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In Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) the criterion for deciding the studies that must be performed is the annual tonnage of the chemical manufactured or imported into the EU. The annual tonnage may be considered as a surrogate for levels of human exposure but this does not take into account the physico-chemical properties and use patterns that determine exposure. Chemicals are classified using data from REACH under areas of health concern covering effects on the skin and eye; sensitisation; acute, repeated and prolonged systemic exposure; effects on genetic material; carcinogenicity; and reproduction and development. We analysed the mandated study lists under REACH for each annual tonnage band in terms of the information they provide on each of the areas of health concern. Using the European Chemicals Agency (ECHA) REACH Registration data base of over 20,000 registered substances, we found that only 19% of registered substances have datasets on all areas of health concern. Information limited to acute exposure, sensitisation and genotoxicity was found for 62%. The analysis highlighted the shortfall of information mandated for substances in the lower tonnage bands. Deploying New Approach Methodologies (NAMs) at this lower tonnage band to assess health concerns which are currently not covered by REACH, such as repeat and extended exposure and carcinogenicity, would provide additional information and would be a way for registrants and regulators to gain experience in the use of NAMs. There are currently projects in Europe aiming to develop NAM-based assessment frameworks and they could find their first use in assessing low tonnage chemicals once confidence has been gained by their evaluation with data rich chemicals., (© 2023. The Author(s).)

Determination of the absorption through the skin is of utmost importance for predictions of benefits and of risks of dermal exposure to xenobiotica. In order to allow for flexibility, the OECD guideline for the determination of skin absorption for different purposes and use conditions (OECD guideline 428 combined with the Technical Guidance Document 28) is inexplicit; hence, different experimental procedures are used which may lead to limited comparability of study results. The here described protocol provides explicit guidance, whereas it does not invalidate other procedures within the frame of the OECD guideline since uncritical versus critical steps are differentiated. Optimizations are presented which finally led to a precisely defined protocol allowing for enhanced comparability of future study results. Some salient properties of this protocol are the storage of the prepared diffusion cell overnight refrigerated in the presence of a protease inhibitor cocktail and include investigation of the integrity of the skin sample as well as the removal of the upper stratum corneum by tape strips under standardized conditions.

Skin absorption testing in vitro is a regulatory accepted alternative method (OECD Guideline 428). Different tests can be applied to evaluate the integrity of the skin samples. Here, we compared the pre- or post-run integrity tests (transepidermal electrical resistance, TEER; transepidermal water loss, TEWL; absorption of the reference compounds water, TWF, or methylene blue, BLUE) and additionally focused on co-absorption of a (3)H-labeled internal reference standard (ISTD) as integrity parameter. The results were correlated to absorption profiles of various test compounds. Limit values of 2kΩ, 10 gm(-2)h(-1) and 4.5∗10(-3)cmh(-1) for the standard methods TEER, TEWL and TWF, respectively, allowed distinguishing between impaired and intact human skin samples in general. Single skin samples did, however, not, poorly and even inversely correlate with the test-compound absorption. In contrast, results with ISTD (e.g. (3)H-testosterone) were highly correlated to the absorption of (14)C-labeled test compounds. Importantly, ISTD did not influence analytics or absorption of test compounds. Therefore, ISTD, especially when adjusted to the physico-chemical properties of test compounds, is a promising concept to assess the integrity of skin samples during the whole course of absorption experiments. However, a historical control dataset is yet necessary for a potential routine application., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Dermal absorption is a critical part in the risk assessment of complex mixtures such as agrochemical formulations. To reduce the number of in vivo or in vitro absorption experiments, the present study aimed to develop an in silico prediction model that considers mixture-related effects. Therefore, an experimental 'real-world' dataset derived from regulatory in vitro studies with human and rat skin was processed. Overall, 56 test substances applied in more than 150 mixtures were used. Descriptors for the substances as well as the mixtures were generated and used for multiple linear regression analysis. Considering the heterogeneity of the underlying data set, the final model provides a good fit (r² = 0.75) and is able to estimate the influence of a newly composed formulation on dermal absorption of a well-known substance (predictivity Q²Ext = 0.73). Application of this model would reduce animal and non-animal testings when used for the optimization of formulations in early developmental stages, or would simplify the registration process, if accepted for read-across.

National legislations for the assessment of the skin sensitization potential of chemicals are increasingly based on the globally harmonized system (GHS). In this study, experimental data on 55 non-sensitizing and 45 sensitizing chemicals were evaluated according to GHS criteria and used to test the performance of computer (in silico) models for the prediction of skin sensitization. Statistic models (Vega, Case Ultra, TOPKAT), mechanistic models (Toxtree, OECD (Q)SAR toolbox, DEREK) or a hybrid model (TIMES-SS) were evaluated. Between three and nine of the substances evaluated were found in the individual training sets of various models. Mechanism based models performed better than statistical models and gave better predictivities depending on the stringency of the domain definition. Best performance was achieved by TIMES-SS, with a perfect prediction, whereby only 16% of the substances were within its reliability domain. Some models offer modules for potency; however predictions did not correlate well with the GHS sensitization subcategory derived from the experimental data. In conclusion, although mechanistic models can be used to a certain degree under well-defined conditions, at the present, the in silico models are not sufficiently accurate for broad application to predict skin sensitization potentials., (Copyright © 2013 Elsevier Inc. All rights reserved.)

