What happens in the skin? Integrating skin permeation kinetics into studies of developmental and reproductive toxicity following topical exposure

Accepted Manuscript Title: What happens in the skin? Integrating skin permeation kinetics into studies of developmental and reproductive toxicity following topical exposure Author: Yuri Dancik Paul Bigliardi Mei Bigliardi-Qi PII: DOI: Reference:

S0890-6238(15)30027-7 http://dx.doi.org/doi:10.1016/j.reprotox.2015.10.001 RTX 7185

To appear in:

Reproductive Toxicology

Received date: Revised date: Accepted date:

10-11-2014 31-8-2015 7-10-2015

Please cite this article as: Dancik Yuri, Bigliardi Paul, Bigliardi-Qi Mei.What happens in the skin? Integrating skin permeation kinetics into studies of developmental and reproductive toxicity following topical exposure.Reproductive Toxicology http://dx.doi.org/10.1016/j.reprotox.2015.10.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

What happens in the skin? Integrating skin permeation kinetics into studies of developmental and reproductive toxicity following topical exposure Yuri Dancik1* [email protected], Paul Bigliardi1, Mei Bigliardi-Qi1 [email protected] 1

Clinical Research Unit for Skin, Allergy and Regeneration & Experimental Dermatology,

Laboratory, Institute of Medical Biology, A*STAR, Singapore *Corresponding author at: Clinical Research Unit for Skin, Allergy and Regeneration & Experimental Dermatology Laboratory, Institute of Medical Biology, A*STAR, Singapore

HIGHLIGHTS  Animal-based studies investigating developmental and reproductive toxicology following skin exposure have not traditionally focused on the skin permeation kinetics of the potential toxicant, which drive toxicity.  Skin permeation kinetics are dependent on a multitude of factors related to the nature of the potential toxicant and solvent, intrinsic skin features and extrinsic exposure factors affecting the skin permeability barrier.  Physiologically-based pharmacokinetic models with a detailed and flexible description of skin and exposure scenarios are the method of choice for the integration of in vivo, in vitro and in silico data from traditional experiments and emerging technologies and the development of alternative methods of toxicity prediction and risk assessment.

ABSTRACT Animal-based developmental and reproductive toxicological studies involving skin exposure rarely incorporate information on skin permeation kinetics. For practical reasons, animal studies cannot investigate the many factors which can affect human skin permeation and systemic uptake kinetics in real-life scenarios. Traditional route-toroute extrapolation is based on the same types of experiments and requires assumptions regarding route similarity. Pharmacokinetic modeling based on skin physiology and structure is the most efficient way to incorporate the variety of intrinsic skin and exposure-dependent parameters occurring in clinical and occupational settings into one framework. Physiologically-based pharmacokinetic models enable the integration of available in vivo, in vitro and in silico data to quantitatively predict the kinetics of uptake at the site of interest, as needed for 21st century toxicology and risk assessment. As demonstrated herein, proper interpretation and integration of these data is a multidisciplinary endeavor requiring toxicological, risk assessment, mathematical, pharmaceutical, biological and dermatological expertise. Abbreviations AUC: Area under the curve BUP: Bitumen Upgrading Product BR: Bayrepel CAP: trans-Capsaicin COM: Complex Organic Mixture CSO: Clarified Slurry Oil DAE: Distillate Aromatic Extract DDT: dichlorodiphenyltrichloroethane DEGBEA: Diethylene glycol (mono) n-butyl ether acetate DEGEE: Diethylene glycol (mono) ethyl ether DGMME: Diethylene glycol (mono) methyl ether DGMBE: Diethylene glycol mono-n-butyl ether DEA: Diethanolamine DGEBPA: Diglycidyl ether of bisphenol A DMF: Dimethylformamide DPGME: Dipropylene glycol (mono) methyl ether EGEEA: Ethylene glycol (mono) ethyl ether acetate EGBE: Ethylene glycol (mono) butyl ether

EGNPE: Ethylene glycol (mono) n-propyl ether EGIPE: Ethylene glycol (mono) iso-propyl ether EGMEA: Ethylene glycol (mono) methyl ether acetate EF: Extrapolation factor 2-EE: 2-ethoxyethanol EG: ethylene glycol 2-EH: 2-Ethylhexanol FA: Formaldehyde HDS kerosene: Hydrodesulfurized kerosene HMB: 2-Hydroxy-4-methoxybenzophenone LAS: Linear alkyle benzene sulphonate 4-MBC: 4-methyl-benzylidene camphor MDEA: N-Methyldiethanolamine MPA: 2-Methoxypropyl-1-acetate MEA: Monoethanolamine 2-ME: 2-Methoxyethanol MMDHCA: methyl-3,4-methylene-dioxy-hydrocinnamic aldehyde MEKP: Methyl ethyl ketone peroxide MS: Methyl salicylate MSK: Methyl trans-styryl ketone NMP: N-methylpyrrolidone NOAEL: No observed adverse effect level OMA: Omadine MDS 2-PE: 2-Phenoxyethanol PEA: Phenylethyl alcohol PBPK: Physiologically-based Pharmacokinetic PEG: Polyethylene glycol RF: Refinery streams RH: Relative humidity SC: Stratum corneum SLS: sodium lauryl sulphate STB: Syntower Bottoms S-23121: Flumipropyn S-53482: Flumioxiazin

TBU: 1,1,1,3-tetrabutylurea TEWL: transepidermal water loss TGA: Thioglycolic acid TMPCC: Trimethylolpropane caprylate caproate TQ: 1,2-dihydro-2,2,4-trimethylquinoline TRE: tretinoin

KEYWORDS: Alternative methods; Dermal exposure; Exposure scenario; Skin penetration; PBPK; Occupational toxicology

1.

INTRODUCTION

Developmental and reproductive toxicity is a complex toxicological endpoint due to the nature of the human reproductive cycle and the large number of effects. Traditional animal-based tests require application of high doses of the potential toxicant and span days [1, 2]. An important requirement is that the results point specifically to evidence of developmental or reproductive toxicity. There should be no confounding results due to local effects (skin irritation) [2, 3]. The dose and effect constraints explain why there have been relatively few skin exposure studies, despite the skin being the principal site of exposure to pesticides [4], a major one for consumer products and industrial solvents [5, 6] and important in therapy. Understanding the kinetics of biophysical processes that yield potentially toxic internal (systemic or organ) concentrations is crucial for the development of toxicity prediction and risk assessment methods. This holds as much true for the assessment of human risk from existing animal data, as in read-across methods [7, 8], as for the development of novel methods based on in vitro assays, computational models and strategies that integrate in vitro, in vivo and in silico data [9]. Animal data, being the only data obtained from consideration of the full reproductive cycle [1, 10, 11], are needed in the development and interpretation of alternative methods. Further, incorporating the relevant kinetics of absorption, distribution, metabolism and elimination is necessary in cases in which new animal experiments may need to be designed to resolve highly specific mechanisms of action leading to toxicity and for the design of alternative methods which capture them [12]. To these ends, an understanding of the impact of intrinsic skin physiology and structure and of the experimental protocols on the kinetics is necessary. The aim of this review is to underline this point with respect to studies of toxicity following skin exposure, because many in- and extrinsic factors can significantly impact skin permeation kinetics and, ultimately, uptake at the site of action [13]. Furthermore, for risk assessment, experimental data should be relevant to occupational conditions [13]. After reviewing the factors that influence cutaneous systemic uptake, we illustrate their quantitative impact in the human and highlight major differences with relevant animal models. Existing developmental and reproductive toxicity animal studies are surveyed and the relevance of the skin exposure protocols to real-life exposure situations is addressed. The compounds in these studies range from cosmetic and therapeutic products to industrial solvents and mixtures. We then review how physiologically-based pharmacokinetic

(PBPK) models with a detailed and flexible description of skin permeation are well-suited to model skin permeation and internal uptake in a variety of occupational and consumer situations. Improvements in skin permeation-PBPK models needed to increase their predictive power are also discussed.

2.

FACTORS AFFECTING SYSTEMIC UPTAKE FOLLOWING SKIN EXPOSURE

2.1. Human skin physiology and structure 2.1.1. Healthy skin The network of blood vessels in the skin is composed of a dense superficial plexus of arterioles and venules in the papillary dermis running along the epidermal-dermal junction and a second, less-dense plexus embedded deep in the reticular dermis, near the hypodermis (Figure 1). The superficial plexus supplies the avascular viable epidermis with nutrients. It is connected to the deep plexus by pairs of ascending arterioles and descending venules, which also supply the skin appendages [14]. Most of the skin microvasculature is found in the papillary dermis (within 1 to 2 mm below the skin surface), with the total number of blood vessels as well as their inner and outer diameters decreasing with depth into the skin [15]. A dense mesh of crisscrossing blood vessels is found surrounding the lower third of the hair follicle [16, 17]. Parallel blood vessels run along the length of the rest of the follicle and connect to those surrounding the sebaceous glands [17]. Eccrine sweat glands and aprocrine glands are also well vascularized [17]. Systemic exposure and distribution to other organs further depend on dermal blood flow rate, itself dependent on neural and local mechanisms, e.g., during thermoregulation [18, 19], and on the inflammation status of the skin. Pharmacokinetic studies, including in vivo microdialysis experiments with volunteers, show that vasodilation enhances systemic uptake and clearance from dermal tissues whereas vasoconstriction has the opposite effect, yielding accumulation in the tissues [20-23]. Certain chemicals such as the insecticide malathion are vasodilators and may increase themselves their systemic uptake [20]. To reach the systemic circulation following skin exposure, compounds must first be absorbed into and permeate through the SC. The thickness of healthy adult SC ranges from about 20 m (cheeks, buttocks, forearms) to about 200 m (palms) [24-26]. It is composed of flat, closely packed interdigitated corneocytes, embedded in a highly organized, dense lipid matrix (Figure 1). They are joined to one another by corneodesmosomes which ensure SC cohesion [27]. The highly effective barrier

property of healthy human skin also arises from the nature and organization of its corneocytes and lipids. Briefly, corneocytes are filled with water and keratin surrounded by a cornified envelope composed of densely cross-linked protein such as filaggrin, loricrin and involucrin [28]. A monolayer of lipids is attached to the cornified envelope and is thought to serve as a template for the formation of the SC’s intercellular lipids, which consist of free fatty acids, ceramides and cholesterol [29]. Ceramides play a key role in the health of the human skin barrier [30]. The primary lateral arrangement of intercellular lipids of the SC is in orthorhombic fashion, imparting order and high packing density [31, 32]. The unique repeat distance (long periodicity phase) of 13 nm of SC lipid lamellae may also contribute to the SC barrier property [28]. Formation of the SC barrier and maintenance of its integrity depend on the SC’s ―acid mantle‖, that is, a pH of about 5.4-6.0 on most body sites [33, 34]. The natural hydration level of healthy SC, dependent on the presence of natural moisturizing factors [35], is essential to the maintenance of the lipid geometry [36]. The SC thickness at any given body site is maintained through the process of desquamation involving continuous cell migration from the basal layer of the epidermis to the SC and shedding of surface corneocytes. In general, the basic physico-chemical properties required for passive absorption into and permeation through the SC are a molecular weight below 500 g/mol, moderate lipophilicity (octanol-water partition coefficient of 1 to 4), adequate lipid and aqueous solubilities, relatively low solubility in the vehicle (if any) and low volatility [13, 37]. Desquamation can prevent highly lipophilic or large molecular weight chemicals from reaching the systemic circulation [38, 39]. Other phenomena that may slow transport through the SC and uptake into the viable tissue include binding to keratin [40] and sequestration, also known as the ―reservoir effect‖. This effect designates the accumulation of xenobiotic molecules in the SC (or in the viable skin layers), rendering the chemical unavailable to the underlying tissues and systemic circulation, or delaying its deeper penetration. A variety of factors may influence the formation of a reservoir including the compound’s molecular weight, lipophilicity, protein binding affinities, aqueous solubility as well as exposure conditions (vehicle, occlusion) [41, 42]. Recent excised skin penetration experiments have provided evidence for the reservoir effect influencing the transport of pesticides [43, 44], fullerenes (C60) in industrial organic solvents [45], cosmetic ingredients [46], phthalates [47]. The cosmetic ingredients study provides an example of delayed reservoir depletion and deeper penetration. While 12.5 to 29% of the application doses of geraniol, DBP and farnesol were recovered in the skin

