A Thesis

entitled

Evaluating UVB and UVA Boosting Technologies for Chemical and Physical Sunscreens

by

An Ngoc Hiep Huynh

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

Master of Science Degree in Pharmaceutical Sciences

Industrial Pharmacy

______Gabriella Baki, Ph.D., Committee Chair

______Jerry Nesamony, Ph.D., Committee Member

______Matthew W. Liberatore, Ph.D., Committee Member

______Dr. Amanda C. Bryant-Friedrich, Dean College of Graduate Studies

The University of Toledo

May 2020 Copyright 2020 An Ngoc Hiep Huynh

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

Evaluating UVB and UVA Boosting Technologies for Chemical and Physical Sunscreens

by

An Ngoc Hiep Huynh

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Master of Science Degree in Pharmaceutical Sciences Industrial Pharmacy

The University of Toledo May 2020

There are currently 14 organic and 2 inorganic UV filters approved in the United

States. Due to coral reef safety concerns, octinoxate and oxybenzone have been banned in

Hawaii, Key West, FL and the US Virgin Islands; and octocrylene is also being studied for its potential impact on coral reef safety, leaving 11 organic UV filters as viable options for sunscreen manufacturers – with limitations on their combination. Since consumers are always looking for sunscreens with high SPF and broad-spectrum protection, the need for

UVB and UVA protection boosting technologies is greater than ever. In a preliminary study, about two dozen emollients were scanned for their SPF boosting capability with selected organic UV filters. In this study, our goal was to evaluate whether and to what extent the in vitro SPF and broad-spectrum protection of three selected organic UV filters

(homosalate, octisalate and avobenzone) and the two inorganic UV filters (zinc oxide and titanium dioxide) can be boosted with three selected emollients (diethylhexyl 2,6- naphthalate, C12-15 alkyl benzoate, and butyloctyl salicylate), and an SPF boosting ingredient (SunSpheresTM). Organic, inorganic and mixed organic/inorganic sunscreens were formulated and tested for stability, viscosity, spreadability (TA.XTPlus texture analyzer), and droplet size; as well as in vitro SPF and broad-spectrum protection

iii (LabSphere 2000S), and water resistance. The results show that C12-15 alkyl benzoate provided the highest in vitro SPF out of three emollients tested for the organic sunscreens.

However, this ingredient did not form a stable emulsion with our inorganic ingredients, therefore, butyloctyl salicylate was selected to be used for this research project. Titanium dioxide had a higher in vitro SPF value; however, zinc oxide provided broader spectrum protection. Therefore, zinc oxide was selected to be combined with the organic UV filters.

We observed an in vitro SPF increase in the case of all sunscreens after exposing the sunscreens to a 20-minute water bath. We believe that the film-former, i.e., polyamide-8 and the drying time contributed to higher SPF values after the water bath. All sunscreens had a shear-thinning behavior, which is typical for creams and lotions. Overall, the organic/inorganic UV filter-based sunscreen containing the SunSpheresTM (CS) performed the best in terms of in vitro SPF, water-resistance, and spreadability.

iv Dedicated to my parents, Lanh Huynh and That Nguyen, my lovely sister, Khanh Huynh, and my partner in everything - Vinh Dang. I would not be here without all of your support.

v Acknowledgements

I would like to acknowledge the faculty at the University of Toledo for sharing knowledge, providing feedback and motivation throughout my journey as an undergraduate and graduate student.

I would like to sincerely thank Dr. Gabriella Baki, my professor and research advisor for providing me with all the knowledge and support that I needed.

I also would like to thank Dr. Jerry Nesamony, Dr. Matthew Liberatore, Dr. Black

Curtis, and Mark Chandler for sharing their knowledge and expertise through my academic journey. I also would like to thank the College of Graduate Studies for their continued financial support throughout my graduate studies.

vi Table of Contents

Abstract ...... iii

Acknowledgements ...... vi

Table of Contents ...... vii

List of Tables ...... x

List of Figures ...... xi

List of Abbreviations ...... xii

List of Symbols ...... xiii

1 Introduction ...... 1

1.1 Skin as a Barrier ...... 1

1.1.1 Skin as a Barrier Against Ultraviolet Rays ...... 4

1.2 Relationship Between Skin Cancer and Sunlight ...... 6

1.2.1 Types of Ultraviolet Radiation ...... 6

1.2.2 How Sunlight Causes Cancer ...... 2

1.3 Sunscreens ...... 3

1.3.1 Importance of Sunscreen Use ...... 3

1.3.1.1 Mechanism of Action of Sunscreens and FDA Regulation of UV

Filters in the US ...... 5

1.3.2 Sunscreen Protection Claims ...... 9

1.3.3 SPF Boosting Technologies ...... 9

vii 1.3.4 UV Filter Combinations in the US...... 11

1.3.5 Sunscreen Dosage Forms ...... 12

1.4 Current Concerns Associated with UV Filters ...... 12

1.4.1 Organic UV Filters ...... 12

1.4.2 Inorganic UV Filters ...... 14

2 Aims of the Research ...... 16

3 Materials and Methods ...... 17

3.1 Materials ...... 17

3.1.1 Active Ingredients ...... 17

3.1.2 Inactive Ingredients ...... 18

3.2 Methods ...... 21

3.2.1 Formulation of Sunscreens ...... 21

3.2.1.1 Formulation Procedure of Sunscreens without SunSpheresTM ...... 25

3.2.1.2 Formulation Procedure of Sunscreens with SunSpheresTM ...... 25

3.2.2 Stability Testing ...... 26

3.2.3 In Vitro SPF Testing and Broad-Spectrum Testing ...... 26

3.2.4 Water Resistance Testing ...... 27

3.2.5 Viscosity Testing ...... 27

3.2.6 Spreadability Testing ...... 28

3.2.7 Droplet Size Analysis ...... 28

4 Results and Discussion ...... 29

4.1 Stability ...... 29

4.2 In Vitro SPF Testing ...... 30

viii 4.3 Water Resistance Testing ...... 33

4.4 Broad Spectrum Testing ...... 34

4.5 Absorbance and Transmittance ...... 36

4.6 Viscosity ...... 39

4.7 Spreadability ...... 42

4.8 Droplet Size ...... 44

5 Conclusion ...... 46

6 Future Studies ...... 48

7 References ...... 49

ix List of Tables

Table 1.1 Electromagnetic spectrum and corresponding wavelengths (8) ...... 1

Table 1.2 Approved UV filters in the US, type of protection provided, and maximum allowed concentration...... 7

Table 3.1 Ingredients used in this study and their functions ...... 20

Table 3.2A Sunscreens and their ingredients formulated in Step 1 of this study...... 23

Table 3.2B Sunscreens and their ingredients formulated in Steps 2 and 3 of this study .. 24

Table 4.1 Stability results ...... 30

Table 4.2 In vitro SPF of sunscreens at 25°C, 40°C, and 45°C on Day 1 and Month 3 ... 33

Table 4.3 Average critical wavelength of sunscreens on Day 1 before and after water bath...... 36

Table 4.4 Droplet size of sunscreens within in one week after formulation ...... 45

x List of Figures

Figure 1-1 A. Structure of the skin, B. Structure of the epidermis, and its main cells...... 2

Figure 1-2 A. Layers of the epidermis, B. Nomenclature of epidermis layers (1) ...... 3

Figure 1-3 Micrograph of the epidermis and upper layer of the dermis, indicating the layers and major cell types (1) ...... 5

Figure 3-1 Formulation steps and decision-making process ...... 22

Figure 4-1 In vitro SPF of sunscreens at 25°C on Day 1 ...... 32

Figure 4-2 In vitro SPF and in vitro SPF after 20 minutes of water bath of sunscreens on

Day 1 ...... 34

Figure 4-3 Absorbance and transmittance graphs of the sunscreens on Day 1 ...... 38

Figure 4-4 Viscosity curve of Newtonian, shear-thinning, shear-thickening, and plastic systems (52)...... 39

Figure 4-5 Viscosity of sunscreens on Day 1 ...... 40

Figure 4-6 Viscosity of sunscreens on Day 1, Month 1, Month 2 and Month 3 at 25 s-1 . 41

Figure 4-7 Typical spreadability test graph...... 43

Figure 4-8 Firmness and stickiness of sunscreens ...... 44

xi List of Abbreviations

6-4PPs pyrimidine ...... 6-4 Pyrimidone Photoproducts

CPDs ...... Cyclobutane Pyrimidine Dimers

DNA ...... Deoxyribonucleic Acid

FDA ...... Food and Drug Administration

INCI ...... International Nomenclature of Cosmetic Ingredients

MED ...... Minimal Erythema Dose

O/W ...... Oil-in-Water

PABA ...... Para Aminobenzoic Acid

SC ...... Stratum Corneum SPF ...... Sun Protection Factor

US ...... United States USAN ...... United States Adopted Name UV ...... Ultraviolet UVA ...... Ultraviolet A UVB ...... Ultraviolet B UVC ...... Ultraviolet C

W/O ...... Water-in-Oil w/w ...... Weight by weight or weight percent

xii List of Symbols

% ...... Percent °C ...... Degree Celsius ® ...... Registered TM...... Trademark

! ...... Viscosity µm ...... Micrometer g...... Gram km ...... Kilometer mL ...... Milliliter mm ...... Millimeter n...... Number of replicates ng...... Nanogram nm ...... Nanometer Pa...... Pascal rpm ...... Rotations per minute s ...... Seconds x...... Times (magnification)

xiii Chapter 1

1 Introduction

1.1 Skin as a Barrier

The skin has been recognized as the largest organ of the human body, where it accounts

for approximately sixteen percent of the human body weight. Wickett et al. suggested that

the major roles of the skin are to protect the body from various environmental factors and

to prevent water loss, among other components of the body. In addition, the skin has

substantial immune and sensory functions. Additionally, the skin is responsible for the

synthesis of Vitamin D as well as thermoregulation (1). The skin has three major layers,

which include the epidermis, dermis, and the subcutis layer as it is shown in

Figure 1.1A(2).

