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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., (© The Author(s) 2021. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society.)

A database of 91 chemicals with published data from both transgenic rodent mutation (TGR) and rodent comet assays has been compiled. The objective was to compare the sensitivity of the two assays for detecting genotoxicity. Critical aspects of study design and results were tabulated for each dataset. There were fewer datasets from rats than mice, particularly for the TGR assay, and therefore, results from both species were combined for further analysis. TGR and comet responses were compared in liver and bone marrow (the most commonly studied tissues), and in stomach and colon evaluated either separately or in combination with other GI tract segments. Overall positive, negative, or equivocal test results were assessed for each chemical across the tissues examined in the TGR and comet assays using two approaches: 1) overall calls based on weight of evidence (WoE) and expert judgement, and 2) curation of the data based on a priori acceptability criteria prior to deriving final tissue specific calls. Since the database contains a high prevalence of positive results, overall agreement between the assays was determined using statistics adjusted for prevalence (using AC1 and PABAK). These coefficients showed fair or moderate to good agreement for liver and the GI tract (predominantly stomach and colon data) using WoE, reduced agreement for stomach and colon evaluated separately using data curation, and poor or no agreement for bone marrow using both the WoE and data curation approaches. Confidence in these results is higher for liver than for the other tissues, for which there were less data. Our analysis finds that comet and TGR generally identify the same compounds (mainly potent mutagens) as genotoxic in liver, stomach and colon, but not in bone marrow. However, the current database content precluded drawing assay concordance conclusions for weak mutagens and non-DNA reactive chemicals., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

The Organisation for Economic Co-operation and Development Test Guideline 488 (TG 488) provides recommendations for assessing germ cell and somatic cell mutagenicity using transgenic rodent (TGR) models. However, important data gaps exist for selecting an optimal approach for simultaneously evaluating mutagenicity in both cell types. It is uncertain whether analysis of germ cells from seminiferous tubules (hereafter, tubule germ cells) or caudal sperm within the recommended design for somatic tissues (i.e., 28 days of exposure plus three days of fixation time, 28 + 3d) has enough sensitivity to detect an effect as compared with the analysis of sperm within the recommended design for germ cells (i.e., 28 + 49d and 28 + 70d for mouse and rat, respectively). To address these data gaps, the Germ Cell workgroup of the Genetic Toxicology Technical Committee of the Health and Environmental Sciences Institute reviewed the available TGR mutagenicity data in male germ cells, and, characterized the exposure history of tubule germ cells for different sampling times to evaluate its impact on germ cell mutagenicity testing using TG 488. Our analyses suggest that evaluating mutant frequencies in: i) sperm from the cauda epididymis at 28 + 3d does not provide meaningful mutagenicity data; ii), tubule germ cells at 28 + 3d provides reliable mutagenicity data only if the results are positive; and iii) tubule germ cells at 28 + 28d produces reliable positive and negative results in both mice and rats. Thus, the 28 + 28d regimen may provide an approach for simultaneously assessing mutagenicity in somatic tissues and germ cells from the same animals. Further work is required to support the 28 + 28d protocol for tissues other than slowly proliferating tissues as per current TG 488. Finally, recommendations are provided to guide the experimental design for germ cell mutagenicity data for regulatory submission, as well as other possible approaches to increase the reliability of the TGR assay., (Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.)

