washington, USA). The following ingredients were received as gifts: hexamethyldisiloxane (Dow Corning, Midland,

Michigan, USA), isohexadecane (Presperse, Somerset, New Jersey, USA), isopropyl stearate (Lubrizol, Wickliffe, Ohio, USA), C12-15 alkyl benzoate (Phoenix Chemical,

Calhoun, Georgia, USA), ethylhexyl methoxycinnamate and Germaben II (INCI:

Propylene glycol, diazolidinyl urea, methylparaben and propylparaben, Ashland,

Covington, Knetucky, USA), propanediol (DuPont Tate & Lyle Bio Products, Loudon,

Tennessee, USA), sorbitan oleate, polysorbate 80 and cetyl alcohol (Croda, Edison, New

10 Jersey, USA), stearic acid (Acme Hardesty, Blue Bell, Pennsylvania, USA), glyceryl monostearate (Corbion, Lenexa, Kansas, USA), natural glycerin (Spectrum, New

Brunswick, New Jersey, USA), sodium lauryl sulfate (SLS) (PCCA, Houston Texas,

USA), and hydroxypropylmethylcellulose (HPMC) (Spectrum Chemical, Gardena,

California, USA). All ingredients were of cosmetic grade.

Animals

Seven-week old female SAS Sprague Dawley (SD) rats (180-200 grams) were obtained from Charles River (Wilmington, Massachusetts, USA) and used for the acute oral, acute dermal and repeated dermal toxicity tests. Twelve rats were used for the acute oral toxicity test, whereas 10 rats were used for the acute dermal toxicity and 10 rats for the repeated dose 28-day dermal toxicity. The toxicity tests were performed according to the Organization for Economic Cooperation and Development (OECD, 2002a) test guideline, i.e., OECD Guideline 423 for the acute oral toxicity test (OECD, 2002b) and

OECD Guideline 404 for the acute dermal toxicity test (OECD, 1981, 2004, 2015), with slight modifications. The injection volume was adjusted to 4 ml/kg depending on the body weight of the rat (Turner, Pekow, Vasbinder, & Brabb, 2011). The female rats were nulliparous and non-pregnant. The rats were given standard rat pellets (Envigo Teklad) and reverse osmosis water ad libitum. They were acclimatized to the laboratory conditions for 7 days before the experiments. The rats were housed in groups, depending on the weight of the animal, in accordance with Institutional Animal Care and Use

Committee (IACUC) guidelines (three to four rats for acute oral toxicity and two to three rats for acute and repeated dermal toxicity). The animals were housed under a 12-hour

11 light/dark cycle, at temperatures of 23 ± 2 ºC and a humidity of 55 ± 5%. The animal protocol for this study was approved by the IACUC of The University of Toledo and was in accordance with all National Institutes of Health guidelines (No. 8023, revised 1978) for animal research, including the Guide for the Care and Use of Laboratory Animals

(Institute of Laboratory Animal Resources, Commission on Life Sciences, 2011).

Chemical Characterization Methods

Establishment of Solubility. Formulating for Efﬁcacy TM (ACT Solutions Corp,

Kirkwood, Delaware, USA), hereafter referred to as ‘FFE’, is an in silico modeling software used in the personal care and the pharmaceutical industry for formulation design. In this project, FFE was utilized to predict the solubility of alkenones based on

Hansen Solubility Parameters (HSPs) using a variety of solvents. In order to add ingredients to FFE, we created a simpliﬁed molecular-input line-entry system (SMILES), as this information was not available in the published literature or chemical databases

(e.g., PubChem). ChemDraw (Release 15.0, CambridgeSoft, Waltham, Massachusetts,

USA), a molecule editing software, was used to create the SMILES.

Determination of Melting Point. Melting point was determined using differential scanning calorimetry (DSC) analysis. Using a Mettler MT 5 microbalance

(Mettler Toledo, Columbus, Ohio, USA), a 6 mg sample was sealed in an aluminum crucible. A pinhole was created on the lid of the crucible to vent gas buildup. DSC was performed at a 10ºC/min ramp from 0–400 ºC, using a DSC 822e instrument (Mettler

Toledo, Columbus, Ohio, USA) attached to a F25-ME refrigerated/heating circulator

(Julabo, Allentown, Pennsylvania, USA). Nitrogen gas was purged at a rate of 10

12 mL/min. Thermal Advantage (TA) Universal analysis software was used to obtain the scans.

Thickening Capability Test. This test was used to determine how alkenones thicken emollients commonly used in personal care and makeup product formulations. In order to select comparators for alkenones, a literature search was done on the melting point of various commonly used waxes. The waxes with melting points close to that of the alkenones were tested using the above-mentioned DSC method to conﬁrm the melting range. Two waxes with similar melting ranges, microcrystalline wax and ozokerite, were identiﬁed as good comparators. The melting point peak was 69 ºC for microcrystalline wax and 74 ºC for ozokerite.

Solvents identiﬁed by FFE were used in this test. To evaluate the thickening of the waxes, 9.0 g of solvent was weighed on an analytical balance, with an accuracy of

0.001 g, into a 25 mL glass beaker. The solvent, while covered with aluminum foil to avoid evaporation, was heated approximately 5 ºC above the melting point of the specific wax. When the solvent reached the desired temperature, it was removed from the heat and 1.0 g of the wax was added to the solvent. Mixing continued until room temperature was reached. The mixture was then reweighed and the evaporated solvent was replaced.

The beaker was left covered with a piece of aluminum foil overnight. The stability of the mixture was checked the following day and if it remained stable, viscosity was measured with a Brookfield viscometer (Brookﬁeld Engineering Laboratories, Middleboro,

Massachusetts, USA). Stability was qualitatively assessed based on signs of separation and any visible change in color.

13 A Brookﬁeld viscometer DV-I, with a concentric cylinder spindle (#21) and a small sample adapter, was used to determine the viscosity of the different wax-solvent mixtures. The tests were performed at 21 ºC. The spindle was rotated from 0–100 rpm.

All measurements were done in triplicate.

Development of the Lipstick and Lip Balm Formulation. Microcrystalline wax and ozokerite were selected as comparators for the alkenones based on the melting point determination of the alkenones. A lipstick formula (Table 1) and a lip balm formula

(Table 2), containing both microcrystalline wax and ozokerite, were selected for the alkenones to be tested as an alternative for either microcrystalline wax or ozokerite. This approach allowed for the testing of the compatibility of the various waxes and the suitability of the alkenones in a lipstick/lip balm formula. For the lipstick, Phase A was added to a glass beaker and heated to 80 ºC to melt the waxes. The pigment dispersion in

Phase B was prepared using an EXACT three-roll mill (EXACT Technologies, Inc,

Oklahoma City, OK, USA). The ingredients of Phase B were added to the melted Phase

A one-by-one and mixed until the color was uniform. The mixture was removed from the heat source. Phase C was added to phase A/B and the mixture was poured into a metal lipstick mold while still hot. When settled, the sticks were topped off and the mold then was put into the refrigerator for 15 min. The lipsticks were then removed from the mold and inserted into plastic lipstick cases. For the lip balm, phase A was heated to 80 ºC while mixing until all ingredients were melted. The mixture was removed from the heat and phase B was added with mixing until a homogenous mixture was obtained. The mixture was then poured into lip balm tubes.

14 Table 1: List of ingredients and their percentages for developing each lipstick formulas.

Table 1. Lipstick formulas INCI Name Lipstick 1 Lipstick 2 Lipstick 3 % (w/w) % (w/w) % (w/w) Phase A Castor oil 25.8 25.8 25.8 Triglyceride 16.0 16.0 16.0 Isoeicosane 17.0 17.0 17.0 Meadowfoam seed oil 5.0 5.0 5.0 Lanolin alcohol 5.0 5.0 5.0 Microcrystalline wax 2.0 2.0 - Ozokerite wax 5.0 - 5.0 Alkenone wax - 5.0 2.0 Candelilla wax 7.0 7.0 7.0 Carnauba wax 3.0 3.0 3.0 Phase B Red 7 (and) castor oil 2.0 2.0 2.0 Mica pearl white 11.0 11.0 11.0 Phase C Tocopherol 0.2 0.2 0.2 Propylene glycol 1.0 1.0 1.0 (and) diazolidnyl urea (and) methylparaben (and) propylparaben

Table 2: List of ingredients and their percentages for developing each lip balm formulas.

