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.1.The TGR mutation assay is based on transgenic rats and mice that contain multiple copies of chromosomally integrated plasmid and phage shuttle vectors that harbour reporter genes for detection of mutation. Mutagenic events arising in the rodent are scored by recovering the shuttle vector and analyzing the phenotype of the reporter gene in a bacterial host. TGR gene mutation assays allow mutations induced in a genetically neutral transgene to be scored in any tissue of the rodent, and therefore circumvent many of the existing limitations to the study of in vivo gene mutation. TGR models for which sufficient data are available to permit evaluation include Muta™ mouse, Big Blue® mouse and rat, LacZ plasmid mouse, and the gpt delta mouse. Mutagenesis in the TGR models is normally assessed as a mutant frequency (MF); however, if required, molecular analysis can provide additional information.2.OECD guidelines exist for a range of in vitro mutation assays that are capable of detecting both chromosomal and gene mutations. In vivo assays are required components of a thorough genetic toxicity testing programme. For somatic cells, those assays that are most commonly conducted, for which OECD guidelines are currently available, assess induced chromosomal mutation. In addition there are non-transgenic assays that can be used for analysis of gene mutation; none of these have an OECD test guideline. Existing in vivo assays are limited by a range of different factors, including cost of the assay, the number of tissues in which genotoxicity may be measured, the state of understanding of the endpoint, and the nature of the chemicals that will be detected.3.As of July 2004, 163 agents have been evaluated using TGR assays. The majority of experimental records have assessed a subset of these chemicals, most of which are strong mutagens and carcinogens. Of the 103 agents whose carcinogenicity has been evaluated 90 are carcinogens and only 13 are noncarcinogens. The following conclusions may be drawn from the existing TGR mutation data.•The ability to use all routes of administration has been demonstrated. Experiments can be tailored to use the most relevant route of administration.•The ability to examine mutation in virtually all tissues has been demonstrated. TGR assays have most commonly examined mutagenicity in the liver and bone marrow.•The majority of the experiments have used shorter administration times than is currently recommended by the International Workshops on Genotoxicity Testing (IWGT); there are limited data available to assess the effects of longer sampling time except at extremely short administration times.•Although it is recognized that a number of factors may influence the tissue specificity of mutation, including cell turnover, DNA repair, toxicokinetics, and the nature of the genetic target, there are currently limited experimental data specific to transgenes that are available to inform the discussion.•Limited data are available to evaluate the results of TGR assays in known target tissues for carcinogenicity. A case-by-case analysis of instances in which discrepancies are apparent suggests that in the majority of cases, factors such as nongenotoxic mechanism of action, inappropriate mode of administration, or inadequate study design may account for the observed negative result in the tissue of interest.•Qualitatively similar results have been obtained in the majority of experiments that have assayed different transgenes using similar experimental parameters.•The spontaneous mutant frequency (SMF) in most somatic tissues of TGR animals is 5–10-fold higher than observed in available endogenous loci using the same animals. Factors such as the age of the animal, the tissue, and the animal model influence the absolute value of the SMF. In most somatic tissues, with the exception of brain, there is an age related increase in mutation frequency throughout the life of the animal. Most, but not all, studies suggest that the SMF in male germline tissues remains low and constant throughout the life of the animal.•Multiple treatments of a mutagen appear to increase mutant frequencies in neutral transgenes in an approximately additive manner. However, extremely long treatment times of 12 weeks or longer may produce an apparent increase in MF through clonal expansion, genomic instability in developing preneoplastic foci or tumours, or through oxidation damage of DNA resulting from chronic induction of cytochrome P-450 monooxygenases.•The time required to reach the maximum mutant frequency is tissue-specific, and appears to be related to the turnover time of the cell population: the optimal sampling time differs according to tissue, with liver and bone marrow at opposite extremes among proliferating somatic tissues: in bone marrow, the mutant frequency appears to reach a maximum at extremely short sampling times and then decreases over 28 days following an acute treatment; in liver the induced mutation frequency increases over the month following exposure, reaches a maximum, and remains relatively constant thereafter. There are insufficient data available for other tissues to support any conclusion regarding optimal sampling time.•The results of studies carried out on a given chemical using similar experimental protocols suggest that the TGR assays show good qualitative reproducibility in both somatic and germ cells, and quantitative reproducibility over a limited range of conditions and laboratories. The data are insufficient to draw conclusions regarding the quantitative reproducibility of the assays over a wider range of conditions.•Although there exists a theoretical possibility that ex vivo and in vitro mutations may arise during the course of a TGR experiment, these types of mutations are expected to be extremely rare in a properly conducted experiment using the major TGR models. For positive selection systems, any such mutations will not be detected.•The weight of evidence suggests that transgenes and endogenous genes respond in approximately the same manner to mutagens in the few instances where direct comparisons are possible. Sensitivity is determined in large part by the SMF: the higher SMF in transgenes, as compared to testable endogenous genes, appears to reduce their sensitivity, especially when acute treatments are used. The sensitivity of transgenes can be enhanced by increasing the administration time.•Mutagens that induce deletions are likely to be detected more easily in certain endogenous genes than in transgenes due to phenotypic selection issues.•A very high proportion of the TGR experiments carried out to date have examined the activity of compounds that are known to be strong mutagens. A limited number of noncarcinogens have been evaluated with TGRs. The specificity of the TGR assay for predicting carcinogenicity is generally higher than other assays evaluated in this paper. However, additional data from TGR assays on noncarcinogens is required.