The Organisation for Economic Co-operation and Development Test Guideline (TG) 488 for the transgenic rodent (TGR) mutation assay recommends two sampling times for assessing germ cell mutagenicity following the required 28-day exposure period: 28 + > 49 days for mouse sperm and 28 + >70 days for rat sperm from the cauda epididymis, or three days (i.e., 28 + 3d) for germ cells from seminiferous tubules (hereafter, tubule germ cells) plus caudal sperm for mouse and rat. Although the latter protocol is commonly used for mutagenicity testing in somatic tissues, it has several shortcomings for germ cell testing because it provides limited exposure of the proliferating phase of spermatogenesis when mutations are fixed in the transgene. Indeed, analysis of sperm at 28 + 3d has generated negative results with established germ cell mutagens, while the analysis of tubule germ cells has generated both positive and either negative or equivocal results. The Germ Cell workgroup of the Genetic Toxicology Technical Committee of the Health and Environmental Sciences Institute modelled mouse and rat spermatogenesis to better define the exposure history of the cell population collected from seminiferous tubules. The modelling showed that mouse tubule germ cells at 28 + 3d receive, as a whole, 42% of the total exposure during the proliferating phase. This percentage increases to 99% at 28 + 28d and reaches 100% at 28 + 30d. In the rat, these percentages are 22% and 80% at 28 + 3d and 28 + 28d, reaching 100% at 28 + 44d. These results show that analysis of tubule germ cells at 28 + 28d may be an effective protocol for assessing germ cell mutagenicity in mice and rats using TG 488. Since TG 488 recommends the 28 + 28d protocol for slow dividing somatic tissues, this appears to be a better compromise than 28 + 3d when slow dividing somatic tissues or germ cells are the critical tissues of interest., (Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.)

ToxCast is a multiyear effort to develop a cost-effective approach for the US EPA to prioritize chemicals for toxicity testing. Initial evaluation of more than 500 high-throughput (HT) microwell-based assays without metabolic activation showed that most lacked high specificity and sensitivity for detecting genotoxicants. Thus, EPA initiated a pilot project to investigate the use of standard genotoxicity endpoints using medium-throughput genotoxicity (MTG) assays in the context of a large testing program. Twenty-five chemicals were selected from the ToxCast program based in part on their known genotoxicity. The two MTG assays used were the Ames II(™) assay and 96-well In Vitro MicroFlow(®) Micronucleus (MN) assay. The Ames II assay showed a reasonable correlation with published Ames test data and industry submissions, though specificity was much better than sensitivity due to restraints on top concentrations as prescribed by ToxCast. Overall concordance was 73% both with and without metabolic activation. The flow MN assay had concordances of 71% and 58% with and without metabolic activation, respectively, when compared to published data and submissions. Importantly, a comparison of results without S9 from the MTG assays to an HT ToxCast p53 activation assay showed a fairly good degree of concordance (67%). The results reported here indicate that assays for genotoxicity endpoints can be conducted in a MT format and have the potential to add to the interpretation of results from large-scale testing programs such as EPA's ToxCast program. Inherent limitations such as the top concentrations used in large scale testing programs are discussed. Environ. Mol. Mutagen. 56:468-476, 2015. © 2014 Wiley Periodicals, Inc., (© 2014 Wiley Periodicals, Inc.)

As part of the international Pig-a validation trials, we examined the induction of Pig-a mutant reticulocytes and red blood cells (RET(CD59-) and RBC(CD59-), respectively) in peripheral blood of male Sprague Dawley(®) rats treated with urethane (25, 100 and 250mg/kg/day) or saline by oral gavage for 29 days. Additional endpoints integrated into this study were: micronucleated reticulocytes (MN-RET) in peripheral blood; chromosome aberrations (CAb) and DNA damage (%tail intensity via the comet assay) in peripheral blood lymphocytes (PBL); micronucleated polychromatic erythrocytes (MN-PCE) in bone marrow; and DNA damage (comet) in various organs at termination (the 29th dose was added for the comet endpoint at sacrifice). Ethyl methanesulfonate (EMS; 200mg/kg/day on Days 3, 4, 13, 14, 15, 27, 28 and 29) was evaluated as the concurrent positive control (PC). All animals survived to termination and none exhibited overt toxicity, but there were significant differences in body weight and body weight gain in the 250-mg/kg/day urethane group, as compared with the saline control animals. Statistically significant, dose-dependent increases were observed for urethane for: RET(CD59-) and RBC(CD59-) (on Days 15 and 29); MN-RET (on Days 4, 15 and 29); and MN-PCE (on Day 29). The comet assay yielded positive results in PBL (Day 15) and liver (Day 29), but negative results for PBL (Days 4 and 29) and brain, kidney and lung (Day 29). No significant increases in PBL CAb were observed at any sample time. Except for PBL CAb (likely due to excessive cytotoxicity), EMS-induced significant increases in all endpoints/tissues. These results compare favorably with earlier in vivo observations and demonstrate the utility and sensitivity of the Pig-a in vivo gene mutation assay, and its ability to be easily integrated, along with other standard genotoxicity endpoints, into 28-day rodent toxicity studies., (© The Author 2015. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