Table 2. Lip Balm formulas INCI Name Lip Balm 1 Lip Balm 2 Lip Balm 3 % (w/w) % (w/w) % (w/w) Phase A Petrolatum 45.5 45.5 45.5 Jojoba oil 22.0 22.0 22.0 Almond oil 13.0 13.0 13.0 Microcrystalline wax 8.0 8.0 - Ozokerite 8.0 - 8.0 Alkenones - 8.0 8.0 Avocado butter 3.0 3.0 3.0 Phase B Tocopherol 0.5 0.5 0.5

Cream Formulation. Four oil-in-water (O/W) emulsions were formulated. One contained the alkenones, while the other three were formulated with cetyl alcohol, stearic

15 acid and glyceryl monostearate as comparators for the alkenones. The composition of the creams can be found in Table 3. All four creams were formulated identically. First, water was heated to 80 ºC and xanthan gum was mixed with propanediol to form a slurry that added to the water. The oil phase (phase B) was combined in a separate beaker and heated to 80 ºC. When both phases reached the same temperature, the oil phase was added to the water phase with propeller mixing. The emulsion was brieﬂy homogenized, and then removed from the heat. The emulsion was allowed to cool with continuous propeller stirring and phase C was added when the emulsion reached 45 ºC. The emulsion was allowed to cool to room temperature under continuous mixing. Water loss was checked by weighing the creams and evaporated water was replaced. The emulsion was mixed again and was homogenized for 10 seconds. Finally, each cream was stored in plastic containers with screw top lids and stored at room temperature.

16 Table 3: List of ingredients and their percentages for developing each cream formulas.

Table 3. Cream formulas INCI Name Cream 1 Cream 2 Cream 3 Cream 4 % (w/w) % (w/w) % (w/w) % (w/w) Phase A Water 68.8 68.8 68.8 68.8 Propanediol 5.0 5.0 5.0 5.0 Xanthan gum 0.2 0.2 0.2 0.2 Phase B Isopropyl isosterate 15 15 15 15 Alkenones 5.0 - - - Cetyl Alcohol - 5.0 - - Stearic Alcohol - - 5.0 - Glyceryl monostearate - - - 5.0 Sorbitan oleate 1.5 1.5 1.5 1.5 Polysorbate 80 3.5 3.5 3.5 3.5 Phase C Propylene glycol 1.0 1.0 1.0 1.0 (and) diazolidnyl urea (and) methylparaben (and) propylparaben

Determination of Viscosity, Thixotropy and Rheology. The rheological properties and viscosity of the emulsions were evaluated using a Discovery Hybrid

Rheometer DHR-3 (TA Instruments, New Castle, DE, USA). A 40 mm 2◦ cone and plate geometry at 25.0 ± 0.1 ºC was used to test ~0.8 mL of each sample. The shear rates ranged from 0.1–100 s−1 in steady state ﬂow for viscosity and thixotropy measurements.

Dynamic viscoelasticity was measured as a function of frequency in the linear viscoelastic region (LVE). First, the LVE range was determined with an amplitude sweep

(Callens, Ceulemans, Ludwig, Foreman, & Remon, 2003). The amplitude sweep established the extent of the material’s linearity. Below the critical stress level, the structure is intact, and G’ (storage modulus) and G” (loss modulus) remain constant.

Increasing the stress above the critical stress level disrupts the network structure. Once

17 above the critical stress, the material’s structure is non-linear and G’ typically decreases.

After the LVE was deﬁned, a frequency sweep at an oscillation stress below the critical value was performed. The frequency sweep provides additional information about the effect of colloidal forces, type of network structure in the cream and the interactions among the droplets (Callens et al., 2003; Madsen, Eberth, & Smart, 1998).

Observing the Liquid Crystalline Structure. Oleosomes are liquid crystal regions that form around oil drops in an emulsion (T. Tadros, Leonard, Taelman,

Verboom, & Wortel, 2006). Oleosomes are multilayers of lamellar liquid crystals surrounding the oil droplets that become randomly distributed as they progress into the continuous phase. The rest of the liquid crystals produce the “gel” phase, which is viscoelastic (T. F. Tadros, 2016). The creams were observed with a polarized light microscope (Accu-Scope® 3000LED, Accu-Scope, Commack, New York, USA). For the microscopic evaluation, a small amount of each emulsion was placed on a microscopic slide and quickly covered with a cover slip. The sample was ﬁnger pressed to spread and create a cream layer as thin as possible. A 40× objective lens was used with cross polarizers in a bright ﬁeld to detect birefringence. Birefringence was determined using a polarized light microscope (Pajdzik & Glazer, 2006). The sample was placed between two polarizers oriented with their planes of vibration being mutually perpendicular. When an isotropic sample was placed between crossed polars, the state of the polarization of light was unchanged and in theory, no light was transmitted through the optical system.

The light can only be transmitted if the state of polarization was changed, i.e., the sample is birefringent (Pajdzik & Glazer, 2006). Oleosomes appear under optical microscopy as

“Maltese crosses” (Haimovici, Gantz, Rumelt, Freddo, & Small, 2001).

18 Stability Testing. The stability of the lipsticks, lip balms and creams were monitored at room temperature (25 ºC) and an elevated temperature (45 ºC) in stability cabinets for 10 weeks. The samples were checked visually for signs of cracks formation, sweating, and changes in hardness of the overall substance at weeks 1, 2, 4, 6, 8, and 10.

Lipsticks and lip balms were assessed in their ﬁnal containers. Hardness was evaluated when the lipstick or lip balm were raised with the dial at the bottom of the container. If rigidity was stable, the overall intact structure of the lipstick of lip balm would remain unchanged as it was raised. Samples of creams were placed into 1.5 mL centrifuge tubes that were placed in stability cabinets. Changes in stability of creams were recorded as either color changes or a clear separation layers occur within the formula.

Animal Study Design

For the oral toxicity study, twelve rats were randomly divided into two groups

(vehicle and vehicle containing alkenones, n=6 per group), whereas ten rats were used in the acute dermal toxicity, and another ten rats were used in the repeated fixed doses 28- day dermal application, n=5 per group. For dermal toxicity, each group had four different skin sites where four different treatments were applied and observed for signs of irritation such as redness. Figure 2a shows the timeline of the experimental design of alkenone administration for different animal groups and Figure 2b and Figure 2c shows the actual overview skin application treatments performed on the rats for both acute and chronic exposure. Each application square was 2.5 x 2.5 cm.

Figure 2a: Experimental design showing the duration of administration of alkenones for three different studies in SAS Sprague Dawley rats.

Figure 2b: Experimental design showing the site (6.25 cm2) of acute dermal applications of different treatments in two stand-alone studies.

Figure 2c: Experimental design showing the site (6.25 cm2) of repeat dermal applications of different treatments in two stand-alone studies.

Suspension Formulation

The alkenones were not soluble in solvents such as dimethyl sulfoxide (DMSO),

Tween 80, Tween 20, Sesame seed oil, and Dulbecco’s modification of Eagle’s medium

(DMEM) media. Therefore, we used a suspension formulation to administer the alkenones to rats using oral gavage. After weighing, the alkenones were pulverized using a mortar and pestle and 600 µL of glycerin was added as a wetting agent to form a paste.

Hydroxypropylmethylcellulose (HPMC) was placed in water heated to 80 °C to yield a

3% w/v solution. The HPMC solution was vortexed and allowed to cool to room temperature. The 3% w/v of HPMC was added to the paste. The resulting suspension was poured into a large test tube and removed using an oral gavage stainless steel needle (3 inches length and ball diameter of 2.25 mm) prior to dosing the rats.