For ethical and regulatory reasons, in vitro tests for scoring potential toxicities of cosmetics are essential. A test strategy for investigating potential skin sensitization using two human keratinocytic and two human dendritic cell lines has been developed (Mehling et al. Arch Toxicol 86:1273–1295, 2012). Since prohaptens may be metabolically activated in the skin, information on xenobiotic metabolizing enzyme (XME) activities in these cell lines is of high interest. In this study, XME activity assays, monitoring metabolite or cofactor, showed the following: all three passages of keratinocytic (KeratinoSens® and LuSens) and dendritic (U937 und THP-1) cells displayed N-acetyltransferase 1 (NAT1) activities (about 6–60 nmol/min/mg S9-protein for acetylation of para-aminobenzoic acid). This is relevant since reactive species of many cosmetics are metabolically controlled by cutaneous NAT1. Esterase activities of about 1–4 nmol fluorescein diacetate/min/mg S9-protein were observed in all passages of investigated keratinocytic and about 1 nmol fluorescein diacetate/min/mg S9-protein in dendritic cell lines. This is also of practical relevance since many esters and amides are detoxified and others activated by cutaneous esterases. In both keratinocytic cell lines, activities of aldehyde dehydrogenase (ALDH) were observed (5–17 nmol product/min/mg cytosolic protein). ALDH is relevant for the detoxication of reactive aldehydes. Activities of several other XME were below detection, namely the investigated cytochrome P450-dependent alkylresorufin O-dealkylases 7-ethylresorufin O-deethylase, 7-benzylresorufin O-debenzylase and 7-pentylresorufin O-depentylase (while NADPH cytochrome c reductase activities were much above the limit of quantification), the flavin-containing monooxygenase, the alcohol dehydrogenase as well as the UDP glucuronosyl transferase activities.

Skin corrosion or irritation refers to the production of irreversible or reversible damage to the skin following the application of a test substance, respectively. Traditionally, hazard assessments are conducted using the in vivo Draize skin test, but recently in vitro tests using reconstructed human epidermis (RhE) models have gained regulatory acceptance. In this study, skin corrosion (SCT) and irritation tests (SIT) using a RhE model were implemented to reduce the number of in vivo tests required by regulatory bodies. One hundred and thirty-four materials were tested from a wide range of substance classes included 46 agrochemical formulations. Results were assessed according to UN GHS, EU-CLP, ANVISA and US EPA classification schemes. There was high correlation between the two in vitro tests. Assessment of the SCT sensitivity was not possible due to the limited number of corrosives in the data set; SCT specificity and accuracy were 89% for all classification systems. Accuracy (63-76%) and sensitivity (53-67%) were low in the SIT. Specificity and concordance for agrochemical formulations alone in both the SCT and SIT were comparable to the values for the complete data set (SCT: 91% vs. 89% specificity, 91% vs. 89% accuracy and SIT: 64-88% vs. 70-85% specificity, 56-75% vs. 63-76% accuracy)., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Skin exposure to sensitizing chemicals can induce allergic reactions. Certain chemicals, so called pro-sensitizers, need metabolic activation to become allergenic. Their metabolic activation occurs in skin cells such as keratinocytes or dendritic cells. These cell types are also incorporated into dermal in vitro test systems used to assess the sensitizing potential of chemicals for humans. In vitrosystems range from single cell cultures to organotypic multi-cellular reconstructed skin models. Until now, their metabolic competence to unmask sensitizing potential of pro-sensitizers was rarely investigated. This review aims to summarize current information on available skin in vitro models and the relevance of xenobiotic metabolizing enzymes for the activation of pro-sensitizers such as eugenol, 4-allylanisole, and ethylendiamine. Among others, these chemicals are discussed as performance standards to validate new coming in vitro systems for their potential to identify pro-sensitizers.

Contact allergies are complex diseases, and it is estimated that 15-20 % of the general population suffers from contact allergy, with increasing prevalence. Evaluation of the sensitization potential of a substance is usually carried out in animal models. Nowadays, there is much interest in reducing and ultimately replacing current animal tests. Furthermore, as of 2013, the EU has posed a ban on animal testing of cosmetic ingredients that includes skin sensitization. Therefore, predictive and robust in vitro tests are urgently needed. In order to establish alternatives to animal testing, the in vitro tests must mimic the very complex interactions between the sensitizing chemical and the different parts of the immune system. This review article summarizes recent efforts to develop in vitro tests for predicting skin sensitizers. Cell-based assays, in chemico methods and, to a lesser extent, in silico methods are presented together with a discussion of their current status. With considerable progress having been achieved during the last years, the rationale today is that data from different non-animal test methods will have to be combined in order to obtain reliable hazard and potency information on potential skin sensitizers.

