Concomitant observations of UDS in the liver and micronuclei in the bone marrow of rats exposed to cyclophosphamide or 2-acetylaminofluorene

Oral dosing of between 5–30 mg/kg of cyclophosphamide (CP) to Alderley Park rats induced micronuclei in the bone marrow between 12 and 36 h after dosing, but failed to induce unscheduled DNA synthesis (UDS) in the liver at similar dose levels and treatment periods. Dose levels of > 30 mg/kg were toxic to the liver. In contrast, 2-acetylaminofluorene (2AAF) induced UDS in the rat liver between 4–36 h after dosing, but gave only a weak response in the bone marrow assay at dose levels between 0.5 and 2 g/kg. Selected observations were made for each chemical using both tissues of the same test animal.

It is concluded that an assessment of the genotoxicity in vivo of chemicals defined as genotoxic in vitro will contribute to an assessment of their possible mammalian carcinogenicity, and that these should involve assays conducted using both the bone marrow and the liver of rodents. Due to its relative ease of commission, the bone marrow micronucleus assay will usually be conducted first; in the case of negative results it is recommended that a liver genotoxicity assay should also be conducted. The case for employing in vivo short-term genotoxicity tests to predict the possible organotropic carcinogenicity or germ cell mutagenicity of a new in vitro genotoxin is discussed.     

The comet assay as an indicator test for germ cell genotoxicity

The in vivo comet assay is a well-established genotoxicity test. It is currently mainly performed with somatic cells from different organs to detect a genotoxic activity of potential carcinogens. It is regarded as a useful test for follow-up testing of positive or equivocal in vitro test results and for the evaluation of local genotoxicity. However, the comet assay also has the potential to detect germ cell genotoxicity and may be used for demonstrating the ability of a substance or its metabolite(s) to directly interact with the genetic material of gonadal and/or germ cells. Such results are important for the classification of germ cell mutagens, e.g. in the context of the "Globally Harmonized System of Classification and Labelling of Chemicals" (GHS). This review summarizes and discusses available information on the use of the comet assay with germ cells and cells from the gonads in genetic toxicology. The literature contains results from in vitro studies, ex vivo studies and in vivo studies. With regard to the assessment of germ cell genotoxicity, only in vivo studies are relevant but the other kind of studies provided important information on various aspects of the methodology. Many comet assay studies with human sperm have been performed in the context of male infertility and assisted fertilization. The results of these studies are not reviewed in detail here but various aspects of the assay modifications used are discussed. Measuring DNA effects by the comet assay in sperm requires additional steps for chromatin decondensation. Many different modifications of the alkaline and the neutral comet assay are in use but a standard protocol has not been established yet. High and variable background levels of DNA effects were reported and there is still need for standardization and validation of the comet assay with sperm. Some human biomonitoring studies with human sperm were published, but it seems to be premature to use these data for hazard identification and classification of chemicals. In contrast, the standard alkaline in vivo comet assay can easily be adapted to investigations with cells from reproductive organs. Tests with cells from the gonads (testis and ovary) seem to be most appropriate and a promising tool for demonstrating that a test compound reaches the gonads and is able to interact with the genetic material of germ cells. However, studies to standardize and validate these methods are necessary before the comet assay can be usefully applied in risk assessment of germ cell mutagens.     

The comet assay in male reproductive toxicology

Due to our lifestyle and the environment we live in, we are constantly confronted with genotoxic or potentially genotoxic compounds. These toxins can cause DNA damage to our cells, leading to an increase in mutations. Sometimes such mutations could give rise to cancer in somatic cells. However, when germ cells are affected, then the damage could also have an effect on the next and successive generations. A rapid, sensitive and reliable method to detect DNA damage and assess the integrity of the genome within single cells is that of the comet or single-cell gel electrophoresis assay. The present communication gives an overview of the use of the comet assay utilising sperm or testicular cells in reproductive toxicology. This includes consideration of damage assessed by protocol modification, cryopreservation vs the use of fresh sperm, viability and statistics. It further focuses on in vivo and in vitro comet assay studies with sperm and a comparison of this assay with other assays measuring germ cell genotoxicity. As most of the de novo structural aberrations occur in sperm and spermatogenesis is functional from puberty to old age, whereas female germ cells are more complicated to obtain, the examination of male germ cells seems to be an easier and logical choice for research and testing in reproductive toxicology. In addition, the importance of such an assay for the paternal impact of genetic damage in offspring is undisputed. As there is a growing interest in the evaluation of genotoxins in male germ cells, the comet assay allows in vitro and in vivo assessments of various environmental and lifestyle genotoxins to be reliably determined.     

