Performance of the DASF compared to other combinations of OECD NAMs for eye hazard identification of surfactants.

Currently, the OECD has adopted three defined approaches (DAs) for eye hazard identification of non-surfactant liquids and solids (TG467) according to the three UN GHS categories (Cat.1, Cat.2, No Cat.). We are now expanding the applicability domain with a new DA for chemicals having surfactant (SF) properties (DASF). It is based on a combination of RhCE test methods (OECD TG492: EpiOcular™ EIT or SkinEthic™ HCE EIT) and a modification of the Short Time Exposure (STE, TG491) method. The aim of the current study was to compare the performance of the DASF with the performance of other NAMs currently included in the OECD TGs and with the classification based on the Draize eye test to identify potential additional DAs. The minimum performance criteria (75% Cat.1, 50% Cat.2, 70% No Cat.) used for the adoption of the DAs currently included in OECD TG467 were used for this purpose. The DASF identified 90.9% of Cat. 1 (N=23), 77.8% of Cat. 2 (N=9) and 76.0% of No Cat. (N=17) surfactants, meeting the minimum performance criteria. Some of the NAMs that are currently included in the OECD TGs seem promising methods to be part of a DA to identify Cat. 1 or No Cat. for eye hazard assessment of surfactants. However, the number of surfactants that have been tested to evaluate reliability and relevance was often too small. To date, the DASF is the only DA that has evaluated a sufficiently large number of surfactants and whose performance met the OECD acceptance criteria.

Three non-animal-based defined approaches (DAs) for eye hazard assessment of non-surfactant liquid and solid chemicals were adopted as full replacements as OECD Test Guideline (TG 467). We now extend the applicability domain to surfactants with a new DA (DASF), which combines OECD-adopted test systems based on human 3D eye models and rabbit 2D corneal cells. The DASF has been shown to provide reliable results in predicting the eye irritation potential of 50 surfactants. The aim of the current study was to compare the performance of the DASF with the performance of other OECD TG new approach methodologies and compare it with the classification based on historical animal test data. Based on this analysis no additional DAs could be derived. Until today, the DASF is the best predicting, human-relevant DA that covers the whole range of eye irritation responses across the different surfactant classes.

The dimer effect: A refinement approach towards skin sensitization assessment in-chemico using Amino acid Derivative Reactivity Assay.

Skin sensitization is a key endpoint for safety assessment, especially for cosmetics and personal care products. The adverse outcome pathway for skin sensitization and the chemical and biological events driving the induction of human skin sensitization are now well understood. Several non-animal test methods have been developed to predict sensitizer potential by measuring the impact of chemical sensitizers on these key events. In this work, we have focused on Key Event 1 (the molecular initiating step), which is based on formation of a covalent adduct between skin sensitizers and endogenous proteins and/or peptides in the skin. There exists three in-chemico assays approved by the Organization for Economic Co-operation and Development-(1) Direct Peptide Reactivity Assay (DPRA), (2) Amino Acid Derivative Reactivity Assay (ADRA), and (3) Kinetic Direct Peptide Reactivity Assay (kDPRA) to quantify peptide/amino acid derivative depletion after incubation with test chemicals. However, overestimated depletion of the cysteine-based peptide/amino acid derivatives is known in such assays because of the dimerization of the thiol group. In this present work, we report the synthesis and structural confirmation of the dimer of N-(2-[1-naphthyl]acetyl)-L-cysteine (NAC) from the ADRA assay to allow simultaneous determination of (a) peptide depletion by quantifying NAC monomer and (b) peptide dimerization by quantifying NAC dimer thereby eliminating the overestimation. We present a case study with three chemicals to demonstrate the importance of this approach. Thus, this simultaneous assay gives a more informed view of the peptide reactivity of chemicals to better identify skin sensitizers.

Deriving a Continuous Point of Departure for Skin Sensitization Risk Assessment Using a Bayesian Network Model.