1 Figure 1-1 A. Structure of the skin, B. Structure of the epidermis, and its main cells.

Copyright © Kellie Holoski, Head & Neck Cancer Guide. All rights reserved. Permission granted for educational purposes. Available at https://headandneckcancerguide.org

The epidermis is the outermost layer of the human skin. It is made up of four types of major cells, including the keratinocytes, melanocytes, Langerhans cells, and Merkel cells, as it is shown in Figure 1.1B. Keratinocytes, which originate from a single layer within the basal layer of the epidermis (Figure 1.2A), are the principal cells in the human skin, contributing approximately 95% of the total cells (3). Keratinocytes multiply and expose the daughter cells to terminal differentiation. This, in turn, leads to the formation of the stratum corneum (SC). Melanocytes are also located in the basal layer and are made up of dendritic cells that are derived from the neural crest (3). These cells are responsible for melanin synthesis, which are then stored in subcellular organelles and later transported to the keratinocytes. In addition, the subcellular organelles are responsible for the formation of the melanin cap that protects the skin from the harmful ultraviolet (UV) radiation (3). The number and size of the subcellular organelles are responsible for determining the skin color (4). The Langerhans cells, which are primarily the antigen-

2 presenting dendritic cells in the epidermis, are derived from the bone marrow and are located throughout the epidermis (3). Merkel cells are involved in the transmission of sensory information from the skin to the sensory nerves.

Figure 1-2 A. Layers of the epidermis, B. Nomenclature of epidermis layers (1). Copyright license number: 4823360982532

The dermis is the second layer of the skin located right below the epidermis. The dermis is approximately 0.5 to 5 millimeters thick, depending on the human body site. For instance, the dermis in the eyelid is thinner than on the back. It is mainly made up of two major layers, which include the papillary and reticular dermis. The papillary dermis is primarily supplied by various sensory nerve endings and blood vessels. On the other hand, the reticular dermis is usually the major layer of the dermis and is usually located above the subcutis layer. The dermis also contains histiocytes, which are antigen-presenting cells

(3). These antigen-presenting cells provide a surface for the phagocytosis and help in the degradation of foreign components that transport antigens to the T cells. In addition, the dermis also contains mast cells located close to the dermal blood vessels. These mast cells are mainly responsible for the secretion of chemotransmitters, such as histamines, which are used during allergic reactions. Additionally, the dermis contains eccrine and apocrine

3 sweat glands, which are responsible for regulating the body temperature (3) and sebaceous glands, which are responsible for the production and secretion of sebum (5).

Subcutis is the innermost layer of the human skin and is made up of lipocytes, which are organized into fat lobules that are disjointed from one another by fibrous septae.

The subcutis layer provides a base for body fats. In an average person, approximately 80 percent of body fat is located in the subcutis (3). It has been shown that fats located in the subcutis are associated with endocrine functions (6). Fats generate hormones, such as leptin, which significantly contribute to the control of metabolic energy as well as the regulation of appetite.

1.1.1 Skin as a Barrier Against Ultraviolet Rays

The epidermis consists of two major cells that act as barriers for UV rays (1). These include the melanocytes and Langerhans cells. Melanocytes are mainly found in the basal layer of the epidermis (Figure 1.3) and produce pigments granules known as melanosomes containing melanin, which are responsible for skin color (1). These melanosomes are then transported from the melanocytes to the keratinocytes, which in turn, protect the cell nucleus from UV light and determines the color of the skin. Furthermore, Wickett et al. outlined the process by which melanin is synthesized constantly. This occurs to enhance the renewal of the epidermis, also known as cornification. However, this process can be sped up in response to UV exposure to produce tanning. Langerhans cells in the epidermis are responsible for allergic reactions of the skin. In essence, these cells are dendritic immune cells and, thus, act as an immune barrier of the epidermis and also provide a substantial contribution to contact allergy.

4 Figure 1-3 Micrograph of the epidermis and upper layer of the dermis, indicating the

layers and major cell types (1). Copyright license number: 4823360982532

Biniek, Levi, and Dauskardt investigated how UV radiation reduces the barrier function of the skin (7). They concluded that the SC, the outermost layer of the epidermis, acts as a barrier against UV radiation. Although UV radiation is associated with some benefits, such as the production of vitamin D, these rays can have severe negative consequences, from wrinkle formation to the development of skin cancer (7). The SC has been found to provide essential mechanical protection as well as a controlled permeable barrier to the external environment. However, despite the SC acting as an efficient barrier against UV rays, increased exposure can affect its function. This consequently can lead to severe damage in the skin, which can be manifested by cracking and chapping of the skin.

In addition, excessive UV exposure can lead to harmful skin responses such as scarring, abnormal desquamation, inflammation, and infection, among others.

5 1.2 Relationship Between Skin Cancer and Sunlight

1.2.1 Types of Ultraviolet Radiation

The sun emits electromagnetic radiation, which consists of a wide range of wavelengths:

50% of visible light, 40% of infrared (IR) radiation, and approximately 10% of ultraviolet

(UV) radiation. There are three types of UV rays: UVA, UVB, and UVC. UVC has the shortest wavelength and is blocked by the ozone layer. UVB has ranged from 290 to

320 nm. With a longer wavelength, UVB can go through the ozone layer and reach the

Earth’s surface. UVA has the longest wavelength range between 320 and 400 nm. With this range of wavelengths, UVA can also reach the Earth’s surface.

6 Table 1.1 Electromagnetic spectrum and corresponding wavelengths (8)

Light spectrum Wavelength Gamma ray Less than 0.01 nm X-ray 0.01–10 nm Ultraviolet 10-400 nm UVC 200-290 nm UVB 290-320 nm UVA 3240-400 nm Visible 400-700 nm Violet 400-450 nm Blue 450-495 nm Green 495-570 nm Yellow 570-590 nm Orange 590-620 nm Red 620-700 nm Infrared-A 700-1400 nm Infrared-B 1400-3000 nm Infrared-C 3000 nm – 1 mm Microwave 1 mm – 1 m Radio 1 mm- 100 km

It is known that the entrance of the UV rays into the skin have significant adverse impacts, which leads to both acute and chronic conditions (9). One of the major acute conditions resulting from harmful UV radiation is the erythema. Examples for chronic conditions due to UV radiation are photoaging, which refers to the changes in the physical appearance of the skin over time based on UV exposure, as well as skin cancer, including cutaneous malignant melanoma, squamous cell carcinoma, and basal cell carcinoma (10).

1 1.2.2 How Sunlight Causes Cancer

The most common cancer in the US is skin cancer. It is estimated that approximately 9,500 people in the US are diagnosed with skin cancer every day, more than 3 million Americans a year, and one in five Americans will develop skin cancer in their lifetime (11).

The process by which UV rays lead to skin cancer involves the induction of damage to the DNA. UV rays can damage DNA through inducing pyrimidine photoproducts, purine photoproducts, and inducing DNA double-strand breaks. UV-induced pyrimidine photoproducts are divided into three major classes of lesions: cyclobutane pyrimidine dimers (CPDs), pyrimidine 6-4 pyrimidone photoproducts (6-4PPs) and their Dewar isomers. UV-induced purine photoproducts involve at least one adenine residue that undergoes photocycloaddition reactions with contiguous adenine or thymine. UV-induced lesions such as CPDs, 6-4PPs, strand breaks, and oxidative products are the most common and persistent lesions leading to compromised functional, mutagenesis, and cell death (12).

During the process of repairing the resulting damage, mistakes are often experienced. This, in turn, results in the inclusion of inappropriate bases in the genetic material. During the process of repairing the DNA, some of the damaged DNA remain unrepaired, which consequently interrupt the cellular processes by blocking the machinery responsible for the synthesis of DNA and RNA. This automatically leads to the incorporation of the wrong bases into the DNA. The resulting errors in the process of repairing the DNA lead to mutation, which consequently results in loss or wrong expression of the affected genes. Various researchers suggested that alterations in the p53 tumor suppressor genes have a substantial impact on the development of skin cancer (13-

15). The protein contained in the p53 suppressor gene also contributes to programmed cell

2 death (also known as apoptosis) and thus, it has been revealed that the p53 suppressor act as a "guardian of the genome" by helping DNA repair and aiding in the removal of cells that contain various damaged DNA. The development of skin cancer occurs when unrepaired DNA damage in the p53 suppressor gene is converted to mutations leading to carcinogenesis. Continuous exposure to UV rays can lead to mutations in the p53 suppressor gene within keratinocytes that can resist apoptosis and other growth regulators.

Studies have also demonstrated that UVB-damaged keratinocytes are removed by the interaction of Fas/Fas-Ligands and that this process is dysregulated during the process of UV-induced skin carcinogenesis that occurs as a result of mutation of the p53 in DNA damaged keratinocytes (16). This demonstrates a relationship between Fas/Fas-ligand and the p53 pathway. In addition, it shows that the dysregulation of these pathways results in the pathogenesis of UV-induced human skin cancer. Velez and Howard argued that UV rays induce inimitable sorts of p53 mutations in skin cancer more frequently as compared to those present in other kinds of human cancer (15). Additionally, they suggested that the p53 mutations are found in the skin that has been exposed to the sun, and thus, the p53 mutation can act as an indicator of prior sun exposure in human skin. The findings from this study suggested that p53 mutations occur right before the development of skin cancer.

