Validation of the 3D reconstructed human skin micronucleus (RSMN) assay: an animal-free alternative for following-up positive results from standard in vitro genotoxicity assays.

In vitro test batteries have become the standard approach to determine the genotoxic potential of substances of interest across industry sectors. While useful for hazard identification, standard in vitro genotoxicity assays in 2D cell cultures have limited capability to predict in vivo outcomes and may trigger unnecessary follow-up animal studies or the loss of promising substances where animal tests are prohibited or not desired. To address this problem, a team of regulatory, academia and industry scientists was established to develop and validate 3D in vitro human skin-based genotoxicity assays for use in testing substances with primarily topical exposure. Validation of the reconstructed human skin micronucleus (RSMN) assay in MatTek Epi-200™ skin models involved testing 43 coded chemicals selected by independent experts, in four US/European laboratories. The results were analysed by an independent statistician according to predefined criteria. The RSMN assay showed a reproducibly low background micronucleus frequency and exhibited sufficient capacity to metabolise pro-mutagens. The overall RSMN accuracy when compared to in vivo genotoxicity outcomes was 80%, with a sensitivity of 75% and a specificity of 84%, and the between- and within-laboratory reproducibility was 77 and 84%, respectively. A protocol involving a 72-h exposure showed increased sensitivity in detecting true positive chemicals compared to a 48-h exposure. An analysis of a test strategy using the RSMN assay as a follow-up test for substances positive in standard in vitro clastogenicity/aneugenicity assays and a reconstructed skin Comet assay for substances with positive results in standard gene mutation assays results in a sensitivity of 89%. Based on these results, the RSMN assay is considered sufficiently validated to establish it as a 'tier 2' assay for dermally exposed compounds and was recently accepted into the OECD's test guideline development program.

Three-tier testing approach for optimal ocular tolerance sunscreen.

Background: Poor ocular tolerance of sunscreens is partially responsible for poor compliance in use of sunscreens. A three-tiered approach for the testing of ocular tolerance for such products is described that includes an in vitro test for ocular irritation, an in vitro test for the activation of pain receptors, and finally a clinical study involving ocular instillation of the product under controlled conditions followed by ophthalmologic and subjective self-evaluation on a graded scale. We report the results for a new water-based facial sunscreen (SCFW) with very good ocular tolerance. Methods: The ocular irritation potential of SCFW was determined using the EpiOcular™ human cell construct which constituted the first-tier testing. Briefly, the tissues were exposed to SCFW and appropriate positive and negative controls for 15 minutes to 24 hours. After treatment, the tissues were rinsed and cytotoxicity determined. The calculated ET50 value (time at which relative viability decreased 50%) was then used to determine the ocular irritation potential. In the second-tier testing, the sting potential of SCFW was determined by employing the NociOcular assay that measures the activation of TRPV1 (transient receptor potential cation channel subfamily V member 1) specific receptors linked to pain sensation in a neuronal model with over-expression of functional TRPV1 channels. Finally, as the third-tier testing, SCFW was tested in a clinical study with instillation of product into the ocular cul-de-sac and ocular irritation was evaluated after 30 seconds, 15 minutes, and 60 minutes by an ophthalmologist. Participating subjects were also asked to score sensation on a scale of 0 to 3 from slight prickliness to severe stinging. Assay control reference product with known good ocular tolerability (10% baby shampoo) was concurrently tested. Results: In the in vitro topical application assay using the EpiOcular™ construct, no significant cytotoxicity was observed in the tissues exposed to SCFW, indicating minimal ocular irritation potential. In the in vitro NociOcular assay, the cells exposed to the prepared dilutions of SCFW showed minimal TRPV1 specific activity, indicating minimal ocular sting potential. In the in vivo study, no statistically significant differences were found in terms of subjective or objective eye irritation assessment between SCFW and 10% baby shampoo. Conclusion: SCFW showed negligible ocular irritation potential in tier 1, minimal potential to activate pain receptors in tier 2, and good ocular tolerability that was comparable to 10% baby shampoo in tier 3 testing. The results suggest that SCFW has good eye tolerance and that the tiered approach can be used to evaluate facial sunscreens for ocular tolerability.

Using the cytosensor microphysiometer to assess ocular toxicity.

Measuring in vitro cytotoxicity is one method currently used to estimate damage to the eye after chemical exposure. The Cytosensor Microphysiometer method evaluates cytotoxicity by measuring the test material-induced reduction in the metabolic rate of L929 cells. Changes in metabolic rate are measured indirectly as a function of changes in the extracellular acidification rate of the cells. During exposure to increasing concentrations of a cytotoxic material, there is a decrease in the release of acid byproducts into the surrounding medium as the cells die. These acidic metabolic byproducts cause a measurable change in the pH of a lightly buffered medium, which can be measured by the Cytosensor Microphysiometer. The change in the pH of the medium over time is then converted into a metabolic rate estimate for the cells. The endpoint measurement from the assay is the metabolic rate decline of 50%, the MRD50 value (in units of mg/ml).