Regulations of cosmetic ingredients and products have been the most advanced in embracing new approach methodologies (NAMs). Consequently, the cosmetic industry has assumed a forerunner role in the development and implementation of animal-free next-generation risk assessment (NGRA) that incorporates defined approaches (DAs) to assess the skin sensitization potency of ingredients. A Bayesian network DA predicting four potency categories (SkinSens-BN) was constructed against reference Local Lymph Node Assay data for a total of 297 substances, achieving a predictive performance similar to that of other DAs. With the aim of optimally informing risk assessment with a continuous point of departure (PoD), a weighted sum of the SkinSens-BN probabilities for four potency classes (non-, weak, moderate, and strong/extreme sensitizer) was calculated, using fixed weights based on associated LLNA EC3-values. The approach was promising, e.g., the derived PoDs for substances classified as non-sensitizers did not overlap with any others and 77% of PoDs were similar or more conservative than LLNA EC3. In addition, the predictions were assigned a level of confidence based on the probabilities to inform the evaluation of uncertainty in an NGRA context. In conclusion, the PoD derivation approach can substantially contribute to reliable skin sensitization NGRAs.

Development of a defined approach for eye hazard identification of solid chemicals according to the three UN GHS categories.

Currently there are two OECD-adopted defined approaches (DA) for eye hazard identification of non-surfactant liquids (OECD TG 467). The current study aimed to develop a DA for eye hazard identification of solid chemicals according to the three UN GHS categories (Cat.1, Cat. 2, No Cat.): the DAS. The DAS combines two test methods described in OECD TG 437 and TG 492. The DAS was developed based on in-depth statistical analysis of a database on solids containing in vitro and historically curated in vivo Draize eye test data. The performance of the DAS was assessed by comparing the predictions with the classification based on in vivo Draize eye test data, on the one hand, and with the performance criteria established by the OECD expert group, on the other hand. In a first tier of the DAS, the SkinEthic™ HCE EIT method (TG 492) is used to distinguish No Cat. from classified substances. For classified substances, the BCOP LLBO method (TG 437) is used to identify Cat. 1, and the remaining solids are predicted Cat. 2. In summary, 77.4% Cat. 1 (N=31), 52.3% Cat. 2 (N=18), and 70.0% of No Cat. (N=60) solids were correctly identified compared to the classification based on the Draize eye test. The percentage of correct predictions met the minimum OECD performance values of 75% Cat. 1, 50% Cat. 2, and 70% No Cat., and the percentage of mispredictions was below the established maximum values. Therefore, inclusion of the DAS in OECD TG 467 has been achieved.

Defined approaches combine information from different non-animal testing methods in a specific way and interpret the results according to a fixed procedure. Such defined approaches are already available as full replacements of animal testing to assess the eye hazard of liquid chemicals (OECD Test Guideline 467). This study used two OECD-adopted in vitro methods, based on human cells and corneas from cattle, to create a defined approach that can be used for solid chemicals. The performance of the procedure was assessed against data from previous animal tests for 109 solid chemicals. The results have already led to this defined approach being adopted by the OECD TGs programme for inclusion in TG 467. With the adoption of the new defined approach, non-animal human relevant strategies are now available for eye hazard assessment of liquids and solids, reducing the need for animal testing.

Skin sensitisation prediction using read-across, an illustrative next generation risk assessment (NGRA) case study for vanillin.

Skin sensitisation is a key adverse human health effect to be addressed in the safety assessment of cosmetic ingredients. Regulatory demands and scientific progress have led to the development of a Next Generation Risk Assessment (NGRA) framework, relying on the use of New Approach Methodologies (NAM) Defined Approaches (DA) and read-across instead of generating animal data. This case study illustrates the application of read-across for the prediction of the skin sensitisation potential of vanillin at the hypothetical use concentration of 0.5% in a shower gel and face cream. A three-step process was applied to select the most suitable analogues based on their protein reactivity, structural characteristics, physicochemical properties, skin metabolism profile and availability of skin sensitisation data. The applied read-across approach predicted a weak skin sensitiser potential for vanillin corresponding with a Local Lymph Node Assay EC3 value of 10%. Based on this EC3 value a point of departure of 2500 µg/cm2 was derived, resulting in an acceptable exposure level (AEL) of 25 µg/cm2. Because the consumer exposure levels (CEL) for the face cream (13.5 µg/cm2) and shower gel (0.05 µg/cm2) scenarios were lower than the AEL, the NGRA concluded both uses as safe.

Off to a good start? Review of the predictivity of reactivity methods modelling the molecular initiating event of skin sensitization.

