Mutagenicity testing with transgenic mice. Part I: Comparison with the mouse bone marrow micronucleus test

As part of a larger literature study on transgenic animals in mutagenicity testing, test results from the transgenic mutagenicity assays (lacI model; commercially available as the Big Blue(R) mouse, and the lacZ model; commercially available as the Mutatrade markMouse), were compared with the results on the same substances in the more traditional mouse bone marrow micronucleus test. 39 substances were found which had been tested in the micronucleus assay and in the above transgenic mouse systems. Although, the transgenic animal mutation assay is not directly comparable with the micronucleus test, because different genetic endpoints are examined: chromosome aberration versus gene mutation, the results for the majority of substances were in agreement. Both test systems, the transgenic mouse assay and the mouse bone marrow micronucleus test, have advantages and they complement each other. However, the transgenic animal assay has some distinct advantages over the micronucleus test: it is not restricted to one target organ and detects systemic as well as local mutagenic effects.     

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.     

Detailed review of transgenic rodent mutation assays

Induced chromosomal and gene mutations play a role in carcinogenesis and may be involved in the production of birth defects and other disease conditions. While it is widely accepted that in vivo mutation assays are more relevant to the human condition than are in vitro assays, our ability to evaluate mutagenesis in vivo in a broad range of tissues has historically been quite limited. The development of transgenic rodent (TGR) mutation models has given us the ability to detect, quantify, and sequence mutations in a range of somatic and germ cells. This document provides a comprehensive review of the TGR mutation assay literature and assesses the potential use of these assays in a regulatory context. The information is arranged as follows. (1) TGR mutagenicity models and their use for the analysis of gene and chromosomal mutation are fully described. (2) The principles underlying current OECD tests for the assessment of genotoxicity in vitro and in vivo, and also nontransgenic assays available for assessment of gene mutation, are described. (3) All available information pertaining to the conduct of TGR assays and important parameters of assay performance have been tabulated and analyzed. (4) The performance of TGR assays, both in isolation and as part of a battery of in vitro and in vivo short-term genotoxicity tests, in predicting carcinogenicity is described. (5) Recommendations are made regarding the experimental parameters for TGR assays, and the use of TGR assays in a regulatory context.     

A report of the U.S. Environmental Protection Agency Gene-Tox Program. Evaluation of mutagenicity assays for purposes of genetic risk assessment

For the vast majority of chemicals, mammalian germ-line (MG) mutation data do not exist. The question was examined of how best to utilize results of non-MG genotoxicity assays that are included in the Gene-Tox data base to provide information of the likelihood that genetic damage might be induced in and transmitted by the reproductive cells of exposed human beings. Two approaches were used to assess the relative value of different assays for genetic hazard identification. (1) Test results were weighted according to parameters by which conditions of an assay resemble those encountered in the potential induction of transmitted genetic damage in mammals. For this purpose, 35 assays were grouped into 16 categories that were assigned weights ranging from 1 to 15; there were 2367 chemicals in the data base. This system was evaluated by comparing the sum of weighted test results for each chemical with the outcome of MG-standard (MGst) tests where such had been reported. (MGst tests used were the specific-locus and heritable-translocation assays [SLT and HTT] for gene mutations and chromosome aberrations, respectively.) The weighting system produced a few false positives with respect to the MGst results. It produced no false negatives, but the available evidence is limited by the circumstance that MGst test have evidently been preferentially performed with chemicals that had already been shown to be positive in several other assays. (2) Findings from each MGst test were compared with those from each of the other assays in turn, provided that at least 10 chemicals had been tested in both of the assays. There were 11 such comparisons involving the SLT, and 14 such comparisons involving the HTT. The observed concordance was above random expectation in several comparisons, particularly those involving certain mammalian in vivo tests, but in only one case (HTT vs. unscheduled DNA synthesis in the testis) did the degree of elevation approach statistical significance.     

Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens III. Appropriate follow-up testing in vivo

There has been much discussion in recent years regarding the most appropriate follow-up testing in vivo when positive results are obtained in vitro but the in vivo micronucleus (MN) test (traditionally the most widely-used test) is negative. Not all rodent carcinogens give positive results in the micronucleus test, and so it has been common practice to include a second in vivo assay such as the unscheduled DNA synthesis (UDS) test. This has proved useful but is usually limited to analysis of rodent (usually rat) liver. With the increased evaluation and use of other in vivo assays, e.g. for transgenic mutations (TG) and DNA damage (Comet assay) it was important to investigate their usefulness. We therefore examined the published in vivo UDS, TG and Comet-assay results for 67 carcinogens that were negative or equivocal in the micronucleus test. Between 30 and 41 chemicals were evaluated in each of the three in vivo tests, with some overlap. In general, the UDS test was disappointing and gave positive results with <20% of these carcinogens, some of which induced tumours in rat liver and produced DNA adducts in vivo. The TG assay gave positive responses with >50% of the carcinogens, but the Comet assay detected almost 90% of the micronucleus-negative or equivocal carcinogens. This pattern of results was virtually unchanged when the in vitro profile (gene mutagen or clastogen) was taken into account. High sensitivity (ability to detect carcinogens as positive) is only really useful when the specificity (ability to give negative results with non-carcinogens) is also high. Based on small numbers of publications with non-carcinogens, the TG and Comet assays gave negative results with non-carcinogens on 69 and 78% of occasions, respectively. Although further evaluation of the Comet and TG assays, particularly with non-carcinogens, is needed, these data suggest that they both should play a more prominent role in regulatory testing strategies than the UDS test.     

Induction of gene mutations by 5-(2-chloroethyl)-2'-deoxyuridine (CEDU), an antiviral pyrimidine nucleoside analogue

5-(2-chloroethyl)-2'-deoxyuridine (CEDU) had been developed for the treatment of herpes simplex infections. In the Salmonella reverse mutation test, the compound was found to be mutagenic in strains TA1535 and TA102 at very high concentrations (> or =2500 micro g/plate), both with and without S9-mix. The mutagenic potential of CEDU was further investigated in vivo and in vitro. It did not induce DNA repair in rat hepatocyte primary cultures, and was negative in the micronucleus test in V79 cells and in the comet assay in human leukocytes. In vivo, CEDU was negative in the bone marrow micronucleus test in CD1 mice. The mouse spot test provided a clearly positive result. Treatment of mice on day 9 of pregnancy with 2000 mg/kg resulted in 5.9% of the F1 animals having genetically relevant spots, whereas the corresponding vehicle control group had a spot rate of 1.9%. Since these data clearly identified CEDU as an inducer of gene mutations in vivo, this potential was further investigated in lacZ transgenic Muta Mouse. Six female animals were treated daily on five consecutive days with 2000 mg/kg/day and sacrificed, after a treatment-free sampling time, 14 days later. The data showed a clear increase in the mutant frequency in the bone marrow, the lung and in the spleen. CEDU is an exception in the group of nucleoside analogues, because it was found to be a strong gene mutagen and, in contrast to the other compounds of this group investigated so far, had no considerable clastogenic effects.     

Testing strategies in mutagenicity and genetic toxicology: an appraisal of the guidelines of the European Scientific Committee for Cosmetics and Non-Food Products for the evaluation of hair dyes

The European Scientific Committee on Cosmetics and Non-Food Products (SCCNFP) guideline for testing of hair dyes for genotoxic/mutagenic/carcinogenic potential has been reviewed. The battery of six in vitro tests recommended therein differs substantially from the batteries of two or three in vitro tests recommended in other guidelines. Our evaluation of the chemical types used in hair dyes and comparison with other guidelines for testing a wide range of chemical substances, lead to the conclusion that potential genotoxic activity may effectively be determined by the application of a limited number of well-validated test systems that are capable of detecting induced gene mutations and structural and numerical chromosomal changes. We conclude that highly effective screening for genotoxicity of hair dyes can be achieved by the use of three assays, namely the bacterial gene mutation assay, the mammalian cell gene mutation assay (mouse lymphoma tk assay preferred) and the in vitro micronucleus assay. These need to be combined with metabolic activation systems optimised for the individual chemical types. Recent published evidence [D. Kirkland, M. Aardema, L. Henderson, L. Müller, 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] suggests that our recommended three tests will detect all known genotoxic carcinogens, and that increasing the number of in vitro assays further would merely reduce specificity (increase false positives). Of course there may be occasions when standard tests need to be modified to take account of special situations such as a specific pathway of biotransformation, but this should be considered as part of routine testing. It is clear that individual dyes and any other novel ingredients should be tested in this three-test battery. However, new products are formed on the scalp by reaction between the chemicals present in hair-dye formulations. Ideally, these should also be tested for genotoxicity, but at present such experiences are very limited. There is also the possibility that one component could mask the genotoxicity of another (e.g. by being more toxic), and so it is not practical at this time to recommend routine testing of complete hair-dye formulations as well. The most sensible approach would be to establish whether any reaction products within the hair-dye formulation penetrate the skin under normal conditions of use and test only those that penetrate at toxicologically relevant levels in the three-test in vitro battery. Recently published data [D. Kirkland, M. Aardema, L. Henderson, L. Müller, 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] suggest the three-test battery will produce a significant number of false as well as real positives. Whilst we are aware of the desire to reduce animal experiments, determining the relevance of positive results in any of the three recommended in vitro assays will most likely have to be determined by use of in vivo assays. The bone marrow micronucleus test using routes of administration such as oral or intraperitoneal may be used where the objective is extended hazard identification. If negative results are obtained in this test, then a second in vivo test should be conducted. This could be an in vivo UDS in rat liver or a Comet assay in a relevant tissue. However, for hazard characterisation, tests using topical application with measurement of genotoxicity in the skin would be more appropriate. Such specific site-of-contact in vivo tests would minimise animal toxicity burden and invasiveness, and, especially for hair dyes, be more relevant to human routes of exposure, but there are not sufficient scientific data available to allow recommendations to be made. The generation of such data is encouraged.     