1.3 Sunscreens

1.3.1 Importance of Sunscreen Use

UVB radiation can penetrate through the epidermis, while UVA radiation can penetrate through both epidermis and dermis. UVB is a major cause of sunburn, skin cancer,

3 thickening of the SC, photoaging, tanning, and immunosuppressive effects. UVA leading to tanning, photoaging, and lead to skin cancer by damaging keratinocytes in the basal cell layer (10). In addition, Sanchez et al. suggested that excessive UV exposure can consequently lead to keratinocyte cancer (17). Sanchez et al. argued that due to the increased changes in consumers’ lifestyles, many people get exposed to excess sunlight and this has been associated with a higher number of cases of keratinocyte cancer (17, 18).

According to the American Academy of Dermatology, the majority of all skin cancer cases are attributable to exposure to natural and artificial UV light. There is an increased risk of squamous cell carcinoma, basal cell carcinoma, and melanoma with increasing intermittent sun exposure in childhood. A person’s chance of developing melanoma can almost double from even just one blistering sunburn during childhood or adolescence. The average annual cost for treatment of skin cancer from 2007 to 2011 was 8.1 billion dollars for an estimated

4.9 million U.S. adults which come to $1,653 per person per year while the annual cost of sunscreens ranging from $30 to $62 (19).

National and international health authorities have proposed protective measures, including the use of sunscreen. It is known that sunscreens can protect the human skin both against the longer wavelength UVA and shorter wavelength UVB rays (20), and therefore, sunscreens are essential in preventing skin damage (21). Regular sunscreen use has been shown to reduce melanoma risk (22). Sunscreens are being developed with active substances (i.e., UV filters) aimed at protecting the skin against some UV radiation (18).

4 1.3.1.1 Mechanism of Action of Sunscreens and FDA Regulation of UV Filters in the US

Sunscreens are considered drugs in the US because they provide protection against a disease, i.e., skin cancer, and therefore, they are regulated by the Food and Drug

Administration (23). UV filters are the active ingredients in sunscreens. There are two types of UV filters available, inorganic (also known as physical) and organic (also known as chemical) UV filters. The mechanism of action of these two types of UV filters is different.

Organic UV filters absorb UV energy and convert the absorbed energy into longer wavelength, lower energy such as infrared energy. Inorganic UV filters scatter, reflect and, absorb radiation. The approved UV filters in the US, their coverage UV region, and maximum allowed concentration are shown in Table 1.2 (24-26).

Sunscreen UV filters work in part by absorbing the energy of UV radiation and converting it to heat. Electrons within the UV filter move to a higher electronic energy state, or excited state, when they absorb the energy from UV photons, which is then quickly converted to heat by non-radiation energy dissipation or other forms of light such as fluorescence, phosphorescence, or infrared rays. Afterwards, the electron returns to a ground state and is ready to receive another UV photon. This whole process happens in a very short amount of time. As long as the chemical structure of the sunscreen is stable during the excited state, then energy can be continuously absorbed and converted without loss of efficacy. However, a few active sunscreen ingredients are not photostable, leading to chemical structural changes when it is in the excited state. When this happens, the original molecules are broken down and unable to reach the excited state again, thus losing the ability to absorb additional UV photons. Degradation of original active ingredient can

5 lead to the free radical formation, which can then react with nearby molecules and form photo-byproducts. The overall efficacy of the sunscreen decreases because there is now less active ingredient to absorb UV photons (27).

6 Table 1.2 Approved UV filters in the US, type of protection provided, and maximum

allowed concentration.

Type of Maximum Type protection allowed of UV USAN Name INCI name concentration filter UVA UVB (% w/w)

Aminobenzoic acid PABA x 15

Butyl Avobenzone methoxydibenzoyl- x 3 methane

Ethoxyethyl Cinoxate x 3 methoxycinnamate

Dioxybenzone Benzophenone-8 x x 3

Homosalate Homomenthyl x 15 salicylate

Meradimate Menthyl x 5 anthranilate

Octocrylene Octocrylene x 10 Organic Octinoxate Octyl x 7.5 methoxycinnamate

Octisalate Ethylhexyl x 5 salicylate

Oxybenzone Benzophenone-3 x x 6

Padimate O Ethylhexyl x 8 dimethyl PABA

Ensulizole Phenylbenzimidaz x 4 ole sulfonic acid

Sulisobenzone Benzophenone-4 x x 10

Trolamine Triethanolamine x 12 salicylate salicylate

Inorganic Titanium dioxide Titanium dioxide x x 25

7 Zinc oxide Zinc oxide x x 25

There are advantages and disadvantages of organic and inorganic filters (28). Some disadvantages of organic filters are that the individual UV filters often have a narrow spectrum of protection, some are photolabile, for example, avobenzone, oftentimes several filters need to be combined to achieve a high SPF, and the oil-soluble filters may feel occlusive and greasy. Additionally, there have been concerns over the safety, irritancy, and environmental impacts of many of the organic UV filters. The advantages of organic filters are that they give a good aesthetic feel, have good efficacy at low concentrations, and are well understood by formulators. The advantages of inorganic filters are that they provide broad-spectrum protection, they are photostable, generally safe/non-irritant, and dispersions are easy to incorporate into product bases. Disadvantages of inorganic UV filters are that they have a perceived poor aesthetic feel due to their white cast, and in their powder form, they can be difficult to formulate with. The white cast can be modified when decreasing the particle size of the UV filters, however, this may also lead to reduced its efficacy.

None of the UV filters available today can provide a high SPF and broad-spectrum protection when used alone, without aesthetic drawbacks. When various organic and inorganic UV filters are combined however, they can have a synergistic effect and help eliminate the poor aesthetics, and create a photostable sunscreen with a high SPF and broad-spectrum protection.

8 1.3.2 Sunscreen Protection Claims

There are two types of UV protection claims that can be made on sunscreens. One is the sun protection factor (SPF) and another one is broad-spectrum protection. SPF indicates how long it takes for UV rays to redden protected skin compared to unprotected skin. To determine the SPF of a sunscreen, Equation 1 is used (29).

Equation 1 where MED refers to minimal erythema dose, MED sunscreen refers to minimal erythema dose of protected skin, MED without sunscreen refers to minimal erythema dose of unprotected skin. The SPF value indicates protection against sunburn, which is caused by both UVB and UVA radiation. UVB rays are more efficient is causing erythema, however,

UVA rays also contribute to this.

The claim broad-spectrum protection can be used on products that provide protection against both UVA and UVB. In the US, “broad-spectrum” can be claimed if the in vitro determined critical wavelength value is ≥370 nm. The critical wavelength is the wavelength below which 90% of the area under the absorbance curve resides (30).

1.3.3 SPF Boosting Technologies

A booster is an ingredient that can help increase the power or effectiveness of UV filters without it having UV absorbing capability (31). Many commercial products utilize boosting technologies today due to the consumer demand to have a lower amount of UV filters in sunscreens, but still, achieve a high SPF and broad-spectrum protection.

9 One approach to provide an SPF boost to UV filters is to increase optical path length by using scattering particles. An example of such particles is SunSpheresTM, which are a styrene/acrylate copolymer manufactured through emulsion polymerization. They can be described as hollow spheres. SunSpheresTM have a different refractive index in their outer shell as compared to their hollow inner core. UV radiation gets bent and scattered as it passes through the two different refractive indices of the SunSpheresTM particles, thus changing its direction. When SunSpheresTM are incorporated into organic or inorganic sunscreens, the effect of bending and scattering radiation helps to keep the UV radiation away from the skin by redirecting it to UV filters, which will both absorb and scatter the

UV radiation away from the skin. Philbin discussed that these boosters work equally well with both inorganic and organic UV filters (32). Additionally, SunSpheresTM are compatible with commonly used sunscreen ingredients, such as emollients. SunSpheresTM have the capacity to boost the efficiency of both UVA and UVB filters. This, in turn, helps formulators to use significantly less UV filter and still produce the same SPF. Given that many organic UV filters are greasy substances, using SunSpheresTM may also allow formulators to create more aesthetically pleasing products

Another approach to boosting SPF is to use emollients that can efficiently dissolve the crystalline UV filters and reduce the free radical generation in the skin from exposure to UV radiation (33). The most widely used crystalline UV filters in the US were the

PABA, benzophenones, oxybenzone, and avobenzone. Therefore, the emollients were added to the formulations at a comparatively higher percentage to enhance the dissolution of the solid UV filters.

10 In this thesis work, we utilized both approaches, separately and combined with each other to investigate their efficiency and potential synergistic effects.

1.3.4 UV Filter Combinations in the US

As it was shown in Table 1.2, there are fourteen organic UV filters approved by the FDA in the US. The currently allowed maximum amount of each UV filter was also shown in the same table. Two of the approved UV filters, i.e., oxybenzone and octinoxate, are now banned in Hawaii, Florida, California, and the US Virgin Islands due to their harmful impact on coral reefs and sea life (34) (35). In the US, the most widely used organic UV filters are/were avobenzone, homosalate, octisalate, octinoxate, and octocrylene. Recently, octocrylene is being studied for its impact on coral reef safety as well (36). This leaves formulators with eleven viable organic UV filter choices. Formulators often prefer using liquid organic UV filters due to their ease of incorporation into formulations. Therefore, organic sunscreens on the market usually contain a combination of several organic liquid

UV filters combined with avobenzone. For this reason, in this study, we selected the two popular and coral-safe liquid organic UV filters, i.e., homosalate, and octisalate combined with avobenzone.

The current FDA regulations do not allow avobenzone to be combined with inorganic UV filters. Therefore, to be in line with the FDA regulations, when we combined organic and inorganic UV filters, avobenzone was removed from the sunscreens.