(SC and viable tissue) 24h after application, these values decreased 2 to 3-fold 72h after application, while the amount permeating the skin increased [46]. The viable epidermis can act as a secondary barrier to the penetration of compounds. It is about 60 to 80 m thick [26, 48, 49] and composed of approximately 40% protein, 40% water and 20% lipids [50]. Its high water content hinders the transport of lipophilic compounds [51, 52]. It is also the site of phase I and phase II metabolites located primarily in the basal layer, just above the cutaneous capillaries and the superficial plexus (Figure 1) [53-55]. The role of skin enzymes in has been extensively reviewed [56-60]. For some chemicals, cutaneous metabolism may lead to detoxification, while for others it may lead to the formation of toxic metabolites and potential allergens, prior to access to the systemic circulation. Further, keratinocytes express a variety of protein transporters [61] which may either promote or hinder the transport of xenobiotics in the viable tissue [62-65]. Similarly to the SC, transport in the viable epidermis (and underlying skin layers) can be delayed due to sequestration mediated by the chemical’s lipophilicity and protein binding affinity, by metabolism, if any, and systemic clearance [42]. The nature of the dermis impacts the access of compounds to the deeper blood vessels. The dermis is approximately 500 m to a few millimeters thick [48, 66] depending on body site. It is composed primarily of collagen bundles, elastic fibers, fibrocytes and ground substance. The main processes that can affect solute transport are binding to these components and sequestration [52, 67]. Metabolism and carrier-mediated transport could also play a role, but may differ from the viable epidermis due to differential protein expression (e.g., the transporter protein p-glycoprotein mainly expressed in the dermis, sweat ducts and muscle tissue [68]). Below the dermis lies the 1 to several millimeters thick hypodermis or subcutaneous fat tissue. The hypodermis consists of fat microlobules interwoven by fibrous strands principally made up of collagen [69]. It protects against mechanical shock, provides heat insulation and energy storage and facilitates skin attachment and mobility over the underlying fascia and muscle tissue [50, 69]. The skin appendages, and in particular the hair follicle, constitute transport pathways through which chemicals may access the network of blood vessels surrounding the appendages as well as penetrate to the viable tissues bypassing the SC (Figure 1). The follicular pathway is a potential route for lipophilic chemicals able to easily partition into the sebum-filled infundibulum [70-72]. From there, toxicant molecules might cross the

relatively permeable boundary of the hair follicle [73] and/or penetrate into the deeper hair follicle. A study comparing blood flow, skin temperature and redness on the forehead, the forearm and the calf of volunteers found the highest baseline levels on the forehead [74]. Application of the vasodilator benzyl nicotinate to each site yielded an increase in each parameter, with the fastest increase, but also the fastest decrease, on the forehead. This study illustrates the potential impact of hair follicles (vellus hairs on the forehead) to both permeation and systemic uptake. The efficacy of follicular transport to the systemic circulation also likely depends on the hair follicle cycle [70] and whether follicular protein transporters [75] would affect transport into the deep follicle. The hair follicle, the hair shaft cuticle and the sebaceous gland are potential reservoir sites [13, 76, 77]. Finally, it should be kept in mind that systemic uptake of toxicants may differ significantly between body sites due to differences in blood flow rate, skin layer thickness (in particular SC thickness), hydration levels, lipid and sebum content and hair follicle density. Corneocyte size, and therefore the length of the SC lipid penetration pathway, correlate linearly with SC thickness [78]. Early studies with volunteers and different chemicals, reviewed in reference [13], showed that skin penetration as measured by permeant urinary recovery correlates with skin layer thickness and hair follicle density. The skin of the scrotum, jaw angle, forehead and scalp was shown to be, respectively, about 40, 13 and 4 to 6 times more penetrable compared to that of forearm [79-81]. In another study forehead skin permeability was 2-fold greater than through postauricular abdomen and arm skin [82].

2.1.2. Diseased skin Dermatological conditions alter the physiology and/or structure of the skin and can significantly affect skin permeation and subsequent systemic uptake. Clinical features of the most common form of psoriasis, plaque psoriasis, are erythematous, scaling plaques, increased keratinocyte proliferation and hyperkeratosis [83, 84]. Depending on age, atopic dermatitis is characterized by pruritus, scaliness, crusts, thickened patches showing lichenification and excoriation [85]. Lipid composition in skin displaying atopic dermatitis, lamellar ichthyosis, psoriasis, is altered compared to healthy skin with, in general, decreases in the amounts of ceramides and increases in short chain lipids, unsaturated lipids, and unsaturated free fatty acids [28, 86]. Lipid organization is impaired due to changes in the long periodicity phase of the lipid lamellae and a

decrease in the orthorhombicity of the lipid arrangement

[28]. Blood flow,

vasoconstriction and vasodilation in plaque psoriatic skin are altered due to elongation, widening, and an increased in the tortuosity of cutaneous blood vessels [87-89]. A recent imaging study shows plaque psoriatic forearm skin displaying thin and loosely packed cells layers in the viable epidermis, and a mean cross-sectional size of blood vessels nearly twice that in healthy skin [90]. The alterations brought on by these diseases yield increased penetration of xenobiotics which under normal conditions do not easily penetrate the skin [13]. Experiments with volunteers with mycosis fungoides (cutaneous T-cell lymphoma) and atopic or seborrheic dermatitis on the penetration of carmustine and hydrocortisone, respectively, recovered in serum or urine revealed a 5- to 6-fold increase in penetration [91, 92]. Other volunteer studies on the skin penetration of 5-methoxypsoralen and a plasminogen activator, measured respectively, via tape-stripping and in biopsies, yielded similar trends [93, 94]. Studies utilizing experimental skin models have shed light on possible mechanisms of altered penetration and systemic uptake in compromised skin. A model of inflamed skin suggested the rate of penetration and systemic absorption of highly protein-bound chemicals could be reduced due to protein leaching into skin tissue during inflammation [95]. Use of an experimental model mimicking inflammatory diseases such as atopic eczema and the extreme variant Netherton syndrome via higher level of mono-unsaturated fatty acids (MUFAs) yielded a 5-fold increase in transepidermal water loss (TEWL), indicated compromised integrity, and a 14-fold higher hydrocortisone flux compared to a model of healthy SC [96]. Mechanistically, higher amounts of MUFAs yielded higher conformational disorder and induced a phase transition

from

orthorhombic

to

hexagonal

packing,

thereby

decreasing

lipid

compactness and reducing the SC barrier function [96]. A study using a model mimicking the lipid composition of psoriatic scales suggested that hydrocortisone flux through psoriatic skin is reduced compared to healthy skin [97]. The authors showed a higher content of phase-separated cholesterol was responsible for the reduced flux, indicating that factors other than altered lipid composition might be equally or more significant in affecting the SC barrier in psoriatic skin. While these experimental models are highly instructive from a mechanistic point of view, from the clinical point of view it should be stressed that there is a wide spectrum of psoriasis phenotypes beyond the most common plaque psoriasis [98, 99]. These phenotypes occur at different body sites, there may be progression from one to another over time, and skin permeability may be

differentially impacted. Inverse psoriasis, for instance, is characterized by thin erythematous plaques, no scaling and occurs at moist, occluded body sites such as the axillary or perineal areas [99]. Depending on the chemical, scars may also affect skin permeation, but results are contradictory. Early studies investigated the penetration of compounds through scarred skin to evidence the role of hair follicles in skin penetration, since scarred skin is appendage-free [100, 101]. Percentages of absorption and accumulation into the skin generally correlated with lipophilicity and were, for the most lipophilic chemical, 3 and 8fold higher in normal skin 8 hours after application [100]. In a later study, TEWL was found to be about 5-fold higher in freshly scarred vs. healthy skin, indicating a loss of the skin barrier integrity [102]. Microscopic observations revealed that more than a change in lipid composition, alteration of the corneocytes’ envelope (loss of hydrophobicity, formation of fine wrinkles) might lead to the decrease in barrier integrity. These observations were used to rationalize the increased penetration of 5-fluorouracilcontraining ethosomes, lipid carriers, in hypertrophic scar vs. healthy tissue [103]. Here also there is a need for clinical distinction between different types of scars. Hypertrophic scars are raised scars due to dermal collagen overproduction, whereas atrophic scars are sunken due to loss of fat or muscle tissue at the affected site. A 2- to 3-fold higher average TEWL was measured in hypertrophic scars vs. atrophic scars of volunteers, although the variability in the hypertrophic scar measurement was large [104]. To our knowledge, there is no study directly comparing the penetration of xenobiotics through different types of scars. Scar inflammation would likely further alter the permeability barrier properties of the affected skin sites.

2.2. Age 2.2.1. Infants Knowledge of age-specific physiological, pharmacokinetic, pharmacodynamics, genetic, behavioral and exposure parameters relevant to the pediatric population risk is required for effective assessment [105]. With regards to structure and physiology, the skin of healthy full-term newborns has smaller corneocytes, faster cell turnover, thicker epidermis, increased hydration, seborrhea and higher skin surface pH compared to child and adult skin [13, 106, 107]. These differences are, however, sufficiently small and short-term for the barrier property of the skin to be considered fully functional upon birth [107-109]. Pre-term infants, on the other hand, have underdeveloped skin that can take

from within a week to several weeks to reach full barrier capability, depending on the infant’s gestational age [13, 106, 110-112]. The impaired barrier in pre-term neonates has been shown from TEWL measurements. Depending on the postnatal age, skin of pre-term infants displays TEWL values of about 30 g/(m2 h) and up to 60 - 70 g/(m2 h) [113, 114]. TEWL values of healthy infants of are on the order of 10 g/(m2 h) [115]. Skin permeability studies show that the permeability of excised pre-term infant skin can be 100 to 1000 times greater than full-term infant skin for GA < 30 weeks. After 32 weeks GA, this ratio decreases to 3 to 4-fold [108]. Mean permeability coefficients of salicylic acid and lidocaine decreased from 76∙10-4 to 1.7∙10-4 cm/h and 370∙10-4 to 0.13∙10-4 cm/h, respectively, with GA increasing from 24-25 to 36-40 weeks [116, 117]. Age-related toxicity following skin exposure in the full- and pre-term neonatal population can occur due to factors not directly related to the skin permeability barrier. These include an underdeveloped subcutaneous fat tissue layer which can yield increased systemic uptake [13], higher skin surface area-to-body weight ratio yielding greater absorption and permeation on a body weight basis [13, 111, 112, 118], as well as lower renal excretion and higher hepatic metabolism, potentially reducing toxicant clearance and/or increasing the production of toxic metabolites in the body [108, 112, 118]. As a result of these factors, topical application of chemicals such as alcohol or iodine in antiseptics, lidocaine-prilocaine cream and diaper dyes or powders can lead to a variety of local and systemic toxicities in the infant and child populations [111]. Increasingly prevalent skin conditions such as atopic dermatitis and irritant contact dermatitis [119121] and trauma to pre-term infants [118] may further exacerbate skin absorption and ensuing toxicity. The barrier property of adult skin measured via TEWL depends to a small extent on body site [122, 123], and it appears reasonable that the same would apply in the newborn. However, there is, to our knowledge, no study proving body-site dependent efficacy of the skin barrier in neonates.