Evaluation of chemicals requiring metabolic activation in the EpiDerm™ 3D human reconstructed skin micronucleus (RSMN) assay.

The in vitro human reconstructed skin micronucleus (RSMN) assay in EpiDerm™ is a promising new assay for evaluating genotoxicity of dermally applied chemicals. A global pre-validation project sponsored by the European Cosmetics Association (Cosmetics Europe - formerly known as COLIPA), and the European Center for Validation of Alternative Methods (ECVAM), is underway. Results to date demonstrate international inter-laboratory and inter-experimental reproducibility of the assay for chemicals that do not require metabolism [Aardema et al., Mutat. Res. 701 (2010) 123-131]. We have expanded these studies to investigate chemicals that do require metabolic activation: 4-nitroquinoline-N-oxide (4NQO), cyclophosphamide (CP), dimethylbenzanthracene (DMBA), dimethylnitrosamine (DMN), dibenzanthracene (DBA) and benzo(a)pyrene (BaP). In this study, the standard protocol of two applications over 48h was compared with an extended protocol involving three applications over 72h. Extending the treatment period to 72h changed the result significantly only for 4NQO, which was negative in the standard 48h dosing regimen, but positive with the 72h treatment. DMBA and CP were positive in the standard 48h assay (CP induced a more reproducible response with the 72h treatment) and BaP gave mixed results; DBA and DMN were negative in both the 48h and the 72h dosing regimens. While further work with chemicals that require metabolism is needed, it appears that the RMSN assay detects some chemicals that require metabolic activation (4 out of 6 chemicals were positive in one or both protocols). At this point in time, for general testing, the use of a longer treatment period in situations where the standard 48h treatment is negative or questionable is recommended.

The reconstructed skin micronucleus assay (RSMN) in EpiDerm™: detailed protocol and harmonized scoring atlas.

The European Cosmetic Toiletry and Perfumery Association (COLIPA), along with contributions from the European Centre for the Validation of Alternative Methods (ECVAM), initiated a multi-lab international prevalidation project on the reconstructed skin micronucleus (RSMN) assay in EpiDerm™ for the assessment of the genotoxicity of dermally applied chemicals. The first step of this project was to standardize the protocol and transfer it to laboratories that had not performed the assay before. Here we describe in detail the protocol for the RSMN assay in EpiDerm™ and the harmonized guidelines for scoring, with an atlas of cell images. We also describe factors that can influence the performance of the assay. Use of these methods will help new laboratories to conduct the assay, thereby further increasing the database for this promising new in vitro genotoxicity test.

International prevalidation studies of the EpiDerm 3D human reconstructed skin micronucleus (RSMN) assay: transferability and reproducibility.

Recently, a novel in vitro reconstructed skin micronucleus (RSMN) assay incorporating the EpiDerm 3D human skin model (Curren et al., Mutat. Res. 607 (2006) 192-204; Mun et al., Mutat. Res. 673 (2009) 92-99) has been shown to produce comparable data when utilized in three different laboratories in the United States (Hu et al., Mutat. Res. 673 (2009) 100-108). As part of a project sponsored by the European cosmetics companies trade association (COLIPA), with a contribution from the European Center for the Validation of Alternative Methods (ECVAM), international prevalidation studies of the RSMN assay have been initiated. The assay was transferred and optimized in two laboratories in Europe, where dose-dependent, reproducibly positive results for mitomycin C and vinblastine sulfate were obtained. Further intra- and inter-laboratory reproducibility of the RSMN assay was established by testing three coded chemicals, N-ethyl-N-nitrosourea, cyclohexanone, and mitomycin C. All chemicals were correctly identified by all laboratories as either positive or negative. These results support the international inter-laboratory and inter-experimental reproducibility of the assay and reinforce the conclusion that the RSMN assay in the EpiDerm 3D human skin model is a valuable in vitro method for assessment of genotoxicity of dermally applied chemicals.

Intralaboratory and interlaboratory evaluation of the EpiDerm 3D human reconstructed skin micronucleus (RSMN) assay.

A novel in vitro human reconstructed skin micronucleus (RSMN) assay has been developed using the EpiDerm 3D human skin model [R. D. Curren, G. C. Mun, D. P. Gibson, and M. J. Aardema, Development of a method for assessing micronucleus induction in a 3D human skin model EpiDerm, Mutat. Res. 607 (2006) 192-204]. The RSMN assay has potential use in genotoxicity assessments as a replacement for in vivo genotoxicity assays that will be banned starting in 2009 according to the EU 7th Amendment to the Cosmetics Directive. Utilizing EpiDerm tissues reconstructed with cells from four different donors, intralaboratory and interlaboratory reproducibility of the RSMN assay were examined. Seven chemicals were evaluated in three laboratories using a standard protocol. Each chemical was evaluated in at least two laboratories and in EpiDerm tissues from at least two different donors. Three model genotoxins, mitomycin C (MMC), vinblastine sulfate (VB) and methyl methanesulfonate (MMS) induced significant, dose-related increases in cytotoxicity and MN induction in EpiDerm tissues. Conversely, four dermal non-carcinogens, 4-nitrophenol (4-NP), trichloroethylene (TCE), 2-ethyl-1,3-hexanediol (EHD), and 1,2-epoxydodecane (EDD) were negative in the RSMN assay. Results between tissues reconstructed from different donors were comparable. These results indicate the RSMN assay using the EpiDerm 3D human skin model is a promising new in vitro genotoxicity assay that allows evaluation of chromosome damage following "in vivo-like" dermal exposures.