ECVAM prevalidation study on in vitro cell transformation assays: general outline and conclusions of the study

The potential for a compound to induce carcinogenicity is a key consideration when ascertaining hazard and risk assessment of chemicals. Among the in vitro alternatives that have been developed for predicting carcinogenicity, in vitro cell transformation assays (CTAs) have been shown to involve a multistage process that closely models important stages of in vivo carcinogenesis and have the potential to detect both genotoxic and non-genotoxic carcinogens. These assays have been in use for decades and a substantial amount of data demonstrating their performance is available in the literature. However, for the standardised use of these assays for regulatory purposes, a formal evaluation of the assays, in particular focusing on development of standardised transferable protocols and further information on assay reproducibility, was considered important to serve as a basis for the drafting of generally accepted OECD test guidelines. To address this issue, a prevalidation study of the CTAs using the BALB/c 3T3 cell line, SHE cells at pH 6.7, and SHE cells at pH 7.0 was coordinated by the European Centre for the Validation of Alternative Methods (ECVAM) and focused on issues of standardisation of protocols, test method transferability and within- and between-laboratory reproducibility. The study resulted in the availability of standardised protocols that had undergone prevalidation [1,2]. The results of the ECVAM study demonstrated that for the BALB/c 3T3 method, some modifications to the protocol were needed to obtain reproducible results between laboratories, while the SHE pH 6.7 and the SHE pH 7.0 protocols are transferable between laboratories, and results are reproducible within- and between-laboratories. It is recommended that the BALB/c 3T3 and SHE protocols as instituted in this prevalidation study should be used in future applications of these respective transformation assays. To support their harmonised use and regulatory application, the development of an OECD test guideline for the SHE CTAs, based on the protocol published in this issue, is recommended. The development of an OECD test guideline for the BALB/c 3T3 CTA should likewise be further pursued upon the availability of additional supportive data and improvement of the statistical analysis.     

Cluster analysis in predicting the carcinogenicity of chemicals using short-term assays

Cluster analysis can be a useful tool for exploratory data analysis to uncover natural groupings in data, and initiate new ideas and hypotheses about such groupings. When applied to short-term assay results, it provides and improves estimates for the sensitivity and specificity of assays, provides indications of association between assays and, in turn, which assays can be substituted for one another in a battery, and allows a data base containing test results on chemicals of unknown carcinogenicity to be linked to a data base for which animal carcinogenicity data are available. Cluster analysis was applied to the Gene-Tox data base (which contains short-term test results on chemicals of both known and unknown carcinogenicity). The results on chemicals of known carcinogenicity were different from those obtained when the entire data base was analyzed. This suggests that the associations (and possibly the sensitivities and specificities) which are based on chemicals of known carcinogenicity may not be representative of the true measures. Cluster analysis applied to the total data base should be useful in improving these estimates. Many of the associations between the assays which were found through the use of cluster analysis could be 'validated' based on previous knowledge of the mechanistic basis of the various tests, but some of the associations were unsuspected. These associations may be a reflection of a non-ideal data base. As additional data becomes available and new clustering techniques for handling non-ideal data bases are developed, results from such analyses could play an increasing role in strengthening prediction schemes which utilize short-term tests results to screen chemicals for carcinogenicity, such as the carcinogenicity and battery selection (CPBS) method (Chankong et al., 1985).     