2.2.2. The elderly Skin aging depends on intrinsic factors (biological aging) and extrinsic factors (e.g., steroid-induced atrophy [124] or UV exposure (see section 2.3.4). Changes to the cutaneous vasculature with biological age may affect the systemic uptake of xeniobiotics. These include increased blood vessel stiffness, reduced reactivity affecting vasodilation and vasoconstriction and impaired angiogenesis (reduction in the number

and density of blood vessels) and arterosclerosis (deposition of fatty material on the interior walls of the arteries) [125]. The effect of biological aging on the structure and physiology of the skin, and in particular on the transcutaneous penetration of chemicals, is still a matter of debate [126, 127]. From a structural and physiological point of view, skin aging is characterized by thinning of the epidermis and the dermis, a slower rate of desquamation and decreased sebaceous gland activity [13, 128]. Other age-related intrinsic changes include reduction in acidification, decreased function of pH-dependent enzymes [128]. As for infant skin, TEWL measurements have been conducted to assess changes in skin integrity, but recent results do not present a consistent picture. A study with 150 women 18 to 80 years old showed a low but significant increase in TEWL with age at the site of the décolleté but none on the cheeks, neck, forearms or hands [129]. SC hydration levels generally increased with age on the forehead, neck and arm. Sebum production was lower in the 60-80 age group than in younger volunteers. Another study with 40 women aged 18 to 70 years showed a pronounced decrease in TEWL with increasing age on the cheek [122]. A slight decrease in TEWL with increasing age was observed on the photo-exposed arm, suggesting an improvement in the skin barrier function, but no change on the photo-protected arm [122]. In this study the SC thickness was found to significantly increase with age on the photo-exposed and photo-protected sides of the arm and at a lower rate on the cheek. Lipid compactness (obtained from spectroscopic Raman data) was found to decrease on the photo-exposed arm site only. The authors argued that increase in SC thickness was due to intrinsic aging, whereas changes in SC composition and lipid compactness depended on extrinsic factors (e.g., sun exposure).

2.3. External factors 2.3.1. Exposure dose Passive skin absorption kinetics can vary either proportionally or inversely to the exposure dose [4, 130, 131]. In the latter case, a higher applied dose can lead to a lower percentage of chemical absorbed into the skin than a lower dose, regardless of the type of species and for a variety of application conditions. A plausible explanation is saturation of the skin [4]. Figure 2 illustrates the inverse dose-absorption relationship in for a series of pesticides [132]. Within the animal studies, differences in applied doses may have led to an inverse dose-absorption relationship. High doses in these studies are 3 [133] to 63 [134] greater than the low doses. Most are 10 to 15 times greater. Such

differences may lead to decreases in skin absorption by factors of 42 or greater [4]. Whether or not a chemical exhibits an inverse dose-absorption relationship for a given dose range can also depend on other exposure factors (presence of a vehicle) and its propensity to damage the skin barrier [4].

2.3.2. Applied vehicles, occlusion and skin damage Vehicle and occlusion effects on skin permeation kinetics are difficult to generalize as they depend on active chemical and vehicle physico-chemical properties, relative concentrations and volatilities and can occur via multiple mechanisms. Surfactants enhance absorption into the skin via disruption of the SC lipid structure, specifically the periodicity phases of the lipid lamellae [135, 136]. SLS is also reported to hinder desquamation by preventing the natural degradation of corneodesmosomes [27]. In

vitro

cell

culture

work

has

shown

that

low-dose

SLS

and

cetyltrimethylammoniumbromide (CTAB) exposure may also directly affect the skin keratinocytes,

yielding

hyperproliferation

[137].

The

penetration

enhancement

mechanisms of the common solvents ethanol and propylene glycol is not well understood, with effects on lipid fluidization, extraction and conformational ordering, as well as effects on the solubility of the penetrant rather than on the SC lipids having been put forward [135, 136]. Mixtures such as hydrocarbons or cutting fluids add an extra layer of complexity due to interactions between the various components of the mixture [138]. In nearly all studies of percutaneous penetration through chemically damaged skin, penetration increases. The consensus is that a compromised SC particularly enhances the penetration of small, hydrophilic molecules. Examples include caffeine (MW = 194 g/mol, log Kow = 0.16), 5-fluorouracil (MW = 130 g/mol, log Kow = -0.95) and glyphosate (MW = 170 g/mol, log Kow = -1.7), for which irritation of full-thickness human skin yielded, respectively, 5, 8- and 24-fold increases in the in vitro permeability coefficient [139-141]. Slight damage to the skin may also yield important penetration increases, as shown with pesticides of varying aqueous solubilities, whose penetration in skin following treatment with 0.1 or 0.3% SLS increased up to 3-fold [142]. Erythema is a first sign of irritation and inflammation by induction of vasodilation in the superficial dermis. It leads to an increase in blood flow, which in turn can yield increased and faster transport of topically applied chemicals throughout the deeper cutaneous tissues [67, 143-145]. Increased urinary concentrations of butoxyethoxyacetic acid, a metabolite of the glycol ether

DGMBE, in the skin of volunteers displaying erythema or scaliness, compared to healthy skin, has been shown [146] The penetration of chemicals of average lipophilicity (log Kow ranging from 1.0 to 4.0) can also increase [140]. A positive correlation between salicylic acid (MW = 138 g/mol, log Kow = 2.3) penetration and the degree of irritation was shown, with penetration into human dermis increasing 146-fold in skin displaying severe dermatitis vs. intact skin [147]. Damaged skin may also yield increased penetration of larger compounds. Increased penetration of higher molecular weight oligomers (up to 1000 g/mol) through acetone-pretreated hairless mouse has been reported [148]. Occlusion of the site of topical exposure generally enhances absorption and penetration of a chemical into the skin. Occlusion can prevent chemical removal from the exposure site via wiping or evaporation as well as decrease TEWL and increase the SC hydration level. Increased hydration leads to swelling of the corneocytes and uptake of water into the SC intercellular lipid pathway [13]. One consequence of occlusion can be the induction of a reservoir of chemical in the skin, depending on the mode of occlusion. A study on the penetration of triamcinolone acetonide into the forearm skin of volunteers yielded 2-fold higher drug accumulation in the SC when the skin was occluded after application compared to occlusion prior to application [149]. The effect of postapplication occlusion could be observed 24h after exposure. This experiment attests to the transient effects of occlusion on skin structure. Occlusion may also increase the skin temperature sufficiently to affect skin penetration through increase in the chemical’s volatility, disruption of the SC lipid structure and/or alteration of local cutaneous blood flow [13]. Table 1 illustrates the effect of a change in vehicle or occlusion on the skin penetration kinetics of chemicals or types of chemicals whose reproductive and/or developmental toxicity following dermal exposure has been investigated (see Section 3, Table 4). Of note are the inverted U-shaped relationship between maximum absorbed flux and the percentage of water in the glycol ether-water mixture for butoxyethanol and ethoxyethanol [150], the decrease in DEGBEA steady-state flux following an increase in vehicle water content, contrary to the trend observed for four other glycol ethers [151] and the non-linear relationship between the DMF metabolic excretion rate and the time of exposure to DMF [152]. Skin flux and cumulative amounts of the petroleum substances benzene, toluene and the sunscreen component benzophenone-3 can vary considerably depending on the vehicle of delivery.

In occupational settings, exposure to combinations of irritants often occurs, possibly also with occlusive conditions. In these cases the skin barrier property can be affected differently than by either factor alone. Kartono and Maibach reviewed the effects of combinations of chemical irritants such as SLS, toluene, ethanol, water and acids on the state of the skin barrier [164]. The net cumulative effects of combinations can depend on the nature of the irritants and on the order of exposure. Cumulative effects the TEWL, cutaneous blood flow and/or the skin visual score can be either additive (equal to the sum of the individual effects), synergistic (greater than the sum of the individual effects) or quenching [164]. The duration of occlusion alone or in combination with an irritant is another important parameter. In a short-term exposure study, TEWL increased significantly after 3 and 4h of water exposure compared to baseline, but not after 2h [165]. Subsequent exposure to SLS increased TEWL in all three cases. The combination of occlusion and SLS-induced irritation increased TEWL only when occlusion lasted 4h. In another set of experiments, neither 6h occlusion nor combined 3h occlusion and 3h water exposure increased TEWL. However, both increased TEWL when they were followed by SLS irritation. The clinical score was significantly different from control for 6h occlusion followed by irritation, but not for 3h occlusion combined with 3h water exposure followed by irritation [165]. In long-term studies, continuous occlusion of healthy skin of volunteers over 72h or for 8h/day over 7 days yielded no differences in the state of the skin [166]. In contrast, 6h/day occlusion over 14 days led to an 40% increase in TEWL on day 14 [167]. Occlusion may act differently on irritated skin depending on the nature of prior irritation [166]. Occlusion of SLS-irritated skin for 72h increased TEWL moderately compared to non-occluded irritated skin. Occlusion following tape-stripping yielded lower TEWL than non-occluded tape-stripped skin. The nature of the damage was suggested as a reason. While SLS may increase lipid synthesis, tape-stripping removes lipids and occlusion may accelerate skin repair due to increased hydration [166]. Another study, however, found no significant difference in the TEWL of SLS-irritated or tape-stripped skin occluded over 46h [168]. The variability in the results suggests that the individual and synergistic effects of chemical exposure and occlusion are time-dependent. Studies using the same skin damage protocols for over different time spans are needed to understand the effects from a more mechanistic point of view. These time-dependencies may be altogether

