GADD45a-GFP GreenScreen HC assay results for the ECVAM recommended lists of genotoxic and non-genotoxic chemicals for assessment of new genotoxicity tests

A recent ECVAM workshop considered how to reduce falsely predictive positive results when undertaking in vitro genotoxicity testing, and thus to avoid unnecessary follow-up with tests involving animals. As it was anticipated that modified versions of existing assays as well as new assays might contribute to a solution, an expert panel was asked to identify a list of chemicals that could be used in the evaluation of such assays. Three categories of test chemicals were chosen comprising a total of 62 compounds. This paper provides test results for these chemicals using the GreenScreen HC assay. All tests were carried out in triplicate, by multiple operators, with and without S9, using invariant protocols. Group 1 chemicals should be detected as positive in in vitro mammalian cell genotoxicity tests: 18/20 (90%) were reproducibly positive in GreenScreen HC. Group 2 chemicals should give negative results in in vitro genotoxicity tests: 22/23 (96%) were reproducibly negative in GreenScreen HC. Overall concordance for Groups 1 and 2 is 93%. Group 3 chemicals should give negative results in in vitro mammalian cell genotoxicity tests, but have been reported to induce chromosomal aberrations or Tk mutations in mouse lymphoma cells, often at high concentrations or at high levels of cytotoxicity: 13/17 (76%) were reproducibly negative in GreenScreen HC. Of the four positive compounds in Group 3, p-nitrophenol was only positive at the top dose (10 mM), 2,4-DCP is an in vivo genotoxin, and two chemicals are antioxidant compounds that may be acting as pro-oxidants in the hyperoxic conditions of cell culture. Overall, these predictive figures are similar to those from other studies with the GreenScreen HC assay and confirm its high specificity, which in turn minimizes the generation of falsely predictive positive results.     

An evaluation of the mouse sperm morphology test and other sperm tests in nonhuman mammals. A report of the U.S. Environmental Protection Agency Gene-Tox Program

The literature on the mouse sperm morphology test and on other sperm tests in nonhuman mammals was reviewed (a) to evaluate the relationship of these tests to chemically induced spermatogenic dysfunction, germ-cell mutagenicity, and carcinogenicity, and (b) to make an interspecies comparison to chemicals. A total of 71 papers were reviewed. The mouse sperm morphology test was used to assess the effects of 154 of the 182 chemical agents covered. 4 other murine sperm tests were also used: the induction of acrosomal abnormalities (4 agents), reduction in sperm counts, (6 agents), motility (5 agents), and F1 sperm morphology (7 agents)). In addition, sperm tests for the spermatogenic effects of 35 agents were done in 9 nonmurine mammalian species; these included analyses for sperm count, motility, and morphology, using a large variety of study designs. For the mouse sperm morphology test, 41 agents were judged by the reviewing committee to be positive inducers of sperm-head shape abnormalities, 103 were negative, and 10 were inconclusive. To evaluate the relationship between changes in sperm morphology and germ cell mutagenicity, the effects of 41 agents on mouse sperm shape were compared to available data from 3 different mammalian germ-cell mutational tests (specific locus, heritable translocation, and dominant lethal). The mouse sperm morphology test was found to be highly sensitive to germ-cell mutagens; 100% of the known mutagens were correctly identified as positives in the sperm morphology test. Data are insufficient at present to access the rate of false positives. Although it is biologically unclear why one might expect changes in sperm morphology to be related to carcinogenesis, we found that (a) a positive response in the mouse sperm morphology test is highly specific for carcinogenic potential (100% for the agents surveyed), and (b) overall, only 50% of carcinogens were positive in the test (i.e., sensitivity approximately equal to 50%). Since many carcinogens do not produce abnormally shaped sperm even at lethal doses, negative findings with the sperm test cannot be used to classify agents as noncarcinogens. We conclude that the mouse sperm morphology test has potential use for identifying chemicals that induce spermatogenic dysfunction and perhaps heritable mutations. Insufficient numbers of chemicals agents have been studied by the other sperm tests to permit similar comparisons. A comparison of 25 chemicals tested with sperm counts, motility, and morphology in at least 2 species (including man, mouse and 9 other mammals) demonstrated good agreement in response among species. With further study, interspecies comparisons of chemically induced sperm changes may be useful for predicting and evaluating human effects.     