21 Acute Oral Gavage Administration of the Alkenones

The alkenones suspension and vehicle suspension were administered to female rats by oral gavage in divided doses of 1 mL/kg body for a total of 4000 mg/kg body weight. The starting dose of 1000 mg/kg of the alkenones in the suspension vehicle (3%

HPMC and glycerin) and the vehicle itself was used on two individual rats (four total) as a starting dose as recommended by Whitehead and Stallard (Whitehead & Stallard,

2004), with modifications. Since that dose did not produce signs of toxicity, including a loss of less than 20% body weight, change in physical appearance or change in behavioral pattern after 48 hours, two more animals were given a dosage of 2000 mg/kg of the alkenones. After 48 hours, the 2000 mg/kg dose did not appear to be toxic to the animals. Twelve female SD rats were randomly divided into two groups (6 rats per group). Group 1 (n=6) was given 4000 mg/kg of the alkenones derived from Isochrysis galbana that was prepared in a suspension of glycerin and 3% HPMC. Group 2 (n=6) received the same dose of vehicle. The rats were observed for general behavioral changes, using the body condition scoring scale based on the following typical signs: failure to eat or drink, ruffled fur, runny eyes, respiratory distress, abdominal pain

(hunched posture), or weight loss > 20% (weighed daily). In addition, the facial expression of the rats were quantitated using the Grimace Scale, as previously described

(Sotocinal et al. 2011), which measures changes in the positions of the ears and whiskers, orbital tightening, and nose/cheek flattening. Symptoms of toxicity (and mortality) were noted after each divided treatment for the first 1 hour, followed by observations made within 30 minutes of treatment, and then at 1, 6, 12, 24 and in parallel, rats in group 2 were treated with vehicle (Glycerin and 3% HPMC) to establish a comparative negative

22 control group according to the OECD (2002) guideline. All animals were observed at least once during the first 30 min in the first 24 h and then 4h or 24 h following vehicle or alkenones suspension administration and then once a day, at approximately 10AM, for 14 days, as indicated in the OECD (2002) guideline. All behavioral and physical observations, including changes in skin, fur, secretions from eyes and mucous membrane, and behavioral pattern changes such as posture, response to handling, motor activity, grip strength and sensory reactivity to stimuli, were recorded for each rat. Food consumption was recorded daily, and the body weights of each animal was recorded immediately before the administration of the test substance and every day throughout the length of the study.

The Quantification of White Blood Cells (WBCs), Red Blood Cells (RBCs),

Platelets (PLTs), Monocytes and Hemoglobin (Hb). At the end of the study, animals were sacrificed (n=12) using CO2 asphyxiation, followed by cardiac puncture.

Subsequently, 1 mL of blood was obtained and placed in a 1.3 mL K3E Microtube

(Sarstedt, Newton, North Carolina). Five hundred microliters of blood was placed in a test tube containing 56 USP units of lithium heparin. Subsequently, in whole blood samples, the number of RBCs, WBCs, PLTs, monocytes and the level of Hb were determined using a VetScan HM5 instrument (Abaxis, Union City, CA, USA).

Determination of Blood Parameters. From the collected blood in the lithium heparin test tube, 120 μL was withdrawn and added to the comprehensive diagnostic profile plate (Abaxis, Union City, California, USA) and run in a VetScan 2 device

(Abaxis, Union City, California, USA). We measured albumin, total protein, globulin, sodium (Na+), potassium (K+), alkaline phosphatase (ALP), alanine aminotransferase

23 (ALT), amylase (AMY), total bilirubin (TBIL), bilirubin urea nitrogen (BUN), total calcium (CA), phosphorus (PHOS), creatinine (CRE) and glucose (GLU) in the whole blood samples using a VetScan 2 instrument.

Histology. The animals were euthanized using carbon dioxide (CO2) asphyxiation, followed by a cardiac puncture to collect blood. Immediately following sacrifice, the major organs of each rat (except the brain) were evaluated by gross necropsy. We evaluated abnormal growth of lesions, necrosis, changes in overall organ size and color. The liver, kidneys, spleen and heart were removed, examined for growth of abnormal lesions, weighed and collected for further histopathological examination.

The lungs, stomach, intestine, and colon (data not shown) were examined as described above and weighed, but not collected for histopathological examination. Subsequently, the organs examined were immediately fixed in 10% neutral buffered formalin for

24 hours, then dehydrated in 70% ethanol, and embedded in paraffin. The block of embedded tissue was cut into 5 µm sections using a microtome (Reichert-Jung Biocut

2030 Microtome, Rankin; Holly, Michigan, USA). The resulting tissue sections were subjected to a modified protocol from www.aladdin-e.com (Aladdin, 2018) for haematoxylin and eosin (H&E) staining. Images of the tissue sections were taken using a

VS 120 Virtual Slide Microscope (Olympus, Pittsburgh, Pennsylvania, USA.) The hematoxylin and eosin-stained images were examined using OlyVIA Ver.2.9 software.

24 Single and Repeated Dermal Application of the Alkenones

Twenty-four hours before treatment, fur was removed by closely clipping the dorsal area of the trunk of the animals according to the OECD guidelines (OECD, 1981,

2004, 2015). On the treatment day, Study 1, the alkenones were weighed for the administration of dermal doses of 750 mg/kg or 1500 mg/kg. The doses were based on

OECD guideline 434 fixed dose flow chart, where the highest dose that could be given was 2000 mg/kg with 1000 mg/kg as the next highest dose. Alkenones, at a dose of 2000 mg/kg, could not be given orally and thus were applied to a skin site area (approximately

6 cm2). This was modified to the highest dosage that could be administered, 1500 mg/kg with a second dose of 750 mg/kg in case severe irritation occurred during the 24 hour contact exposure. The alkenones were pulverized in a mortar using a pestle. The powder was mixed with a fixed amount of glycerin (0.6 mL) to form a paste-like substance. Five rats in Study 1 were given 1500 mg/kg, positive control (1% SLS), negative control

(0.9% sodium chloride, saline), and vehicle (Glycerin) on four different skin sites of each individual animal. Another five rats in Study 2 were given positive control, vehicle, 1500 mg/kg, and 750 mg/kg at different skin sites on the shaved backs of the rats. The test compound (300 grams of the paste) was applied to a small area (approximately 6 cm2) of skin and covered with a gauze patch that was held in place with non-irritating tape. Other treatments applied at different test sites were: a negative control (0.9% sodium chloride), a positive control (1% sodium lauryl sulfate (SLS: dissolved sodium chloride) and glycerin. The rats had ad libitum access to standard rat food pellets that were kept in small paper cups and water throughout the study. To further prevent the rats from removing the covered gauze patch, an Elizabethan collar was used and each rat was kept

25 in a separate cage for the 24-hour test period. At the end of the test period, the covering was removed and 1) images of the skin sites were taken using a Panasonic Lumix DMC-

ZS1 digital camera; 2) the reaction of the skin was graded based on the Draize skin reaction numerical scale 35 and 3) a Tewameter reading from each skin site was recorded before washing the skin sites with water. Following the above tests, the rats were returned to their cages.

The clipping of the fur was done as described above for the repeated dermal experiment. On the treatment day, either 50, 200 or 1000 mg/kg were dermally applied and these doses were based on the OECD guideline 434 fixed dose flow chart: 50, 200,

1000 and 2000 mg/kg. Since we could not use the 2000 mg/kg dose, we opted to use the other three doses for our repeated study. Study 1 consisted of five rats that were given the positive control, 1000 mg/kg, the negative control and the vehicle on four different skin sites. Study 2 consisted of five rats that were given the positive control, 1000 mg/kg, 200 mg/kg and 50 mg/kg on four different skin sites. The procedure for preparing, applying the test substances, housing of animals and tests performed after treatment were the same as those mentioned for the acute dermal administration of the alkenones.

However, for chronic administration, the treatment time was lowered to a 6 hour period. For repeated dermal toxicity 6 hours following application, each site was gently wiped with a dry gauze pad to remove any remaining paste residue and allowed to breathe for 2-3 minutes before reading with the probe occurred. Afterwards, the skin sites were washed with water to remove any remaining substances on the skin and wiped down again gently with a dry gauze pad. This procedure was done once a day for 28 consecutive days.

26 Skin Irritation Index Score. The irritation of the application site was graded based on a scale for erythema, eschar and edema formation following the OECD Test

Guideline 404. This scale has been used previously for testing 176 chemicals (Bagley et al., 1996), with the highest grade of 8 indicating the most severe irritation. Although the guideline recommends using rabbits, we used SD female rats since they have been used in both acute and repeated dermal toxicity studies (Han, Lee, & Park, 2012; M. J. Kim et al., 2015; Ryu et al., 2014), including one study that tested a supplement derived from marine algae (Marone, Yasmin, Gupta, & Bagchi, 2010).

Determination of Skin Water Loss. A Tewameter® TM 300, a non-invasive device, was used to measure the amount of water loss through the skin. The measurement unit for reading the rate of loss is known as transepidermal water loss (TEWL) (El-

Chami, Haslam, Steward, & O'Neill, 2014). It has two pairs of sensors in the probe that receive information on the temperature and moisture readings of each TEWL-value during the complete measurement (GmbH 2018). The head of the probe is small (10 mm diameter and 20 mm height), limiting the influence of air turbulence inside the probe.