This workshop reviewed the current science to inform and recommend the best evidence-based approaches on the use of germ cell genotoxicity tests. The workshop questions and key outcomes were as follows. (1) Do genotoxicity and mutagenicity assays in somatic cells predict germ cell effects? Limited data suggest that somatic cell tests detect most germ cell mutagens, but there are strong concerns that dictate caution in drawing conclusions. (2) Should germ cell tests be done, and when? If there is evidence that a chemical or its metabolite(s) will not reach target germ cells or gonadal tissue, it is not necessary to conduct germ cell tests, notwithstanding somatic outcomes. However, it was recommended that negative somatic cell mutagens with clear evidence for gonadal exposure and evidence of toxicity in germ cells could be considered for germ cell mutagenicity testing. For somatic mutagens that are known to reach the gonadal compartments and expose germ cells, the chemical could be assumed to be a germ cell mutagen without further testing. Nevertheless, germ cell mutagenicity testing would be needed for quantitative risk assessment. (3) What new assays should be implemented and how? There is an immediate need for research on the application of whole genome sequencing in heritable mutation analysis in humans and animals, and integration of germ cell assays with somatic cell genotoxicity tests. Focus should be on environmental exposures that can cause de novo mutations, particularly newly recognized types of genomic changes. Mutational events, which may occur by exposure of germ cells during embryonic development, should also be investigated. Finally, where there are indications of germ cell toxicity in repeat dose or reproductive toxicology tests, consideration should be given to leveraging those studies to inform of possible germ cell genotoxicity., (Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (HESI) Project Committee on the Relevance and Follow-up of Positive Results in In Vitro Genetic Toxicity (IVGT) Testing established an Emerging Technologies and New Strategies Workgroup to review the current State of the Art in genetic toxicology testing. The aim of the workgroup was to identify promising technologies that will improve genotoxicity testing and assessment of in vivo hazard and risk, and that have the potential to help meet the objectives of the IVGT. As part of this initiative, HESI convened a workshop in Washington, DC in May 2008 to discuss mature, maturing, and emerging technologies in genetic toxicology. This article collates the abstracts of the New and Emerging Technologies Workshop together with some additional technologies subsequently considered by the workgroup. Each abstract (available in the online version of the article) includes a section addressed specifically to the strengths, weaknesses, opportunities, and threats associated with the respective technology. Importantly, an overview of the technologies and an indication of how their use might be aligned with the objectives of IVGT are presented. In particular, consideration was given with regard to follow-up testing of positive results in the standard IVGT tests (i.e., Salmonella Ames test, chromosome aberration assay, and mouse lymphoma assay) to add weight of evidence and/or provide mechanism of action for improved genetic toxicity risk assessments in humans., (Copyright © 2010 Wiley-Liss, Inc.)

The toxicological relevance of the micronucleus (MN) test is well defined: it is a multi-target genotoxic endpoint, assessing not only clastogenic and aneugenic events but also some epigenetic effects, which is simple to score, accurate, applicable in different cell types. In addition, it is predictive for cancer, amenable for automation and allows good extrapolation for potential limits of exposure or thresholds and it is easily measured in experimental both in vitro and in vivo systems. Implementation of in vitro micronucleus (IVMN) assays in the battery of tests for hazard and risk assessment of potential mutagens/carcinogens is therefore fully justified. Moreover, the final draft of an OECD guideline became recently available for this test. In this review, we discuss the prerequisites for an acceptable MN assay, including the cell as unit of observation, importance of cell membranes, the requirement of a mitotic or meiotic division and the assessment of cell division in the presence of the test substance. Furthermore, the importance of adequate design of protocols is highlighted and new developments, in particular the in vitro 3D human skin models, are discussed. Finally, we address future research perspectives including the possibility of a combined primary 3D human skin and primary human whole blood culture system, and the need for adaptation of the IVMN assays to assess the genotoxic potential of new materials, in particular nanomaterials.