11 1.3.5 Sunscreen Dosage Forms

Many dosage forms are available today on the market for sunscreens to cater to the varying needs of consumers. Examples for dosage forms available for sunscreens include lotion, cream, and stick. Emulsions are the most popular dosage form for sunscreen applications due to their many benefits. These include aesthetics, compatibility, the inclusion of both hydrophilic and hydrophobic actives is possible, lower cost, and formulation flexibility

(28). The dosage form emulsion includes both lotions and creams. The difference between lotions and creams is viscosity. Lotions tend to be less viscous than creams. There are two basic types of emulsions, i.e., water-in-oil (W/O) and oil-in-water(O/W) emulsions. O/W emulsion-based sunscreens have gained more popularity among consumers as daily skincare products and products for men due to their lighter and non-greasy skin feels, while

W/O emulsion-based sunscreens tend to be more favored as sun protection products for the entire body and baby sunscreens due to their improved water resistance. In this study, W/O emulsions were formulated as opposed to O/W because the inorganic filters were more stable and dispersed better in this type of emulsion.

1.4 Current Concerns Associated with UV Filters

1.4.1 Organic UV Filters

Despite their significance in protecting the skin against harmful UVB and UVA radiation,

UV filters are associated with various concerns. Although UV filters are designed for topical application, many organic UV filters can absorb into the blood after application and lead to systemic exposure (37). As a result, the photoprotection on the skin gets lost, and

12 the skin becomes vulnerable to UV rays. The fact that sunscreen actives are able to absorb into the blood causes concerns to many consumers. A recent study performed by Matta et al. assessed the systemic absorption of six organic UV filters, including avobenzone, oxybenzone, octocrylene, homosalate, octisalate, and octinoxate in lotion, aerosol spray, non-aerosol spray and pump spray dosage forms (38). It was found that all tested sunscreen active ingredients were absorbed systemically, remained in plasma for at least 3 days after the last application, and exceeded the FDA threshold of 0.5 ng/mL. The most common adverse event was rash, which developed in 14 participants. More studies will need to be done in the future on this topic, potentially including more active ingredients as well. The authors emphasized that their findings did not indicate that consumers should refrain from using sunscreens.

A challenge, from a formulation perspective related to organic UV filters, is the photosensitivity of some organic UV filters. A well-known example is avobenzone. UV light can degrade avobenzone, which prevents it from functioning normally, essentially making it inactive. Photostabilizers are ingredients that can substantially improve the photostability of photoinstable UV filters. A common approach to photostabilization is to quench the excited state of the UV filter and quickly return the UV filter to the ground state. An example of this mechanism is polyester-8, an ingredient we used in our formulations.

Another concern with the use of some organic UV filters is their potentially damaging effect on the tropical coral reefs (39). Certain cities and regions, including Key

West, banned the use of two organic UV filters – namely oxybenzone and octinoxate – as mentioned in section 1.3.5 of this paper. A lower amount of active ingredients used could

13 decrease the amount absorbed into the body and the amount leaking into the ocean, which could lead to decrease in the harmful effect of sea life. In order to decrease the amount of

UV filters in sunscreens but still have the same SPF, SPF boosting technologies can be used.

1.4.2 Inorganic UV Filters

The major characteristic of an inorganic UV filter is the capacity to absorb, reflect, and scatter UV rays over the entire UVA and UVB range. These properties are primarily determined by the refractive index, particle size, sunscreen film thickness, as well as the product base (40). Titanium dioxide and zinc oxide are available as pigmentary grade and attenuation grade materials. Pigmentary grade titanium dioxide and zinc oxide have a larger particle size (i.e., 200-500 nm), while the attenuation grade, also known as microfine or micronized powders, have a much smaller particle size, i.e., 10-100 nm. The pigmentary grade particles reflect and scatter UV radiation into the visible spectrum (>400 nm), leaving a white cast appearance on the skin. This is the main disadvantage of inorganic UV filters causing consumer dislike. The pigmentary grade inorganic UV filters are therefore used in makeup products. Particle size reduction eliminates this absorption in the visible spectrum and enables formulators to create invisible products. Decreasing the particle size too much to a too-small size can lead to less broad protection, which is undesired. The formulator, therefore, needs to choose the particle size of titanium dioxide and zinc oxide carefully so that the product has the benefits and aesthetics desired by the particular consumer for whom it is intended.

14 Inorganic UV filters have the capacity to produce and release reactive oxygen species and metal ions into the aquatic environment. This can consequently have negative effects on aquatic organisms (41). Additionally, Hazeem et al. pointed out that inorganic

UV filters can have various impacts on marine algae based on factors such as the size and form of the crystals as well as the particles’ morphology (42). Surface treatment for inorganic UV filters can reduce the production and release of reactive oxygen species and metal ions, and prevent the photocatalytic activity of these UV filters. Additionally, surface treatment can also help with dispersibility, and improve the skin feel. Considering everything regarding inorganic UV filters, they are still deemed very safe by the FDA.

15 Chapter 2

2 Aims of the Research

The aims of this study were to develop a sunscreen formula that can be used for both organic and inorganic UV filters, and evaluate the SPF boosting effect of emollients for organic UV filters and SunSpheresTM for organic and inorganic UV filters. In this study, the maximum allowed amount of three organic filters, i.e., homosalate, octisalate, and avobenzone was used. The inorganic filters were used in a monographed, but less than maximum allowed amount.

An additional aim was to evaluate whether organic UV filters alone, or inorganic

UV filters alone, or their combination can lead to a more stable and/or better performing product. We also studied the effect of a film-former and evaluated the water-resistance properties of the sunscreens.

16 Chapter 3

3 Materials and Methods

3.1 Materials

3.1.1 Active Ingredients

Organic UV filters

Homosalate and avobenzone were purchased from Making Cosmetics (Snoqualmie, WA,

USA). Homosalate is a clear viscous liquid with a faint odor. This ingredient was of

cosmetic grade. Avobenzone is an off-white to yellowish, crystalline powder with a weak

odor. It was a cosmetic grade ingredient. Octisalate (Neo Heliopan OS) was received from

Symrise (Branchburg, NJ, USA). It is a clear, colorless to light yellowish liquid, and has a

slightly floral odor. It was of USP grade.

Inorganic UV filters

SolaveilTM CZ-300 (INCI: zinc oxide (and) caprylic/capric triglyceride (and)

polyhydroxystearic acid (and) isostearic acid, referred to as zinc oxide dispersion in this

thesis) and SolaveilTM CT-300 (INCI: titanium dioxide (and) caprylic/capric triglyceride

(and) polyhydroxystearic acid (and) aluminum stearate (and) alumina, referred to as

titanium dioxide dispersion in this thesis) were received from Croda (Newark, NJ, USA).

17 The zinc oxide dispersion contained 57% of zinc oxide (i.e., solid content), while the titanium dioxide dispersion contained 33% of titanium dioxide.

3.1.2 Inactive Ingredients

Glycerin (INCI: glycerin), triglyceride (INCI: Caprylic/capric triglyceride), magnesium aluminum silicate (INCI: magnesium aluminum silicate), EDTA (INCI: tetrasodium

EDTA), Paraben-DU (INCI: Propylparaben (and) methylparaben (and) diazolidinyl urea

(and) propylene glycol) were purchased from Making Cosmetics (Snoqualmie, WA, USA).

The following ingredients were received as gifts: Corapan® TQ (INCI: diethylhexyl 2,6- naphthalate, Symrise, Branchburg, NJ, USA); xanthan gum (INCI: xanthan gum, CP

Kelco, Atlanta, GA); Finsolv® TN (INCI: C12-15 alkyl benzoate, Innospec, Littleton, CO);

EsterlacTM Care+ (INCI: sodium isostearoyl lactylate, Corbion, Lenexa, KS); Hallbrite®

BHB (INCI: butyloctyl salicylate) and Polycrylene® (INCI: polyester-8, Hallstar, Chicago,

IL); SunSpheresTM (INCI: styrene/acrylic copolymer, Dow Inc., Midland, MI), ArlacelTM

1690 (INCI: sorbitan isostearate (and) polyglyceryl-3 polyricinoleate), and OleocraftTM LP

20 (INCI: polyamide-8, Croda, Newark, NJ). All ingredients were of cosmetic grade.

Deionized water was provided by the University of Toledo Health Science Campus.

Table 3.1 displays the ingredients that were used in the sunscreens in this study.

A goal of this study was to develop a simple sunscreen formula that would work with both organic and inorganic UV filters. A basic sunscreen formula typically includes the UV filter(s), emollients that can act as solvent, enhance the sensory properties and may photostabilize as well, water, humectant to retain moisture, emulsifier to form the emulsion and stabilize it, photostabilizer if a phostounstable UV filter is used, dispersing agent to

18 help powder ingredients disperse well in the formula, film-former to create an even sunscreen layer on the skin, thickener to build viscosity into the sunscreen, and preservative to protect from microbial growth (43). The starting formula was developed in this study, for the specific UV filter blend we used, based on compatibility of the ingredients.

19 Table 3.1 Ingredients used in this study and their functions

INCI name Function Water Solvent Glycerin Humectant Xanthan Gum Thickener EDTA Chelating agent Magnesium Aluminum Silicate Thickener Styrene/acrylic copolymer SPF booster Homosalate Organic UV filter Octisalate Organic UV filter Avobenzone Organic UV filter Zinc oxide (and) caprylic/capric triglyceride (and) Inorganic UV filter polyhydroxystearic acid (and) isostearic acid Titanium dioxide (and) caprylic/capric triglyceride (and) Inorganic UV filter polyhydroxystearic acid (and) aluminum stearate (and) alumina Emollient / SPF C12-15 alkyl benzoate booster Emollient / SPF Butyloctyl salicylate booster Emollient / SPF Diethylhexyl 2,6-naphthalate booster Polyester-8 Photostabilizer Caprylic / capric triglyceride Emollient Sorbitan isostearate and polyglyceryl-3 Emulsifier Sodium isostearoyl lactylate Dispersing agent Polyamide-8 Film-former Propylparaben, methylparaben, diazolidinyl urea, propylene Preservative glycol

20 3.2 Methods

3.2.1 Formulation of Sunscreens

Eleven sunscreens were formulated (Figure 3.1). In step 1, three sunscreens (O1, O2, O3) were formulated using a combination of three organic UV filters and a different emollient in each, namely C12-15 alkyl benzoate (O1), butyloctyl salicylate (O2) and diethylhexyl-

2,6-naphthalate (O3). The best organic sunscreen based on in vitro SPF and broad- spectrum protection was selected to be combined with SunSpheresTM (OS).