Finally, the probe’s light weight makes it easy to handle and it has no significant effect on the skin structure. The Tewameter® TM 300 is a globally used method for measuring

TEWL and has been used in both animals (Indra & Leid, 2011) and humans (Eo et al.,

2016; Gardien, Baas, de Vet, & Middelkoop, 2016). The open chamber probe was held in the middle of each skin site application for 20 seconds before and after each treatment.

27 Statistical Analysis

The normally distributed data were expressed as the mean ± standard deviation

(SD). The statistical analysis of the data was performed using GraphPad Prism (Version

7.0, GraphPad Software, La Jolla, California, USA). For the body weight and food consumption for the acute oral, dermal, and chronic test, the data were not normally distributed and were expressed as the modes and analyzed using the Mann-Whitney test.

The data from the Vet Scan HM5 and VS2 values were all normally distributed except for TBIL, BUN, and potassium. Therefore, the TBIL, BUN, and potassium data were analyzed using the Mann-Whitney test, whereas the other blood chemistry data were analyzed using Welch’s t test. The post-mortem organ weight data were analyzed using

Welch’s t test. The Tewameter data for acute dermal skin site application was analyzed using a two-way ANOVA, with the treatment and baseline measurements as the fixed effect and ID as the random effect. For repeated Tewameter data, a mixed effects model is applied for the treatment, baseline and days (treated as categorical variable) as the fixed effect and ID as the random effect. Tukey post hoc analyses were used for the pairwise difference for the dermal Tewameter values. The a priori significance level was p < 0.05.

Chapter Three

Results

The Determination of Solubility and Thickening Capability Test

The three individual alkenones and a mixture as previously described (G.W.

O’Neil et al., 2017), were entered into FFE to generate HSPs based on the SMILES. FFE calculated that the individual components and the overall alkenones blend had a high

MVol (Table 4), which was predicted based on the composition of the alkenones (ref.

Figure 1).

Using 53 solvents found in the FFE, the solubility of the alkenones was predicted.

Based on the HSPs, five solvents were selected for the thickening capability test that were defined as “good” by the FFE for the alkenones. The ﬁve solvents were: hexamethyldisiloxane, isohexadecane, isopropyl isostearate, C12-15 alkyl benzoate and ethylhexyl methoxycinnamal. The main groups of emollients commonly used in personal care and makeup applications include silicones, hydrocarbons and esters (Douguet et al.,

2017). A silicone, a hydrocarbon, a light ester and a higher molecular weight ester were selected in terms of providing a light, easy to spread, non-greasy and non-sticky feel on the skin. The ﬁfth ingredient was a liquid sunscreen, ethylhexyl methoxycinnamal. The alkenones were incompatible with the liquid silicone, hexamethyldisiloxane and the

29 hydrocarbon, isohexadecane as these solvent-wax mixtures separated into two layers overnight. The alkenones were compatible with the rest of the solvents, and no signs of separation were observed at room temperature within 24 hours. All mixtures had a shear thinning behavior. The viscosity data is shown in Figure 3. The alkenones thickened the three solvents in a similar manner as the other two waxes and viscosities were comparable. The viscosity data indicate that the alkenones were also highly effective in thickening the liquid sunscreen.

Figure 3: Viscosity of wax-emollient mixtures 24 h after formulation (average ± SD). Viscosities are compared at 9.3 s−1 for emollients 1–4, and at 0.93 s−1 for emollient 5. MW: microcrystalline wax, O: ozokerite, A: alkenones. Reprinted from (McIntosh K. et al., 2018).

Melting Point Determination

The melting point of the alkenones was in the range of 71.1–77.4 °C. The DSC thermogram exhibited a sharp, characteristic endothermic melting peak at 73.8°C.

Examples of waxes commonly used in personal care products include beeswax, candelilla

30 wax, carnauba wax, castor wax, lanolin alcohol, sunﬂower wax, ozokerite and microcrystalline wax. The melting point of these waxes ranged from 61–89 °C.

31 Table 4: Parameters of the Hansen Solubility components (δD, δP, δH), molar volume (MVol), Ingredient-Skin Gap (ISG), Solubility in Skin (SolS) and LogK were calculated by Formulating for Efficacy™.

Table 4. Parameters calculated by Formulating for Efficacy™

Ingredient δD δP δH MVol ISG SolS LogK

37:3 methyl 16.3 0.6 2.5 613 57.2 0.3 16.81 alkenone 37:2 methyl 16.2 0.7 2.2 621 58.8 0.3 15.58 alkenone 38:2 ethyl 16.1 0.8 2.2 638 60.1 0.3 16.27 alkenone Mixture 16.2 0.7 2.3 622 58.5 0.3 - combined according to O’Neil, 2012

Lipstick and Lip Balm Stability

The surface of the lipsticks and lip balms formulated with the alkenones (i.e.,

Lipsticks 2 and 3, and Lip Balms 2 and 3) was more matte than that of the lipstick and lip balm made without the alkenones (i.e., Lipstick 1 and Lip Balm 1). The color was uniform for all lipsticks. The various waxes were compatible with one another. The alkenones were compatible with the rest of the ingredients as well, and no visible changes were observed during the course of the stability study. The lipsticks showed no signs of change at room temperature during the 10 weeks (Table 5). The ﬁrst sign of instability was observed at Week 4 at an elevated temperature, when Lipstick 1 and 2 had signs of sweating. At Week 10, at high temperature, Lipsticks 1 and 2 were considered unstable as they had signs of severe sweating. Lipstick 3 showed no signs of sweating, even at Week

10 at elevated temperature, making it the most resistant formulation to high temperature.

32 For the lip balms, the ﬁrst signs of a slight change were observed at Week 2. Lip

Balm 2 (i.e., made with microcrystalline wax and the alkenones) was softer than the other two balms at 25 ºC and was extremely soft, especially at the center of the balm, at 45 ºC.

This softness was considered a sign of instability. The melting point peak is 68.8 °C for microcrystalline wax, 73.8 °C for the alkenones and 74.2 °C for ozokerite (measured in- house with the above-described DSC method).

33 Table 5: Stability of lipsticks and lip balms (n=3) in cabinets set at 25 ºC and 45 ºC for 10 weeks.

Time Lipstick Lip Balm Point 1 2 3 1 2 3 Temperat 25 45°C 25° 45°C 25° 45° 25° 45° 25° 45°C 25°C 45°C ure °C C C C C C C Day 1 ------Week 1 ------+ - + soft - + soft soft Week 2 ------+ - ++ - + soft very soft soft Week 4 - + - + - - - + ++ ++ + + slight slight soft very very (verti soft sweati sweati soft soft cal ng ng crack s on stick’ s side) Week 6 - + - + - - - + ++ ++ + + slight slight soft very very (verti soft sweati sweati soft soft cal ng ng crack s on stick’ s side) Week 8 - + - + - - - + ++ ++ + + slight slight soft very very (verti soft sweati sweati soft soft cal ng ng crack s on stick’ s side) Week 10 - ++ - ++ - - - + ++ ++ + + severe severe soft very very (verti soft sweati sweati soft soft cal ng ng crack s on stick’ s side) Markings: -: no change +: slight change (i.e., reversible separation) ++: significant change (i.e, irreversible separation)

Viscosity, Thixotropy and Rheology

All four creams were opaque/white. The viscosity of each sample is reported using two shear rates (s−1) in Table 6. The two selected shear rates were 1 and 100 s−1, representing a low shear and a high shear application, respectively. The viscosity varied more than 10-fold. All four creams contained the same emulsifier system, a combination of poly-sorbate 80, a nonionic emulsifier with a high hydrophyl lipophyl balance (HLB) value (HLB: 15.0) and sorbitan oleate, a nonionic emulsifier with a low HLB value

(HLB: 4.3). As both emulsiﬁers were liquid ingredients, they did not significantly thicken the emulsions. Differences in ﬂow and viscosity between different creams were due to the different waxes and their capacity to thicken the emulsions. All four creams were shear- thinning, as is expected for a cosmetic lotion/cream (Figure 3a). All four creams fit the

Power Law model (Viscosity = k × shear raten) (using viscosity measurements with increasing shear rate, which is discussed below). Cream 1 had the lowest viscosity, whereas Cream 2 and Cream 3 had very similar viscosities and Cream 4 had the highest viscosity, congruent with the k parameter of the Power Law model (Table 6). The shear thinning index, n, varied from 0.14–0.26. To determine whether any of the samples were thixotropic, viscosity was plotted for increasing and decreasing shear rate (Figure 3b).