Review of literature on chemical-induced aneuploidy in mammalian male germ cells

80 papers published between 1970 and 1984 were evaluated for results pertaining to chemical-induced aneuploidy in mammalian male germ cells. Diverse assays and end points were represented. The assays considered to involve direct measures of aneuploidy were based upon chromosome counts in premeiotic, meiotic, and embryonic cells, and the male pronucleus, or upon phenotypic expression of X-linked genetic markers. Assays in which indirect measures were interpreted as evidence for aneuploidy included those primarily assessing chiasma frequencies, univalent frequencies, and spermatid/sperm sex chromosome body counts. An initial screening to reject studies with insufficient data and those which did not involve a single chemical test agent led to the elimination of 39 papers from further review. The remaining 41 papers reported effects from 46 different chemicals. These papers were rigorously assessed for adequacy of experimental protocols, relevance of end points as direct measures of aneuploidy, and completeness of data presentation and statistical analysis. Criteria specific to each assay were also considered. 4 chemical tests were considered to provide reliable positive or negative aneuploidy data. Cyclophosphamide and chloral hydrate each caused metaphase II hyperploidy when injected into mice. Very limited analyses of trenimon and isoniazid provided negative results. Test findings for 44 chemicals were viewed as inconclusive. It was concluded that standardization of tests to evaluate chemical-induced aneuploidy in male germ cells and the application of these tests towards increasing the data base are badly needed.     

Reduction of misleading ("false") positive results in mammalian cell genotoxicity assays. II. Importance of accurate toxicity measurement

In a previous publication, Fowler et al. [4] demonstrated that the seemingly high rate of false or misleading positive results obtained in in vitro cytogenesis assays for genotoxicity - when compared with in vivo genotoxicity or rodent carcinogenicity data - was greater when rodent cell lines were used that were also reported to have mutant or non-functional p53. As part of a larger project for improvement of in vitro mammalian cell assays, we have investigated the impact of different toxicity measures, commonly used in in vitro cytogenetic assays, on the occurrence of misleading positive results. From a list of 19 chemicals that produce "false" positive results in in vitro mammalian cell assays [10], six substances that had given positive responses in CHO, CHL and TK6 cells [4], were evaluated for micronucleus induction in vitro, with different measures of toxicity for selection of the top concentration. The data show that estimating toxicity by relative cell count (RCC) or replication index (RI) consistently underestimates the toxicity observed by other measures (Relative Population Doubling, RPD, or Relative Increase in Cell Count, RICC). RCC and RI are more likely to lead to selection of concentrations for micronucleus scoring that are highly cytotoxic and thus could potentially lead to artefacts of toxicity being scored (elevated levels of apoptosis and necrosis), generating misleading positive results. These results suggest that a further reduction in the frequency of misleading positive results in in vitro cytogenetic assays can be achieved with this set of chemicals, by avoiding the use of toxicity measures that underestimate the level of toxicity induced.     

Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens II. Further analysis of mammalian cell results, relative predictivity and tumour profiles