Further development of the EpiDerm 3D reconstructed human skin micronucleus (RSMN) assay.

The upcoming ban on testing of cosmetics in animals by the European Union's 7th Amendment to the Cosmetics Directive will require genotoxicity safety assessments of cosmetics ingredients and final formulations to be based primarily on in vitro genotoxicity tests. The current in vitro test battery produces an unacceptably high rate of false positives, and used by itself would effectively prevent the use and development of many ingredients that are actually safe for human use. To address the need for an in vitro test that is more predictive of genotoxicity in vivo, we have developed an in vitro micronucleus assay using a three-dimensional human reconstructed skin model (EpiDerm) that more closely mimics the normal dermal exposure route of chemicals. We have refined this model and assessed its ability to predict genotoxicity of a battery of chemicals that have been previously classified as genotoxins or non-genotoxins based on in vivo rodent skin tests. Our reconstructed skin micronucleus assay correctly identified 7 genotoxins and 5 non-genotoxins, demonstrating its potential to have a higher predictive value than currently available in vitro genotoxicity tests, and its utility as part of a comprehensive in vitro genotoxicity testing strategy.

Use of the cytosensor microphysiometer to predict results of a 21-day cumulative irritation patch test in humans.

The cytosensor microphysiometer (mu phi) was investigated as a rapid, relatively inexpensive test to predict performance of skin cleansing wipes on the human 21-day cumulative irritation patch test (21CIPT). It indirectly measures metabolic rate changes in L929 cells as a function of test article dose, by measuring the acidification rate in a low-buffer medium. The dose producing a 50% reduction in metabolic rate (MRD50), relative to the baseline rate, is used as a measure of toxicity. The acute toxicity of the mu phi assay can be compared to the chronic toxicity of the 21CIPT, which is based largely on the exposure of test agents to the epidermal cells, resulting in damage and penetration of the stratum corneum leading to cell toxicity. Two series of surfactant-based cleansing wipe products were tested via the mu phi assay and 21CIPT. The first series, consisting of 20 products, was used to determine a prediction model. The second series of 38 products consisted of routine product development formulas or marketed products. Comparing the results from both tests, samples with an MRD50 greater than 50 mg/ml provided a 21CIPT score consistent with a product that performs satisfactorily in the market. When the MRD50 was greater than 78 mg/ml, the 21CIPT score was usually zero. The mu phi may be more sensitive than the 21CIPT for ranking minimally irritating materials. The mu phi assay is useful as a screen for predicting the performance of a wet wipes formula on the 21CIPT, and concurrently reduces the use of animals for safety testing in a product development program for cleansing wipes.

Development of a method for assessing micronucleus induction in a 3D human skin model (EpiDerm).

To meet the requirements of the EU 7th Amendment to the Cosmetics Directive, manufacturers of cosmetics products will need to ascertain the safety of ingredients using non-animal methods. Starting in 2009, in vivo genotoxicity tests for cosmetics ingredients will not be allowed. Skin is a target area of interest for many cosmetic products because of its relatively high exposure. Therefore, it would be beneficial to have a non-animal, skin-based genotoxicity assay, especially one that utilized human skin in vitro. In this paper, we describe the development of a reproducible micronucleus assay that uses EpiDerm engineered human skin constructs (MatTek Corp., Ashland, MA). We describe methods for isolating single cells from the 3D skin model and for processing the cells for microscopic analysis of micronuclei (MN). In addition, since little was known about the kinetics of the dividing keratinocytes in the EpiDerm model, we evaluated whether cytochalasin B (Cyt-B) could be used to distinguish the population of dividing cells allowing the development of a micronucleus assay in binucleated cells. We found that the frequency of binucleated cells increased both with time and with increasing concentration of Cyt-B. After a 48-h exposure, 30-50% binucleated cells were reproducibly obtained. Finally, we evaluated micronucleus induction using the model genotoxicants mitomycin C (MMC) and vinblastine sulfate (VB). The background frequency of MN is very low and reproducible in this model, and statistically significant increases in the frequency of micronucleated cells were induced by both MMC and VB. These are initial steps in developing a routine "in vivo-like" assay for chromosomal damage in human tissue. It is hoped that other investigators utilize these methods to further the understanding of this potentially valuable new non-animal method.