Differences between the up and down curves indicates the extent of the thixotropy.

Cream 1, made with the alkenones, was shear-thinning without being thixotropic. The area between the two curves increased in the following order: Cream 2 < Cream 3 <

Cream 4. The largest difference in viscosity at 1 s−1 was measured for Cream 4 as 11,500 cP or 60 % decrease. The alkenones did not yield any thixotropic property to Cream 1.

Figure 4: (a) Viscosity-shear rate data and power law fitting for the four creams (b) Viscosity-shear rate data showing the extent of thixotropy in the four creams. Reprinted from (McIntosh K. et al., 2018).

36 During the viscoelastic measurements, the sample was not rotated, and an oscillation shear flow was applied. Oscillatory flow may be considered a non-destructive test. G’ represents the elastic behavior of a sample, while G” represents the viscous portion (Mason, Bibette, & Weitz, 1995). If G’ is above G”, the samples have a more elastic or gel-like behavior. In the case of all four creams, G’ > G”, indicating that the formulations were more elastic than viscous, i.e., the creams were gel-like. Cream 4 had the highest G’. An almost constant value of G’ and G” was observed over the entire frequency range with the value of G’ exceeding that of G”. The frequency independence of G’ and G” indicates gel-like behavior for the creams. Overall, both viscosity and G’ increased in the same order: Cream 1 < Cream 2 < Cream 3 < Cream 4 (Table 6).

Table 6. Viscosity and storage modulus (average ± SD) of the creams at room temperature. Sample Viscosity at 1 s-1 Viscosity at 100 s-1 G’ at 10 rad/s (Pa) (cP) (cP) Cream 1 1470 ± 10 54 ± 1 11 ± 1

Cream 2 6950 ± 110 147 ± 1 45 ± 10

Cream 3 8460 ± 570 169 ± 1 1560 ± 1100

Cream 4 19400 ± 2500 400 ± 10 4710 ± 4300

Liquid Crystalline Structure

The formation of the liquid crystal structure is the result of an ordered arrangement of the emulsiﬁer, oil and wax molecules at the oil-water interface.

Birefringence was detected in Creams 2– 4. No birefringence was observed for Cream 1, suggesting that the alkenones did not produce any liquid crystal structure at the water-oil

37 interface. Unlike the other waxes, which migrated to the water-oil interface, the alkenones most likely stayed in the oil phase.

Cream Stability

The stability of the samples was evaluated for ten weeks or until any irreversible change was noticed. All samples remained stable at room temperature without any physical signs of instability (i.e. clear oil separation) for ten weeks. At the higher temperature, Cream 1 started to show signs of a reversible separation (creaming) very early in the stability test. Cream 2 started showing signs of creaming at Week 2. Creams

3 and 4 showed irreversible separation at the higher temperature at Week 2 and were considered unstable.

Table 7: Stability of creams. Cream 1: alkenones; Cream 2: cetyl alcohol; Cream 3: stearic acid; Cream 4: glyceryl monostearate.

Table 7: Stability of creams. Time point Cream 1 Cream 2 Cream 3 Cream 4

Temperature 25°C 45°C 25°C 45°C 25°C 45°C 25°C 45°C

Week 1 - + ------

Week 2 - + - + - ++ - ++

Week 4 - + - + - -

Week 6 - + - + - -

Week 8 - + - + - -

Week 10 - + - + - -

Markings: -: no change +: slight change (i.e., reversible separation) ++: significant change (i.e, irreversible separation)

38 The Effects of the Acute Oral Administration from Alkenones

Statistical analysis by the Mann-Whitney test indicated that the administration of

4000 mg/kg of the alkenone formulation did not significantly alter the body weight compared to animals treated with vehicle (Figure 5). Furthermore, no significant difference in food consumption between the alkenone- and vehicle-treated animals was detected by Mann-Whitney analysis (Figure 5).

Figure 5: Determination of (a) body weight, and (b) food consumption, following one- time oral administration of alkenones (4000 mg/kg) compared to vehicle in female SAS Sprague Dawley rats (n=6). No significant differences between treatments were detected by a Mann-Whitney test for either of these measurements.

The acute administration of 4000 mg/kg of the alkenone formulation did not significantly increase changes in behavior based on the facial grimace scale nor body postures or signs of stress compared to animals treated with vehicle (data not shown).

The gross necropsy results indicated that neither the administration of 4000 mg/kg of the alkenone formulation nor vehicle produced apparent signs of pathology (Figure 6). No histopathological changes were present in the liver, kidneys, spleen, heart, stomach, intestine and colon of animals treated with 4000 mg/kg of the alkenones or vehicle group

39 (data not shown). The brain tissue was not evaluated as the alkenones, which consist of long 37 hydrocarbon chain, would not cross the blood brain barrier intact. However, the metabolites of the alkenones could potentially enter through one of the many fatty acid transport proteins (FATPs) (Mitchell, On, Del Bigio, Miller, & Hatch, 2011). Further studies must be done to verify this hypothesis.

The acute administration of the 4000 mg/kg formulation of the alkenones did not significantly alter the number of WBC, RBC, PLT, HGB, and monocytes or the level of hemoglobin (p < 0.05) compared to vehicle–treated animals (Figure 7). There was no significant difference between the alkenone- and vehicle-treated animals in the whole blood levels of albumin, total protein, globulin, Na+, K+, ALP, ALT, AMY, TBIL, BUN,

CA, PHOS, CRE, and GLU (p < 0.05) (Figure 8).

Figure 6: Histopathological evaluation of the (a) liver, (b) spleen, (c) heart, (d) kidney and (e) skin of SAS Sprague Dawley rat after acute oral exposure to vehicle control and alkenones (4000 mg/kg) after 14 days. (C1) Represents vehicle control rat 1, while (T1) represents treated rat 1. The treated samples were stained with haematoxylin and eosin (H&E) stains and observed at a magnification of 20× (n=6). There were no significant pathological changes in the various tissue sections between the vehicle and treated groups.

Figure 7: Blood chemistry profile in SAS Sprague Dawley rats after one-time oral exposure to vehicle control and alkenones (4000 mg/kg) for 14 days. The determination of (a) white blood cells (WBC), (b) red blood cells (RBC), (c) platelets (PLT), (d) hemoglobin (HGB), and (e) monocytes in rats that were treated with vehicle or alkenones suspensions at the day 14. There were no significant differences between the treatments detected by Welch’s t test for either of these measurements.

Figure 8: Blood chemistry profile to determine organ toxicity in SAS Sprague Dawley rats after a one-time oral exposure to vehicle control and alkenones (4000 mg/kg) for 14 days. (A) Albumin, total protein, and globulin levels in grams per deciliter (g/dL), (B) sodium and potassium levels, millimoles per liter (mMol/L), (C) alkaline phosphatase (ALP), alanine aminotransferase (ALT) and amylase (AMY) levels in units per liter (U/L), (D) total bilirubin (TBIL), blood urea nitrogen (BUN), total calcium (CA), phosphorus (PHOS), creatinine (CRE) and glucose (GLU) levels in milligrams per deciliter (mg/dl), collected from rats treated acutely with oral vehicle and alkenone suspensions. There were no significant differences between the treatments detected by Welch’s t test for either of these measurements. No significant differences were detected by Mann-Whitney analysis between the TBIL, BUN, and K+ measurements.

43 Acute Dermal Treatment

We conducted experiments to determine the effect of the topical administration of either 1,500 mg/kg or 750 mg/kg alkenone formulation for 24 h to rats over 14 days. The body weights, as well as the food consumption, of the animals in Study 1 and Study 2 were not significantly altered (p < 0.05 for both measurements, Mann-Whitney U test) compare to animals treated with vehicle (Figure 9). The exposure of the skin to the positive control, 1% SLS, for 24 h, significantly increased (Sidak’s multiple comparison, p < 0.0001) skin water loss compared to the alkenones (1,500 mg/kg) and glycerin

(Figure 10). The open cylinder chamber probe of the Tewameter® TM 300 was held against each skin with light pressure for at least 20 seconds to get an average reading.

The TEWL values ranged from 0-10 (very healthy conditions) to above 30, indicating critical skin barrier dysfunction. If the alkenones produce skin damage to the skin, then similar to the positive control, it should yield high TEWL values. Four out of ten rats had increased TEWL readings compared to their positive controls, while all other topical application sites had TEWLs ranging from 0-10 (i.e. very healthy skin condition) (Figure

11).