A random mutation capture assay to detect genomic point mutations in mouse tissue

Herein, a detailed protocol for a random mutation capture (RMC) assay to measure nuclear point mutation frequency in mouse tissue is described. This protocol is a simplified version of the original method developed for human tissue that is easier to perform, yet retains a high sensitivity of detection. In contrast to assays relying on phenotypic selection of reporter genes in transgenic mice, the RMC assay allows direct detection of mutations in endogenous genes in any mouse strain. Measuring mutation frequency within an intron of a transcribed gene, we show this assay to be highly reproducible. We analyzed mutation frequencies from the liver tissue of animals with a mutation within the intrinsic exonuclease domains of the two major DNA polymerases, δ and ε. These mice exhibited significantly higher mutation frequencies than did wild-type animals. A comparison with a previous analysis of these genotypes in Big Blue mice revealed the RMC assay to be more sensitive than the Big Blue assay for this application. As RMC does not require analysis of a particular gene, simultaneous analysis of mutation frequency at multiple genetic loci is feasible. This assay provides a versatile alternative to transgenic mouse models for the study of mutagenesis in vivo.     

Refinement and reduction of acute oral toxicity testing: a critical review of the use of cytotoxicity data

Acute oral toxicity testing is still required for the classification and labelling of chemicals, agrochemicals and related formulations. There have been increasing efforts over the last two decades to reduce the number of animals needed for this testing, according to the Three Rs concept. To evaluate the utility of an in vitro cytotoxicity test in our routine testing for acute oral toxicity, we have implemented in our laboratory the neutral red uptake (NRU) method, with Balb/c 3T3 fibroblasts after a 48-hour exposure, which was recommended in ICCVAM Report 07-4519, 2006. Initially, we tested 16 substances that had existing in vivo and in vitro data available, to prove our technical proficiency with the in vitro test. Then, testing was performed with 187 test substances, including a broad variety of chemicals, agrochemicals and formulations. The starting dose for acute oral systemic toxicity assays in rats (LD50) was estimated by using the prediction model presented in the ICCVAM validation study, and subsequently compared to the results obtained by in vivo testing performed according to, or similar to, OECD Test Guideline 423. Comparison of all of the 203 predicted LD50 values that were deduced from the in vitro IC50 values, with the in vivo results from oral toxicity studies in rats, resulted in a low overall concordance of 35%. The in vitro cytotoxicity assay achieved a good concordance of 74%, only for the weakly toxic substances (EU-GHS Cat. 4). However, it must be noted that 71% of the substances tested (i.e. 145/203) were classified as being weakly toxic in vitro. We further analysed the utility of the in vitro test for predicting the starting dose for an in vivo study, and the potential reduction in animal usage that this would engender. In this regard, the prediction by the cytotoxicity test was useful for 59% of the substances. However, the use of a standard starting dose of 300 mg/kg bw by default (without previous cytotoxicity testing) would have been almost as useful (50%). In contrast, the prediction by an experienced toxicologist was correct for 95% of the substances. However, this was only performed for 40% of the substances, mainly those of no to low toxicity. Calculating the theoretical animal numbers needed in several scenarios supported these results. The additional analysis, considering some physicochemical data (solubility, molecular weight, log POW), substance class and mode of action, revealed no specific applicability domains. In summary, the use of the 3T3 NRU cytotoxicity data alone did not sufficiently contribute to refinement and reduction in the acute oral toxicity testing of the substance portfolio tested routinely in our laboratory.     

Application of in vitro cell transformation assays in regulatory toxicology for pharmaceuticals, chemicals, food products and cosmetics

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.     

Testing strategies in mutagenicity and genetic toxicology: An appraisal of the guidelines of the European Scientific Committee for Cosmetics and Non-Food Products for the evaluation of hair dyes