different for the elderly who display slower recovery of skin barrier function following irritation than young people [169]. Moreover, these findings are not only applicable to solvents and surfactants. The tandem effect of irritation from a hand cleanser and occlusion from protective equipment (gloves) has been reported [170]. 2.3.3. Environmental conditions The efficacy of the skin barrier depends on environmental conditions, mainly ambient temperature, humidity, airflow and UV exposure. The effects of temperature have recently been reviewed [171]. Aside from potentially increasing a chemical’s intrinsic molecular diffusion and/or its solubility in a vehicle [171, 172], heat intrinsically modifies the skin. Temperatures around 40C and above can cause fluidization of the lipids and transitioning from the orthorhombic to the hexagonal packing configuration [171, 173]. Heat causes vasodilation and increased blood flow [174, 175]. Increased skin penetration and systemic uptake through local heating is the basis of heat-aided drug delivery patches [171]. Mean plasma concentrations of fentanyl and testosterone in volunteers were 3- and 2-fold larger, respectively, compared to delivery without heat [176, 177]. Local blood flow and nitroglycerine plasma concentration increased more than 2-fold in volunteers whose skin around the patch was heated by an infrared source [178]. In a nicotine delivery study, blood flow and plasma concentration increased 9- and 13-fold, respectively with local skin heating at 43C [179]. Studies focusing on the effect of an internal (e.g., blood or body core) temperature increase due to physical exertion or heated environments such as saunas showed 2 to 6- and 1.5-fold increases in nitroglycerin and nicotine maximum plasma concentrations [180, 181]. Comparison of the nicotine results suggests local skin heating could have a significantly greater effect on the systemic uptake of chemicals from skin than ambient temperature changes. In a toxicological context, the in vitro flux of haloacetonitriles and chloral hydrate through human cadaver skin increased 57 to 170% following an ambient temperature change from 25 to 40C [182]. This has clinical importance for the assessment of toxicity in cold or tropical climates. Effects of increased relative humidity (RH) include greater SC fluidity due to insertion of unbound water molecules within the SC lipids and altered protein (keratin) conformation [183]. A 90% RH coupled with a temperature increase yielded a 3- to 4-fold increase in the in vitro penetration of the pesticide parathion through pig skin compared to the baseline conditions of 60% RH, with the magnitude of the effects dependent on the applied parathion dose [184, 185]. Beyond the penetrant’s dose, effects of changes in

the environment depend on the vehicle, if any. In an in vitro skin penetration experiment with caffeine dissolved in ethanol, an increase in air temperature at high (70%) RH yielded a decrease in mean caffeine penetration due to crystallization of the later on the skin surface [186]. On the other hand, at low (28%) RH, the same temperature increase combined with ethanol desiccation led to a 2-fold increase in mean penetration. Abrupt environmental changes may also cause SC barrier impediment. An rapid switch from a humid (80% RH) to a dry environment (< 10% RH) led to a 5- to 6-fold increase in the TEWL of mouse skin within 2 days, before a return to baseline levels on day 7 [187]. The authors discuss the potential implications for individuals repeatedly exposed to drastic temperature and humidity changes in high-humidity environments, particularly those who already have an intrinsic skin condition such as atopic dermatitis. Airflow (wind velocity) is of significance for volatile compounds, affecting the balance of evaporation to absorption at the skin surface [188, 189]. UV exposure to the skin causes both acute effects and chronic conditions which compromise the barrier properties of the skin and can affect percutaneous penetration and systemic uptake of xenobiotics. Acute effects in human skin include thickening of the SC and viable epidermis [190-194], epidermal pleomorphy and spongiosis (intercellular edema) [191] and alterations of SC lipids leading to decreased intercellular lipid cohesion, increased lipid fluidization and SC hydration [195]. Local cutaneous blood flow is affected through telangiectasia (dilation of blood capillaries) [196, 197]. In areas with constant low dose UVA exposure, cutis rhomboidalis nuchae (deep furrowing of the skin) [198] and increased epidermal and dermal thickness are observed, particularly on the dorsal sides of the neck and hands. The overall consequence of UV exposure is a weakening of the skin barrier. Nicotinic acid absorption into the severely UV-damaged skin of rats was equivalent to absorption through tape-stripped skin, that is, with a severely deficient SC barrier [199]. In another study, the cumulative amount of 5-aminolevulinic penetrated acid through chronically UVB-exposed mouse skin was 10 times greater than through healthy skin 8 hours after application, but 20 times less than through tape-stripped skin in which much or all of the SC was removed [200]. Recent studies have sought to link the specific effects of UVA and UVB irradiation to the permeation profiles of chemicals of varying lipophilicities. The increase in permeation of hydrophilic and moderately lipophilic chemicals through mouse skin was shown to be greater than for a strongly lipophilic chemical, especially following UVA exposure, due to damage to the SC and viable epidermis caused by the irradiation

[201, 202]. For some chemicals, UVB irradiation may have no significant effect or may decrease the flux through and retention in the skin [202]. Further work with a greater number of chemicals is required to generalize these observations and extrapolate them to human skin.

3.

HUMAN VS. ANIMAL SKIN

3.1. Physiological / structural differences Assessing physiological and structural differences between human and animal skin is challenging because of the high variability in human skin due to body site, age, and exposure to a variety of external factors. This is particularly the case for parameters such as human skin thickness and blood flow [203]. Table 2 compares important human and animals parameters that influence transcutaneous permeation, focusing on body sites most likely to be exposed to toxicants in typical occupational settings. The average human skin thicknesses listed are from studies conducted with volunteers whose range from the 20s to the 70s. For animals, the focus is on rat, rabbit and mouse skin as these are the animal models most commonly used in reproductive and developmental toxicology. While the periodical structure of the lipid lamellae is nearly same in the SC of different mammalian species [204, 205], differences are seen in the packing organization and lipid composition. Lipids in rat SC are predominantly hexagonally, less orthorhombically, and therefore less densely packed than human SC lipids [206]. Rat and rabbit SC lipids include a lower percentage of ceramides than human SC. The rat skin layer thicknesses are comparable to those of human on many body sites, but mouse and rabbit skin are significantly thinner. Follicular densities differ vastly, especially when animal skin is compared to human body sites other the scalp. Cutaneous blood flow rates also differ significantly across species and across body sites within a species [207]. Human vs. animal differences in enzyme expression and in the impact of metabolism on xenobiotic skin permeation is difficult to assess, particularly quantitatively, due to the scarcity of animal data [208]. Qualitatively, it appears clear that rat and mouse skin are average models of human skin, with similar activities of oxidoreductases, hydrolases and conjugating enzymes, but porcine skin is a better model.

3.2. Skin permeability differences

Permeability of compounds through healthy mouse, rabbit and rat skin is generally greater than through healthy human skin. The nature of animal skin appears more significant in determining differences than the physico-chemical properties of chemicals. Mouse, specifically hairless mouse skin, is deemed an unsuitable model for human skin permeability work because of the thinness of the layers, the need to hydrate it thoroughly to make it comparable to human skin, and its higher susceptibility to damage [209]. The rabbit vs. human skin comparison yields inconsistent results which depend on the chemical’s lipophilicity [209]. It should be noted, however, than percutaneous penetration studies use rabbit ear skin, which on the whole is thinner than on the animals’ back (Table 1). There is, to our knowledge, insufficient data to make a clear statement comparing permeation through rabbit back and human skin. Though rat skin is a common model for skin permeation both in pharmaceutical and toxicological research, its permeability generally overestimates that of human skin [209-211]. For only about a fifth of the chemicals reviewed in [209] is rat skin considered an approximate model for human skin. About half of the chemicals surveyed permeate rat skin faster than human skin, that is, with a 3- or greater fold difference. Table 3 provides the rat-to-human factor of difference for some chemicals for which developmental and/or reproductive toxicity following skin exposure has been studied (see Section 3, Table 4) A formula for calculating the percentage of human skin penetration from in vitro human and rat and in vivo rat data has been proposed [209, 211]. However, the utility of such a formula has been called into question in light of method-specific variability in the percentages obtained [212]. Moreover, percentages of skin permeation can be potentially ill-suited to specific occupational scenarios and exposure conditions [213]. Despite the high skin exposure doses used in animal toxicity experiments and the generally higher permeability of rodent skin, there is little evidence suggesting that pre-term infant skin permeation can be approximated by an animal model [110]. The effect of age on rodent skin permeation appears to be chemical-dependent [214] and there is no evidence that animal models can help predict skin permeation in the elderly. Adequate use of an animal skin model also requires an understanding of the effects of skin damage due to chemical exposure and occlusion across species. To date, little experimental work has addressed this issue. One study on cutaneous effects following petrolatum exposure to various species revealed that New Zealand white rabbits displayed mild acanthosis, hyperkeratosis, edema, erythema and inflammation, whereas Wistar-Han rats and two mice strains presented no effects [215]. These results imply

that a study incorporating skin absorption and permeation in different species may need to ―normalize‖ the kinetics results for the response of the species’ skin to the treatment. In the next section we review published in vivo (animal) developmental and reproductive toxicology studies. The focus is on the range of exposure conditions used in the studies, their relevance to occupational exposure scenarios, and on the ways in which observed skin damage was addressed.

4.

CURRENT PRACTICES IN IN VIVO SKIN DEVELOPMENTAL / REPRODUCTIVE TOXICITY STUDIES

Table 4 provides an overview of experimental protocols used in in vivo developmental and/or reproductive toxicology studies in which toxicity following skin exposure was investigated. The focus is on the elements of the exposure scenarios which are most likely to impact the amount of chemical reaching the systemic circulation. This includes not only the doses of the potential toxicants applied to the skin of animals, but also that of additional chemicals applied as vehicle, the frequency of application, whether the exposure site was occluded, and the clinical damage to the skin resulting from the treatments. The references in Table 4 were obtained from the Fertility and Developmental Toxicity in experimental animals (FeDTeX) database [242] and a PubMed search spanning the years 1982 to 2014. The PubMed search was conducted using combinations of the search terms ―dermal‖, ―developmental‖, ―estrogen‖, ―estrogenic‖, ―reproductive‖, ―skin‖, ―teratogen‖, ―teratogenic‖ and ―toxicity‖, in September 2014 and in March 2015. Exposure dose ranges used in the animal developmental toxicity studies correspond to very high human equivalent doses, the goal being to obtain a demonstrable effect at the highest dose of exposure and a NOAEL at the lowest dose [300]. The presence or lack of a removal step affects daily exposure doses. Among the studies which include a removal step, removal times range from approximately 2 to 6 hours after application. The choice of 6 hours agrees with Kimmel and Francis’ suggestion (based on convenience, not scientific evidence) [2] but the reasons for other application durations are unclear. In particular, ―at least‖ 3 hours is not defined precisely in [246] and, in [267], no reason for a difference in application durations in the rat and rabbit experiments is given. The daily application durations in the two glycol ether studies [261, 262] are significantly different, since the DGMME study did not include a removal step, while the DGMBE study included daily removal of the chemical 4 hours after application. Such