Figure 9: Determination of (a) body weight, and (b) food consumption, following acute dermal exposure of Study 1 compared to Study 2 in female SAS Sprague Dawley rats (n=5). No significant differences by a Mann-Whitney test for either of these groups.

Figure 10: The transepidermal water loss (TEWL) was measured before and 24 hours after one-time dermal application in (a) Study 1 rats treated with 1% SLS (positive control), saline (negative control), glycerin (vehicle) and 1500 mg/kg alkenones (AK); (b) Study 2 rats treated with 1% SLS (positive control), vehicle (glycerin), 750 mg/kg or 1500 mg/kg alkenones (AK); (c) Combined data from Studies 1 and 2 rats to show any significant change in TEWL measurements.

Figure 11: The transepidermal water loss (TEWL) was measured before and after one- time application of 1% SLS (sodium lauryl sulphate), alkenones (1500 mg/kg) and vehicle (glycerin) on dermal skin in SAS Sprague Dawley rats. Statistical significance between baseline and 24 hr readings was determined by two-way ANOVA analysis followed by Tukey’s multiple comparisons tests.

Repeated Dermal Applications from Alkenones-Containing Paste

The different repeated dermal treatment applications of Study 1 or Study 2 for 6 hours per day for 28 days did not significantly alter body weight (Mann-Whitney test) and food consumption (Mann-Whitney test), compared to animals treated with vehicle

(Figure 12). Furthermore, the repeated dermal application of the alkenones did not

47 significantly alter the relative weights for the liver, kidneys, spleen, heart, stomach, intestines and colon (data not shown).

Figure 12: The determination of (a) body weight, and (b) food consumption, following repeated dermal exposure of Study 1 compared to Study 2 in female SAS Sprague Dawley rats (n=5). No significant differences were detected by a Mann-Whitney test for either of these measurements.

Figure 13: The transepidermal water loss (TEWL) was measured before and after 6 hours repeated dermal exposure every day for 28 days on skin area exposed to (a) 1% SLS (positive control), (b) 1000 mg/kg alkenone, (c) 200 mg/kg alkenone, (d) 50 mg/kg alkenone, (e) normal saline (negative control), and (f) glycerin. The rats were randomly divided into two study groups. Study 1 Rats (13, 14, 17, 18, and 20) were treated with 1% SLS, saline, glycerin (vehicle) and 1000 mg/kg alkenones, respectively. Study 2 Rats (11, 12, 15, 16, and 19) were treated with 1% SLS, 50 mg/kg alkenones, 200 mg/kg alkenones, and 1000 mg/kg alkenones, respectively

Over the 28-day exposure to the alkenones, the 1000 mg/kg dose of the alkenones only increased the TEWL value to 10 in one rat on day 1 in Study 1 (Figure 13). In

49 addition, the application of saline (the negative control) also increased the TEWL in the aforementioned animal. The TEWL values for the positive control (1% SLS) were the highest on day 2, with eight out of ten rats having TEWL values greater than 10. Overall, the 1g/kg paste formulation of the alkenones significantly (Sidak’s multiple comparison, p < 0.05 and p < 0.01, respectfully) lowered the TEWL values throughout the 28-day period (Fig. 14). The positive control always significantly increased the water loss.

Figure 14: The transepidermal water loss (TEWL) measurements before and after 6 hours repeated dermal exposure of different treatments each day at (a) Day 1, (b) Day 2, (c) Day 8, (d) Day 14, (e) Day 21, and (f) Day 28.

Chapter Four

Discussion

Physical characteristics

The majority of personal care products are shear thinning and therefore, a wax like the alkenones that has a similar characteristic, is desirable. The Ingredient-Skin Gap

(ISG) is defined as the compatibility of the ingredient with the stratum corneum (i.e., the outer layer of the epidermis)(Abbott, 2012). A small ISG indicates that the ingredient is more compatible with the stratum corneum and has a greater likelihood of penetrating the skin (the MVol also plays an essential role in determining skin penetration) (Abbott,

2012). All three individual alkenones and the mixture were on the higher end of the scale

(certain ingredients have an ISG as low as 2), as seen in Table 4. The solubility in the skin (SolS) is estimated from the HSP distance. The alkenones had a low SolS value in all cases. A proper formulation strategy can be used to produce higher SolS values for skin penetration or lower SolS values for prolonged residence time on the skin which is required for delivering a topical agent. The octanol/water partitioning coefﬁcient (Log K) of the three individual alkenones were very high, suggesting that they could accumulate in the fatty adipose tissue.

52 Out of 53 solvents that were in FFE as default, ﬁve solvents were selected for the thickening capability test based on the HSPs. These ﬁve solvents were: hexamethyldisiloxane, isohexadecane, isopropyl isostearate, C12-15 alkyl benzoate, and ethylhexyl methoxycinnamal. (Benazzouz, Moity, Pierlot, Molinier, & Aubry, 2014) The

ﬁve emollients were located close to the alkenones in the Hansen Space, indicating that they are good comparable solvents, already used in personal care products, for the alkenones. The main groups of emollients commonly used in personal care and makeup applications include silicones, hydrocarbons, and esters and these were selected in terms of skin feel, i.e., providing a light, easy to spread, non-greasy, non-sticky feel on the skin, which most consumers are looking for when purchasing personal skin care products (Hee

Yeon Kim & Chung, 2011; Parente, GÁMbaro, & Ares, 2008). The ﬁfth ingredient was a liquid sunscreen, ethylhexyl methoxycinnamal. We selected a sunscreen as a solvent because we wanted to determine the thickening capability of the alkenones in a liquid organic sunscreen. As mentioned before, the alkenones were incompatible with the liquid silicone (hexamethyldisiloxane) and the hydrocarbon (isohexadecane). Incompatibility may result from a more complex interaction between solvents and waxes. These selected solvents were very different in structure; therefore, it would be predicted that the different waxes interacted differently with the different solvents. We did not expect to see a general pattern in solvent-wax compatibility due to the possible interactions. However, the alkenones were compatible with the rest of the solvents, showing no signs of separation at room temperature within 24 hours. All mixtures had a shear thinning behavior. This is characteristic of microcrystalline wax and ozokerite; therefore, this type of behavior was expected from the alkenones as well.

53 When analyzing the viscosity data, a single shear rate (s−1) was used to obtain a reliable reading for all three waxes and the viscosity was compared at this shear rate.

Again, based on the possible interactions between the components, we did not expect to see a clear pattern for the viscosity values. The alkenones thickened the three solvents in a similar fashion compared to the other two waxes and viscosities were comparable. The fact that the alkenones were stable suggesting they may be used in liquid sunscreen, was very promising. Previous reports indicate that having a thick and even layer of sunscreen on the skin is essential to provide the claimed SPF (W. Liu et al., 2012; Teramura et al.,

2012). The viscosity data indicate that the alkenones were highly effective in thickening the liquid sunscreen, ethylhexyl methoxycinnamate. The melting point of the alkenones was in the range of 71.1–77.4 °C, with a sharp characteristic endothermic melting peak at

73.8 °C. The melting point range of the alkenones was in the range of the other commonly used waxes, making them a viable candidate as a personal care product wax.

Lipstick, Lip Balm and Cream Stability

The surface of the lipsticks and lip balms formulated with the alkenones (i.e.,

Lipsticks 2 and 3, and Lip Balms 2 and 3) was more matte than that of the lipstick and lip balm made without the alkenones (i.e., Lipstick 1 and Lip Balm 1). The matte finish was probably the result of an interaction between the emollients and the wax. If a shiny finish is desired for a lipstick or lip balm, the emollients could be substituted with other types of emollients. In this study, we did not optimize the formula to a shiny or matte finish and we chose a relatively simple starting formula that contained all the waxes of interest and formulated the products. These various waxes were compatible with one another and the

54 color was uniform for all lipsticks. The lipsticks showed no signs of change at room temperature during the 10 weeks (Table 5). The ﬁrst sign of instability was observed at

Week 4 at an elevated temperature when Lipstick 1 and 2 had signs of sweating.

Sweating is a quality problem when oil droplets appear on the surface of the stick (A.C.

Dweck, 1981). The cause of the problem may be an incompatibility between individual ingredients in the formulation, imbalanced composition such as high oil content, or inferior oil-blending capacity of the wax composition (A. C. Dweck & Burnham, 1980).

At Week 10, at high temperature, Lipsticks 1 and 2 were considered unstable as they had signs of severe sweating. Lipstick 3 showed no signs of sweating, even at Week 10 at elevated temperature, making it the most resistant formulation to high temperature.