•Molecular analysis of induced mutations in transgenic targets is possible and provides additional information in situations where high interindividual variation is observed and clonal expansion is suspected, when weak responses are obtained, or when mechanistic information is desired. However, DNA sequence analysis of mutants is laborious and adds to the cost of the experiment; sequencing would not normally be required when testing drugs or chemicals for regulatory applications, particularly where a clear positive or negative result is obtained.4.Analysis of the predictivity of TGR assays for carcinogenicity is hindered somewhat by the fact that TGR data are available for only a small number of noncarcinogens. Of the 90 carcinogens and 13 noncarcinogens that have been assessed using TGR assays, the following conclusions can be drawn regarding the predictivity and complementarity of TGR assays in comparison to a range of other OECD in vitro and in vivo genotoxicity tests.•The TGR assay has high sensitivity and positive predictivity, meaning that most carcinogens have positive results in TGR and there is a high probability that a chemical with a positive result in TGR is a carcinogen.•As is the case with most genotoxicity assays, the TGR assay exhibits low specificity and negative predictivity, meaning that relatively few noncarcinogens were negative in TGR and there is a low probability that a chemical with a negative result in TGR is a noncarcinogen; however, it was no worse than the Salmonella mutagenicity assay in this regard.•Considering all the test batteries and single assays examined using the current dataset, best positive and negative predictivity was obtained from the TGR assay alone, the Salmonella mutagenicity assay alone, and a battery in which a positive result in TGR or Salmonella was considered positive and negative results in both assays was considerd negative. Despite the lack of substantial increases in predictive values of the test batteries compared with the component assays alone, the test batteries had a much lower false negative rate.•TGR and the in vivo micronucleus (MN) assay exhibited significant complementary – i.e. they offered greater predictivity for the detection of mutagens when combined than when alone – consistent with the fact that these two assays measure different genotoxic endpoints.•TGR was usually positive for those carcinogens that were positive in Salmonella and the in vitro chromosomal aberration (CA) assay. In contrast, in vivo MN had a much higher false negative rate for the same chemicals. If in vivo confirmation of positive results from both Salmonella and in vitro CA is warranted, TGR is likely a better choice than in vivo MN.•For chemicals having positive Salmonella and negative in vitro CA results (presumptive gene mutagens), selecting either TGR or in vivo MN as the in vivo confirmation assay did not markedly affect the proportion of correct carcinogenicity predictions.•For chemicals having positive in vitro CA and negative Salmonella results (presumptive clastogens), selecting in vivo MN as the in vivo confirmation assay led to a slightly higher proportion of correct carcinogenicity predictions than did selecting TGR.•For those carcinogens with negative results in both Salmonella and in vitro CA, adding either TGR or in vivo MN to the test battery did not improve the overall predictivity, since neither assay identified the carcinogens missed by the in vitro assays.5.Recommendations, based on internationally harmonized criteria, are made regarding the proper conduct of a TGR assay. These recommendations relate to accepted characteristics of a transgenic rodent mutation assay, treatment protocols, and post treatment sampling procedures. Of particular importance in optimizing TGR protocols are two experimental variables—the administration time and the sampling time. Based on observations that mutations accumulate with each treatment, a repeated-dose regimen for a period of 28 days is strongly encouraged, with sampling at 3 days following the final treatment. If slowly proliferating tissues are of particular importance, then a longer sampling time may be more appropriate.Additional confidence in the recommended test protocol will be provided by research that examines the following:•The influence of the administration time on the observed mutation frequency for weak mutagens. It has not conclusively been determined if data (especially negative results) from experiments using an administration time of less than 28 days should be discounted, if a 28 day treatment period is sufficiently long to permit the detection of weak mutagen-induced mutations in all tissues, or if weak mutagens could in fact be detected using treatment times shorter than 28 days.•The influence of the frequency of treatment on the observed mutation frequency. The difference between weekly and daily administrations on mutant frequency and on the ultimate conclusions of transgenic rodent experiments has not yet been thoroughly investigated.•The influence of sampling time following repeat administrations on the mutant frequency in both slowly and rapidly dividing tissue, particularly when examining weak mutagens. At the current time there are insufficient comparative data available for a range of tissues.Recommendations are made regarding how a TGR assay might be used within a short-term test battery for assessing new compounds. The test battery consists of various combinations of four assays—Salmonella, in vitro CA, in vivo MN and TGR. This proposed strategy is based on the conclusions obtained from the predictivity analysis, and the relative costs of the in vivo assays.TGR assays may also be used to resolve conflicts between in vitro and in vivo tests that are currently components of the standard genotoxicity test battery—Salmonella, in vitro CA and in vivo MN. In situations where the standard test battery has been conducted and there are conflicting results—particularly in situations where Salmonella has a positive result but in vivo MN is negative—TGR may be conducted as an additional test to resolve the conflict.Recommended test strategies are based on an analysis of the existing data. Confidence in these recommendations would be enhanced by additional experimental data in the following areas.•TGR data for additional non-carcinogens to increase the proportion of non-carcinogens in the data set.•Additional testing to fill data gaps for chemicals having known TGR assay but missing data from the Salmonella, in vitro CA, or in vivo MN assays.•The testing of additional chemicals using an accepted test guideline for TGR mutation assays.Based on the information and analyses in this review, there is sufficient evidence to support the recommendation that the OECD undertake the development of a Test Guideline on Transgenic Rodent Gene Mutation Assays. Accordingly, it is recommended that the OECD establish an Expert Working Group to develop such a Test Guideline, and serve as an international forum for undertaking any additional research that would lead to the development of a fuller understanding of the variables surrounding the conduct of TGR mutation assays.
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