In step 2, three inorganic UV filter-based sunscreens were formulated. One contained the zinc oxide dispersion and the best emollient from step 1 (IO1). Another contained the titanium dioxide dispersion and the best emollient from step 1 (IO2). The third contained a combination of the zinc oxide and titanium dioxide dispersions and the best emollient from step 1 (IO3). The best inorganic UV filter-based sunscreen was selected to be combined with SunSpheresTM (IOS).

In step 3, two organic UV filters and an inorganic UV filter were combined using the best emollient from step 1 (C1 and C2). Then SunSpheresTM were added to the best combination product (CS).

21 Figure 3-1 Formulation steps and decision-making process

22 Table 3.2A Sunscreens and their ingredients formulated in Step 1 of this study.

O1 O2 O3 OS % (w/w) Phase A Water 55.3 55.3 55.3 50.3 Glycerin 3 3 3 3 Xanthan Gum 0.1 0.1 0.1 0.1 EDTA 0.1 0.1 0.1 0.1 Magnesium aluminum silicate 1 1 1 1 Styrene/acrylic copolymer - - - 5 Phase B Homosalate 15 15 15 15 Octisalate 5 5 5 5 Avobenzone 3 3 3 3 C12-15 alkyl benzoate 5 - - - Butyloctyl salicylate - 5 - 5 Diethylhexyl 2,6-naphthalate - - 5 - Polyester-8 2 2 2 2 Caprylic / capric triglyceride 3 3 3 3 Sorbitan isostearate and 3 3 3 3 polyglyceryl-3 Sodium isostearoyl lactylate 2 2 2 2 Polyamide-8 2 2 2 2 Phase C Propylparaben, methylparaben, diazolidinyl urea, propylene 0.5 0.5 0.5 0.5 glycol

23 Table 3.2B Sunscreens and their ingredients formulated in Steps 2 and 3 of this study

IO1 IO2 IO3 IOS C1 C2 CS % (w/w) Phase A Water 55.3 55.3 55.3 50.3 35.3 35.3 30.3 Glycerin 3 3 3 3 3 3 3 Xanthan Gum 0.1 0.1 0.1 0.1 0.1 0.1 0.1 EDTA 0.1 0.1 0.1 0.1 0.1 0.1 0.1 Magnesium aluminum silicate 1 1 1 1 1 1 1 Styrene/acrylic copolymer - - - 5 - - 5 Phase B Homosalate - - - - 15 15 15 Octisalate - - - - 5 5 5 Zinc oxide (and) caprylic/capric triglyceride (and) 23 - 11.5 23 23 11.5 23 polyhydroxystearic acid (and) isostearic acid Titanium dioxide (and) caprylic/capric triglyceride (and) - 23 11.5 - - 11.5 - polyhydroxystearic acid (and) aluminum stearate (and) alumina Butyloctyl salicylate 5 5 5 5 5 5 5 Polyester-8 2 2 2 2 2 2 2 Caprylic / capric triglyceride 3 3 3 3 3 3 3 Sorbitan isostearate and 3 3 3 3 3 3 3 polyglyceryl-3 Sodium isostearoyl lactylate 2 2 2 2 2 2 2 Polyamide-8 2 2 2 2 2 2 2 Phase C Propylparaben, methylparaben, 0.5 0.5 0.5 0.5 0.5 0.5 0.5 diazolidinyl urea, propylene glycol

24 3.2.1.1 Formulation Procedure of Sunscreens without SunSpheresTM

Xanthan gum was wet with glycerin then added to the water. Then the rest of phase A was added and heated to 75°C with appropriate stirring using a heating magnetic stir plate at

500 rpm. Phase B was combined in a beaker then heated to 75°C. Then Phase A was slowly added to phase B with continuous mixing using IKE RW 20 (IKA Works, Inc.,

Wilmington, NC) overhead mixer, and the emulsion was allowed to cool with continuous mixing using overhead mixer. Phase C was added at 45°C. Finally, the water loss was calculated by reweighing the batch, then the amount of water missing was replaced.

3.2.1.2 Formulation Procedure of Sunscreens with SunSpheresTM

Xanthan gum was wet with glycerin then added to the water. Then the rest of phase A except for the SunSpheresTM powder was added with appropriate mixing using a magnetic stir plate at 500 rpm. SunSpheresTM were then added to phase A and homogenized for

10 minutes at 7200 rpm using IKA T-18 Ultra Turrax (IKA Works, Inc., Wilmington, NC).

After this, phase A was heated to 75°C. Phase B was combined in a beaker and heated to

75°C. Phase A was slowly added to Phase B with continuous mixing using a IKA RW20 overhead mixer and propeller stirrer, then the emulsion was allowed to cool with continuous mixing using the same overhead mixer. Phase C was added at 45°C. Finally, the water lost during the heating process was replaced and the sunscreen was homogenized for one minute at 7200 rpm using the above-mentioned homogenizer.

25 3.2.2 Stability Testing

Samples of sunscreens were placed into 1.5 mL centrifuge tubes (n=4 for each sunscreen at one temperature, total of 12 tubes per sunscreen). The tubes were placed into stability cabinets (10-140 Analog Incubator, Quincy Lab, Chicago, IL) at room temperature (25°C), and elevated temperature (40°C and 45°C) for 3 months. Samples were checked visually on day 1, month 1, 2, and 3 for any signs of instability. Viscosity was checked at day 1, month 1, 2, 3. In vitro SPF and broad-spectrum protection were checked on day 1, and month 3.

3.2.3 In Vitro SPF Testing and Broad-Spectrum Testing

A polymethylmathacrylate (PMMA) plate (Labsphere, North Sutton, NH, USA) was tared on an analytical balance (Veritas M124A, Santa Clara, CA) with a readability of 0.001 g.

Per FDA 2011 Final Rule on over-the-counter sunscreen testing (FDA regulation), 0.050 g of the sunscreen was placed on each PMMA plate. Using a finger cot, the sunscreen was spread in a circular motion for thirty seconds, in a vertical motion for fifteen seconds, and then in a horizontal motion for another fifteen seconds to ensure the plate was completely covered as evenly as possible. The plate was placed in the dark for fifteen minutes and then taken to the in vitro SPF tester (LabSphere 2000S, Labsphere, North Sutton, NH, USA).

The in vitro SPF was scanned in five different locations on each plate. The test was repeated three times for each sunscreen.

In the US, in order to label a sunscreen broad-spectrum, the product has to have a critical wavelength of at least 370 nm. Critical wavelength is the wavelength below which

90% of the area under the absorbance curve resides (24). The same PMMA plates and the

26 same amount of UV filter-solvent mixture were used for broad-spectrum testing. The critical wavelength was calculated by the LabSphere software.

3.2.4 Water Resistance Testing

The sunscreens were applied to the PPMA plate as described above. The PMMA plate was suspended in a beaker of DI water using binder clips for 20 minutes. The water’s temperature was kept at skin temperature (32 ± 0.5°C), and the water was stirred continuously at 460 rpm using a magnetic stir bar. After 20 minutes, the PMMA plate was removed from the beaker and placed on a solid surface to let air dry. After 1.5 hours, the in vitro SPF method described above was used to measure the SPF. This technique will be referred to as “20-minute water bath” in this thesis.

3.2.5 Viscosity Testing

A TA Instruments Discovery Hybrid Rheometer DHR-3 (TA Instruments, New Castle,

DE, USA) was used with a 40 mm flat plate probe was used to measure the viscosity of the sunscreens. The tests were performed at 25°C. The shear rates ranged from 0.1 to 100 s-1.

The trim gap distance was 600 µm, and the geometry gap distance was 500 µm.

Inertia, friction, and rotation mapping were calibrated at the beginning of each test.

An approximately 1 mL sample was placed on the station. The probe was then displayed at 600 µm above the stationary base (trim gap distance) in order to remove the excess sample. The geometry distance was then displayed at 500 µm and the test was performed to obtain the viscosity.

27 3.2.6 Spreadability Testing

The spreadability of each sunscreen was determined using a TA.XTPlus texture analyzer

(Texture Technologies Corp., Hamilton, MA) with a TTC spreadability fixture. To determine spreadability, each sample was placed into the female cone and pressed down using a metal spatula to eliminate air pockets. The test mode was set to ‘measure compression’ and target mode was set to ‘distance’. Trigger force was 2.3 g, and the male cone’s penetration distance was 11 mm. The pre-test, test, and post-test speeds were set to

3.0, 3.0, and 3.0 mm/s, respectively. Exponent stable micro systems software (version

6.1.10.0) was used to generate spreadability curves.

3.2.7 Droplet Size Analysis

Droplet size analysis was done on all sunscreens using the AmScope MD35 microscope

(AmScopeTM, Irvine, CA). After appropriate dilution, a small amount of each formulation was placed on the glass slide and covered with a glass cover slide. The slide was then observed under different magnifications (4x, 10x, and 40x). The AmScope 3.4 software was used to capture the images. For each sample, thirty droplets were measured, then the average was calculated. Light microscopy is an easy tool to observe droplet size distribution and measure droplet dimensions. It does not require a significant dilution of the sample. Viscosity can also be used as an indirect indicator of droplet size. These two techniques combined were used to analyze droplet size (44, 45).

28 Chapter 4

4 Results and Discussion

4.1 Stability

All sunscreens were glossy and off-white. All sunscreens were stable on Day 1 at 25°C.