The European Scientific Committee on Cosmetics and Non-Food Products (SCCNFP) guideline for testing of hair dyes for genotoxic/mutagenic/carcinogenic potential has been reviewed. The battery of six in vitro tests recommended therein differs substantially from the batteries of two or three in vitro tests recommended in other guidelines. Our evaluation of the chemical types used in hair dyes and comparison with other guidelines for testing a wide range of chemical substances, lead to the conclusion that potential genotoxic activity may effectively be determined by the application of a limited number of well-validated test systems that are capable of detecting induced gene mutations and structural and numerical chromosomal changes.We conclude that highly effective screening for genotoxicity of hair dyes can be achieved by the use of three assays, namely the bacterial gene mutation assay, the mammalian cell gene mutation assay (mouse lymphoma tk assay preferred) and the in vitro micronucleus assay. These need to be combined with metabolic activation systems optimised for the individual chemical types.Recent published evidence [D. Kirkland, M. Aardema, L. Henderson, L. Müller, 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] suggests that our recommended three tests will detect all known genotoxic carcinogens, and that increasing the number of in vitro assays further would merely reduce specificity (increase false positives). Of course there may be occasions when standard tests need to be modified to take account of special situations such as a specific pathway of biotransformation, but this should be considered as part of routine testing.It is clear that individual dyes and any other novel ingredients should be tested in this three-test battery. However, new products are formed on the scalp by reaction between the chemicals present in hair-dye formulations. Ideally, these should also be tested for genotoxicity, but at present such experiences are very limited. There is also the possibility that one component could mask the genotoxicity of another (e.g. by being more toxic), and so it is not practical at this time to recommend routine testing of complete hair-dye formulations as well. The most sensible approach would be to establish whether any reaction products within the hair-dye formulation penetrate the skin under normal conditions of use and test only those that penetrate at toxicologically relevant levels in the three-test in vitro battery.Recently published data [D. Kirkland, M. Aardema, L. Henderson, L. Müller, 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] suggest the three-test battery will produce a significant number of false as well as real positives. Whilst we are aware of the desire to reduce animal experiments, determining the relevance of positive results in any of the three recommended in vitro assays will most likely have to be determined by use of in vivo assays. The bone marrow micronucleus test using routes of administration such as oral or intraperitoneal may be used where the objective is extended hazard identification. If negative results are obtained in this test, then a second in vivo test should be conducted. This could be an in vivo UDS in rat liver or a Comet assay in a relevant tissue. However, for hazard characterisation, tests using topical application with measurement of genotoxicity in the skin would be more appropriate. Such specific site-of-contact in vivo tests would minimise animal toxicity burden and invasiveness, and, especially for hair dyes, be more relevant to human routes of exposure, but there are not sufficient scientific data available to allow recommendations to be made. The generation of such data is encouraged.     

Evaluation of a genotoxicity test measuring DNA-strand breaks in mouse lymphoma cells by alkaline unwinding and hydroxyapatite elution

A rapid genotoxicity test, based on the measurement of the proportion of single- to double-stranded DNA by alkaline unwinding and hydroxyapatite elution in mouse lymphoma cells treated in vitro with various chemicals, was evaluated. Seventy-eight compounds from diverse chemical groups, including commonly tested mutagens, toxic compounds not usually tested for genotoxicity and non-toxic compounds not thought to be genotoxic were tested. The results obtained were compared with those from the mouse lymphoma TK locus forward-mutation assay, providing a basis for assessing the relative sensitivity of the 2 assays using the same cells exposed to chemicals under similar conditions. Clear evidence of DNA-damaging activity was obtained with 43 of the compounds, while 4 gave equivocal results. Of the remaining 31 compounds, 14 were toxic without inducing DNA damage while the rest were non-toxic and did not induce any DNA damage. Results were available from both the alkaline unwinding assay and the mouse lymphoma assay for 61 compounds; they showed a concordance between the 2 assays of 77%. Of the 47 compounds that were positive or equivocal in the alkaline unwinding assay, only carbon tetrachloride and prednisolone were negative in the mouse lymphoma assay, while 12 of the 19 compounds that were negative in the alkaline unwinding assay were positive in the mouse lymphoma assay. These included 3 compounds that interfere with nucleic acid metabolism, and 3 crosslinking agents, which would be expected to produce mutations to a greater extent than strand breaks. The other 6 compounds were anthranilic acid, benzoquinone, p-chloroaniline, diethylmaleate, glucose and procarbazine HCl. Of these only the last is a known carcinogen. It is concluded from the present study that there was good overall agreement between the results of the DNA alkaline unwinding and mouse lymphoma TK locus assays, but that the sensitivity of the alkaline unwinding assay is lower for some classes of compounds. Bearing this in mind, the alkaline unwinding assay is considered suitable as a rapid screen for genotoxic activity in eukaryotic cells.     