A core in vitro genotoxicity battery comprising the Ames test plus the in vitro micronucleus test is sufficient to detect rodent carcinogens and in vivo genotoxins

In vitro genotoxicity testing needs to include tests in both bacterial and mammalian cells, and be able to detect gene mutations, chromosomal damage and aneuploidy. This may be achieved by a combination of the Ames test (detects gene mutations) and the in vitro micronucleus test (MNvit), since the latter detects both chromosomal aberrations and aneuploidy. In this paper we therefore present an analysis of an existing database of rodent carcinogens and a new database of in vivo genotoxins in terms of the in vitro genotoxicity tests needed to detect their in vivo activity. Published in vitro data from at least one test system (most were from the Ames test) were available for 557 carcinogens and 405 in vivo genotoxins. Because there are fewer publications on the MNvit than for other mammalian cell tests, and because the concordance between the MNvit and the in vitro chromosomal aberration (CAvit) test is so high for clastogenic activity, positive results in the CAvit test were taken as indicative of a positive result in the MNvit where there were no, or only inadequate data for the latter. Also, because Hprt and Tk loci both detect gene-mutation activity, a positive Hprt test was taken as indicative of a mouse-lymphoma Tk assay (MLA)-positive, where there were no data for the latter. Almost all of the 962 rodent carcinogens and in vivo genotoxins were detected by an in vitro battery comprising Ames+MNvit. An additional 11 carcinogens and six in vivo genotoxins would apparently be detected by the MLA, but many of these had not been tested in the MNvit or CAvit tests. Only four chemicals emerge as potentially being more readily detected in MLA than in Ames+MNvit--benzyl acetate, toluene, morphine and thiabendazole--and none of these are convincing cases to argue for the inclusion of the MLA in addition to Ames+MNvit. Thus, there is no convincing evidence that any genotoxic rodent carcinogens or in vivo genotoxins would remain undetected in an in vitro test battery consisting of Ames+MNvit.     

Heritable and cancer risks of exposures to anticancer drugs: inter-species comparisons of covalent deoxyribonucleic acid-binding agents

In the past years, several methodologies were developed for potency ranking of genotoxic carcinogens and germ cell mutagens. In this paper, we analyzed six sub-classes of covalent deoxyribonucleic acid (DNA) binding antineoplastic drugs comprising a total of 37 chemicals and, in addition, four alkyl-epoxides, using four approaches for the ranking of genotoxic agents on a potency scale: the EPA/IARC genetic activity profile (GAP) database, the ICPEMC agent score system, and the analysis of qualitative and quantitative structure-activity and activity-activity relationships (SARs, AARs) between types of DNA modifications and genotoxic endpoints. Considerations of SARs and AARs focused entirely on in vivo data for mutagenicity in male germ cells (mouse, Drosophila), carcinogenicity (TD₅₀s) and acute toxicity (LD₅₀s) in rodents, whereas the former two approaches combined the entire database on in vivo and in vitro mutagenicity tests. The analysis shows that the understanding and prediction of rank positions of individual genotoxic agents requires information on their mechanism of action. Based on SARs and AARs, the covalent DNA binding antineoplastic drugs can be divided into three categories. Category 1 comprises mono-functional alkylating agents that primarily react with N7 and N3 moieties of purines in DNA. Efficient DNA repair is the major protective mechanism for their low and often not measurable genotoxic effects in repair-competent germ cells, and the need of high exposure doses for tumor induction in rodents. Due to cell type related differences in the efficiency of DNA repair, a strong target cell specificity in various species regarding the potency of these agents for adverse effects is found. Three of the four evaluation systems rank category 1 agents lower than those of the other two categories. Category 2 type mutagens produce O-alkyl adducts in DNA in addition to N-alkyl adducts. In general, certain O-alkyl DNA adducts appear to be slowly repaired, or even not at all, which make this kind of agents potent carcinogens and germ cell mutagens. Especially the inefficient repair of O-alkyl—pyrimidines causes the high mutational response of cells to these agents. Agents of this category give high potency scores in all four expert systems. The major determinant for the high rank positions on any scale of genotoxic of category 3 agents is their ability to induce primarily structural chromosomal changes. These agents are able to cross-link DNA. Their high intrinsic genotoxic potency appears to be related to the number of DNA cross-links per target dose unit they can induce. A confounding factor among category 3 agents is that often the genotoxic endpoints occur closed to or toxic levels, and that the width of the mutagenic dose range, i.e., the dose area between the lowest observed effect level and the LD₅₀, is smaller (usually no more than 1 logarithmic unit) than for chemicals of the other two categories. For all three categories of genotoxic agents, strong correlations are observed between their carcinogenic potency, acute toxicity and germ cell specificity.     