differences make it difficult to compare the relative toxicity following skin exposure of these glycol ethers. There are disparities in the use of vehicles in the animal studies. In studies of heavy industrial mixtures (BUP, CSO, crude oil, COM, DAE and STB) lack of a vehicle makes sense, as these compounds are likely insoluble in many solvents and can be expected to come directly into contact with the skin in industrial settings. Diethylene glycol monoethyl ether was used to dissolve trans-capsaicin in Chanda et al’s study because this is the solvent contained in the NGX-400 capsaicin patch designed for human use [246, 301], but capsaicin is also used as a fungicide and insecticide [302]. Omadine MDS was dissolved in shampoo blank because it is an antimicrobial and preservative used in shampoos and cosmetic products [303]. The use of olive oil as a vehicle for 4– MBC [278] mimics the lipophilic nature of sunscreen components. For other chemicals, complementary studies on the impact of the vehicle used, or the lack of one, would provide a fuller picture of cutaneous uptake and potential toxicity of the chemical. DMF is used in various industrial settings because of its high solubility in water and investigations of toxicity following exposure to aqueous solutions of DMF, in addition to undiluted, would provide valuable information [156]. Glycol ethers such as DGMME and DGMBE are industrial solvents that are soluble either completely (DGMME) or in proportions (DGMBE) with a variety of substances - water, alcohols, ketones (ex: acetone), ethers, aromatic hydrocarbons and halogenated hydrocarbons [304]. In some studies, vehicles appear to have been used to facilitate the experimental procedure. DEA and MEA were applied in aqueous solution, but are used in a variety of personal care products, including shampoos and soaps and cosmetic products as well as in detergents and industrial products [155, 305, 306]. HDS kerosene was dissolved in mineral oil to reduce irritation to the animals’ skin and loss of kerosene due to evaporation [250, 251]. Other studies appear to have used solvents such as acetone [259, 276], ethanol [288, 296] or corn oil [297] for improved dissolution of the compounds of interest. These solvents, however, do not seem relevant to the personal care product contexts. Another element that varies significantly is occlusion of the site of application. For many of the industrial compounds listed in Table 4, the site of application was un-occluded. When occlusion is used in animal experiments, it is to prevent evaporation of volatile compounds, avoid disturbance of the exposure area during grooming as well as inhalation or ingestion of the compound by the animal [300]. These measures are

needed for the reliable observation of effects. It should be kept in mind, however, that the animal protocol may not correspond to real-life occupational exposure, which may be compound- and/or context-specific, for example when protective gear is worn [5]. There is a significant range in the severity of skin damage due to effects of the chemicals of interest and/or the solvents used. The shaving or clipping of the hair on the site of application may have itself induced irritation or inflammation [307]. The observed clinical signs range from mild to severe and include erythema, edema, scaling, fissuring and lesions. One or more of these observations are reported in nearly all studies. Some reported no clinical effects on skin and one [284] did not provide any information. Among the studies reporting severe irritation, the severity and the number of animals affected was often dose-dependent and in some cases species-dependent (Table 4). Tyl et al also indicated a progression in severity during the gestational period in their rabbit experiment [296]. Twelve studies reported erythema. Among these, descriptions of the severity of erythema vary or are omitted. Some reported low levels, grade 1 erythema or ―mild‖ erythema for some of the rats and at some doses, respectively. Inclusion of results from such experiments is warranted [2]. On the other hand, two studies [263, 266] reported ―severe‖ erythema in their rabbit studies at high doses; those results should be excluded. In total 13 studies provide data on skin permeation and/or systemic uptake kinetics (Table 5). Not all, however, reported kinetic information relevant to the exposure scenario of the toxicological study. In addition, the studies do not discuss how the observed clinical damage to the skin might have affected the kinetics of absorption and permeation or how it might compare to human skin barrier disruption.

5.

PBPK MODELLING

5.1. PBPK models to predict in vivo human toxicity The preceding sections underline the number of factors that can impact the systemic uptake of a potential toxicant following skin exposure. Aside from the physico-chemical nature and dose of the compound, exposure conditions, clinical effects of exposure on healthy human skin, and possible pre-existing conditions in target populations, age and body site can influence cutaneous and systemic bioavailability. When comparing human vs. animal skin permeation, intrinsic skin physiology and structure differences and variations in clinical damage at the site of exposure should be accounted for. Effects are often non-linear, synergistic and species-dependent. Additionally, many effects are

understood only from in vitro experiments conducted with excised human or animal skin, with limited knowledge on the in vivo situation. This makes the establishment of general rules capturing the quantitative impact of these factors, and for the interpretation of animal studies, very difficult. Oral or respiratory route to skin route extrapolation methods rely on default absorption values and apply under strict conditions only [310, 311]. For many chemicals and mixtures, capturing all relevant effects for a comprehensive study of toxicity and occupational risk assessment in animal experiments appears unrealistic, particularly in developmental and reproductive toxicology. Physiologically-based pharmacokinetic (PBPK) modeling represents a promising method to achieve an approximate, but reliable quantitative understanding of the impact of the abovementioned factors. PBPK models are used to predict the disposition of chemicals in the body through mathematical description of the processes of absorption, distribution, metabolism and elimination (ADME). PBPK models can handle a variety of input parameters related to the physico-chemistry of the chemical of interest, body and organ physiological parameters (cardiac output, organ volumes, blood flow rates), effects of environmental conditions on these parameters, route(s) of administration and exposure scenario, as well as population variability [312]. PBPK models can thus be used to establish in vivo and in vitro animal-to-human correlations, as well as integrate available in vitro, in vivo and in silico data for extrapolation to the human in vivo situation of interest [313, 314]. They are useful in hypothesis-testing and design of more targeted in vivo studies [315]. Studies of the drug tretinoin (all-trans-retinoic acid) conducted during the 1990s illustrate how PBPK modelling could utilize past data to predict internal human exposure with relevant skin exposure scenarios and application conditions. Clewell et al developed a whole-body PBPK model for the disposition of tretinoin following oral and dermal exposure [316]. The model was parameterized for humans and animals (mouse, rat, monkey) using in vivo data. Simulations of animal oral and human oral and dermal exposure, where the later corresponded to application of tretinoin in an emollient cream, were run to obtain maximum plasma concentrations Cmax and AUCs in each case. Results showed the Cmax to be the more reliable surrogate metric for extrapolation between species, in agreement with a comprehensive experimental pharmacokinetic and teratogenic study in hamsters [317]. The PBPK model enabled the authors to explain differences in internal exposure between animals and humans due to differences in the basic detoxification pathway of tretinoin as well as dose-dependent differences in

clearance. In a later paper, in vitro embryonic stem cell test EC50 data was extrapolated to an in vivo plasma EC50 value [318]. Reverse dosimetry was then conducted using Clewell et al’s PBPK model to predict an equivalent in vivo oral tretinoin dose leading to toxicity. This last value was comparable to a LOAEL obtained from an in vivo rat study [318]. Proper simulation of the effect of exposure scenarios goes hand-in-hand with a sufficiently sophisticated, but also flexible mathematical description of the internal, cutaneous physiological factors impacting skin permeation. This was illustrated early on [319] and based on fundamental work on mass transfer in skin [320]. To simulate dermal uptake of chloroform and subsequent exhaled breath concentration, a whole-body PBPK model incorporating either a lumped parameter or a distributed parameter SC model was used. In the former, the SC or SC and viable tissue are modelled as homogeneous compartments, in which permeant concentration is equal throughout the compartments (Figure 3). In the later, passive molecular diffusion through the SC is modelled using Fick’s 1D equation and associated initial/boundary conditions. Permeant concentration is a function of time and distance away from the air-skin boundary; resistance to permeability is distributed throughout the SC, not localized at the air-SC boundary (Figure 3). The differences in the description of internal uptake of chloroform affect the way the external environmental conditions, allowing the applied chemical to evaporate, are handled, because the evaporation-diffusion equilibrium is handled more precisely. The overall outcome was that the ―more physiological‖ PBPK model incorporating the distributed parameter SC model yielded better predictions of exhaled chloroform concentration. In particular, more precise values of maximum dose absorbed into the skin and systemic dose (predicting a lag time for systemic uptake) following short-term exposure were obtained. Other studies showing the need for a physiological description of skin permeation processes within whole-body PBPK models include references [321326]. One PBPK model includes the number of corneocyte layers in the SC, corneocyte thickness and corneocyte overlap used to calculate permeant flux into the SC and viable tissue and absorption lag time [326]. The sensitivity of the penetration lag time to the stratum geometrical parameters suggests the need not only to model macroscopic transport in a physiological manner, but also microscopic transport at the level of the corneocytes and lipid phases of the SC.

5.2. Combining whole-body PBPK and microscopic skin permeability models for greater predictive value Several skin transport models incorporating microscopic properties of the skin layers directly into calculations of flux and concentration in the skin have been developed. These include models from research groups at the University at Buffalo and the University of Cincinnati (UB/UC), the Massachusetts Institute of Technology (MIT) and the China Agricultural University (CAU) [327]. We have recently developed a whole-body PBPK framework connecting a generic whole-body PBPK model to the UB/UC model [41]. With respect to skin permeation, the model’s significant features are a detailed structural description of the skin layers, including a two-dimensional microscopic description of the SC, first-principles based transport equations and partition and diffusion coefficient estimation based on empirical correlations. Permeant flux, cumulative amount and concentration in the SC and viable tissues are calculated as a function of time and depth into the skin. The model simulates evaporation of volatile chemicals, a variety of exposure scenarios and the effect of skin surface temperature and airflow. Using this whole-body PBPK framework we showed quantitatively the impact of differences in skin exposure scenario and application conditions that are prevalent in occupational settings (single vs. repeated application, finite vs. infinite dose application, occlusion of the exposure area, presence of a vehicle in which the toxicant is dissolved) on skin permeation kinetics and the ensuing uptake into the systemic circulation [328]. Figure 4 shows the differences in calculated steady-state plasma concentrations for different exposure scenarios and application conditions. Application of an infinite dose, which does not deplete over the duration of exposure, vs. a finite dose, and simulation of occlusion of the exposure area vs. non-occlusion had the largest impacts on the steady-state plasma concentrations, with increases up to 30 (for 2-ME) and 40-fold (for DGMME), respectively. We later used an updated version of this PBPK framework to estimate the external exposure dose of four reproductive toxicants from in vitro data using reverse dosimetry and simulate different skin exposure scenarios [213]. These results showed that, due to differences in skin permeation kinetics, different external dermal doses could be equivalent to a given internal plasma concentration leading to toxicity. Predicting skin permeation and systemic uptake following exposure to certain active chemicals and/or vehicles may necessitate modelling damaged or diseased skin. PBPK models for pathologies such a liver cirrhosis and renal disease, and data needed to

parameterize these models, exist [329-331]. To our knowledge, no equivalent studies for impaired and diseased skin exist to date. Simulations using a whole-body PBPK model incorporating detailed skin transport appear to be a good starting point to obtain orderof-magnitude changes in chemical uptake at the target site due to a compromised skin barrier and/or changes in cutaneous blood flow. This problem has been approached using the UB/UC model by simulating the transport of 1,2-dichloroethane, a widely used industrial chemical [332]. The steady-state skin flux calculated a priori using nominal SC geometric parameters underestimated experimental values by a factor of 150-250. The predicted value was much closer to the experimental value once the SC thickness in the model was reduced, simulating stripped-off SC. PBPK models with an adequate description of the skin appear well-suited to simulate the absorption and fate of chemicals in the body in cases of skin disorders (e.g., atopic dermatitis) associated with chronic kidney disease, reviewed in references [333, 334]. The flexibility afforded by detailed skin permeability models also makes them useful for the estimation of whole-body drug distribution in high-risk populations such as pre-term infants and the elderly. The adjusted skin permeability model needs to be combined with a PBPK model parameterized for the population of interest, that is, with relevant data on height, body and organ weights, hepatic and renal metabolism/clearance, plasma protein content and tissue blood flow rates [331, 335-338]. A PBPK model was used to predict the plasma concentration of bisphenol A (BPA) in children less than 2 year old following relevant feeding schedules [339]. The model was used to estimate the effects of infant vs. adult physiology and of differing oral exposure scenarios on plasma concentrations of BPA and its glucuronated metabolite. Changes in the glucuronidation process within the first 2-3 months yielded 11-fold higher BPA concentration in newborns vs. adults. A switch in BPA exposure via polycarbonate bottles (4.0 to 8.3 g/kg/day) in the 3 to 6month age range to an intake scenario via foods and beverages for adults (1.5 g/kg/day) produced a 5-fold decrease. Similarly, plasma concentrations of the antiarrhythmic drug sotalol were simulated over the full pediatric range, from newborns to adolescents [340]. While the model predicted experimental plasma concentrations in the infant to adolescent ranges within two-fold error, the error was greater for the neonatal data prediction. This was attributed to insufficient anatomical and physiological data on processes affecting sotalol absorption as opposed to elimination [340]. Depending on a compound’s physico-chemical properties and the exposure scenario, the flux of permeant cleared from the skin into the systemic circulation predicted by the

UB/UC model is sensitive to skin layer thicknesses, the skin hydration level [41, 328, 332] and well as the SC lipid fraction and blood flow (unpublished results). Experimental values for these parameters in specific populations (neonates, adults or children with specific skin conditions, workers in a given occupational setting, etc.) could be utilized in a whole-body PBPK framework parameterized for the population of interest. Such a PBPK framework would be useful towards estimating variability in internal dose across populations and identifying sub-populations at risk, an important goal in chemical risk assessment as well as clinical pharmacology [341-344].

