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Organophosphate compounds, which induce organophosphate poisoning, were originally used as pesticides. But this type of product has also been used as warfare nerve agent like sarin, soman, Russian VX, or tabun. HI-6-dimethanesulfonate is a salt of the oxime HI-6 used in the treatment of nerve-agent poisoning. It is known to be the best re-activator component of inactivated acetyl cholinesterase. HI-6-dimethanesulfonate has shown a higher level of solubility with similar potency to reactivate acetyl cholinesterase and a similar pharmacokinetics profile compared with HI-6 dichloride. HI-6 dimethanesulfonate was tested for its mutagenic and genotoxic potential by use of the standard ICH S2R (1) battery for the evaluation of pharmaceuticals. HI-6-dimethanesulfonate was mutagenic in the Ames test only in the presence of metabolic activation. In the mutation assay at the Tk locus in L5178Y mouse-lymphoma cells, HI-6-dimethanesulfonate showed mutagenic activity both with and without metabolic activation, with a significant increase in small colonies. The effects were in favour of a clastogenic activity. It was concluded that the compound was mutagenic and possibly clastogenic in vitro. In contrast, the in vivo micronucleus test in rat bone-marrow did not demonstrate any genotoxic activity and the Comet assay performed in rat liver did not show any statistically or biologically significant increases in DNA strand-breaks. The results of both in vivo studies performed on two different organs with two endpoints are sufficient to conclude the absence of a genotoxic hazard in vivo and to consider that there is no genotoxic concern in humans for HI-6-dimethanesulfonate., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Nowadays, there is clear progress in using the threshold concept in genetic toxicology, but its demonstration and acceptance in risk assessment is still under debate. Although it has been accepted for some non-DNA-reactive agents for which mechanisms of action were demonstrated, there is a growing weight of evidence to also support the existence of thresholded dose-responses for DNA-reactive agents. In this context, we have recently shown in human TK6 lymphoblastoid cells, that DNA-oxidizing agents [potassium bromate, bleomycin and hydrogen peroxide (via glucose oxidase)] produced non-linear dose-responses in the in vitro micronucleus test, thus allowing the determination of No-Observed-Genotoxic-Effect-Levels (NOGELs). Therefore, the aim of the present study was to focus on the analysis of thresholded dose-response curves in order to further investigate the existence of NOGELs for these same directly DNA-damaging agents, by use of other genotoxicity endpoints. Mutation frequency was determined after a 1-h treatment in the thymidine kinase (TK) gene-mutation assay. Primary DNA damage, especially oxidative DNA damage, was also assessed after 1h of treatment, followed - or not - by a 23-h recovery period, with the modified version of the comet assay (i.e. with the glycosylases Fpg and hOgg1). Overall, our analysis demonstrates that there is convincing evidence to support the existence of thresholded dose-responses for DNA-oxidizing agents. The determination of NOGELs depends on the genotoxic endpoint studied and consequently requires different genotoxicity assays performed concurrently. NOGELs could only be defined for the induction of chromosomal aberrations and gene mutations, i.e. for an effect-endpoint but not for primary DNA damage, i.e. for an exposure-endpoint. Further statistical analyses of these data are now required in order to draw conclusions on the exact level of the thresholds., (Copyright © 2011 Elsevier B.V. All rights reserved.)