For organic UV filter-based sunscreens a reversible separation was observed. OS was

stable during the 3 months at 25°C and 40°C, and only reversible separation was observed

at 45°C at Month 3. The inorganic UV filter-based sunscreens were stable at Month1,

except for IO1 at 25°C, however, they became separated at Month 3. Similar to OS, IOS

was also more stable than the rest of the inorganic UV filter-based sunscreens. In the case

of the combined organic/inorganic sunscreens only reversible separation was observed

during the 3-month stability testing (Table 4-1).

29 Table 4.1 Stability results

Day 1 Month 1 Month 3 25°C 25°C 40°C 45°C 25°C 40°C 45°C O1 + +/- +/- +/- +/- +/- +/- O2 + +/- +/- +/- +/- +/- +/- O3 + +/- +/- +/- +/- +/- +/- OS + + + + + + +/- IO1 + - + + - - - IO2 + + - - +/- - - IO3 + + + + - - - IOS + +/- + + +/- + + C1 + + + + + +/- +/- C2 + + + + + + +/- CS + +/- + + +/- + +

+ stable +/- reversible separation - irreversible separation

4.2 In Vitro SPF Testing

The in vitro SPF of each sunscreen was measured on Day 1, and Month 3 at 25°C, 40°C, and 45°C. These results are summarized in Table 4.2. The three emollients tested in this study provided a very similar in vitro SPF, which was unexpected. There was no statistically significant difference between O1, O2, and O3. Based on our previous study with these emollients, diethylhexyl-2,6-naphthalate was expected to give the highest SPF with this organic UV filter combination, and C12-15 alkyl benzoate the lowest SPF. In our previous study, we tested the emollients as mixtures with the UV filters, not in realistic sunscreens. Any component of the sunscreens, such as the emulsifier, film-former,

30 photostabilizer could have affected the SPF, which lead to unexpected results. The highest in vitro SPF value would be normally selected for the next steps. Although O1 had the highest SPF out of three emollients we tested, butyloctyl salicylate was selected due to its better stability with the inorganic UV filters. The inorganic UV filter-based sunscreens showed signs of instability within 24 hours after formulation when made with C12-15 alkyl benzoate. OS had a significant, about a 3-fold increase in in vitro SPF compared to O1,

O2, and O3 (p<0.05).

The in vitro SPF of IO2 was three times higher than for IO1. IO1 contained 13.1% of zinc oxide, while IO2 contained 7.6% of titanium dioxide. IO3 contained 10.4% of total solid content (6.6% zinc oxide and 3.8% of titanium dioxide. Therefore, the SPF of IO3 was expected to be in between that of IO1 and IO2. IO1 was selected to be combined with the SunSpheresTM due to its broad-spectrum protection. IO1 had better coverage in the

UVA-I region, compared to IO2 and IO3, as seen in Figure 4-3. IOS and IO3 had a similar in vitro SPF.

The SPF of the combined organic and inorganic UV filter-based sunscreens was significantly higher than the SPF of any other sunscreen. In the case of C1, it was close to

SPF 50. C1 was selected to be combined with the SunSpheresTM because it provided higher

SPF and had broader spectrum protection. The SunSpheresTM boosted the SPF of C1and the broad-spectrum protection even more, as it was expected.

The SPF of the sunscreens containing SunSpheresTM (i.e., OS, IOS, and CS) was higher than the SPF of the same sunscreen without the SunSpheresTM – the SPF of OS is higher than O2, IOS is higher than IO1 and CS is higher than C1. As described in Chapter 1,

31 the SunSpheresTM are very effective SPF boosting ingredients, which we confirmed in this study.

At Month 3, the in vitro SPF value of most sunscreens increased significantly. A potential explanation is that a small amount of water could have evaporated from the product, resulting in a relatively more concentrated product (46). In addition, at 40 and

45°C, the water evaporation rate is faster than at room temperature. The increasing trend of SPF value was observed for most sunscreens at Month 3.

60

50

40 SPF 30

In vitro vitro In 20

10

0 O1 O2 O3 OS IO1 IO2 IO3 IOS C1 C2 CS

Figure 4-1 In vitro SPF of sunscreens at 25°C on Day 1

32 Table 4.2 In vitro SPF of sunscreens at 25°C, 40°C, and 45°C on Day 1 and Month 3

Day 1 Month 3 25°C 25°C 40°C 45°C O1 12 ± 1 19 ± 3 17 ± 1 18 ± 2 O2 10 ± 1 18 ± 1 21± 1 21 ± 2 O3 9 ± 0 17 ± 0 19 ± 3 18 ± 1 OS 33± 4 59 ± 10 55 ± 3 92 ± 8 IO1 11 ± 0 - - - IO2 30 ± 6 37 ± 5 - - IO3 21 ± 2 - - - IOS 20± 2 65 ± 3 85 ± 6 98 ± 7 C1 49 ± 1 53 ± 7 54. ± 7 76 ± 3 C2 44 ± 5 54 ± 4 59 ± 4 45 ± 2 CS 57 ± 2 88 ± 4 91 ± 6 112 ± 6 - Indicates instability, which prevented in vitro SPF testing

4.3 Water Resistance Testing

The in vitro SPF value after a 20-minute water bath increased compared to the in vitro SPF before the water bath in the case of all sunscreens. We believe this increase in SPF was due to the film-former, i.e., polyamide-8, which is an oil-structuring polymer that has good compatibility with low to medium polarity oils (47). Polyamide-8 is claimed to deliver water-resistance to sunscreens (48). After scanning a PMMA plate for the initial in vitro

SPF, the plate was immersed into a warm circulated water bath for twenty minutes. This process helped the oil layer spread evenly on the plate, which created a better coverage

(49). After the immersion, the PMMA plate was air-dried for more than one hour until the water completely evaporated, leaving an oil layer on the plates. In hours, more water could

33 have evaporated from the plate than for 15 minutes, which was the wait time before the water bath (for the initial reading). In addition, the water bath’s temperature was around

32°C, which is higher than room temperature when the plate was initially spread. This could have contributed to the higher in vitro SPF measured after the water bath.

150

120

90 SPF

In vitro vitro In 60

30

0 O1 O2 O3 OS IO1 IO2 IO3 IOS C1 C2 CS

SPF SPF after 20 min waterbath

Figure 4-2 In vitro SPF and in vitro SPF after 20 minutes of water bath of sunscreens on

Day 1

4.4 Broad Spectrum Testing

Broad-spectrum protection refers to protection for UVB, UVA-II, and UVA-I area.

Homosalate and octisalate are organic UV filters used in this study and they provide UVB protection only. In order to provide broad-spectrum protection, these organic UVB filters

34 needed to be combined with avobenzone, which is an organic UVA filter. Titanium dioxide and zinc dioxide are the inorganic UV filters we used in this study, they both provide both

UVB and UVA protection, therefore, they can provide broad-spectrum protection by themselves.

Sunscreens with a critical wavelength of 370 nm or higher would be considered to pass the in vitro broad-spectrum protection test. The critical wavelength is identified as the wavelength at which the integral of the spectral absorbance curve reaches 90% of the integral over the UV spectrum from 290 to 400nm (50). Table 4-3 summarizes the critical wavelength of sunscreens on Day 1 before and after the water bath. Before the water bath, all the sunscreens except for C1 and C2 had a critical wavelength higher than 370 nm.

After the water bath, most sunscreens’ critical wavelength decreased slightly or remained unchanged, but it was not significant except for IO2, a sunscreen that contains titanium dioxide. Titanium dioxide is more effective in the UVB region and also offers protection in the UVA-II area, but not good protection in the UVA-I area (51). During the water bath, some titanium dioxide could have been washed off leading to a significant decrease in critical wavelength value.

35 Table 4.3 Average critical wavelength of sunscreens on Day 1 before and after water

bath.

Critical wavelength Critical wavelength Sunscreens before the water bath after the water bath (nm) (nm) O1 380 ± 0 380 ± 0 O2 380 ± 0 379 ± 1 O3 380 ± 0 380 ± 0 OS 380 ± 1 379 ± 0 IO1 372 ± 0 372 ± 0 IO2 369 ± 2 362 ± 2 IO3 370 ± 0 369 ± 0 IOS 374 ± 0 373 ± 0 C1 369 ± 0 368 ± 0 C2 367 ± 0 365 ± 0 CS 370 ± 0 369 ± 1

4.5 Absorbance and Transmittance

The three organic sunscreens showed a low but constant absorbance between 290 nm to

380 nm.

IO1, the zinc oxide-based sunscreen had more constant protection between 280 to

360 nm. All sunscreens had the small dip in the absorption curve in the UVA-II region, which means that they provided weaker protection in this UV region. IO2, the titanium dioxide-based sunscreen had a higher absorbance than IO1 in the UVB region, and the protection fall below that of IO1 at about 350 nm. It means that IO2 provided a better UVB protection – which was observed in the in vitro SPF, but slightly narrower protection

36 overall. The absorption of IO3 was in between IO1 and IO2, as it was expected based on the IO1 and IO2 results.

In the C series (i.e., combined organic/inorganic sunscreens) a better UVB protection was observed than in any organic or inorganic sunscreen before. C1 had better

UVA protection as well than any other sunscreen before. These results confirm the synergistic effect between the organic and inorganic UV filters.

The transmittance results were basically the opposite of the absorbance results, as one would expect. Absorbance refers to how much light is being absorbed. Transmittance shows how much light passes through the sample. Since sunscreens absorb UV light in the

UVB and most of the UVA region, the transmittance was low for most sunscreens between

290 nm to 370 nm, except for IO2, which had a higher transmittance starting at around 352 nm. Most sunscreens had a small peak in the UVA-II region, which can be associated with slightly lower absorption in this region.