5.3. Improving PBPK models for skin exposure Currently in the UB/UC model, the effect of occlusion and application of aqueous or ethanol-based vehicles on SC physiology and transport parameters can be modelled by switching from a partially hydrated to a fully hydrated state. This is achieved through the use of two different sets of values for the geometrical and structural SC input data affected by the hydration level (SC thickness, corneocyte horizontal period, water, protein, lipid, corneocyte,

and keratin fractions) and to a rescaling of the SC lipid

diffusion and transbilayer permeability coefficients [41]. Comparison of simulations and experimental data from solvents such as ethanol and acetone, which are highly soluble in SC, suggest modelling transient SC hydration and swelling would be more appropriate [332]. Observed skin penetration can be more prolonged than predicted by a Fickian diffusion model [189, 345, 346] due to solute accumulation in the tissue (the reservoir effect). Comparisons of experimental and simulation results suggest that the traditional description of diffusion and partition coefficients as constant within a skin layer and at a given interfacial boundary, respectively, may be inappropriate for certain chemicals and/or under certain exposure conditions. In such cases, the diffusion and partitioning coefficient might depend on the chemical’s local concentrations in the skin and vary with time [189, 345, 346]. More experimental data is needed to formulate a sound theory. Improving systemic uptake simulations calls for a more sophisticated description of the effect of blood flow on skin permeation. In vivo skin permeation models, including the UB/UC model, generally incorporate blood flow as a 1st order elimination process following passive diffusion to the viable tissues. Analyses of in vivo human experimental data show that dermis and deeper tissue concentrations cannot always be solely explained by passive diffusion. Depending on the permeant’s physico-chemical

properties, transport to and redistribution within the deep tissues may occur as a result of blood flow-mediated convective transport [67, 347]. Modelling these biophysical processes necessitates different partial differential equations than those used to model SC transport, but they can be coded into a multi-layer model such as the UB/UC model. The main hurdle at present may be the lack of deep tissue human skin experimental data allowing for a generalization of the phenomena described in references [67, 347]. Revisiting older in vivo human skin penetration data and animal-to-human extrapolation using the animal pharmacokinetic models developed by Roberts and colleagues (e.g., [348, 349]) appears to be the way forward. For greater physiological realism, skin permeation models incorporated into whole-body PBPK models also need to include cutaneous metabolism. Mathematical descriptions of metabolism in skin transport models are summarized in reference [350]. MichaelisMenten kinetics was used to describe cutaneous metabolism in a PBPK model for the disposition of the cosmetic ingredient hydroquinone, but additional experimental data might lead to changes in the mathematical formulation [321]. Incorporating skin metabolism also helps to model a potentially important effect of skin irritation or disease. In psoriatic skin, a number of enzymes (cyclooxygenase-2 (COX-2), cytochrome P450 reductase (CPR), heme oxygenase-1 (HO-1), gluthathione S-transferase M1 (GSTM1), gluthathione S-transferase P1 (GSTP1) gluthathione peroxidase-1 (GPx-1), MRP1, NADPH and CYP2E1) have elevated activity compared to healthy skin [351, 352]. Expression of certain cytochrome P450 enzymes, CYP1B1 and CYP3A5 is decreased in psoriatic skin [351]. Although cutaneous metabolism is lower than hepatic metabolism, it is not negligible for chemicals that are substantially metabolized within the time course of permeation through the viable tissue, particularly since the skin is the largest organ and in cases in which large surface areas could be exposed. Models incorporating the effect of non-ionizing visible and UV light in combination with cutaneous metabolism, possible sequestration of UV-absorbing filters in the dermis and realistic exposure scenarios could help resolve controversies in the area of systemic toxicity from sunscreens [353]. With respect to pediatric populations, skin layer thicknesses, water, lipid and protein contents, and pH have been described [109, 354, 355] and can be incorporated into a sufficiently detailed skin model. To our knowledge, however, no study has addressed the metabolic enzyme content of neonatal skin and its evolution with age, or proposed an extrapolation method from adult skin data. For general pediatric PBPK modeling, there is a need for refinement in the extrapolation of anatomical and physiological parameters

between age ranges [340, 356] and to better identify relevant covariate parameters for a given physiological process, including age-dependent changes in covariates [335, 356]. Finally, skin permeation models that can predict flux and concentration profiles of mixtures components are needed. Quantitative structure-permeability relationships (QSPRs) have been developed to predict steady-state permeability coefficients [138]. These relationships yield an understanding of the effects of component interaction in the steady-state, but do not reveal their effects in the short term following exposure. An important step towards this goal was achieved with the recent development of an extension to the UB/UC model for multiple components [357]. The model extension yielded improved predictions of the transient in vitro absorption of vanillylnonanamide (a CAP derivative) from a propylene glycol vehicle for a small applied (finite) dose, in particular.

6.

OUTLOOK

Animal-based developmental and reproductive toxicity studies are meant to identify the potential for adverse effects due to exposure and to determine a NOAEL or NOEL [300, 358]. Doses applied to the skin of animals in developmental and reproductive toxicity studies generally exceed by orders of magnitude the doses that humans would be exposed to in the same time frame. Results indicating no effect in the animal, combined with skin permeation studies that show greater penetration through animal skin than through healthy human skin, may lead to the conclusion that the compound is not a developmental or reproductive toxicant for humans. This conclusion, however, presupposes rapid clearance of the compound, as shown in a comprehensive study of PEA, which is highly volatile (limiting its absorption into skin) and highly excreted in the urine [237, 291]. For other chemicals, studies showing no effects in animals, or only at intermediate and high doses, may not account for long-term effects in humans. These can result from accumulation of the chemical in the fat tissue (e.g., for DDT [359]), or in populations suffering from diseases affecting elimination (e.g., chronic kidney disease). Reliability of toxicity predictions requires the investigation of relevant exposure routes and support from well-defined kinetics [360]. In the case of skin exposure, a multitude of factors in various populations and in different clinical and occupational settings can influence the kinetics of skin permeation and systemic uptake. Animal-based studies cannot realistically test for all factors [13]. In vitro assays, being designed to eventually replace animal tests, focus on sensitive targets identified in the animal studies and

toxicity mechanisms at the cellular level [1, 10, 11]. Organotypic 3D skin cultures present significant advantages over cells cultured in monolayers by mimicking the in vivo physiology and cellular organization [361, 362]. They show high potential for reasonable approximation of not only skin permeability [363] and cutaneous metabolism [208], but also intrinsic and environment-mediated skin pathologies [361, 362]. Non-invasive in vivo imaging methods, e.g., multiphoton microscopy, photoacoustic imaging and Raman spectroscopy, are being developed to visualize healthy and diseased skin and quantify skin permeation of chemicals with and without vehicles [364-367]. Data from these various methods should be integrated to further validate in vitro and in silico methods of toxicity prediction and risk assessment. The method of choice to predict safe human dermal doses based on the relevant kinetics are animal-to-human and in vitro-to-in vivo extrapolation via PBPK modelling [9]. To this end, advanced PBPK models with a detailed and flexible description of skin structure and physiology, yielding transient skin permeation and target site concentration profiles are needed. The models should accommodate any dermal dose and realistic exposure scenarios to be used in a life stage approach to risk assessment [368, 369]. This review highlights the interdisciplinary nature of toxicology and risk assessment. In the context of toxicity resulting from skin exposure, there is need for collaboration between not only toxicologists, risk assessors and mathematical modelers or physicists, but also molecular biologists, pharmaceutical scientists and dermatologists who can assess the consequences of exposure to chemicals on the skin barrier and subsequent clinical effects.

7.

ACKNOWLEDGMENTS

YD’s work was funded by the A*STAR Strategic Positioning Fund.

8.

REFERENCES

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FIGURE CAPTIONS Figure 1: Schematic overview of the skin layers and the vascular network in human skin, and factors influencing the penetration of chemicals into and through the skin.

Figure 2: Comparison of ratios of skin absorption following high dose vs. following low dose skin exposure for 19 pesticides. Data obtained from Thongsinthusak et al [132]. For 15 of the 19 chemicals (filled-in dots), an inverse absorption to applied dose ratio was observed.

Figure 3: Schematic illustrating the fundamental difference between modelling a skin layer as a homogenous compartment vs. as a membrane in which concentration is calculated as a function of depth and time using Fick’s diffusion equation (adapted from [319]). For volatile chemicals, the mathematical treatment of solute disposition within the tissue impacts the way application conditions (e.g., occlusion vs. non-occlusion) are handled, since the PBPK models cited herein model the evaporation-diffusion equilibrium at the air-skin interface.

Figure 4: Comparison of steady-state plasma concentrations obtained from human skin permeation simulations of 9 chemicals with 1 to 3 different applied doses and different application conditions. (a) Applied human equivalent doses derived from the animal doses as described in [328]. (b), (c) Scenario ―A‖ designates the scenario used the toxicological studies (Table 1 in [328]). The other scenarios are identical to ― ‖ but for one modification. ―B‖: application of an infinite dose instead of a finite dose; ― occluded‖ and ―― non-occluded‖: simulation of occlusion instead of non-occlusion or vice-versa; ― 50% water‖ and ―

0% water‖: simulation of the chemical in a 50% and

aqueous solution instead of neat; ― instead of in a vehicle.