For the lip balms, the ﬁrst signs of a slight change were observed at Week 2. Lip

Balm 2 (i.e., made with microcrystalline wax and the alkenones) was softer than the other two lip balms at 25 °C and was extremely soft, especially at the center of the balm, at

45 °C. This softness was considered a sign of instability. A lip balm that cannot hold its shape at room temperature and higher temperature is unlikely to pass quality testing

(Dayan, 2017). When twisting the lip balm upwards, as one would do to apply the balm to the lips, the balm did not hold its shape and it was a semi-solid, greasy mass. Lip balms are expected to have a certain hardness and resistance to higher temperature in case they are left at elevated temperature, e.g., in the car in the summertime (Ribeiro

Fernandes et al., 2013). This behavior of lip balm 2 can be explained based on the melting point of the waxes. The melting point peak is 68.8 °C for microcrystalline wax,

73.8 °C for the alkenones, and 74.2 °C for ozokerite (measured in-house with the above- described DSC method). Overall, the stability of the lipsticks and lip balms made with the

55 alkenones was comparable to that of the lipstick and lip balm made without the alkenones. During formulation and stability testing, the sticks and balms were identical and had similar qualities. The combination of the two lower melting point waxes resulted in a softer product. The difference between the melting point peak of ozokerite and alkenones was not signiﬁcant, as it was less than 1 °C, but the change in product hardness was noticeable. When looking at product hardness, the interaction among all ingredients, should be taken into account, as an interaction may modify product softness (Fernandes et al., 2013). At an elevated temperature, all lip balms were softer starting at Week 1.

Higher temperature softens the consistency of the waxes and butters used in the lip balms, making the lip balms softer. These lip balms would probably regain their original hardness if taken out of the stability cabinet and allowed to cool to room temperature.

The stability of the cream samples was evaluated for ten weeks or until any irreversible change was noticed. All samples remained stable at room temperature without any physical signs of instability for ten weeks. At the higher temperature, Cream

1 started to show signs of a reversible separation (creaming) very early in the stability test. Cream 2 started showing signs of creaming at Week 2. Creams 3 and 4 showed irreversible separation at the higher temperature at Week 2, and were therefore considered unstable.

Viscosity, Thixotropy, Rheology and Liquid Crystalline Structure

The viscosity of each cream sample was reported at two shear rates, a low (1 s−1) and high (100 s−1) shear application, respectively, i.e., when a cream is applied to the skin. A Power Law model (Viscosity = k × shear raten) showed all four creams were

56 shear-thinning. Cream 1 made with the alkenones had the lowest viscosity, followed by

Cream 2 and Cream 3 with similar viscosities and Cream 4 had the highest viscosity. A shear thinning flow behavior is a common rheological similarity between diverse fluid systems that would include gels, pastes and creams. The shear thinning index, n, varied from 0.14–0.26, which is similar to other personal care products (Arshad, Khan, &

Akhtar, 2016; AZoM; Kurzmann, 2019; Park & Song, 2010). Creams without the alkenones showed thixotropic properties. Thixotropic materials lose structure during shear, which may rebuild upon standing (Brummer, 2006). This behavior is a key factor for the ease of application of a cosmetic cream to a surface (through structure breakdown in spreading) and then rebuild its structure and viscosity, preventing it from dripping and running after application (Miner, 1993). Differences between the increasing and decreasing shear rate curves of the viscosity indicated the extent of the thixotropy. Cream

1, made with the alkenones, was shear-thinning without being thixotropic. The area between the two curves increased in the following order: Cream 2 < Cream 3 < Cream 4.

The magnitude of thixotropy was related to the wax. The alkenones did not provide any thixotropic property to Cream 1; however, when combined with another wax, thixotropy could be built into the product if desired.

In the case of all four creams, G’ > G”, indicating that the formulations were more elastic than viscous, i.e., the creams were gel-like. Overall, both viscosity and G’ increased in the same order: Cream 1 < Cream 2 < Cream 3 < Cream 4 (Table 6). Cream

4 had the highest G’. Creams exhibiting G’ greater than G’’ may be more stable than formulations with G’’ values higher than G’ since they can recover their structure faster

57 and more efficiently and are less susceptible to gravitational forces that can lead to phase separation of emulsions (Alam & Aramaki, 2009).

Lastly, the formation of the liquid crystal structure is the result of an ordered arrangement of the emulsiﬁer, oil and wax molecules at the oil-water interface. The stability of cosmetic emulsions can be increased by forming liquid crystals in the continuous phase. The alkenones did not produce any liquid crystal structure at the water- oil interface. Fatty acids, such ascetyl alcohol and stearic acid, are known to form liquid crystalline structures (Mcgee, Sgaramella, & Vedantam, 2008). The other waxes migrated to the water-oil interface while the alkenones did not transition to water phase.

Toxicity

Compounds tested between 2000 mg/kg < LD50 ≤ 5000 mg/kg are defined as a class 5 toxicity category, based on the criteria of the Globally Harmonized Hazard

Classification and Labelling Scheme (Nations, U., 2011). This facilitates the identification of substances that produce toxicity at low doses (OECD, 2002a). Although optional, we included this category as the alkenones, if used to formulate personal care products or topical drugs, will directly contact human skin. This is the first pilot toxicity study with alkenones, as there have been no previous reports on the evaluation of alkenones toxicity in animals. In this study, our results with the alkenones derived from commercial Isochrysis algae indicated that they are in the class 5 toxicity category following oral administration to SD female rats. The findings of our study indicate that alkenones have no acute toxicity under the present experimental conditions, and its lowest lethal dose is above 4000 mg/kg in female SD rats. There were no significant

58 changes in food consumption for the 4000 mg/kg alkenones group compared to the vehicle control group. During the acute oral observation study period, no deaths were recorded in either the control or the 4000 mg/kg group that were not due to accidental oral gavage injury or handling. The behavioral observational data indicated that only grooming and partial eye closure occurred in the first 10 minutes after treatment in both groups, which was considered to be a change attributable to the handling of oral gavage.

No other significant alterations in behavioral or physical parameters were noted during the 14-day study.

The oral administration of 4000 mg/kg alkenones suspensions did not induce significant abnormalities based on gross necropsy after the 14-day observation. This acute oral administration also did not significantly alter the weight of the organs compared the vehicle control group. The histology data showed no significant changes in the alkenone-treated group compared to the vehicle control group. Thus, even at a high dose, alkenones did not induce any organ-specific damage.

The blood analysis results indicated a slight, non-significant decrease in monocytes in the animals treated with 4000 mg/kg alkenones. However, there was no significant difference between the alkenone- and vehicle-treated animals in the number of

WBC, RBC, PLT or HGB levels or the 14 chemical parameters tested. However, the number of monocytes were slightly decreased in the animals treated with 4000 mg/kg of the alkenones compared to the vehicle control group. Isochrysis galbana has been reported to have no significant effect on diabetic rats (Nuño et al., 2013). The number of red or white blood cells showed no significant effects on a different set of rat fed with

Isochrysis galbana (Herrero, Abalde, & Fabregas, 1993). Polysaccharides from the

59 Iscochrysis sp. can produce immunomodulatory effects by increasing the levels of IL-1β in macrophages (Yu et al., 2010). Although the body temperature was not recorded to determine if a fever was occurring after the rats had been exposed to the alkenones, it is well established that IL-1β plays a role in pain and inflammation (Ren & Torres, 2009).

Our behavior recordings indicated slight increase in pain levels only within the two hours after rats were treated with vehicle or alkenone treatment, but returned back to baseline the for the remaining 14-day observation. The alkenones were specifically isolated from

Isochrysis galbana and thus, no polysaccharides should have been present in the oral formula. Overall, our results suggest that the acute oral administration of alkenones (4000 mg/kg) did not significantly alter the blood chemistry parameters measured in this study.

Dermal acute and chronic dermal exposure was performed to determine if the alkenones directly affect the skin. When determining the fixed doses, we followed OECD

Test No. 434 guidelines with modifications for the use of five animals at 1500 mg/kg in

Study 1, the highest dose used. A second dose of 750 mg/kg was also included in Study 2 animal group in case there were effects with a different amount of alkenones and the vehicle used to determine the effects of the alkenones on a larger skin surface area. The

1500 mg/kg and the 750 mg/kg doses of the alkenones did not produce erythema, eschar or edema formation during the 24 hours of contact at the skin sites. Thus, Study 2 rats tolerated the alkenones on a larger surface area of the skin and the different amounts of alkenones with the vehicle showed no significant change in effects at a single treatment.