The assessment of skin sensitizing properties of chemicals has moved away from animal methods to new approach methodologies (NAM), guided by qualitative mechanistic understanding operationalized in an adverse outcome pathway (AOP). As with any AOP, the molecular initiating event (MIE) of covalent binding of a chemical to skin proteins is particularly important. This MIE has been modelled by several test methods by measuring the reaction of a test chemical with model peptides in chemico. To better understand the similarities and differences, a data repository with publicly available data for the direct peptide reactivity assay (DPRA), amino acid derivative reactivity assay (ADRA) and kinetic DPRA (kDPRA), as well as the peroxidase peptide reactivity assay (PPRA) was assembled. The repository comprises 260 chemicals with animal and human reference data, data on four relevant physicochemical properties, and between 161 to 242 test chemical results per test method. First, an overview of the experimental conditions of the four test methods was compiled allowing to readily compare them. Second, data analyses demonstrated that the test methods' predictivity was consistently reduced for poorly watersoluble chemicals and that the DPRA and ADRA can be used interchangeably. It also revealed new categorization thresholds for the DPRA and ADRA that are potentially relevant for strategic uses. In summary, a detailed assessment of reactivity test methods is provided, highlighting their potential and limitations. The results presented are intended to stimulate scientific discussion around test methods modelling the MIE of the skin sensitization AOP.

Applying a next generation risk assessment framework for skin sensitisation to inconsistent new approach methodology information.

Cosmetic products must be safe for their intended use. Regulatory bans on animal testing for new ingredients have resulted in a shift towards the use of new approach methodologies (NAMs) such as in silico predictions and in chemico / in vitro data. Defined approaches (DAs) have been developed to interpret combinations of NAMs to provide information on skin sensitization hazard and potency, three having been adopted within OECD Test Guideline 497. However, the challenge remains as to how DAs can be used to derive a quantitative point of departure for use in next generation risk assessment (NGRA). Here we provide an update to our previously published NGRA framework and present two hypothetical consumer risk assessment scenarios (rinse-off and leave-on) on one case study ingredient. Diethanolamine (DEA) was selected as the case study ingredient based on the existing NAM information demonstrating differences with respect to the outcomes from in silico predictions and in chemico / in vitro data. Seven DAs were applied, and these differences resulted in divergent DA outcomes and reduced confidence with respect to the hazard potential and potency predictions. Risk assessment conclusion for the rinse-off exposure led to an overall decision of safe for all applied DAs. Risk assessment conclusion for the higher leave-on exposure was safe when based on some DAs but unsafe based on others. The reasons for this were evaluated as well as the inherent uncertainty from the use of each NAM and DA in the risk assessment, enabling further refinement of our NGRA framework.

How to facilitate the implementation of 3D models in China by applying good in vitro method practice for regulatory use.

The Organization for Economic Co-operation and Development (OECD) Guidance Document No. 34 and No. 286 on Good In Vitro Method Practices (GIVIMPs) for the development and implementation of in vitro methods for regulatory use in human safety assessment have been endorsed. Considering that China is accelerating the development of alternative approaches in both research and acceptance, early application of these principles is beneficial to the implementation and acceptance of in vitro alternative methods in China. To promote the replacement of animal testing for regulatory use, L'Oréal initiated the EpiSkin™ skin irritation test (SIT) implementation program in China. More than 50 external scientists participated, and the method has been established in 34 organizations including authorities, industries, and testing service laboratories. Taking two collaborations with Guangdong CDC and Shanghai SGS for in vitro SIT as examples, we demonstrated a method implementation process in good alignment with the OECD principles. The current study illustrated the practical way in which both OECD Guidance documents assisted in the transfer and establishment of in vitro approaches and further promoted the future scientific recognition and acceptance of new OECD-accepted alternative testing methodologies in China.

Transferability and reproducibility of the EpiSkin™ Micronucleus Assay.

A novel in vitro 3D micronucleus assay was developed in China using the EpiSkin™ 3D human skin model. This EpiSkin™ Micronucleus Assay showed good predictivity and reproducibility during internal validation and is expected to contribute to in vitro genotoxicity testing as a follow-up for positive results from 2D micronucleus assay. Having developed the assay in one laboratory, further work focused on the transferability and inter-laboratory reproducibility in two additional Chinese authority laboratories (Guangdong Provincial Center for Disease Control and Prevention and Zhejiang Institute for Food and Drug Control). Formal training was provided for both laboratories, which resulted in good transferability based on the results of two positive compounds, such as mitomycin C and vinblastine. Independent experiments were then performed, and inter-laboratory reproducibility was checked using 2-acetylaminofluorene, 5-fluorouracil, 2,4-dichlorophenol, and d-limonene. The dose-responses of the positive control chemical, mitomycin C, were similar to those of the developing laboratory, and all test chemicals were correctly classified by all laboratories. Overall, there was a good transferability as well as intra- and inter-laboratory reproducibility of the EpiSkin™ Micronucleus Assay. This study further confirmed the assay's robustness and provided confidence to enter following validation stages for scientific acceptance.