One of the consequences of the low specificity of the in vitro mammalian cell genotoxicity assays reported in our previous paper [D. Kirkland, M. Aardema, L. Henderson, L. Muller, Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens. I. Sensitivity, specificity and relative predictivity, Mutat. Res. 584 (2005) 1-256] is industry and regulatory agencies dealing with a large number of false-positive results during the safety assessment of new chemicals and drugs. Addressing positive results from in vitro genotoxicity assays to determine which are "false" requires extensive resources, including the conduct of additional animal studies. In order to reduce animal usage, and to conserve industry and regulatory agency resources, we thought it was important to raise the question as to whether the protocol requirements for a valid in vitro assay or the criteria for a positive result could be changed in order to increase specificity without a significant loss in sensitivity of these tests. We therefore analysed some results of the mouse lymphoma assay (MLA) and the chromosomal aberration (CA) test obtained for rodent carcinogens and non-carcinogens in more detail. For a number of chemicals that are positive only in either of these mammalian cell tests (i.e. negative in the Ames test) there was no correlation between rodent carcinogenicity and level of toxicity (we could not analyse this for the CA test as insufficient data were available in publications), magnitude of response or lowest effective positive concentration. On the basis of very limited in vitro and in vivo data, we could also find no correlation between the above parameters and formation of DNA adducts. Therefore, a change to the current criteria for required level of toxicity in the MLA, to limit positive calls to certain magnitudes of response, or to certain concentration ranges would not improve the specificity of the tests without significantly reducing the sensitivity. We also investigated a possible correlation between tumour profile (trans-species, trans-sex and multi-site versus single-species, single-sex and single-site) and pattern of genotoxicity results. Carcinogens showing the combination of trans-species, trans-sex and multi-site tumour profile were much more prevalent (70% more) in the group of chemicals giving positive results in all three in vitro assays than amongst those giving all negative results. However, single-species, single-sex, single-site carcinogens were not very prevalent even amongst those chemicals giving three negative results in vitro. Surprisingly, when mixed positive and negative results were compared, multi-site carcinogens were highly prevalent amongst chemicals giving only a single positive result in the battery of three in vitro tests. Finally we extended our relative predictivity (RP) calculations to combinations of positive and negative results in the genotoxicity battery. For two out of three tests positive, the RP for carcinogenicity was no higher than 1.0 and for 2/3 tests negative the RP for non-carcinogenicity was either zero (for Ames+MLA+MN) or 1.7 (for Ames+MLA+CA). Thus, all values were less than a meaningful RP of two, and indicate that it is not possible to predict outcome of the rodent carcinogenicity study when only 2/3 genotoxicity results are in agreement.     

The ability of plant genotoxicity assays to predict carcinogenicity

A number of assays have been developed which use higher plants for measuring mutagenic or cytogenetic effects of chemicals, as an indication of carcinogenicity. Plant assays require less extensive equipment, materials and personnel than most other genotoxicity tests, which is a potential advantage, particularly in less developed parts of the world. We have analyzed data on 9 plant genotoxicity assays evaluated by the Gene-Tox program of the U.S. Environmental Protection Agency, using methodologies we have recently developed to assess the capability of assays to predict carcinogenicity and carcinogenic potency. All 9 of the plant assays appear to have high sensitivity (few false negatives). Specificity (rate of true negatives) was more difficult to evaluate because of limited testing on non-carcinogens; however, available data indicate that only the Arabidopsis mutagenicity (ArM) test appears to have high specificity. Based upon their high sensitivity, plant genotoxicity tests are most appropriate for a risk-averse testing program, because although many false positives will be generated, the relatively few negative results will be quite reliable.     

Practical aspects of mutagenicity testing strategy: an industrial perspective

Genetic toxicology studies play a central role in the development and marketing of new chemicals for pharmaceutical, agricultural, industrial, and consumer use. During the discovery phase of product development, rapid screening tests that require minimal amounts of test materials are used to assist in the design and prioritization of new molecules. At this stage, a modified Salmonella reverse mutation assay and an in vitro micronucleus test with mammalian cell culture are frequently used for screening. Regulatory genetic toxicology studies are conducted with a short list of compounds using protocols that conform to various international guidelines. A set of four assays usually constitutes the minimum test battery that satisfies global requirements. This set includes a bacterial reverse mutation assay, an in vitro cytogenetic test with mammalian cell culture, an in vitro gene mutation assay in mammalian cell cultures, and an in vivo rodent bone marrow micronucleus test. Supplementary studies are conducted in certain instances either as a follow-up to the findings from this initial testing battery and/or to satisfy a regulatory requirement. Currently available genetic toxicology assays have helped the scientific and industrial community over the past several decades in evaluating the mutagenic potential of chemical agents. The emerging field of toxicogenomics has the potential to redefine our ability to study the response of cells to genetic damage and hence our ability to study threshold phenomenon.     