With the perspective to use human reconstructed skin models for genotoxicity testing which require metabolic activation of xenobiotics, this study aimed to characterize activities of biotransforming enzymes within two human reconstructed skin models, the epidermis model EpiDerm™ (MatTek) and the Phenion® Full-Thickness skin model Phenion®FT (Henkel). According to existing gene expression profiles, Cytochrome P450 (CYP) enzymes, Flavin-dependent monooxygenases (FMO), N-acetyltransferases (NAT) and UDP-glucuronyltransferases (UDP-GT) were investigated in S9 or microsomal fractions. CYP-catalyzed monooxygenation was assayed using 7-ethoxyresorufin, pentoxyresorufin and benzyloxyresorufin as substrates. FMO activity was tested using benzydamine. Conjugating activities of NAT and UDP-GT were determined by acetylation of p-aminobenzoic acid or glucuronation of 4-methylumbelliferone, respectively. Although CYPs were detected by expression profiling, no CYP activity was detected in either the epidermal nor the full-thickness reconstructed skin model while expression and activity of FMO, UDP-GT and NAT were demonstrated in both., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In vitro skin penetration rates in rat and man were compared to those obtained in vivo in rats. Saturation of absorption was frequently observed at higher exposure levels in in vitro and in vivo. Lipophilic compounds showed the highest penetration rates through rat skin in vitro. In all cases in vitro dermal penetration through rat skin was higher than in vivo. Thus, the in vitro study may serve as a first tier test. The in vivo data suggest an inverse relationship between molecular weight and the rate of dermal absorption for lipophilic as well as hydrophilic compounds. Rat skin was more permeable to all tested substances than human skin (mean difference 10.9-fold). Thus, the systemic exposure of humans may be significantly overestimated if risk assessment is based only on the results of an in vivo rat study, because human skin is less permeable than rat skin. It would appear, therefore, that an estimate of actual dermal penetration through human skin should be based on the combined use of in vivo and in vitro data, using the following equation: % Human dermal penetration = (% rat in vivo dermal penetration) x (in vitro rate dermal penetration human / in vitro rate dermal penetration rat).

The present paper reviews the comparative rates of skin penetration between rat and man for a total of 14 chemicals in in vitro absorption studies. The results showed that in vitro absorption assays are capable of demonstrating large differences in the rate of skin penetration. Saturation of absorption was also frequently observed at higher exposure levels. The highest absorption rates through rat and human epidermis were observed with compounds with a molecular weight of approximately 300, an aqueous solubility of approximately 1–6 mg/l, and a log10 (POCTANOL/WATER) of approximately 3–4. When the absorption data for 3 compounds with a log10 (POCTANOL/WATER) of 2.9–3.0 were compared, there appeared to be an inverse relationship between molecular weight/aqueous solubility and the rate of dermal absorption. Lipophilic compounds with low aqueous solubility (<4 mg/l) showed the highest penetration rates through rat skin, but this was not always the case for human skin. The human skin was invariably less permeable to all tested substances than rat skin, though no constant factor of difference could be identified. The factor of difference would not appear to be determined by molecular weight, lipophilicity, or aqueous solubility. The actual systemic exposure of humans may be significantly overestimated if risk assessment is based only on the results of an in vivo rat study. It would appear that dermal penetration through human skin should be based on the combined use of in vivo and in vitro data, using the following equation: %Human dermal penetration=[% dermal penetration rat (in vivo)]×[rate dermal penetration human (in vitro)]/[rate dermal penetration rat (in vitro)] [Copyright &y& Elsevier]

Abstract: Microfine metallic oxides such as titanium dioxide or zinc oxide have been found to be highly protective against harmful UV rays. Because their long-term use could potentially lead to health effects if significant amounts of these microfine metallic oxides would be absorbed through the skin, the in vitro absorption of microfine zinc oxide and titanium oxide in cosmetic formulations through porcine skin was investigated. In the experiments with a microfine zinc oxide formulation, the mean total recoveries of Zn were in the range from 102% to 107% of the total Zn applied. Virtually the total amount of applied Zn was recovered in the first five tape strips. The amounts of Zn found in the skin membrane and the receptor fluid were comparable in untreated, vehicle treated or test substance treated skin preparations. The absorption-time plots from diffusion cells treated with the vehicle did not differ from those treated with the ZnO containing formulation. In the experiments with microfine titanium dioxide formulations T-Lite SF-S and T-Lite SF, mean total recoveries of Ti ranged from 98% to 100% and 86% to 93% of the total Ti applied, respectively. Virtually the total amount of applied Ti could be removed from the skin surface by washing. The amounts of titanium found in the tape strips and skin preparations were in the order of the analytical determination limit. No Ti was found in the receptor fluid at any sampling time. The results show that neither zinc or titanium ions nor microfine zinc oxide or titanium dioxide particles were able to penetrate porcine stratum corneum. Therefore, from the absence of internal exposure we conclude that their use in sunscreens does not pose a health risk. [Copyright &y& Elsevier]