Expansion of the Cosmetics Europe skin sensitisation database with new substances and PPRA data.

The assessment of skin sensitisation is a key requirement in all regulated sectors, with the European Union's regulation of cosmetic ingredients being most challenging, since it requires quantitative skin sensitisation assessment based on new approach methodologies (NAMs). To address this challenge, an in-depth and harmonised understanding of NAMs is fundamental to inform the assessment. Therefore, we compiled a database of NAMs, and in vivo (human and local lymph node assay) reference data. Here, we expanded this database with 41 substances highly relevant for cosmetic industry. These structurally different substances were tested in six NAMs (Direct Peptide Reactivity Assay, KeratinoSens™, human Cell Line Activation Test, U-SENS™, SENS-IS, Peroxidase Peptide Reactivity Assay). Our analysis revealed that the substances could be tested without technical limitations, but were generally overpredicted when compared to reference results. Reasons for this reduced predictivity were explored through pairwise NAM comparisons and association of overprediction with hydrophobicity. We conclude that more detailed understanding of how NAMs apply to a wider range of substances is needed. This would support a flexible and informed choice of NAMs to be optimally applied in the context of a next generation risk assessment framework, ultimately contributing to the characterisation and reduction of uncertainty.

Amending the U-SENS™ skin sensitization test method for interfering auto-fluorescent chemicals.

Limitations of the applicability domain of new approach methodologies (NAM) present a major challenge for the testing of cosmetic ingredients in Europe, as the regulation does not allow to resort to in vivo test method. Therefore, research focused on overcoming such limitations of established in vitro test methods is frequently conducted. Here, we address a limitation of the U-SENS™, an in vitro skin sensitization test method that addresses the key event 3 on activation of dendritic cells of the adverse outcome pathway (AOP) for skin sensitization. The applicability domain of the U-SENS™ excludes autofluorescent substances that can interfere with the measurement of the expression of CD86, i.e., the primary readout. An evaluation of several fluorochromes identified APC as most suitable for testing auto-fluorescent chemicals. Acceptance criteria were reproducibly met when using the APC-labelled antibody. Equivalent performance in terms of reproducibility and skin sensitisation hazard assessment of the standard FITC-labelled antibodies and the APC-labelled antibodies was demonstrated by testing 40 substances. Finally, the value of the expanded technical applicability domain was highlighted with a case study using sulfuretin. In conclusion, we successfully demonstrated the expansion of the U-SENS™ applicability domain to interfering auto-fluorescent chemicals by using APC-labelled antibodies.

Assessment of the skin sensitization potential of fragrance ingredients using the U-SENS™ assay.

The U-SENS™ assay was developed to address the third key event of the skin sensitization adverse outcome pathway (AOP) and is described in OECD test guideline 442E, Annex II. A dataset of 68 fragrance ingredients comprised of 7 non-sensitizers and 61 sensitizers was tested in the U-SENS™ assay. The potential for fragrance ingredients to activate dendritic cells, measured by U-SENS™, was compared to the sensitization potential determined by weight of evidence (WoE) from historical data. Of the non-sensitizers, 4 induced CD86 cell surface marker ≥1.5-fold while 3 did not. Of the sensitizers, 50 were predicted to be positive in U-SENS™, while the remaining 11 were negative. Positive and negative predictive values (PPV and NPV) of U-SENS™ were 93% and 21%, respectively. No specific chemical property evaluated could account for misclassified ingredients. Assessment of parent and metabolite protein binding alerts in silico suggests that parent chemical metabolism may play a role in CD86 activation in U-SENS™. Combining the U-SENS™ assay in a "2 out of 3" defined approach with the direct peptide reactivity assay (DPRA) and KeratinoSens™ predicted sensitization hazard with PPV and NPV of 97% and 24%, respectively. Combining complementary in silico and in vitro methods to the U-SENS™ assay should be integrated to define the hazard classification of fragrance ingredients, since a single NAM cannot replace animal-based methods.