Mutation tests in Neurospora crassa. A report of the U.S. Environmental Protection Agency Gene-Tox Program

Many mutation tests have been developed in Neurospora crassa during the almost 40 years of its use in mutation research. These tests detect two major classes of mutation: gene mutation and meiotic nondisjunction. Within the first class, forward- and reverse-mutation tests have been used. The forward-mutation tests include those that detect mutations at many loci and at specific loci. Both kinds of forward-mutation tests have been done in homokaryons (n) and heterokaryons (n + n'). From the publications that were not rejected by our pre-established criteria, data were extracted for 166 chemicals that had been tested for mutagenicity. Only 6 of the 166 chemicals have been tested in one or more gene mutation test and the meiotic nondisjunction test; these 6 chemicals were positive in the first and negative in the second. Of the 102 chemicals tested in one or more gene mutation tests, 94 were positive and 8 were negative. Of the 70 chemicals tested in the meiotic nondisjunction test, 7 were positive and 63 were negative. Two tests, the ad-3 forward-mutation test and the meiotic nondisjunction test, have been used most frequently. These two tests are especially important for hazard evaluation, because each detects a class of mutations that is likely to be deleterious or lethal in the F1 - disomics by the meiotic nondisjunction test and multilocus deletions by the ad-3 forward-mutation test in heterokaryons. Generally, direct-acting chemicals are mutagenic in the gene mutation tests, but few chemicals that required metabolic activation have been tested. Only 31 of the 166 chemicals tested in N. crassa have been tested for carcinogenicity. Among these chemicals, there is a good association between mutagenicity in gene mutation tests and carcinogenicity but a poorer association between meiotic nondisjunction and carcinogenicity; however, only a small number of chemicals has been tested in the meiotic nondisjunction test. Further use and development of certain mutation tests in N. crassa are desirable.     

Assessing the use of known mutagens to calibrate the Salmonella typhimurium mutagenicity assay: I. Without exogenous activation

There has been an increasing need in genetic toxicology to progress from strictly qualitative tests to more quantitative tests. This, in turn, has increased the need to develop better quality assurance and comparative bioassay methods. In this paper, two laboratories tested 10 Salmonella mutagens in order to determine the usefulness of selected chemicals as potential reference materials to calibrate the Salmonella assay. If variance within a bioassay is sufficiently low and the rankings of the compounds are of acceptable consistency, the chemicals later could be evaluated for use as standard control compounds, as audit materials, and as standard reference materials for comparative bioassay efforts. The results demonstrated that the chosen chemicals (with the possible exception of dimethylcarbamylchloride) provide such consistent results in the Salmonella mutagenicity bioassay that they can be used for semi-quantitative calibration and as possible bioassay controls, special audit chemicals, and potentially as reference standards in comparative bioassay efforts. Reference standards, whether used as audit materials or in comparative bioassays, must be used concurrently with the test substances of interest; used without bias; used in a standardized, highly controlled bioassay; and be tested across an appropriate dose range. The study also shows that when these compounds are used as reference standards much care must be given to the number and spacing of doses if highly reproducible slope values are to be generated. We recommend use of a pilot test to establish a dose range for definitive tests and the placement of doses for the definitive tests within the first half of the linear dose-response curve. For appropriate comparisons, one should replicate the tests using the defined dose range and analyze the results in a non-biased statistical manner.     