For the assessment of genotoxic effects of cosmetic ingredients, a number of well-established and regulatory accepted in vitro assays are in place. A caveat to the use of these assays is their relatively low specificity and high rate of false or misleading positive results. Due to the 7th amendment to the EU Cosmetics Directive ban on in vivo genotoxicity testing for cosmetics that was enacted March 2009, it is no longer possible to conduct follow-up in vivo genotoxicity tests for cosmetic ingredients positive in in vitro genotoxicity tests to further assess the relevance of the in vitro findings. COLIPA, the European Cosmetics Association, has initiated a research programme to improve existing and develop new in vitro methods. A COLIPA workshop was held in Brussels in April 2008 to analyse the best possible use of available methods and approaches to enable a sound assessment of the genotoxic hazard of cosmetic ingredients. Common approaches of cosmetic companies are described, with recommendations for evaluating in vitro genotoxins using non-animal approaches. A weight of evidence approach was employed to set up a decision-tree for the integration of alternative methods into tiered testing strategies., (Copyright 2010 Elsevier Inc. All rights reserved.)

Dextromethorphan (DMP) is an effective and widely used antitussive drug. While DMP has over a 50 year safe-marketing history, the only available genotoxicity data was an unpublished, negative Ames assay (Roche). Lack of a complete genotoxicity profile on DMP, specifically covering the chromosomal damage endpoint, prompted a regulatory request for an in vitro chromosome aberration assay. In accordance with EC and CPMP Guidance, we evaluated data for a number of chemicals with a structural relationship to DMP. DMP contains no structural alerts for genotoxicity or carcinogenicity using the Deductive Estimation of Risk from Existing Knowledge (DEREK) software tool, confirming the negative results obtained in the existing Ames assay. This is also consistent with the mostly negative genotoxicity and carcinogenicity data available on structurally related chemicals including morphine, codeine, nalbuphine, buprenorphine, naloxone, hydromorphone, levorphanol, and oxycodone. A state-of-the-science, in vitro chromosome aberration assay was also conducted, which demonstrated a lack of genotoxicity for DMP. The overall weight of evidence for DMP and its structural analogues, supports the conclusion that this class of phenanthrene-based chemicals, and DMP, in particular, are not genotoxic in vitro or in vivo, and do not represent a carcinogenic risk to patients.

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.

In this report, results are presented from an international study of the in vitro micronucleus assay using Chinese hamster ovary cells. This study was coordinated by an organizing committee supported by the SFTG (the French branch of the European Environmental Mutagen Society). Test chemicals included mannitol, bleomycin, cytosine arabinoside, urethane and diethylstilboestrol. Mitomycin C was used as a positive control. Each chemical was evaluated in at least two laboratories following a variety of different protocols (short and long exposures, varying recovery times, with and without cytochalasin B) in order to help determine a standard protocol for routine testing in Chinese hamster ovary cells. Mannitol and urethane were negative, while bleomycin, cytosine arabinoside and diethylstilboestrol induced a dose dependent increase in micronucleated cells. In the presence of cytochalasin B, increases in micronuclei were observed in binucleated as well as mononucleated cells in cultures treated with bleomycin, cytosine arabinoside or diethylstilboestrol. Importantly, all three of these chemicals were detected in each of the different treatment/recovery regimens. No differences were seen in the sensitivity or accuracy of the responses in the presence of absence of cytochalasin B. Overall, these results demonstrate the suitability of Chinese hamster ovary cells for the in vitro micronucleus assay.