37 Figure 4-3 Absorbance and transmittance38 graphs of the sunscreens on Day 1 4.6 Viscosity

Figure 4-4 shows the viscosity curve of a Newtonian, shear-thinning, shear-thickening, and plastic system. When fluid has the same viscosity at different shear rates, it is called a

Newtonian system. Shear rate is the rate at which the shearing force is applied. Shear- thinning or pseudoplastic behavior is when a material’s viscosity decreases with an increase in shear rate. Shear-thickening or dilatant behavior is when a material’s viscosity increases with an increase in shear rate. Plastic systems typically exhibit yield stress, which is a specific shear stress value that must be exceeded in order to make a structured fluid flow. Below the yield stress, the fluid cannot flow, thus viscosity is unchanged until this threshold is exceeded.

Figure 4-4 Viscosity curve of Newtonian, shear-thinning, shear-thickening, and plastic

systems (52).

39 All sunscreens were shear-thinning as their viscosity decreased when the shear rate increased (Figure 4-5). This behavior is typical for cream and lotion and is what expected result (53).

10000.0

1000.0

100.0

10.0

Viscosity (Pa.s) Viscosity 1.0

0.1

0.0 0.1 1.0 10.0 100.0 Shear rate (1/s)

O1 O2 O3 OS IO1 IO2 IO3 IOS C1 C2 CS

Figure 4-5 Viscosity of sunscreens on Day 1

Figure 4.6 summarizes the viscosity of the sunscreens on Day 1, Month 1, Month 2, and Month 3 at a shear rate of 25 1/s. Organic sunscreens had a higher viscosity compared to inorganic sunscreens. When organic UV filters were combined with inorganic UV filters, the viscosity significantly increased. For O1, and O3, the viscosity was very similar on Day 1, Month 1, and Month 2. At Month 3, the viscosity decreased for both O1 and O3.

For O2, the viscosity continuously decreased from Day 1 to Month 3. For inorganic sunscreens, the viscosity minimally increased over time. When organic and inorganic UV

40 filters were combined, the viscosity was not constant over the testing period. For C1, the viscosity increased from Day 1 to Months 1 and 2 and decreased to the initial value by

Month 3. For C2, the viscosity dramatically decreased from Day 1/Month 1 to Month 2/3.

When SunSpheresTM were added to the sunscreens, the viscosity increased in the case of inorganic sunscreens, and organic/inorganic combined sunscreens, however, the viscosity was much lower for organic sunscreens. SunSpheresTM are a powder ingredient, which was expected to increase the viscosity – as it was observed for the inorganic and combined sunscreens. The viscosity decrease we experienced for the organic sunscreen was unexpected. A potential explanation is that interaction could have occurred between the SunSpheresTM and organic filters and/or other ingredients used.

14 Day 1 12 Month 1 Month 2 Month 3 10

8

6

Viscosity (Pa.s) Viscosity 4

2

0 O1 O2 O3 OS IO1 IO2 IO3 IOS C1 C2 CS

Figure 4-6 Viscosity of sunscreens on Day 1, Month 1, Month 2 and Month 3 at 25 s-1

41 4.7 Spreadability

We utilized an instrumental analysis for this purpose, which is commonly done in the industry. Figure 4-7 shows a typical spreadability testing graph. Spreadability refers to the ease of which a product can be spread in a thin and even layer on the skin during application. Penetration and compression style tests are simple methods for testing the hardness of a sample. During the test, the male cone penetrates the sample in the female cone to the desired depth, and the penetration force increases until the point of the maximum desired penetration, which results in a positive peak. The male probe then withdraws from the sample, which results in a negative peak. The positive peak refers to the firmness of a sample, i.e., the force required to obtain a given deformation. The negative peak refers to the stickiness of a sample, i.e., the force necessary to overcome the attractive forces between the surface of the product and the surface of the probe. The lower the firmness value, the more spreadable the product; and the higher the stickiness, the more cohesive the product.

Due to their low viscosity, we were unable to measure the firmness and stickiness of OS, and IOS. It can be seen in Figure 4-8 that O2 had the highest firmness among all sunscreens. There is no optimal number for what is considered good spreading. This is usually dictated by consumer acceptance.

CS had the lowest number for both firmness and stickiness, which means that it was easy to spread, and did not seem to have a sticky nature. For sunscreens to perform well and have high effectiveness, they should be able to spread well to create an even layer on the skin. Lower firmness and stickiness value correlated to better spreadability and higher SPF. CS had the highest in vitro SPF and also had favorable spreading

42 characteristics. O2 had higher firmness and stickiness values compare to O1 and O3, but it had a very similar in vitro SPF with O3, which was unexpected. There is a high correlation between the measurements of firmness and spreadability but the relationship is not perfect

(54).

Figure 4-7 Typical spreadability test graph.

43 1000.00

800.00

600.00

400.00

200.00

0.00

Force (g) Force -200.00

-400.00

-600.00

-800.00

-1000.00 O1 O2 O3 IO1 IO2 IO3 C1 C2 CS

Firmess Stickiness

Figure 4-8 Firmness and stickiness of sunscreens

4.8 Droplet Size

Table 4.4 summarizes the sunscreens’ droplet size. Droplet size ranged between 2.6 and

10.7 µm, which is typical for regular emulsions. The similar particle size and monodisperse distribution confirm the uniformity of the formulation method.

44 Table 4.4 Droplet size of sunscreens within in one week after formulation

Droplet size Sample (µm) O1 2.6 ± 0.6 O2 3.3 ± 0.7 O3 3.2 ± 0.6 OS 7.2 ± 1.5 IO1 4.0 ± 1.1 IO2 4.4 ± 1.4 IO3 4.3 ± 1.6 IOS 3.0 ± 0.6 C1 3.5 ± 1.0 C2 3.7 ± 0.7 CS 10.7 ± 3.7

45 Chapter 5

5 Conclusion

The aims of this study were to develop a sunscreen formula that can be used for both organic and inorganic UV filters, and evaluate the SPF boosting effect of emollients for organic UV filters and SunSpheresTM for organic and inorganic UV filters. Eleven sunscreens were formulated and tested for in vitro SPF, water resistance, broad-spectrum protection, viscosity, spreadability, and droplet size.

All sunscreens were glossy and off-white. They all had a monodisperse droplet size distribution and the average droplet size was in the range of 2.6 and 10.7 µm.

In a preliminary study, diethylhexyl-2,6-naphthalate had the highest in vitro SPF when mixed with organic UV filters. It was followed by butyloctyl salicylate, and then

C12-15 alkyl benzoate. Our results in this study showed that when diethylhexyl-2,6- naphthalate was used in a realistic sunscreen formulation, the in vitro SPF value was the lowest, while that of C12-15 alkyl benzoate was the highest. We can conclude that when emollients are mixed with liquid organic UV filters and other ingredients needed to formulate emulsions, the protection can be different than it is in a simple oily mixture due to potential interaction, and/or synergism between the ingredients.

O2, i.e., the sunscreen with butyloctyl salicylate was selected to be combined with the SunSpheresTM due to its SPF and good compatibility with the inorganic UV filters. As for inorganic UV filter, titanium dioxide provided a higher in vitro SPF than zinc oxide,

46 but zinc oxide had more broad-spectrum protection throughout UVB and UVA regions.

Therefore, zinc oxide was selected to be combined with organic UV filters and with

SunSpheresTM. C1, i.e., combined organic UV filters (without avobenzone) and zinc oxide had a higher in vitro SPF than C2, i.e., combined organic UV filters (without avobenzone) with zinc oxide and titanium dioxide. C1 was therefore combined with the SunSpheresTM.

All sunscreens that contained the SunSpheresTM had a higher in vitro SPF than the same sunscreens without SunSpheresTM and had broad-spectrum protection throughout UVB and

UVA regions.

We observed an in vitro SPF increase in the case of all sunscreens after exposing the sunscreens to a 20-minute water bath. We believe that the film-former, i.e., polyamide-

8 and the drying time contributed to higher SPF values after the water bath. All sunscreens had a shear-thinning behavior, which is typical for creams and lotions. Spreadability can influence whether consumers have a good experience applying the product to their skin.

CS had the lowest number for both firmness and stickiness, which means that it was easy to spread, and did not have a sticky nature. CS had the highest in vitro SPF and also had favorable spreading characteristics.

Overall, the organic/inorganic UV filter-based sunscreen containing the

SunSpheresTM (CS) performed the best in terms of in vitro SPF, water-resistance and spreadability.

47 Chapter 6

6 Future Studies

This research could be used as a control study for future studies. In this study, we used the maximum allowed amount of the three organic UV filters, and the in vitro SPF, in vitro water resistance, and broad-spectrum protection were evaluated. To take a step forward, certain modifications could be done. For example, different film-formers or different dispersions of inorganic UV filters could be used. Additionally, the amount of UV filters used in the sunscreens could be decreased and it could be studied whether the same SPF and broad-spectrum protection can be achieved by utilizing different SPF boosting technologies. SunSpheresTM are physical SPF boosting particles, which are plastic particles, and for this reason more sustainable alternatives may need to be utilized in the future. These technologies could be studied using our sunscreens as control.

48 7 References

1. Wickett RR, Visscher MO. Structure and function of the epidermal barrier.

American Journal of Infection Control. 2006;34(10):S98-S110.

2. Understand the Anatomy: Head & Neck Cancer Guide; [Available from: https://headandneckcancerguide.org/adults/introduction-to-head-and-neck-cancer/skin- cancer/anatomy/.

3. Lai-Cheong JE, McGrath JA. .

Mediciine. 2009;37(5):223-6.

4. d'Ischia M, Wakamatsu K, Cicoira F, Di Mauro E, Garcia-Borron JC, Commo S, et al. Melanins and melanogenesis: from pigment cells to human health and technological applications. Pigment Cell Melanoma Res. 2015;28(5):520-44.

5. Baki G, Alexander KS. Skin care products. Skin care products. First edition ed:

Wiley; 2015. p. 133.