Comparison of a forward and a reverse mutation assay in Salmonella typhimurium measuring L-arabinose resistance and histidine prototrophy

A forward and a reverse mutation assay designed to detect environmental mutagens have been compared in Salmonella typhimurium. The forward mutation assay scored resistance to L-arabinose and the reverse assay, reversion of histidine auxotrophy. Eighteen chemicals of different structural groups, all known to be mutagenic in the histidine reverse assay, were applied to strains carrying the genetic markers needed to perform both mutation assays. The mutagenicity of each chemical was determined by both plate and liquid tests. The plate test counted absolute numbers of surviving mutants and the liquid test separately measured survival and frequency of mutants among the survivors. All the chemicals used were found to be mutagenic in both mutation assays. The response of the L-arabinose assay was equal to or larger than the response of the histidine assay in the case of 16 chemicals. The two other compounds, 2-nitrofluorene and sodium azide, were detected more efficiently by the histidine assay. Sodium azide, a non-carcinogenic compound, is a potent mutagen in the histidine assay, but very weak in the L-arabinose assay.     

Mammalian in vivo assays for aneuploidy in female germ cells

This paper presents an evaluation of and offers recommendations for assays to detect chemically induced aneuploidy in mammalian female germ cells. 72 papers on female germ cell aneuploidy, published from 1970 to 1984, were reviewed. 28 papers were selected for critical evaluation; the other 44 papers were rejected according to pre-established criteria. Salient points emerging from the information reviewed allow an assessment of the current status of mammalian female germ cell tests for aneuploidy. The majority of data have been obtained by analyzing metaphase II mouse oocyte chromosomes following superovulation. Various classes of chemicals were administered usually around the time of ovulation. Dose-response relationships have not been obtained for the majority of chemicals evaluated. The method of data reporting and analysis usually was not conducive to comparisons among different studies. Few of the 16 chemicals studied can be regarded as negative for their ability to induce aneuploidy, whereas an even smaller number should be considered as positive. Certainly, a need exists to identify the chemicals and the dosages that could increase the incidence of aneuploidy in mammalian female germ cells. Obtaining such data definitely is feasible in cytogenetic laboratories. However, the mammalian female germ cell aneuploid assay should not be perceived as a rapid, inexpensive, routine procedure. The assay is capable of detecting aneuploidy following anaphase I when metaphase II oocytes are studied and following anaphases I and II when first-cleavage zygotes are studied.     

Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens I. Sensitivity, specificity and relative predictivity