During the safety evaluation process of new drugs and chemicals, a battery of genotoxicity tests is conducted starting with in vitro genotoxicity assays. Obtaining positive results in in vitro genotoxicity tests is not uncommon. Follow-up studies to determine the biological relevance of positive genotoxicity results are costly, time consuming, and utilize animals. More efficient methods, especially for identifying a putative mode of action like an indirect mechanism of genotoxicity (where DNA molecules are not the initial primary targets), would greatly improve the risk assessment for genotoxins. To this end, we are participating in an International Life Sciences Institute (ILSI) project involving studies of gene expression changes caused by model genotoxins. The purpose of the work is to evaluate gene expression tools in general, and specifically for discriminating genotoxins that are direct-acting from indirect-acting. Our lab has evaluated gene expression changes as well as micronuclei (MN) in L5178Y TK(+/-) mouse lymphoma cells treated with six compounds. Direct-acting genotoxins (where DNA is the initial primary target) that were evaluated included the DNA crosslinking agents, mitomycin C (MMC) and cisplatin (CIS), and an alkylating agent, methyl methanesulfonate (MMS). Indirect-acting genotoxins included hydroxyurea (HU), a ribonucleotide reductase inhibitor, taxol (TXL), a microtubule inhibitor, and etoposide (ETOP), a DNA topoisomerase II inhibitor. Microarray gene expression analysis was conducted using Affymetrix mouse oligonucleotide arrays on RNA samples derived from cells which were harvested immediately after the 4 h chemical treatment, and 20 h after the 4 h chemical treatment. The evaluation of these experimental results yields evidence of differentially regulated genes at both 4 and 24 h time points that appear to have discriminating power for direct versus indirect genotoxins, and therefore may serve as a fingerprint for classifying chemicals when their mechanism of action is unknown.

There has been much discussion on acceptable spontaneous mutant frequencies in the mouse lymphoma assay (MLA). This culminated in the International Workshop on Genotoxicity Testing (IWGT) recommended control limits for the microtitre version of 50-170 mutants/106 viable cells, which has now been included in the draft Organization for Economic Co-Operation and Development guideline for assays investigating mammalian cell gene mutation at the tk locus. Some of the factors affecting mutant frequency have been investigated. It was shown that when culturing methotrexate cleansed TK+/− cells, a spontaneous mutant frequency of ∼100 mutants/106 viable cells was achieved after only 26 doublings. However, after further culturing for ∼6 months the spontaneous mutant frequency only gradually increased. Culturing for this time did not affect the karyotype of the cell in so much as the modal chromosome number remained stable. The spontaneous mutant frequency could effectively be manipulated by cleansing with various concentrations of methotrexate. The necessity for using appropriately heat-inactivated horse serum was confirmed. Finally, following treatment with 4-nitroquinoline- N-oxide, cells did not preferentially survive when plated at high cell densities (1.6 cells plus 2,000 feeder cells/well) versus cells at low density (1.6 cells/well). It was considered that these findings confirm that the dynamics of spontaneous mutant formation in the MLA are complex. However, the karyotype of L5178Y cells is remarkably stable and assuming investigators are using cells with appropriate provenance and good culturing technique, it is clear that the IWGT recommendations are achievable. Environ. Mol. Mutagen. 55:35-42, 2014. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Examines the relationship between parasite infestation and chemical mutagen metabolism. Experimentation of the schistosoma hematobium parasite; Reduction of dichlorobenzidine mutagenicity; Alteration of chemical mutagen activity in an infested hamster.

Abstract: Two year rodent bioassays play a key role in the assessment of carcinogenic potential of chemicals to humans. The seventh amendment to the European Cosmetics Directive will ban in 2013 the marketing of cosmetic and personal care products that contain ingredients that have been tested in animal models. Thus 2-year rodent bioassays will not be available for cosmetics/personal care products. Furthermore, for large testing programs like REACH, in vivo carcinogenicity testing is impractical. Alternative ways to carcinogenicity assessment are urgently required. In terms of standardization and validation, the most advanced in vitro tests for carcinogenicity are the cell transformation assays (CTAs). Although CTAs do not mimic the whole carcinogenesis process in vivo, they represent a valuable support in identifying transforming potential of chemicals. CTAs have been shown to detect genotoxic as well as non-genotoxic carcinogens and are helpful in the determination of thresholds for genotoxic and non-genotoxic carcinogens. The extensive review on CTAs by the OECD (OECD (2007) Environmental Health and Safety Publications, Series on Testing and Assessment, No. 31) and the proven within- and between-laboratories reproducibility of the SHE CTAs justifies broader use of these methods to assess carcinogenic potential of chemicals. [Copyright &y& Elsevier]