0% (v/v)

undiluted‖: simulation of the chemical applied neat

Tables Table 1: Changes in skin penetration or permeation kinetic parameters corresponding to changes in the vehicle or an element of the exposure scenario. Chemical

Change in exposure parameter

Effect on kinetic parameter

Butoxyethanol

Increase in water vehicle content:

Mean maximum flux:

Ethoxyethanol

CAP

DEA

- from 0 to 50 % - from 50 to 90 % Increase in water vehicle content:

7.0-fold increase 4.1-fold decrease

[150]

Mean maximum flux:

- from 0 to 50 % - from 50 to 90 % Change in vehicle from

Mean AUC and

isopropyl alcohol to:

concentration decrease:

- Propylene glycol - Mineral oil Change from shampoo, bath

3.0 to 3.3-fold 2.7-fold Mean cumulative

and emulsion to hair dye

amount permeated:

formulations and applications

Reference

2.4-fold increase 30-fold decrease

[153]

[154]

1.9 to 18-fold increase

Change in vehicle from water to synthetic oil

Mean absorbed dose: 8.6-fold decrease

EGNPE

Increase in water vehicle content

Mean steady-state flux:

EGIPE

from 0 to 50 %

[155]

DPGME

1.4- to 2.7-fold increase

[151] EGMEA Increase in water vehicle content

Mean maximum flux:

DEGBEA from 0 to 50 % Increase in exposure duration: rate: DMF

2.5-fold decrease

Metabolite excretion

increase: - from 2 to 10 min - from 10 to 20 min

8.5-fold 4.4-fold

[152]

Increase in water vehicle content:

Mean steady-state flux increase:

4-MBC

- from 0 to 50 % - from 50 to 90 % Change from an emulsion- to an

Mean % of applied dose

alcohol-based sunscreen

recovered in SC

formulation

2-PE

[156]

7.0-fold 8.4-fold

[157]

1.6-fold increase

Change from non-occlusion

Mean initial flux and

to occlusion

permeability coefficient:

[158]

2.7-fold increase

Petroleum substance (Benzene)

Change from neat application

Mean steady-state flux

to a vehicle:

decrease:

-

Saturated water solution Hexane Hexadecane Isooctane Gasoline

2.9-fold 5.3-fold 13-fold 3.4-fold 9.0-fold

[159] cited in [160]

Change from no vehicle to: permeability

Steady-state

coefficient increase:

Petroleum substance (Toluene)

-

Methanol Ethanol 1-Pentanol 1-Ooctanol 2-Metoxyethanol 2-Butoxyethanol Benzyl alcohol Propylene glycol Glycerol Ether Acetone Dimethylsulfoxide N,N-Dimethylacetamide N,N-Diemthylformamide

7.4-fold 2.0-fold 1.1-fold 1.2-fold 1.3-fold 1.3-fold 1.1-fold 1.8-fold 1.1-fold 1.5-fold 1.4-fold 9.0-fold 4.0-fold 6.5-fold

[161] cited in [160]

Steady-state permeability coefficient decrease: - 1-Propanol - Isobutanol - Cyclohexanol - Ethylene glycol Change in vehicle from

1.1-fold 1.1-fold 3% 1.4-fold Mean cumulative

liquid paraffin to:

amount penetrated through skin at 8h decrease:

Sunscreen component (Benzophenone-3)

-

Coconut oil Ethanol:coconut oil Aqueous cream Oily cream

5.0-fold 2.0-fold 2.2-fold 1.5-fold Mean % retained in skin at 8h increase:

- Coconut oil - Ethanol:coconut oil

3% 14.5-fold

[162]

- Aqueous cream - Oily cream

5% 1.8-fold

Concurrent application of

Recovery amounts in

DEET and BP-3 vs. BP-3

upper SC and upper

alone increase:

viable epidermis [163] 77 - 97% and 231% Plasma Cmax and AUC: 60 - 63% increase

Table 2: Important human and animal skin parameters affecting transcutaneous permeation and systemic uptake SC lipid composition

Skin blood

[% of total epidermal weight]

flow rate [mL/min/kg]

Forehead and scalp: ~500-1000

Ceramides: 40-50

4.3 [220] **

[217, 218]

Cholesterol: 20-33

Mean blood flow in the face/forehead is at least several-fold higher than in the forearm [221].

Average thickness [m] Species | body site SC

Face, forehea d

Forearm s

N/A [24, 25]

[24-26, 48, 222]

Whole skin 1500 (♀)

13-17

18-23 Human

Viable epidermis

2000 (♂) [216]

57-77

1200-1400

[26, 48]

[48, 216]

N/A

N/A

Follicular density [count/cm2]

Free fatty acids: 7-13 18 [223]

[219]

173-208 Palms

0 [217]

[24, 25] Sphingolipids: ~24 Free sterols: ~16 Mouse

5-9

13-29

700-800

[209, 224]

[209, 224]

[209, 224]

Back

5045-6220 (scapula)

Free fatty acids: ~10

21 [220]

[225]

Overall, similar to human SC

206 [207]

[209, 226, 227]

Inner ear

11-12

17-19

276

[207, 209]

[207, 209]

[209]

80 [209]

7

11

2000-2200

1728-2034 (scapula)

[207]

[207]

[229]

[225]

Rabbit Back

Lower ceramide, higher cholesterol ester and triglyceride content than pig skin, the closest to human skin

84 [207]

55 [207]

[209, 228]

8000 [209]

Back

18 (♀)

31 (♀)

2040 (♀)

35 (♂) [230]

61 (♂)

2800 (♂)

[230]

[230]

32

2090

18 Rat

[209, 224]

[209, 224]

1500-1598 (scapula) [225] 8000 [209]

[209, 224]

Free fatty acids: 11.1 Triglycerides: 7.6 96 [207] Cholesterol: 5.6 23 [220] Ceramide 3: 0.3

7*

Primary follicle: 336.1

10 * /

[231]

[231]

Secondary follicle: 5872.2 [231]

* Mean of rat abdominal and back skin values. ** Body site and measurement methods are not specified in [220].

[231]

Table 3: Comparison of human and rat skin permeation parameter values Compound

Parameter

CAP

Steady-state permeability coefficient

Rat skin / human skin ratio

Reference

Hydrophilic CAP: 5.9 [232] Commercial CAP: 3.6

DEA

Steady-state permeability coefficient

1.8

[233]

DGMBE

Steady-state permeability coefficient

1.7

[234]

2-EE

Steady-state permeability coefficient

2-EH

Steady-state permeability coefficient

MEA

Cumulative dose absorbed

Undiluted: 2.0 In methanol vehicle: 3.9

5.8

[235]

[234]

Undiluted: 9.8 [233] In water vehicle: 1.2

NMP

Information not available

4.0

[236]

2-PE

Steady-state permeability coefficient

1.3

[158]

PEA

Cumulative dose recovered in urine

10

[237]*

Petroleum substance (toluene)

Steady-state permeability coefficient

6.0

[238]

Petroleum substance

Steady-state permeability

12

[239]

82

(o-xylene)

coefficient

TGA

Information not available

3.0

*Difference exposure protocols used for humans and rats

83

[240] cited in [241]

Table 4: Overview of skin exposure protocols in in vivo animal studies of occupational compounds and mixtures Exposure protocols in in vivo skin studies Compound

Animals

Reported skin effects Application days

BUP

Applied doses [mg/kg/day]

Daily removal after application

Vehicle

Rats

Daily for 4 and 13 weeks

25, 100, 400 8, 20, 50, 125

No

No

Unspecified

Rats

GD 0-19

50, 200, 400

No

No

No

BR Rabbits

GD 0-28

50, 100, 200

No

No

No

Rats

GD 7-17

16, 24, 32 mg/rat/day

At least 3h later

Diethylene glycol monoethyl ether

Yes

CAP

COM

Rabbits

GD 7-19

3, 6.5, 13 L/cm2

At least 3h later

Diethylene glycol monoethyl ether

Yes

Rats

GD 11-15

500, 1500

No

Acetone

No

84

Ref.

Occluded application site  Dryness and crustiness after 1st week of treatment.  Progressive thickening.  Localized ulcerations in some animals.  Other, non-specified milder changes.  Scaling and sloughing in 14/30 animals at lowest dose, in all animals at the higher doses.  Squamous and/or cracked skin in ―virtually‖ all animals, duration of which increased with dose.  Erythema, with number of affected animals and severity increasing with dose.  Grade 1 flaking and erythema, scabbing and some skin redness, with number of incidences generally increasing with the dose.  Flaking (grades 1-3), erythema (grades 1-2), edema (grades 1-2), wrinkling and lesions. No evident dose-dependency.  High dose: dermal or subdermal edema, either moderate, i.e., slight

[243, 244]

[245]

[246]

[247]

thickening and translucence around neck and forelegs (not application site) or severe, i.e., swelling and reddening over the whole body.

Crude oils

HDS kerosene

CSO

Mice

GD 11-15

500, 1500

No

Acetone

No

Rats

5 days/week for 13 weeks (systemic toxicity study) GD 0-19 (developmental toxicity study)

125, 500, 1000, 2000 30, 125, 500

No

No

No

 Slight to moderate irritation [248].  Minimal irritation [249].

[248, 249]

[250]

GD 0-20

165, 330, 494

No

2 USP mineral oils

No

 Irritation among males varied from slight to mild to moderate with increasing doses and was most severe in the high-dose group.  Mild to moderate irritation in females at the highest concentration.

GD 0-20

135, 330, 494

No

Squib 340 mineral oil

No

 Slight to moderate irritation at the highest dose.

[251]

5 days/week for 13 weeks (subchronic toxicity study)

8, 30, 125, 500

No

No

No

 Congestion and reactive lymphoid hyperplasia at site of application.  Overall, little effect on skin.

[134]

GD 0-19

4, 8, 30, 125, 250

Information not available

No

No

Information not available

[252]

GD 0-19

1st study: 0.05, 1, 10, 50, 250 2nd study: 1, 50, or 25

Undiluted in 1st study for doses 1, 10, 50 250

No

None.

[253]

Rats

Rats

None reported.

1st study: No 2nd study: 6h later for each

85

Mice

DAE

DEGEE

Rats

Rats

―pulse‖ exposure every 2 to 3 days

mg/kg/day, dissolved in acetone otherwise.

0-14 (female), 70 days (male)

0.1, 1, 10, 50, 250

6h later

No

No

GD 9-12

10, 100, 1000

No

No

No

GD 0-19

5, 25, 50

6h later

No

No

None.  Flaking and scabbing at site of application in some animals, at highest dose. None.  Extensive chronic inflammation, epidermal hyperkeratosis / hyperplasia.  Three mice displayed malignant squamous-cell carcinoma histologically.

[254] [255] [256]

5 days/week up to 10 weeks

1000

No

No

No

5 days/week for 13 days (subchronic toxicity study) GD 0-19, 0-16, 10-12, depending on dose, in developmental toxicity study

30, 125, 500, 1250 8, 30, 125

No

No

No

None.

[258]

GD 6-19

5, 25, 150, 450

6h later

Acetone

No

 Higher incidences of desquamation at 25, 150, and 450 mg/kg/day compared to vehicle controls.

[259]

7-16

0.35 mL

4x daily applications

No

No

None.

[260]

86

[257]

DGMME

DGMBE

Rabbits

GD 6-18

50, 250, 750

No

No

Yes

Rabbits

GD 7-18

100, 300, 1000

4h later

Water

No

Rats

GD 6-15

150, 500, 1500

6h later

Water (4 mL/kg/day)

Yes

DEA

DEP

Rabbits

GD 6-18

35, 100, 350

Mice

GD 0-17

500, 1600, 5600

Water (2 mL/kg/day)

6h later

Yes

Information not available

None.  6/20 animals exhibited slight erythema and 5/20 desquamation at 300 mg/kg/day.  Moderate irritation, including edema, fissuring and hardening at 1000 mg/kg/day.  Effects were observed after 1 week and carried on for the rest of the study.  Crusting, ecchymosis, necrosis and erythema, dose-dependent in incidence and severity mainly for 500 and 1500 mg/day/day.  No significant effects at low dose.  Excoriation, exfoliation, crusting, ecchymosis, necrosis and scabbing at highest dose.  Slight to well-defined edema and severe erythema at highest dose.