The performance of a battery of three of the most commonly used in vitro genotoxicity tests--Ames+mouse lymphoma assay (MLA)+in vitro micronucleus (MN) or chromosomal aberrations (CA) test--has been evaluated for its ability to discriminate rodent carcinogens and non-carcinogens, from a large database of over 700 chemicals compiled from the CPDB ("Gold"), NTP, IARC and other publications. We re-evaluated many (113 MLA and 30 CA) previously published genotoxicity results in order to categorise the performance of these assays using the response categories we established. The sensitivity of the three-test battery was high. Of the 553 carcinogens for which there were valid genotoxicity data, 93% of the rodent carcinogens evaluated in at least one assay gave positive results in at least one of the three tests. Combinations of two and three test systems had greater sensitivity than individual tests resulting in sensitivities of around 90% or more, depending on test combination. Only 19 carcinogens (out of 206 tested in all three tests, considering CA and MN as alternatives) gave consistently negative results in a full three-test battery. Most were either carcinogenic via a non-genotoxic mechanism (liver enzyme inducers, peroxisome proliferators, hormonal carcinogens) considered not necessarily relevant for humans, or were extremely weak (presumed) genotoxic carcinogens (e.g. N-nitrosodiphenylamine). Two carcinogens (5-chloro-o-toluidine, 1,1,2,2-tetrachloroethane) may have a genotoxic element to their carcinogenicity and may have been expected to produce positive results somewhere in the battery. We identified 183 chemicals that were non-carcinogenic after testing in both male and female rats and mice. There were genotoxicity data on 177 of these. The specificity of the Ames test was reasonable (73.9%), but all mammalian cell tests had very low specificity (i.e. below 45%), and this declined to extremely low levels in combinations of two and three test systems. When all three tests were performed, 75-95% of non-carcinogens gave positive (i.e. false positive) results in at least one test in the battery. The extremely low specificity highlights the importance of understanding the mechanism by which genotoxicity may be induced (whether it is relevant for the whole animal or human) and using weight of evidence approaches to assess the carcinogenic risk from a positive genotoxicity signal. It also highlights deficiencies in the current prediction from and understanding of such in vitro results for the in vivo situation. It may even signal the need for either a reassessment of the conditions and criteria for positive results (cytotoxicity, solubility, etc.) or the development and use of a completely new set of in vitro tests (e.g. mutation in transgenic cell lines, systems with inherent metabolic activity avoiding the use of S9, measurement of genetic changes in more cancer-relevant genes or hotspots of genes, etc.). It was very difficult to assess the performance of the in vitro MN test, particularly in combination with other assays, because the published database for this assay is relatively small at this time. The specificity values for the in vitro MN assay may improve if data from a larger proportion of the known non-carcinogens becomes available, and a larger published database of results with the MN assay is urgently needed if this test is to be appreciated for regulatory use. However, specificity levels of <50% will still be unacceptable. Despite these issues, by adopting a relative predictivity (RP) measure (ratio of real:false results), it was possible to establish that positive results in all three tests indicate the chemical is greater than three times more likely to be a rodent carcinogen than a non-carcinogen. Likewise, negative results in all three tests indicate the chemical is greater than two times more likely to be a rodent non-carcinogen than a carcinogen. This RP measure is considered a useful tool for industry to assess the likelihood of a chemical possessing carcinogenic potential from batteries of positive or negative results.     

Mutagenicity testing with transgenic mice. Part II: Comparison with the mouse spot test

The mouse spot test, an in vivo mutation assay, has been used to assess a number of chemicals. It is at present the only in vivo mammalian test system capable of detecting somatic gene mutations according to OECD guidelines (OECD guideline 484). It is however rather insensitive, animal consuming and expensive type of test. More recently several assays using transgenic animals have been developed. From data in the literature, the present study compares the results of in vivo testing of over twenty chemicals using the mouse spot test and compares them with results from the two transgenic mouse models with the best data base available, the lacI model (commercially available as the Big Blue(R) mouse), and the lacZ model (commercially available as the Mutatrade mark Mouse). There was agreement in the results from the majority of substances. No differences were found in the predictability of the transgenic animal assays and the mouse spot test for carcinogenicity. However, from the limited data available, it seems that the transgenic mouse assay has several advantages over the mouse spot test and may be a suitable test system replacing the mouse spot test for detection of gene but not chromosome mutations in vivo.     

Evaluation of five hepatitis C virus screening tests and two supplemental assays: performance when testing sera from sexually transmitted diseases clinic attendees in the USA

The performances of five screening tests (recombinant peptide-based first and second generation tests from Abbott and Ortho, and a synthetic peptide-based test from Biochem Immunosystems) and two supplemental tests: recombinant peptide- based, Abbott neutralization test and Chiron second generation recombinant immunoblot assay (RIBA 2), were evaluated for their ability to detect hepatitis C virus (HCV) antibodies in a population of 276 individuals attending a sexually transmitted diseases (STD) clinic in the USA. Although the five screening tests produced a variable number (35-62) of repeatedly reactive samples, only 13% (36/276) were classified as true positives by the supplemental tests. Thirty-four of the 36 were reactive by all screening tests and 32 of the true positives were reactive by both supplemental tests, while 2 did not neutralize but were reactive in the RIBA 2 test. Of the remaining 2 of the true positives which were discordant by several of the screening assays, 1 was confirmed by both supplemental assays but the other required a chemiluminescent enhancement technique to show positivity in RIBA 2. The sensitivities of the first and second generation Abbott and Ortho tests ranged from 97% to 100% and that of the Biochem test was 94%. The specificities of these tests ranged from 89.2% to 99.6%. The second generation Ortho test presented 9.4% (26/276) false positives. The use of second generation Ortho as a screening test would lead to an excessive number of confirmatory false positives. the positive predictive values of the screening tests ranged from 58.1% to 97.1%. Although the synthetic peptide based Biochem test exhibited the best overall indices, the presence of 2 false negative results would prevent its use as a singular screening test. Nevertheless its high specificity may lend itself to be used as a second screening test before confirmatory testing with RIBA 2.     