SkinEthic HCE Time-to-Toxicity on solids: A test method for distinguishing chemicals inducing serious eye damage, eye irritation and not requiring classification and labelling.

This study describes the development of a Time-to-Toxicity approach for solids (TTS) based on the SkinEthic™ HCE tissue construct, capable to distinguish chemicals that do not require classification for serious eye damage/eye irritation (No Cat.) from chemicals that require classification for eye irritation (Cat. 2), and serious eye damage (Cat. 1). Briefly, the time-to-toxicity of 69 solids was evaluated by exposing SkinEthic™ HCE tissue constructs to the test chemical for two different time periods (30-min, and 120-min). Based on the viability observed for the different exposure periods, a classification was assigned. The within laboratory reproducibility in terms of concordance in classifications (3 UN GHS categories), based on a set of 48 solids, was 93.7%. Furthermore, 73.6% Cat. 1 (N = 24), 55.6% Cat. 2 (N = 15) and 72.2% No Cat. (N = 30) were correctly identified with the SkinEthic™ HCE TTS test method. This study provides evidence that the SkinEthic™ HCE Time-to-Toxicity method (multiple exposure times) can distinguish Cat. 2 solids from Cat. 1 solids. This is an added value compared to the SkinEthic™ HCE EITS method (single exposure time) that can distinguish No Cat. chemicals from chemicals that do require classification and labelling for eye irritation/serious eye damage (Cat. 2/Cat. 1).

CATMoS: Collaborative Acute Toxicity Modeling Suite.

BACKGROUND: Humans are exposed to tens of thousands of chemical substances that need to be assessed for their potential toxicity. Acute systemic toxicity testing serves as the basis for regulatory hazard classification, labeling, and risk management. However, it is cost- and time-prohibitive to evaluate all new and existing chemicals using traditional rodent acute toxicity tests. In silico models built using existing data facilitate rapid acute toxicity predictions without using animals. OBJECTIVES: The U.S. Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) Acute Toxicity Workgroup organized an international collaboration to develop in silico models for predicting acute oral toxicity based on five different end points: Lethal Dose 50 (LD50 value, U.S. Environmental Protection Agency hazard (four) categories, Globally Harmonized System for Classification and Labeling hazard (five) categories, very toxic chemicals [LD50 (LD50≤50mg/kg)], and nontoxic chemicals (LD50>2,000mg/kg). METHODS: An acute oral toxicity data inventory for 11,992 chemicals was compiled, split into training and evaluation sets, and made available to 35 participating international research groups that submitted a total of 139 predictive models. Predictions that fell within the applicability domains of the submitted models were evaluated using external validation sets. These were then combined into consensus models to leverage strengths of individual approaches. RESULTS: The resulting consensus predictions, which leverage the collective strengths of each individual model, form the Collaborative Acute Toxicity Modeling Suite (CATMoS). CATMoS demonstrated high performance in terms of accuracy and robustness when compared with in vivo results. DISCUSSION: CATMoS is being evaluated by regulatory agencies for its utility and applicability as a potential replacement for in vivo rat acute oral toxicity studies. CATMoS predictions for more than 800,000 chemicals have been made available via the National Toxicology Program's Integrated Chemical Environment tools and data sets (ice.ntp.niehs.nih.gov). The models are also implemented in a free, standalone, open-source tool, OPERA, which allows predictions of new and untested chemicals to be made. https://doi.org/10.1289/EHP8495.

Next generation risk assessment for skin sensitisation: A case study with propyl paraben.

Skin sensitisation is a key adverse health effect to be addressed in the safety assessment of cosmetic ingredients. Regulatory demands have urged the development of Next Generation Risk Assessment (NGRA) using New Approach Methodologies (NAM) and Defined Approaches (DA) instead of animal models. An illustrative NGRA case study shall demonstrate if the use of propyl paraben at 0.2% in a face cream was safe for consumers. A sequential stacking tier testing DA based on NAM data predicted propyl paraben to be a non-sensitiser, while some NAM input data showed positive results. To increase confidence, structurally related parabens were considered, which revealed NAM and DA hazard predictions similar to those of propyl paraben, non-sensitiser classifications in animal models and very rare cases of human skin allergy. Based on a weight of evidence it was decided that propyl paraben should be considered a non-sensitiser leading to a favourable NGRA conclusion, in line with traditional risk assessment. Examination of an ab initio NGRA based on NAM and metabolism data resulted in a more conservative weak sensitiser consideration as point of departure, which still led to a favourable conclusion.