6. Rivera-Gonzalez G, Shook B, Horsley V. Adipocytes in skin health and disease.

Cold Spring Harb Perspect Med. 2014;4(3).

7. Biniek K, Levi K, Dauskardt RH. Solar UV radiation reduces the barrier function of human skin. Proc Natl Acad Sci U S A. 2012;109(42):17111-6.

8. Lawrence K, Al-Jamal M, Kohli I, Hamzavi I. Clinical and Biological Relevance of Visible and Infrared Radiation. In: Wang SQ, Lim HW, editors. Principles And Practice

Of Photoprotection: Springer International Publishing AG Switzerland; 2016. p. 3.

49 9. Pearse AD, Gaskell SA, Marks R. Epidermal changes in human skin following irradiation with either UVB or UVA. J Invest Dermatol. 1987;88(1):83-7.

10. Baki G, Alexander KS. Skin care products. Skin care products. First edition ed:

Wiley; 2015. p. 282.

11. Skin Cancer: American Academy of Dermatology; [Available from: https://www.aad.org/media/stats-skin-cancer.

12. Rastogi RP, Richa, Kumar A, Tyagi MB, Sinha RP. Molecular mechanisms of ultraviolet radiation-induced DNA damage and repair. J Nucleic Acids.

2010;2010:592980.

13. Dai C, Gu W. p53 post-translational modification: deregulated in tumorigenesis.

Trends Mol Med. 2010;16(11):528-36.

14. Rivlin N, Brosh R, Oren M, Rotter V. Mutations in the p53 Tumor Suppressor

Gene: Important Milestones at the Various Steps of Tumorigenesis. Genes Cancer.

2011;2(4):466-74.

15. Abreu Velez AM, Howard MS. Tumor-suppressor Genes, Cell Cycle Regulatory

Checkpoints, and the Skin. N Am J Med Sci. 2015;7(5):176-88.

16. Ananthaswamy HN. Sunlight and Skin Cancer.pdf. Journal of Biomedicine and

Biotechnology. 2001.

17. Sanchez G, Nova J, Rodriguez-Hernandez AE, Medina RD, Solorzano-Restrepo C,

Gonzalez J, et al. Sun protection for preventing basal cell and squamous cell skin cancers.

Cochrane Database Syst Rev. 2016;7:CD011161.

50 18. Klimová Z, Hojerová J, Pažoureková S. Current problems in the use of organic UV filters to protect skin from excessive sun exposure. Acta Chimica Slovaca. 2013;6(1):82-

8.

19. Johal R, Leo MS, Ma B, Sivamani RK. The economic burden of sunscreen usage.

Dermatology Online Journal. 2014;20(6).

20. Schulz J, Hohenberg H, Pflucker F, Gartner E, Will T, Pfeiffer S, et al. Distribution of sunscreens on skin. Advance Drug Delivery Reviews. 2002.

21. Serpone N, Dondi D, Albini A. Inorganic and organic UV filters: Their role and efficacy in sunscreens and suncare products. Inorganica Chimica Acta. 2007;360(3):794-

802.

22. Lane ME, Hadgraft J, Oliveira G, Vieira R, Mohammed D, Hirata K. Rational formulation design. 2012;34(6):496-501.

23. Wiechers J. Clinical Proof for Validity of the »Formulating for Efficacy« Concept:

Enhanced Skin Delivery Results inEnhanced Skin Efficacy and Both Can Be Predicted.

IFSCC Magazine. 2011:29-38.

24. Electronic Code of Federal Regulations: Food and Drug Adminitration; [updated

April 16, 2020. Available from: https://www.ecfr.gov/cgi-bin/text- idx?SID=cacd92e0a44451fa5eb26797b6bf6321&mc=true&node=sp21.5.352.b&rgn=div

6&mod=article_inline - se21.5.352_110.

25. Herzog B. Photoprotection of Human Skin. In: Albini A, Fasani E, editors.

Photochemistry2012. p. 258-9.

51 26. Daly S, Ouyang H, Maitra P. Chemistry of Sunscreens. In: Wang SQ, Lim HW, editors. Priciples and Practice of Photoprotection: Springer International Publishing AG

Switzerland; 2016. p. 162-5.

27. Daly S, Ouyang H, Maitra P. Chemistry of Sunscreens. In: Wang SQ, Lim HW, editors. Principles And Practice Of Photoprotection: Springer International Publishing AG

Switzerland; 2016. p. 160.

28. Hewitt JP. Introduction to Sun Protection. 2019.

29. Michelle. WHAT DOES SPF MEAN? THE SCIENCE OF SUNSCREEN: Lab

Muffin; 2017 [Available from: https://labmuffin.com/what-does-spf-mean-the-science-of- sunscreen/?utm_source=newsletter&utm_medium=email&utm_campaign=new_post_wh at_does_spf_mean_the_science_of_sunscreen&utm_term=2017-08-24.

30. McCall MJ, Gulson B, Andrews D. Consumer Use of Sunscreens Contaning

Nanoparticles. Nanotechnology Environamental Health and Safety: William Andrew

Applied Science 2018. p. 389-423.

31. Sohn M. UV Booster and Photoprotection. In: Wang SQ, Lim HW, editors.

Principles and Practice of Photoprotection: Springer International Publishing AG

Switzerland; 2016. p. 228.

32. Philbin MT, inventorSkin care formulation. United States2015.

33. Agrapids-Paloympis LE, Nash RA, Shaath NA. The effect of solvents on the ultraviolet absorbance of sunscreens J Soc Cosmet Chem. 1987;38:209-21.

34. Grabenhofer R. Key West Joins Hawaii in the Ban of Octinoxate and Oxybenzone.

Cosmetics & Toiletries. 2019.

52 35. Czajka K. THE U.S. VIRGIN ISLANDS BANS POTENTIALLY DANGEROUS

SUNSCREEN CHEMICALS. Pacific Standard Magazine. 2019.

36. Shukla P. First Sunscreen, Now Makeup And Hair Spray, May Be Harming Corals.

Forbes. 2019.

37. Chisvert A, Leon-Gonzalez Z, Tarazona I, Salvador A, d’imosthenis GiokasIschia

M. An overview of the analytical methods for the determination of organic ultraviolet filters in biological fluids and tissues. Analytica Chimica Acta. 2012;752:11-29.

38. Matta MK, Florian J, Zusterzeel R, Pilli NR, Patel V, Volpe DA, et al. Effect of

Sunscreen Application on Plasma Concentration of Sunscreen Active Ingredients: A

Randomized Clinical Trial. JAMA. 2020;323(3):256-67.

39. Downs CA, Kramarsky-Winter E, Fauth JE, Segal R, Bronstein O, Jeger R, et al.

Toxicological effects of the sunscreen UV filter, benzophenone-2, on planulae and in vitro cells of the coral, Stylophora pistillata. Ecotoxicology. 2014;23(2):175-91.

40. GM M. Sunblock Mechanisms of action. Photodermatol Photoimmunol Photomed.

1999(15):34-6.

41. D M, G L, A M, A VG. Potential effects of TiO2 nanoparticles and TiCl4 in saltwater to Phaeodactylum tricornutum and Artemia franciscana. Science of the Total

Environment. 2017;579:1379-86.

42. Hazeem LJ, Bououdina M, Rashdan S, Brunet L, Slomianny C, Boukherroub R.

Cumulative effect of zinc oxide and titanium oxide nanoparticles on growth and chlorophyll a content of Picochlorum sp. Environ Sci Pollut Res Int. 2016;23(3):2821-30.

43. Osterwalder U, Sohn M, Herzog B. Global state of sunscreens. Photodermatol

Photoimmunol Photomed. 2014;30(2-3):62-80.

53 44. Rafati H, Ebrahimi SN, Rezadoost H. Preparation of a delayed releasing device of hydroxypropyl methylcellulose phthalate macrocapsules containing peppermint oil.

Iranian Polymer Journal. 2008:273-80.

45. Hu Y-T, Ting Y, Hu J-Y, Hsieh S-C. Techniques and methods to study functional characteristics of emulsion systems. Journal of Food and Drug Analysis. 2017;25:16-26.

46. Finga M, Fieldhouse B. STUDIES OF WATER-IN-OIL EMULSIONS AND

TECHNIQUES TO MEASURE EMULSION TREATING AGENTS Proceedings of the

Arctic Marine Oilspill Program. 1994:213-44.

47. Larsson IM. A Water Resistant Film Forming Emulsion. Lunds University: Lunds

University; 2017.

48. OleoCraft™ LP-20: Croda; [Available from: https://www.crodapersonalcare.com/en-gb/products-and-applications/product- finder/product/2462/OleoCraft_1_LP-20.

49. Rarah M, Oliveira R, Caldas J, Rajagopal K. Viscosity of water-in-oil emulsions:

Variation with temperature and water volume fraction. Journal of Petroleum Science and

Engineering. 2005:169-84.

50. Electronic Code of Federal Regulation Title 21 FDA: FDA; 2019 [Available from: https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfCFR/CFRSearch.cfm?fr=201.327.

51. Rai R, Shanmuga SC, Srinivas C. Update on photoprotection. Indian J Dermatol.

2012;57(5):335-42.

52. Coussot P. Introduction to the rheology of complex fluids. Understanding the

Rheology of Concrete: Woodhead Publishing Series in Civil and Structural Engineering;

2012. p. 3-22.

54 53. Rheology in Use: Anton Paar; [Available from: https://wiki.anton-paar.com/us- en/basics-of-rheology/rheology-in-use/.

54. Texture Analysis Professionals Blog [Internet]. Stable Micro Systems: Stable

Micro Systems. 2014. [cited 2020]. Available from: https://textureanalysisprofessionals.blogspot.com/2014/11/ten-ways-to-measure-cheese- texture_18.html.

55