Incorporation of mammalian metabolism into mutagenicity testing

In the 1950's and 1960's it became obvious that many chemicals in daily use were mutagenic or carcinogenic, but there seemed to be little relation between the two activities. As scientists were debating the cause of this discrepancy, it was hypothesized that mammalian metabolism could form highly reactive intermediates from rather innocuous chemicals and that these intermediates could react with DNA and were mutagenic. This commentary presents the historical development of metabolic activation in mutagenicity tests, beginning with Udenfriend's hydroxylation system, which mimics aspects of mammalian metabolism in a purely chemical mixture, and extending through procedures that moved closer and closer to incorporating actual mammalian metabolism into the test systems. The stages include microsomal activation systems, host-mediated assays, incorporation of human P450 genes into the target cells or organisms, and detecting mutations in single cells in vivo. A recent development in this progression is the insertion of recoverable vectors containing mutational targets into the mammalian genome. Since the target genes of transgenic assays are in the genome, they are not only exposed to active metabolites, but they also undergo the same repair processes as endogenous genes of the mammalian genome.     

Validation of a genotoxicity test based on p53R2 gene expression in human lymphoblastoid cells

Genotoxicity assessment is important for predicting the carcinogenicity of chemical substances. p53R2 is a p53-regulated gene that is induced by various genotoxic stresses. We previously developed a p53R2-dependent luciferase reporter gene assay in the MCF-7 human breast adenocarcinoma cell line, and demonstrated its ability to detect genotoxic agents. In this paper, we investigate the applicability of the p53R2-based genotoxicity test in the human lymphoblastoid cell line TK6. TK6 cells that express wild-type p53 have been widely used for genetic toxicology studies. To evaluate the performance of the test system in TK6 cells, we referred to 61 of the chemicals on the list of 20 genotoxic and 42 non-genotoxic chemicals recommended for the evaluation of modified or new mammalian cell genotoxicity tests by the European Centre for the Validation of Alternative Methods. The overall accordance, sensitivity, and specificity of our results with the ECVAM list were 90% (55/61), 85% (17/20), and 93% (38/41), respectively. These results indicate that the p53R2-based genotoxicity test can detect various types of genotoxic chemicals without compromising its specificity. This test will be a valuable tool for rapid screen for identifying chemicals that may be genotoxic to humans.     

The mouse spot test. Evaluation of its performance in identifying chemical mutagens and carcinogens

The published results on 60 chemicals and X-rays investigated in the mouse spot test were compared with data on the same chemicals tested in the bacterial mutation assay (Ames test) and lifetime rodent bioassays. The performance of the spot test as an in vivo complementary assay to the in vitro bacterial mutagenesis test reveals that of 60 agents, 38 were positive in both systems, 6 were positive only in the spot test, 10 were positive only in the bacterial test and 6 were negative in both assays. The spot test was also considered as a predictor of carcinogenesis; 45 chemicals were carcinogenic of which 35 were detected as positive by the spot test and 3 out of 6 non-carcinogens were correctly identified as negative. If the results are regarded in sequence, i.e. that a positive result in a bacterial mutagenicity test reveals potential that may or may not be realized in vivo, then 48 chemicals were mutagenic in the bacterial mutation assay of which 38 were active in the spot test and 31 were confirmed as carcinogens in bioassays. 12 chemicals were non-mutagenic to bacteria of which 6 gave positive responses in the spot test and 5 were confirmed as carcinogens. These results provide strong evidence that the mouse coat spot test is an effective complementary test to the bacterial mutagenesis assay for the detection of genotoxic chemicals and as a confirmatory test for the identification of carcinogens. The main deficiency at present is the paucity of data from the testing of non-carcinogens. With further development and improvement of the test it is probable that the predictive performance of the assay in identifying carcinogens should improve, since many of the false negative responses may be due to inadequate testing.     