Real world-like simulations show efficient predictive power of in vitro skin corrosion tests used as stand-alone and in combination and how can toxicologists take advantage of them.

Historically, performance of in vitro toxicology test methods has been evaluated and considered for regulatory purposes based on sensitivity & specificity values derived from validation studies. Other indicators are however useful to evaluate in vitro tests such as positive & negative predictive values (PPV & NPV), likelihood ratios (LRs) or odds ratio (OR). These indicators, which are routinely used in diagnostic tests, if adapted and adequately applied to in vitro tests would help determine their realistic predictive power in real world-like situation and help risk assessors know how they can rely on in vitro tests used for their safety assessments. This paper performs a series of simulations considering the actual distribution in ECHA C&L inventory of skin corrosive chemicals to calculate several of these indicators of performance (PPV, NPV, LRs, OR). It shows applied examples of predictive power on EpiSkin™ and SkinEthic™ RHE two validated in vitro skin corrosive tests, explains how to build testing strategies based on these examples, compares so called 'bottom-up' and 'top-down' approaches, and demonstrates the number of tests required, and how risk assessors can practically take advantage of this.

The EyeIRR-IS assay: Development and evaluation of an in vitro assay to measure the eye irritation sub-categorization of liquid chemicals.

Several alternative methods have been developed and regulatory adopted by OECD as in vitro alternatives to the Draize eye irritation assay either to detect chemicals not requiring classification (No Category) or inducing serious damage to the eye (Category 1) but none are sensitive enough to identify chemicals inducing reversible eye effects (category 2) which are categorised by default. Therefore, the discriminatory power of a genomic approach applied to the SkinEthic™ Human Corneal Epithelium (HCE) model was investigated to allow subcategorization capacity according to UN GHS classification. An algorithm based on gene expression modulation on a training (62) and a test (31 liquids) chemical set, tested neat and at 30%was evaluated in an assay called EyeIRR-IS. Its accuracy prediction to distinguish Cat1/Cat2 from No Cat was 95% with a specificity of 89% and a sensitivity of 98%. For subcategorization into the 3 GHS classes the accuracy reached 84% with 94% Cat1, 67% Cat2 and 89% No Cat correctly predicted. No Cat.1 chemicals were underestimated as negative with a majority of misclassified Cat2 over predicted as Cat 1. In conclusion, the performance of the assay suggests its added value in a defined approach for liquids to replace the Draize assay.

A new 3D model for genotoxicity assessment: EpiSkin™ Micronucleus Assay.

The European Regulation on Cosmetics (no. 1223/2009) has prohibited the use of animals in safety testing since March 2009 for ingredients used in cosmetics. Irreversible events at the chromosome level (clastogenesis and aneugenesis) are commonly evaluated by scoring either micronuclei or chromosome aberrations using cell-based genotoxicity assays. Like most in vitro genotoxicity assays, the 2D in vitro micronucleus assay exhibits a poor specificity and does not mimic the dermal route. To address these limitations, the current project aims to develop and validate a 3D micronucleus assay using the EpiSkin™ model. This project is scientifically supported by the Cosmetics Europe Genotoxicity Task Force. In a first step, two key criteria for the development of micronucleus assay, namely, the sufficient yield of cells from the EpiSkin™ model and an acceptable proliferation rate of the basal layer, were assessed and demonstrated. Subsequently, six chemicals (vinblastine, n-ethylnitrosourea, ß-butyrolactone, 2-acetylaminofluorene, 2,4-dichlorophenoland d-limonene) were evaluated in the EpiSkin™ Micronucleus Assay. At least two independent experiments using 48- and 72-h incubations were performed for each chemical. Results showed good inter-experimental reproducibility, as well as the correct identification of all six tested chemicals. The metabolism of 2-acetylaminofluorene on the EpiSkin™ model was also investigated and confirmed by the formation of an intermediate metabolite (2-aminofluorene). These preliminary results from the EpiSkin™ Micronucleus Assay indicate that it is a promising in vitro assay for assessing genotoxicity. The availability and suitability of this test method contribute significantly to the development of non-animal testing methods in China and its impact on the worldwide field.