MicroRNA in lung cancer diagnostics and treatment

The measurement of serine139-phosphorylated histone H2AX (γH2AX) provides a biomarker of DNA double-strand breaks (DSBs) and may identify potential genotoxic activity. In order to evaluate a flow cytometry assay for γH2AX detection (hereafter termed the γH2AX by flow assay), 6 prototypical (3 pro- and 3 proximate) genotoxins, i.e. dimethylbenz[a]anthracene (DMBA), 2-acetylaminofluorene (2-AAF), benzo[a]pyrene (B[a]P), methyl methane sulphonate (MMS), methyl nitrosourea (MNU) and 4-nitroquinoline oxide (4NQO), were selected to define assay evaluation criteria. In addition, 3 non-genotoxic cytotoxins (phthalic anhydride, n-butyl chloride and hexachloroethane) were included to investigate the influence of cytotoxicity on assay performance. At similar cytotoxicity levels (relative cell counts; RCC 75-40%) all prototypical genotoxins induced marked concentration-dependent increases in γH2AX compared with the non-genotoxins. As a result, assay evaluation criteria for a positive effect were defined as >1.5-fold γH2AX @ RCC >25%. Twenty five additional chemicals with diverse structures and genotoxic activity were selected to evaluate the γH2AX by flow assay. Results were compared with Ames bacterial and in vitro mammalian genotoxicity tests (mouse lymphoma assay and/or chromosome aberration assay). γH2AX by flow assay results were highly predictive of Ames (sensitivity 100%; specificity 67%; concordance 82%) and in vitro mammalian genotoxicity tests (sensitivity 91%; specificity 89%; concordance 91%) and provide additional evidence that γH2AX is a biomarker of potential genotoxic activity, underpinned mechanistically by the cellular response to DSBs. Discordant findings were predominately attributed to differences in specificity for some mammalian cell genotoxins that are Ames non-mutagens or for "biologically-irrelevant" positives in the mammalian tests. Simple anilines were classified as genotoxic following rat liver S9-mediated bioactivation, however, effects on γH2AX were atypical and limited to a small sub-population of S-phase nuclei. Nevertheless, the γH2AX by flow assay represents a novel genotoxicity assay with the potential to flag both pro- and proximate genotoxins.     

Genetic toxicology: can we design predictive in vivo assays?