The use of yeast cultures for the detection of environmental mutagens using a fluctuation test.

A microbial fluctuation test, modified for the detection of environmental mutagens has been evaluated using a number of strains of the yeast Saccharomyces cerevisiae. Auxotrophic diploid cultures of yeast which produce prototrophic colonies by both mitotic gene conversion and mutation have been extensively utilized for the detection and evaluation of chemicals showing genetic activity. A number of the yeast strains utilized were shown to be suitable for use in the fluctuation test although the time scales of the experiments were considerably extended (up to 16 days) compared to those involving bacteria. The yeast strains respond to doses of mutagens at least a 100-fold lower than that required in a conventional short exposure treat and plate experiment. In experiments involving the induction of mitotic gene conversion at the tryptophan-5 and histidine-4 loci in the fluctuation test significant increases in prototrophic cells were produced in the presence of the insecticide Lindex (0.05 microng/ml), the preservative Thiomersal (0.0001 microng/ml), a mahogany hair dye (0.01 microng/ml), the herbicide Paraquat (0.02 microng/ml) and the alkylating agent ethyl methane sulphonate (0.1 microng/ml). The results demonstrate that the fluctuation test provides an extremely sensitive assay for the detection of chemicals which show genetic activity in yeast at non-toxic concentrations.     

Recommended lists of genotoxic and non-genotoxic chemicals for assessment of the performance of new or improved genotoxicity tests: a follow-up to an ECVAM workshop

At a recent ECVAM workshop considering ways to reduce the frequency of irrelevant positive results in mammalian cell genotoxicity tests [D. Kirkland, S. Pfuhler, D. Tweats, M. Aardema, R. Corvi, F. Darroudi, A. Elhajouji, H.-R. Glatt, P. Hastwell, M. Hayashi, P. Kasper, S. Kirchner, A. Lynch, D. Marzin, D. Maurici, J.-R. Meunier, L. Müller, G. Nohynek, J. Parry, E. Parry, V. Thybaud, R. Tice, J. van Benthem, P. Vanparys, P. White, How to reduce false positive results when undertaking in vitro genotoxicity testing and thus avoid unnecessary followup animal tests: Report of an ECVAM Workshop, Mutat. Res. 628 (2007) 31-55], recommendations for improvements/modifications to existing tests, and suggestions for new assays were made. Following on from this, it was important to identify chemicals that could be used in the evaluation of modified or new assays. An expert panel was therefore convened and recommendations made for chemicals to fit three different sets of characteristics, namely: This paper therefore contains these three recommended lists of chemicals and describes how these should be used for any test-evaluation programme.     

Bacterial assays for recombinagens.

Two principal strategies have been used for studying recombinagenic effects of chemicals and radiation in bacteria: (1) measurement of homologous recombination involving defined alleles in a partially diploid strain, and (2) measurement of the formation and loss of genetic duplications in the bacterial chromosome. In the former category, most methods involve one allele in the bacterial chromosome and another in a plasmid, but it is also possible to detect recombination between two chromosomal alleles or between two extrachromosomal alleles. This review summarizes methods that use each of these approaches for detecting recombination and tabulates data on agents that have been found to be recombinagenic in bacteria. The assays are discussed with respect to their effectiveness in testing for recombinagens and their potential for elucidating mechanisms underlying recombinagenic effects.