A review and analysis of the Chinese hamster ovary/hypoxanthine guanine phosphoribosyl transferase assay to determine the mutagenicity of chemical agents. A report of phase III of the U.S. Environmental Protection Agency Gene-Tox Program

Published literature on the Chinese hamster ovary cell/hypoxanthine guanine phosphoribosyl transferase (CHO/HGPRT) assay from mid-1979 through June 1986 was reviewed and evaluated. Data from the papers considered acceptable include test results on 121 chemicals belonging to 25 chemical classes. A total of 87 chemicals were evaluated positive, 3 negative, and 31 inconclusive. Mutagenicity data on 49 of the 121 chemicals evaluated could also be compared with in vivo animal carcinogenicity data. 40 of the 43 reported animal carcinogens were considered mutagenic. Caprolactam, the only definitive noncarcinogen in the group of 49, was not mutagenic. The CHO/HGPRT assay was concluded to be an appropriate assay system for use in the screening of chemicals for genotoxicity.     

Mutagenic activity of 27 dyes and related chemicals in the salmonella/microsome and mouse lymphoma TK+/- assays

A total of 27 dyes and related chemicals were tested for mutagenicity in both the Salmonella typhimurium plate-incorporation and FMN-modified assays as well as the mouse lymphoma TK+/- assay. Half of the compounds tested were monoazo dyes (14); the remainder consisted of disazo (3), aminotriphenylmethane derivatives (4), and other miscellaneous (6) color compounds. The results obtained in this study are compared with data from dyes of the same batch tested in other laboratories in the Salmonella plate-incorporation assay and in both in vitro and in vivo/in vitro UDS assays. Agreement of results from the various assays that could be compared (excluding results that were equivocal or indeterminate) ranged from 80 to 91%. Sufficient data were available to provide an overall index of in vitro activity for 15 chemicals; of these, 14 compounds could be compared to and agreed with reports of their carcinogenic potential in the literature.     

On the origin of unusual mutations recovered from the lacI transgenic assay

Amongst approximately 25,000 mutants recovered from tissues of the lacI mouse and rat transgenic mutation assay, we identified seven mutants that carry changes that are unlike the majority of mutations that are normally recovered in these systems. The recovered mutants feature replacements and insertions of sequences that originate in the animal's genome, in the bacteriophage lambda construct that harbors the lacI gene, and in the genome of the E. coli plating host. These mutants demonstrate that mutations resulting from diverse mechanisms, in addition to the normal point mutations, can be recovered. In addition, the data indicate that such mutations may often not be of animal origin.     

Genotoxicity of aloeemodin in vitro and in vivo

The present in vitro and in vivo experiments were undertaken to clarify the genotoxic potential of the hydroxyanthrachinone aloeemodin which can be found in different plant derived products for therapy of constipation. The results demonstrate that aloeemodin is able to induce mutagenic effects in vitro. Positive results were obtained in the chromosomal aberration assay with CHO cells, as well as in the Salmonella reverse mutation assay (frameshift mutations in strains TA 1537, TA 1538 and TA 98). No mutagenic potential of aloeemodin, however, was observed in the gene mutation assay with mammalian cells in vitro (HPRT assay in V79 cells). Each assay was performed in the presence and absence of an extrinsic metabolic activation system (S9-mix). In in vivo studies (micronucleus assay in bone marrow cells of NMRI mice; chromosome aberration assay in bone marrow cells of Wistar rats; mouse spot test [DBA/2J × NMRI]) no indication of a mutagenic activity of aloeemodin was found. Information about a possible reaction of aloeemodin with DNA was derived from an in vivo UDS assay. Hepatocytes of aloeemodin-treated male Wistar rats did not show DNA damage via repair synthesis. All these data suggest that aloeemodin is able to interact with DNA under certain in vitro conditions. However, in vivo the results that were negative did not indicate a genotoxic potential. Therefore, it may be assumed that a genotoxic risk for man might be unlikely.     

Histological complexities of pancreatic lesions from transgenic mouse models are consistent with biological and morphological heterogeneity of human pancreatic cancer

Although pancreatic cancer is the fourth leading cause of cancer death, it has received much less attention compared to other malignancies. There are several transgenic animal models available for studies of pancreatic carcinogenesis, but most of them do not recapitulate, histologically, human pancreatic cancer. Here we review some detailed molecular complexity of human pancreatic cancer and their reflection in histomorphological complexities of pancreatic lesions developed in various transgenic mouse models with a special concern for studying the effects of chemotherapeutic and chemopreventive agents. These studies usually require a large number of animals that are at the same age and gender and should be either homozygote or heterozygote but not a mixture of both. Only single-transgene models can meet these special requirements, but many currently available models require a mouse to simultaneously bear several transgene alleles. Thus it is imperative to identify new gene promoters or enhancers that are specific for the ductal cells of the pancreas and are highly active in vivo so as to establish new single-transgene models that yield pancreatic ductal adenocarcinomas for chemotherapeutic and chemopreventive studies.