The present analysis examines the assumptions in, the perceptions and predictivity of and the need for short-term tests (STTs) for genotoxicity in light of recent findings that most noncarcinogens from the National Toxicology Program are genotoxic (i.e., positive in one or more in vitro STTs). Reasonable assumptions about the prevalence for carcinogens (1-10% of all chemicals), the sensitivity of these STTs (ca. 90% of all carcinogens are genotoxic) and their estimated "false positive" incidence (60-75%) imply that the majority of chemicals elicit genotoxic responses and, consequently, that most in vitro genotoxins are likely to be noncarcinogenic. Thus, either the usual treatment conditions used in these in vitro STTS are producing a large proportion of artifactual and meaningless positive results or else in vitro mutagenicity is too common a property of chemicals to serve as a useful predictor of carcinogenicity or other human risk. In contrast, the limited data base on in vivo STTs suggests that the current versions of these assays may have low sensitivity which appears unlikely to improve without dropping either their 'short-term' aspect or the rodent carcinogenicity benchmark. It is suggested that in vivo genotoxicity protocols be modified to take into consideration both the fundamentals of toxicology as well as the lessons learned from in vitro genetic toxicology. In the meantime, while in vivo assays are undergoing rigorous validation, genetic toxicology, as currently practiced, should not be a formal aspect of chemical or drug development on the grounds that it is incapable of providing realistic and reliable information on human risk. It is urged that data generated in new, unvalidated in vivo genotoxicity assays be exempted from the normal regulatory reporting requirements in order to encourage industry to participate in the laborious and expensive development of this next phase of genetic toxicology.     

International Commission for Protection against Environmental Mutagens and Carcinogens. ICPEMC working paper No. 5. Genotoxicity tests as predictors of carcinogens: an analysis

Differences between the results of numerical validation studies comparing in vitro and in vivo genotoxicity tests with the rodent cancer bioassay are leading to the perception that short-term tests predict carcinogenicity only with uncertainty. Consideration of factors such as the pharmacokinetic distribution of chemicals, the systems available for metabolic activation and detoxification, the ability of the active metabolite to move from the site of production to the target DNA, and the potential for expression of the induced lesions, strongly suggests that the disparate sensitivity of the different test systems is a major reason why numerical validation is not more successful. Furthermore, genotoxicity tests should be expected to detect only a subset of carcinogens, namely genotoxic carcinogens, rather than those carcinogens that appear to act by non-genetic mechanisms. Instead of relying primarily on short-term in vitro genotoxicity tests to predict carcinogenic activity, these tests should be used in a manner that emphasizes the accurate determination of mutagenicity or clastogenicity. It must then be determined whether the mutagenic activity is further expressed as carcinogenicity in the appropriate studies using test animals. The prospects for quantitative extrapolation of in vitro or in vivo genotoxicity test results to carcinogenicity requires a much more precise understanding of the critical molecular events in both processes.     

Genotoxicity studies with mineral oils; effects of oils on the microbial mutagenicity of precursor mutagens and genotoxic metabolites

In vitro genotoxicity assays are extensively used to predict carcinogenic activity in vivo. The standard microbial mutagenicity assays however often fail to yield positive results with mineral oils which are carcinogenic to mice in long-term skin-cancer studies. A comprehensive programme of studies has therefore investigated the basis of this apparently anomalous behaviour. This investigation has addressed the possible effects of oils on the bioactivation of precursor mutagens and the disposition of mutagenic metabolites by studying the microbial mutagenicity of selected precursor mutagens (benzo[a]pyrene, benzo[a]anthracene, 2-aminoanthracene and 2-naphthylamine) and intrinsically reactive mutagens [+/- )-benzo[a]pyrene-4,5-oxide and (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene) in the presence and absence of mineral oils. Notably the mutagenicity associated with the deliberate additions of these mutagens or precursor mutagens to oils was readily detected by the microbial assays. The mutagenicity of only one of the precursor mutagens, benzo[a]pyrene, was significantly reduced by the oils, and then only in the standard plate-incorporation assay. Interestingly the degree of suppression appeared to be related to the polycyclic aromatic hydrocarbon content of the oils. In the case of 2-aminoanthracene large enhancements in its mutagenicity were observed in the presence of oils. These latter findings appear to be due to effects of oils on the bioactivation of precursor mutagens rather than on the disposition of their bioactivation products. The mutagenicity of intrinsically reactive mutagens, of a type generated by bioactivation of polycyclic aromatic hydrocarbons, was not significantly reduced in the presence of mineral oils. This indicates that it is unlikely that components in oils trap or facilitate the deactivation of ultimate mutagens whether these pre-exist in the oil or are formed from precursors by bioactivation in the in vitro test system. Viewed overall these results suggest that mineral oils judged to be carcinogenic on the basis of in vivo studies in mouse skin may possess only very weak genotoxic potential. While this potential is likely to be a prerequisite for carcinogenic action, the current results cause attention to be focussed on other factors, e.g. promotion, as potentially important determinants of the carcinogenic potencies of mineral oils in mouse skin.     