[262]

[263]

Information not available

[264] cited in [265] [266]

[267]

DGEBPA

Rabbit

6-18

30, 100, 300

No

PEG 400

Yes

 In varying numbers of animals, dose-dependent levels of erythema, exfoliation and fissuring, hemorrhage and edema.

DMF

Rats

GD 6-10 and 1315

94, 472, 994

3h later

No

No

 Dermal irritancy.

87

[261]

Rabbits

2-EE

EG

Day 6-18 postinsemination

100, 200, 400

6h later

No

Semi

GD 6-15

0.1 mL

NA

NA

NA

GD 7-16

0.5 mL

4x daily applications

No

6-15

404, 1677, 3549

6h later

No

4x daily applications

 Irritation. None.

[268] cited in [269]

None reported.

[270]

Yes

Information not available

[271]

No

No

None.

[260]

Rats

Mice

0.35 mL

EGEEA Rats

7-16

EGBE

0.12 mL

2-EH

Rats

GD 6-15

252, 840, 2520

6h later

No

Yes

 Exfoliation, encrustation.  Erythema at doses 840 mg/kg/day.

[272]

FA

Hamster

GD 8-11

0.5 mL

2h later

FA solution (37%)

No

 Animals scratched at the treated area for 1 or 2 days post treatment.

[273]

6h later

No

No

 LCCO at highest dose: slight to moderate erythema, very slight to slight oedema and desquamation.

[274]

Information not available

Acetone and/or lotion

Information not available

None.

[275]

Gas oils

Rats

GD 0-19

Rats 2- and 13-week studies

HMB Mice

100, 300, 600 (ULSD) 100, 450, 750 (LCCO) 1.25 to 20 mg HMB (2 weeks) 12.5 to 200 mg/kg (13 weeks) 0.5 to 8 mg HMB (2 weeks) 22.75 to 364 mg/kg

88

(13 weeks)

Mice (male)

5 days/week, 91 days

10, 20, 100, 400

Group 2: 30 min later 2x daily application at 34h interval

LAS

Rats

GD 0-20

Group 1: 1, 2, 10, Group 2: 20, 100, 400

4-MBC

Rats

Postnatal day 2126

137.5, 275, 412.5 mg

No

Acetone

No

None.

[276]

Alkylbenzene, ash and water solution

No

 Slight skin discoloration, slight erythema, skin thickening, fissuring in some animals.  Dose-dependent incidence and duration of effects.

[277]

Olive oil

No

None reported.

[278]

MDEA

Rats

GD 6-21

250, 500, 1000

6h later

Water

Yes

MMDHCA

Rats

13-week study

50, 150, 300

6-7 h later

No

Semi

MEA

Rats

GD 6-15

10, 25, 75, 225

6h later

Water

Yes

89

 Exfoliation, excoriation, crusting, ecchymoses, necrosis, erythema and edema observed at 500 and 1000 mg/kg/day.  Dose-dependent severity of erythema and incidence, duration of all effects.  Slight to moderate erythema, edema, dequamation, atonia and fissuring, inflammation, thickening, abnormal cornification, epidermal ulcers and erosions, accumulation of an exudate, eosinophilic infiltration.  Dose-dependent incidence and severity of effects.  Significant increases in the incidence of skin irritation/lesions

[279]

[280]

[281]

Rabbits

GD 6-18

2- and 13-week studies Mice

MPA

2-ME

Rabbits

10, 25, 75

2-week study:  Extensive coagulative necrosis of epidermis and dermis, epidermal regeneration, hyperplasia.

2-week study: 50.6, 101.3, 202.5, 405, 810 mg/rat 13-week study: 1.07, 3.57, 10.7, 35.7, 107 mg/rat

Rats

MEKP

and maternal body weight effects at highest doses.  The dermal irritation observed at the high dose was progressive, beginning with erythema and leading to necrosis, scabs, and scar formation.  Rabbits: doses of 25 mg/kg/day produced only minor irritation.

No

Dimethyl phthalate

No

2-week study: 112.5, 225, 450, 900, 1800 mg/mouse 13-week study: 0.357, 1.19, 3.57, 11.9, 35.7 mg/mouse

13-week study:  Termination of experiments at the two highest doses due to severity of effects.  10.7 mg/rat, 3.57 mg/mouse doses: necrosis, inflammation and epidermal hyperplasia.  Lower doses: epidermal hyperplasia and hyperkeratosis.

[282]

GD 6-18

1000, 2000

6h later

No

Semi

Mild skin irritation.

[283]

GD 6-17

10 mL/kg

6h later

Physiological saline

Yes

None reported.

[284]

GD 6-15

840

6h later

No

Yes

None.

[272]

Rats

90

MS

Rats

GD 12 (dosedependence) GD 10-14 (GDdependence)

2000 250, 500, 1000

No

No

No

None.

[285]

GD 6-15

1000, 3000, 6000

Information not available

Petroleum-based grease

Information not available

Information not available.

[286] cited in [287]

22, 44, 87.5, 175, 350 (3-month study) 10, 30, 90 (2-year study)

Rats

5 days/week for 14 and 105 weeks

MSK

No

95% Ethanol

No

87.5, 175, 350, 700, 1400 (3-month study) 10, 30, 90 (2-year study)

Mice

NMP

Rats

GD 6-15

75, 237, 750

8h later

No

No

OMA

Rats

GD 6-15

1, 10, 30

2h later

Shampoo blank

No

91

3-month study:  At doses 175 and 350 mg/kg/day: skin irritation, thickening, ulceration, epidermal hyperplasia, hyperkeratosis, inflammation, epidermal necrosis, sebaceous gland hypertrophy. 2-year study:  At 30 and 90 mg/kg/day: epidermal hyperplasia and hyperkeratosis. 3-month study:  Dose-related epidermal hyperplasia, hyperkeratosis, inflammation, epidermal necrosis, sebaceous gland hypertrophy and hair follicle hyperplasia. 2-year study:  Dose-related epidermal hyperplasia and hyperkeratosis, inflammation and melanocyte hyperplasia.  Skin dryness.  Dose-dependent increase in severity.  Erythema, edema, atonia, desquamation, fissuring, coriaceous skin.

[288]

[289]

[133]

2-PE

PEA

STB

RF

Rabbits

GD 6-18

0.45, 1.5, 5.0

2h later

Shampoo blank

No

Rabbit

GD 6-18

300, 600, 1000

No

Neat

Yes

Rats

6-15

140, 430, 1400 70, 140, 280, 430, 700 (corroborative study)

No

No

Yes

Rats

5 days/week for 13 weeks (systemic toxicity. study) GD 0-19 (developmental toxicity study)

Rats

GD 0 -15

8, 30, 125, 500

[290]

[291]

Minimal skin irritation (flaking). No

No

No

4, 8, 30, 125 Different ranges depending on the stream

 Skin irritation.  For some effects, dose-dependent increase in severity.  Erythema, edema, atonia, desquamation, fissuring, coriaceous skin.  Dose-dependent increase in incidence and severity of coriaceous skin at 5.0 mg/kg/day compared to vehicle control.  Slight to moderate reddening.  In some cases, darkened skin areas.  Local irritation at application site observed in some rats early in the treatment period, especially in the high-dosage group.  Localized, low levels of erythema and/or desquamation at intrascapular application sites.  Dose-dependent severity of the reactions.

[292] None

No

No

92

No

Skin irritation.

[293]

S-23121 S-53482

Rats

GD 6-15

30, 100, 300 200, 400, 800

6h later

Corn oil

Yes

None.

[294]

TBU

Rats

GD 6-15

25, 50, 100 mg/rat/day

No

Dimethyl phtalate

No

 Mild to moderate skin irritation.  Dose-dependent severity at 50 and 100 mg/rat/day.

[295]

Rats

GD 6-19

50, 100, 200

6h later

Ethanol-water

No

 Discoloration and scaliness.  At 200 mg/kg/day, slight edema and erythema.

Rabbits

GD 6-29

10, 15, 25, 65

6h later

Ethanol-water

No

 Slight to well-defined erythema, petechial, edema, dryness, flakiness,  Brown patches at 65 mg/kg/day.  Dose-dependent incidence and severity of erythema.

Rats

GD 6-15

200, 600, 2000

6 h later

Corn oil suspension

Yes

 Flaking and scabbing.  Dose-dependent increase in erythema, flaking, edema.

TGA

TMPCC

13-week study 2-year study

TQ Mice

[297]

 Skin discoloration, epidermal hyperplasia, hyperkeratosis.  Dose-dependent increase in incidence and severity of effects.

5, 20, 50, 100, 200 (13-week study) 2.5, 5, 10, 20, 50 (2year study)

Rats

[296]

No

Acetone

2.5, 5, 10, 20, 50 (13-week study) 3.6, 6, 10 (2-year study)

93

No

13-week study:  Epidermal hyperplasia, hyperkeratosis, parakeratosis, fibrosis and subchrinic inflammation.  Dose-dependent increase in incidence and severity of effects. 2-year study: none.

[298]

Rats TRE

3.7 2-day study

Rabbits

Airol cream (0.05%)

No 3.0

No (group 1) Yes (group 2) Yes

94

None.  Marked erythema, skin efflorescence, desquamation and fissure formation.

[299]

Table 5: Overview of skin permeation / systemic uptake results obtained or discussed in the studies listed in Table 4. Compound and reference(s)

Kinetics data

CSO [134]

 45 % of applied carbazole and 3-13 % of applied 2-5 ring polycyclic aromatic hydrocarbons penetrated rat skin in vitro and in vivo.  Exposure conditions in the skin penetration experiments did not match those of the toxicological experiments.

HDS kerosene [250, 251]

 11-15% of applied dose penetrated through the rat skin under conditions reproducing the toxicity study.  Initial penetration of neat HDS kerosene was faster than in the mineral oil vehicles.

Industrial oil components [257, 258, 292]

 Studies cite reference [134] for evidence of skin penetration.

2-ME [285]

 Rapid penetration of 2-ME through human cadaver skin (mean steadystate flux of 2.82 mg/cm2/h), citing reference [308].

MDEA [279, 309]

 Rat skin penetration following 6h and 3 days exposure yielded 20% and 50% of applied dose absorbed. MDEA was sequestered in rat skin.  MDEA elimination is mainly via urine and is slow.

MMDHCA [280]

 Penetration through excised human skin was moderate; 42-50% of applied dose.  Skin penetration protocol did not reproduce toxicity study conditions (uncovered vs. semi-occluded animal application area and dissolution in ethanol vs. neat application to animals)

PEA [237, 291]

 PEA metabolism and excretion in rats and humans were comparable.  The percentage absorbed through rat and human skin following a single topical dosage of 14C-PEA was 77 and 7.6%, respectively.  Lower systemic absorption in humans in a consumer scenario compared to rats lead to the conclusion that PEA is not a developmental toxicant under consumer exposure conditions.

TRE [299]

 Plasma concentration in rats and rabbits following skin penetration were quantified as part of the toxicological study. Plasma values following dermal exposure were below the limit of detection.  This results contrasts with others which obtained measurable plasma concentrations following a single higher application dose.

95