For the repeated treatments, the exposure of Study 2 rats to 1000 mg/kg, 200 mg/kg and

50 mg/kg alkenones also did not produce erythema, eschar, or edema during the 6 hours of contact per day on the skin sites early in the trial. Only on day 25 was there a sharp

60 increase in the irritation scoring, as indicated by the reddening of the skin at the 1000 mg/kg dose of the alkenones, but this score was lower than that of the positive control, whose values were higher than all the other treatments used. The positive control, 1%

SLS, did produce erythema and eschar formation on the skin in some, but not all rats. The

Tewameter device was used as a secondary confirmation of skin damage due to water loss.

TEWL can be produced by environmental stressors, such as ultraviolet radiation, which can damage the skin by inducing the formation of reactive oxygen species (Heck,

Joseph, & Kim, 2014). Skin aging can increase vulnerability to dryness (Kottner,

Lichterfeld, & Blume-Peytavi, 2013). In the acute dermal study, there was no significant difference in the TEWL values between the alkenone- and vehicle-treated animals. The

Tewameter data indicated that only the positive control, 1% SLS, significantly increased water evaporation before and after 24 hours of treatment (Fig. 8). The Draize skin reaction numerical scale showed no redness at the other treatment sites compared to the positive control. It was noted that scabs from the positive skin site application lasted only four days post-treatment in some SD rats. Alkenones applied to the skin both acutely and repeatedly for 28 days did not produce significant skin damage compared to the positive control, based on TEWL readings taken both before and after the 6-hour daily applications. In either Study 1 or 2 animals, the application of the 1000 mg/kg alkenone formulation for 6 hours per day for 28 days did not produce erythema, edema or eschar formation during the first two weeks of exposure. The positive control produced greater water evaporation after 6 hours compared to the highest amount of alkenones and vehicle treated areas combined. During the third and fourth week, erythema was present in two

61 rats of Study 2 after 1000 mg/kg of the alkenone formulation was applied on the skin.

However, erythema was seen after the application of both the negative control and vehicle, possibly as a result of the bandage being too tight on a specific day, although this remains to be determined.

There were limitations in this study. The alkenones could not be evaluated in vitro due their poor solubility. The alkenones would either precipitate out of DMSO,

Dulbecco’s modification of Eagle’s medium, Tween 20, Tween 80, and sesame seed oil, or the solution became cloudy and opaque after sonication for 15 seconds. Consequently, we could only conduct in vivo studies with the alkenones using a suspension formulation.

The solvent dichloromethane completely dissolves the alkenones, but dichloromethane is extremely toxic in various cell lines and animals (Schlosser, Bale, Gibbons, Wilkins, &

Cooper, 2014). Another limitation was the delivery of the alkenones to the rats through oral gavage. The use of water was not feasible as the alkenones are insoluble in water. A suspension of alkenones greater than 200 w/v % was too viscous to pass through the stainless-steel gavage needle. Therefore, a suspension of 100 w/v % was given to the rats four times within a 24-hour period. While corn oil was an option, when mixed with the alkenones, it took a longer time to pass through the needle with more force than should be required to deliver the solution into the stomach. We subsequently used a suspension solution, as previously suggested (Turner et al., 2011), to decrease the stress to the animals.

Glycerin is a common ingredient used in cosmetic products (Draelos, 2010). In addition, it is important to note that glycerin is a humectant that attracts and keeps moisture at the site of application (Stout & McKessor, 2012). Although it is safe for skin

62 use, it may have masked some of irritating properties of the alkenones. However, by applying a high concentration of the alkenones (more than what may be present in a typical skin care product), a large amount was left on the skin site after 6 and 24 hours.

Nonetheless, our toxicity study provided new information regarding skin tolerance to alkenones for extended periods of time.

As mentioned previously, Isochrysis has been grown as animal feed for both farm

(Lemahieu et al., 2013) and marine animals (Chen, Tseng, & Huang, 2015). Other nutrient ingredients sought after from Isochrysis are eicosapentaenoic acid, an omega-3 fatty acid, (Fidalgo et al., 1998) and docosahexaenoic acid, another omega-3 fatty acid that is the primary structural component of the neurons in the human brain and retina (J.

Liu, Sommerfeld, & Hu, 2013). Perhaps using alkenones derived from the commercially available Isochrysis sp. algae in topically applications may represent another promising source for pharmaceutical or personal care product companies.

Chapter Five

Conclusion

Highlights

Alkenones derived from Isochyrsis sp. were evaluated for certain physical characteristics. Alkenones were compared to commonly used waxes and tested for potential personal care product topical applications. Our results indicated that the alkenones could be viable ingredients for lip products. First, the melting point of the alkenones was similar to that of commonly used waxes for lip balms and lipsticks.

Second, the lipsticks and lip balms prepared with the alkenones remained stable at room temperature for ten weeks and at an elevated temperature, they showed similar signs of instability as the lipsticks made without the alkenones. Lastly, the alkenones displayed non-thixotropic properties that could be further investigated to develop other paste-like products. In any case, the final finish of the product could be changed by selecting other emollients. Our goal was to evaluate whether a lipstick and lip balm could be successfully formulated with the alkenones. Product optimization was not pursued in this study. Future studies could evaluate the use of the alkenones in lip balm application area more objectively. Performing stick hardness and breaking strength studies could also confirm the enormous potential of the alkenones for this area with quantitative results.

64 Alkenones in cream formulas had an acceptable performance in emulsions. They did not thicken the emulsions to the same extent as the comparator waxes (i.e., cetyl alcohol, stearic acid, and glyceryl monostearate). In terms of viscoelastic characteristics,

Cream 1 made with the alkenones had similar rheology as Creams 2-4 (made without the alkenones). An essential characteristic of a successful formulation is product stability. All creams were stable at room temperature for ten weeks, while at an elevated temperature,

Cream 1 was more stable than Creams 3–4. Waxes are commonly used in sunscreens to thicken products. The alkenones formed a stable mix with the liquid organic sunscreen and thickened the sunscreen quite well. Although not part of this study, these alkenones derived from Isochrysis sp. were studied in more detail for sunscreen applications

(Huynh A et al., 2019). The in vitro results (Huynh A et al., 2019) suggested the alkenones were an excellent, plant derived compound that signiﬁcantly boosted the SPF of the three “reef-safe” UV ﬁlters (homosalate, octocrylene, and octyl salicylate) compared to the control formulas.

Finally, the toxicity studies indicated that the alkenones can be classified as level

5 LD50 toxicant according to GHS, ranging between 2000 to 5000mg/kg. This indicated the alkenones were practically non-toxic as they appeared to be safe after acute oral and dermal administration and after 28 days of dermal application exposure. Although the

Federal Food, Drug and Cosmetic Act (FFDCA) do not require personal care product ingredients to be approved by the United States Food and Drug Administration (FDA) before they are marketed, the ingredients must be safe for consumers under the labeled conditions of use. A follow-up study with clinical trials using alkenones should be assessed with healthy volunteers to confirm two things. First that no other toxic effects

65 are seen that differ with the animal studies. Second to confirm the safety of using alkenones in human application products. Once the correct formulation for product optimization has been determined, alkenones should prove to be a useful ingredient in products from personal care companies that are continually searching for natural, green, sustainable ingredients. In summary, our results suggest that the alkenones can be a non- toxic, great green choice, a renewably-sourced compound that may be used as a structuring agent for lip products and creams when combined with other waxes.

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Appendix A

A List of Manuscripts in Preparation and Conference Presentations

based on this Dissertation

1. McIntosh K, Smith A, Young L, Leitch M, Tiwari AK, Reddy C, O’Neil G,

Liberatore M, Changler M and Baki G. Alkenones as a promising green

alternative for waxes in cosmetics and personal care products. Cosmetics, 2018, 5,

34; doi:10.3390/cosmetics5020034

Submitted:

2. McIntosh K, Sarver J, Mell K, Terreo DJ, Ashby CR Jr., Reddy C, O’Neil G and

Tiwari AK. Oral and dermal toxicity of alkenones in Sprague Dawley rats.

(Revisions submitted to International Journal of Toxicology)

Conference presentations:

1. Kyle. McIntosh. Safety toxicity evaluation with alkenones from Isochrysis

species in female SAS Sprague Rats. Student College of Clinical Pharmacy

(SCCP) April 14, 2019; (Won Best Panel presentation award by SCCP)