Structure/activity relationships of the genotoxic potencies of sixteen pyrrolizidine alkaloids assayed for the induction of somatic mutation and recombination in wing cells of Drosophila melanogaster.

Sixteen pyrrolizidine alkaloids (PAs) were examined for their genotoxic potency in the wing spot test of Drosophila melanogaster following oral application. This in vivo assay tests for the induction of somatic mutation and mitotic recombination in cells of the developing wing primordia. All PAs tested except the C9-monoester supinine were clearly genotoxic. Depending on their chemical structure, however, genotoxicity of the PAs varied widely in a range encompassing about three orders of magnitude. In general, macrocyclic diester-type PAs were the most and 7-hydroxy C9-monoester types the least genotoxic representatives studied, while open diesters were intermediate in this respect. Stereoisomeric PAs mostly showed similar, but sometimes also clearly unequal genotoxicity. An increasing number of hydroxy groups in the PA molecule seemed to reduce its genotoxic potency. With respect to the structure/activity relationships, there appears to be a good correlation between hepatotoxicity of PAs in experimental rodents and genotoxicity in the wing spot test of Drosophila. This suggests that PAs are bioactivated along similar pathways in the mammalian liver and in the somatic cells of Drosophila. The genotoxic potential of PAs in the Drosophila wing spot test and their carcinogenic potential in mammals also seem correlated, although the information in the literature on carcinogenicity of the non-macrocyclic PAs with moderate to low genotoxic potency is concededly limited. Comparisons with other genotoxicity tests suggest that the wing spot test is particularly suitable for genotoxins like PAs, on the one hand because of the versatile metabolic bioactivation system of Drosophila and on the other hand also because of its excellent sensitivity to the crosslinking agents among the genotoxins.     

Development and validation of a high-content screening in vitro micronucleus assay in CHO-k1 and HepG2 cells

In the present study an automated image analysis assisted in vitro micronucleus assay was developed with the rodent cell line CHO-k1 and the human hepatoma cell line HepG2, which are both commonly used in regulatory genotoxicity assays. The HepG2 cell line was chosen because of the presence in these cells of a functionally active p53 protein, a functionally competent DNA-repair system, active enzymes for phase-I and -II metabolism, and an active Nrf2 electrophile responsive system. These properties may result in an assay with a high predictivity for in vivo genotoxicity. The assays with CHO-k1 and HepG2 cells were both evaluated by testing a set of compounds recommended by the European Centre for the Validation of Alternative Methods (ECVAM), among which are in vivo genotoxins and non-genotoxins. The CHO-k1 cell line showed a high sensitivity (percentage of genotoxic compounds that gave a positive result: 80%; 16/20) and specificity (percentage of non-genotoxic compounds that came out negative: 88%; 37/42). Although the sensitivity of the HepG2 cell line was lower (60%; 12/20), the specificity was high (88%; 37/42). These results were confirmed by testing an additional series of 16 genotoxic compounds. For both the CHO-k1 and the HepG2 cell line it was possible to size-classify micronuclei, enabling distinguishing aneugens from clastogens. It is concluded that two high-throughput micronucleus assays were developed that can detect genotoxic potential and allow differentiation between clastogens and aneugens. The performance scores of the CHO-k1 and HepG2 cell lines for in vivo genotoxicity were high. Application of these assays in the early discovery phase of drug development may prove to be a useful strategy to assess genotoxic potential at an early stage.     

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.     

