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.

Implementation of New Test Methods into Practical Testing.

New toxicology test methods, especially those using in vitro methods, are continually being developed. Some are used by industry for screening purposes; others are eventually validated for regulatory use. However, for a new test method to be firmly adopted by industry it must be readily available, generally through an in-house industry laboratory, an academic laboratory, or a contract research organization. Regardless of the type of laboratory which intends to implement the test method, certain steps must be taken to ascertain that the method that is put into place is reproducible and performs identically to the test method that was published or has undergone validation. This involves developing protocols and standard operating procedures, training staff, developing historic positive and negative control data, establishing acceptable performance with proficiency chemicals, and addressing all the safety concerns that may accompany the assay. From experience within a contract research laboratory, we provide guidance on how to most efficiently accomplish these tasks.

Local tolerance testing under REACH: Accepted non-animal methods are not on equal footing with animal tests.

In general, no single non-animal method can cover the complexity of any given animal test. Therefore, fixed sets of in vitro (and in chemico) methods have been combined into testing strategies for skin and eye irritation and skin sensitisation testing, with pre-defined prediction models for substance classification. Many of these methods have been adopted as OECD test guidelines. Various testing strategies have been successfully validated in extensive in-house and inter-laboratory studies, but they have not yet received formal acceptance for substance classification. Therefore, under the European REACH Regulation, data from testing strategies can, in general, only be used in so-called weight-of-evidence approaches. While animal testing data generated under the specific REACH information requirements are per se sufficient, the sufficiency of weight-of-evidence approaches can be questioned under the REACH system, and further animal testing can be required. This constitutes an imbalance between the regulatory acceptance of data from approved non-animal methods and animal tests that is not justified on scientific grounds. To ensure that testing strategies for local tolerance testing truly serve to replace animal testing for the REACH registration 2018 deadline (when the majority of existing chemicals have to be registered), clarity on their regulatory acceptance as complete replacements is urgently required.

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.

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.

Report of an ISRTP workshop: progress and barriers to incorporating alternative toxicological methods in the U.S.

The workshop objectives were to explore progress in implementing new, revised and alternative toxicological test methods across regulatory evaluation frameworks and decision-making programs in the United States, to identify barriers and to develop recommendations to further promote adoption of approaches that reduce, refine, or replace the use of animal methods. The workshop included sessions on: (1) current research, development, and validation of alternative methods within the U.S. federal government; (2) emerging alternative methodologies with potential applications to a broad spectrum of toxicity evaluation strategies; (3) tiered evaluation ("intelligent testing") strategies; and (4) identification of, and recommendations to address, critical barriers that affect adoption and use of new, revised alternative toxicological test methods by U.S. regulatory agencies. Through facilitated discussion, a list of barriers and recommendations were developed and grouped into categories of economic/financial, scientific/technical, and regulatory/policy. Overall, participants from all sectors collectively supported catalyzing actions to promote more meaningful and rapid progress for research to develop alternative methods focused for use in regulatory programs, accelerated lab investigations to validate such alternative methods and adoption of regulatory frameworks which embrace and incorporate these validated alternatives.

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.

Evaluation of some in vitro tests to reduce and replace the sub-acute animal toxicity studies.

The toxicologic and carcinogenic potential of chemicals is usually determined through a sequence of acute, sub-acute (14-day), sub-chronic (90-day) and chronic (two-year) studies in rats and mice of both sexes. The US National Toxicology Program (NTP) does not conduct acute toxicity studies. Dose levels for 14-day toxicity studies are typically estimated from information in the literature, if available. The toxicology information obtained from 14-day studies is used in the selection of doses for 90-day studies. The protocol for 14-day studies consists of five doses and control groups and five animals per group of each sex and species, resulting in the use of 120 animals per study. At present, in addition to refining the current testing protocols, the NTP is evaluating the potential for in vitro test methods to partially or completely avoid the need for 14-day toxicity studies, especially for chemicals where the dermal route of exposure is used. The in vitro assays used were the EpiDerm bioassay to estimate dermal irritation, the neutral red uptake (NRU) bioassay to estimate systemic toxicity and the primary rat hepatocyte cytotoxicity (PRHC) assay to estimate hepatotoxicity. The purpose of using these assays was to assess their potential for predicting relative in vivo toxicity and to support dose selection decisions for 90-day studies. In general, based on these limited number of studies, the EpiDerm and NRU tests were predictive of the responses observed in in vivo studies. However, a larger comparative database is needed to derive definitive conclusions regarding the value of in vitro tests in the prediction of in vivo effects.

Follow-up to the ECVAM prevalidation study on in vitro tests for acute skin irritation. The European Centre for the Validation of Alternative Methods Skin Irritation Task Force report 2.

The European Centre for the Validation of Alternative Methods (ECVAM) Skin Irritation Task Force was established in 1996, to review the status of the development and validation of alternative tests for skin irritation and corrosion, and to identify appropriate non-animal tests for predicting human skin irritation that were sufficiently well-developed to be prevalidated and validated by ECVAM. The EpiDerm method, based on a reconstituted human skin model, was proposed as being sufficiently well advanced to enter a prevalidation (PV) study. Based on a review of test protocols, prediction models (PMs), and data submitted by test developers on ten specified chemicals, with 20% sodium lauryl sulphate as a reference standard, the task force recommended the inclusion of four other tests: EPISKIN and PREDISKIN, based on reconstituted human epidermis or on human skin; the non-perfused pig-ear test, based on pig skin; and the skin integrity function test (SIFT), with ex vivo mouse skin. The prevalidation study on these methods was funded by ECVAM, and took place during 1999-2000. The outcome of the PV study was that none of the methods was ready to enter a formal validation study, and that the protocols and PMs of the methods had to be improved in order to increase their predictive abilities. Improved protocols and PMs for the EpiDerm and EPISKIN methods, the pig ear test, and the SIFT were presented at an extended Task Force meeting held in May 2001. It was agreed that, in the short term, the performance of the revised and harmonised EpiDerm and EPISKIN methods, as well as the modified SIFT, should be evaluated in a further study with a new set of 20 test chemicals. In addition, it was decided that the SIFT and the pig ear test would be compared to see if common endpoints (transepidermal water loss, methyl green-pyronine stain) could be identified.

Ocular safety: a silent (in vitro) success story.

Ocular irritation testing has been one of the animal test methods most criticised by animal welfare advocates. Additional criticism has arisen from within the scientific community, based on the variability of the animal test results and the questionable relevance of the extremely high dose levels employed. As a result, the Draize eye irritation test has been one of the main targets for in vitro replacement. Despite extensive efforts, however, there is still no in vitro method that is fully validated as a regulatory replacement. In spite of this, many individual companies are using diverse in vitro ocular irritation tests to gain important safety and efficacy information about their products and raw materials, eliminating the need for animal testing in the process. This is done in a safe fashion by applying intelligent testing paradigms. ECVAM has played a major role in this success, through its many programmes that have emphasised the importance of understanding the true toxicological need, and then using in vitro tests to provide that information. Thus, even in the absence of a successfully validated regulatory assay, the desired result of reducing animal testing is being met.