Genotoxic activity of organic chemicals in drinking water

The information summarized in this review provides substantial evidence for the widespread presence of genotoxins in drinking water. In many, if not most cases, the genotoxic activity can be directly attributed to the chlorination stage of drinking water treatment. The genotoxic activity appears to originate primarily from reactions of chlorine with humic substances in the source waters. Genotoxic activity in drinking water concentrates has been most frequently demonstrated using bacterial mutagenicity tests but results with mammalian cell assay systems are generally consistent with the findings from the bacterial assays. There is currently no evidence for genotoxic damage following in vivo exposures to animals. In some locations genotoxic contaminants of probable industrial and/or agricultural origin occur in the source waters and contribute substantially to the genotoxic activity of finished drinking waters. The method used for sample concentration can have an important bearing on study results. In particular, organic acids account for most of the mutagenicity of chlorinated drinking water, and their recovery from water requires a sample acidification step prior to extraction or XAD resin adsorption. Considerable work has been done to determine the identity of the compounds responsible for the mutagenicity of organic concentrates of drinking water. Recently, one class of acidic compounds, the chlorinated hydroxyfuranones, has been shown to be responsible for a major part of the mutagenic activity. Strategies for drinking water treatment that have been evaluated with respect to reduction of genotoxins in drinking water include granular activated carbon (GAC) filtration, chemical destruction, and the use of alternative means of treatment (i.e., ozone, chlorine dioxide, and monochloramine). GAC treatment has been found to be effective for removal of mutagens from drinking water even after the GAC is beyond its normal use for organic carbon removal. All disinfectant chemicals appear to have the capacity of forming mutagenic chemicals during water treatment. However, the levels of mutagenicity formed with the alternative disinfectants have been generally less than those seen with chlorine and, especially in the case of ozone, highly dependent on the source water.(ABSTRACT TRUNCATED AT 400 WORDS)     

Genotoxic effects in the Eastern mudminnow (Umbra pygmaea L.) after exposure to Rhine water, as assessed by use of the SCE and Comet assays: a comparison between 1978 and 2005

Surface water used for drinking-water preparation requires continuous monitoring for the presence of toxic compounds. For monitoring of genotoxic compounds fish models have been developed, such as the Eastern mudminnow (Umbra pygmaea L.) because of its clearly visible 22 meta-centric chromosomes. It was demonstrated in the late seventies that Rhine water was able to induce chromosome aberrations and sister chromatid exchange in this fish species. Although in vitro mutagenicity studies of the RIWA (Rhine Water Works, The Netherlands) have shown that the genotoxicity of the river Rhine steadily decreased during the last decades, there is still concern about the presence of some residual mutagenicity. In addition, in most studies the water samples have been tested only in in vitro test systems such as the Salmonella-microsome test. For this reason, and in order to be able to make a comparison with the water quality 27 years ago, a study was performed with the same experimental design as before in order to measure the effect of Rhine water on the induction of SCE in the Eastern mudminnow. As a new test system the single cell gel electrophoresis assay (Comet assay) was performed. Fish were exposed to Rhine water or to groundwater for 3 and 11 days in flow-through aquaria. Fish exposed for 11 days to Rhine water had a significantly higher number of SCE and an increased comet tail-length compared with control fish exposed to groundwater. After exposure for three days to Rhine water there was no difference in SCE and a slightly increased comet tail-length compared with the control. It was concluded that genotoxins are still present in the river Rhine, but that the genotoxic potential has markedly decreased compared with 27 years ago. Furthermore, the Comet assay appears to be a sensitive assay to measure the genotoxic potential of surface waters in fish.     

Chemical structure, Salmonella mutagenicity and extent of carcinogenicity as indicators of genotoxic carcinogenesis among 222 chemicals tested in rodents by the U.S. NCI/NTP

A survey has been conducted of 222 chemicals evaluated for carcinogenicity in mice and rats by the United States NCI/NTP. The structure of each chemical has been assessed for potential electrophilic (DNA-reactive) sites, its mutagenicity to Salmonella recorded, and the level of its carcinogenicity to rodents tabulated. Correlations among these 3 parameters were then sought. A strong association exists among chemical structure (S/A), mutagenicity to Salmonella (Salm.) and the extent and sites of rodent tumorigenicity among the 222 compounds. Thus, a approximately 90% correlation exists between S/A and Salm. across the 115 carcinogens, the 24 equivocal carcinogens and the 83 non-carcinogens. This indicates the Salmonella assay to be a sensitive method of detecting intrinsic genotoxicity in a chemical. Concordance between S/A and Salm. have therefore been employed as an index of genotoxicity, and use of this index reveals two groups of carcinogens within the database, genotoxic and putatively non-genotoxic. These two broad groups are characterized by different overall carcinogenicity profiles. Thus, 16 tissues were subject to carcinogenesis only by genotoxins, chief among which were the stomach, Zymbal's glands, lung, subcutaneous tissue and circulatory system. Conclusions of carcinogenicity in these 16 tissues comprised 31% of the individual chemical/tissue reports of carcinogenicity. In contrast, both genotoxins and non-genotoxins were active in the remaining 13 tissues, chief among which was the mouse liver which accounted for 24% of all chemical/tissue reports of carcinogenicity. Further, the group of 70 carcinogens reported to be active in both species and/or in 2 or more tissues contained a higher proportion of Salmonella mutagens (70%) than observed for the group of 45 single-species/single-tissue carcinogens (39%). 30% of the 83 non-carcinogens were mutagenic to Salmonella. This confirms earlier observations that a significant proportion of in vitro genotoxins are non-carcinogenic, probably due to their non-absorption or preferential detoxification in vivo. Also, only 30% of the mouse liver-specific carcinogens were mutagenic to Salmonella. This is consistent with tumors being induced in this tissue (and to a lesser extent in other tissues of the mouse and rat) by mechanisms not dependent upon direct interaction of the test chemical with DNA. Detection of 103 of the 115 carcinogens could be achieved by use of only male rats and female mice.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

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.     

The in vivo and in vitro genotoxicity of aromatic amines in relationship to the genotoxicity of benzidine.

Benzidine and 12 related aromatic amines have been studied for the effects of substituent groups and pi orbital conjugation on their genotoxicity as measured by their mutagenicity in vitro with Salmonella and by chromosomal aberrations (CA) in vivo in the bone-marrow cells of mice. The in vitro studies indicated increases in mutagenicity with increases in the electron withdrawing ability of para' substituents. Mutagenicity also increases with increased conjugation as shown by the degree of planarity of the biphenyl compounds and by comparing the mutagenicities of biphenyl amines to stilbenes as well as to ethylene bridged diphenyl compounds. The relative in vitro mutagenicity results were not predictive of relative in vivo CA results. The 3 most genotoxic compounds in vivo were the conjugated amines without substituents in the para' position. The CA values for 4-aminostilbene were exceptionally high. These in vivo results indicate increased genotoxicity for benzidine analogs without substitution in the para' position.     

The genotoxicity of enantiomeric aliphatic epoxides

The (R)- and (S)-optical isomers of 9 epoxides, benzyloxymethyloxirane, epichlorohydrin, glycidol, glycidyl 3-nitrobenzenesulfonate, glycidyl 4-nitrobenzoate, glycidyl tosylate, styrene oxide, glycidyl 1-naphthyl ether and glycidyl 4-nitrophenyl ether, have been compared for their in vivo and in vitro genotoxicity. The in vitro short-term test employed was the Ames mutagenicity assay with Salmonella strain TA100. The in vivo tests were chromosomal aberrations (CA) as well as sister-chromatid exchange (SCE) in bone-marrow cells of mice following intraperitoneal administration of these epoxides. Differences in mutagenicity between isomers were established with TA100 for all the compounds. While 13 of the isomers were genotoxic compared to a negative control by CA measurements, only in the case of glycidyl 4-nitrobenzoate could a significant difference be found between isomers by this test. However, with SCE evaluations, differences were detected between the (R)- and (S)-isomers for all the pairs of compounds with the exception of those for benzyloxymethyloxirane and glycidyl 4-nitrophenyl ether. At least in part, differences in the patterns of genotoxicity among compounds can be related to their differences in reaction pathways.