At the 2009 International Workshop on Genotoxicity Testing in Basel, an expert group gathered to provide guidance on suitable follow-up tests to describe risk when basic in vivo genotoxicity tests have yielded positive results. The working group agreed that non-linear dose-response curves occur in vivo with at least some DNA-reactive agents. Quantitative risk assessment in such cases requires the use of (1) adequate data, i.e., the use of all available data for the selection of reliable in vivo models to be used for quantitative risk assessment, (2) appropriate mathematical models and statistical analysis for characterizing the dose-response relationships and allowing the use of quantitative and dose-response information in the interpretation of results, (3) mode of action (MOA) information for the evaluation and analysis of risk, and (4) reliable assessments of the internal dose across species for deriving acceptable margins of exposure and risk levels. Hence, the elucidation of MOA and understanding of the mechanism underlying the dose-response curve are important components of risk assessment. The group agreed on the need for (i) the development of in vivo assays, especially multi-endpoint, multi-species assays, with emphasis on those applicable to humans, and (ii) consensus about the most appropriate mathematical models and statistical analyses for defining non-linear dose-responses and exposure levels associated with acceptable risk., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The reference genotoxic agents Cytosine arabinoside, Vinblastine, Diethylstilboestrol and 2-Aminoanthracene were tested in the in vitro micronucleus assay, in human lymphoblastoid TK6 cells, without cytokinesis block, at the laboratories of Institut Pasteur de Lille, France. This was done in support of the toxicity measures recommended in the late 2007 version of the draft OECD Test Guideline 487 for the testing of chemicals. All four reference agents were positive in the assay at concentrations giving approximately 50% toxicity or less as assessed by draft Test Guideline 487 recommended measures, relative population doublings and relative increase in cell counts. Accordingly, this work supports the premise that relative population doublings and relative increase in cell counts are appropriate measures of toxicity for the non-cytokinesis blocked in vitro micronucleus assay., (Copyright © 2010 Elsevier B.V. All rights reserved.)

During the last decade, there has been clear progress in using threshold in risk assessment but its acceptance by scientists is still under debate. Contrary to indirect DNA-damaging agents, DNA-reactive agents have been assumed to have a non-threshold mode of action, as they directly induce DNA lesions that potentially can be converted into mutations. However, in recent years there is a growing number of data establishing threshold doses even for these DNA-reactive compounds. Indeed, there are several defence and repair mechanisms that provide protection and that may be responsible for genotoxic thresholds. In this context, we recently showed that DNA-oxidizing agents exhibit a thresholded dose-response in vitro with respect to chromosomal alterations. We have hypothesized the involvement of different cellular responses whose nature and efficiency depend on the stress level. The aim of this study was to develop a more complete understanding of these underlying mechanisms. We investigated global gene expression profiles of human lymphoblastoid TK6 cells after exposure to potassium bromate and hydrogen peroxide (via glucose oxidase). Cells were treated for 1h and mRNAs were isolated either immediately at the end of the treatment or after a 23-h recovery period. Our results showed that cells have developed elaborate cellular responses to oxidative stress in order to maintain genomic integrity. Many of altered genes were redox-sensitive transcription factors such as p53, NF-kappaB, AP-1 and Nrf2. Their downstream target genes and signalling pathways were subsequently activated leading mainly to the induction of antioxidant defenses, inflammation, cell cycle arrest, DNA repair and cell death. Overall, our study allowed the identification of key events involved in the thresholded response observed after DNA-oxidizing agents exposure and shows the usefulness of the combination of standard in vitro genotoxicity assays with gene expression profiling technology to determine modes of action, particularly for critical risk assessment., (Copyright 2010 Elsevier B.V. All rights reserved.)

Methylchavicol (or estragole), a natural flavouring substance present in tarragon, was confirmed as a genotoxic chemical in the in vitro UDS test in cultured rat hepatocytes and in the in vivo UDS test in hepatocytes of exposed rats. Deep-frozen tarragon was clearly less genotoxic than methylchavicol at equivalent dose levels, and desiccated tarragon was negative. Both forms of tarragon tested in vitro have the ability to decrease significantly the genotoxicity of methylchavicol added to the culture medium at concentrations

An increase in the number of revertant colonies in the Ames test is generally taken as a strong indication of mutagenic activity of a test compound. However, irrelevant positive findings may constitute a major problem in regulatory drug testing. In this study, mixtures containing only amino acids such as glycine, lysine, arginine and isoleucine, routinely used as peptide preservatives in polypeptide pharmaceutical products, were investigated for mutagenesis in the Ames Salmonella typhimurium test. The results demonstrated that in the presence of metabolic activation, all the solutions containing arginine induced an increase in the number of revertant colonies in strains TA98, TA100 and TA1535 compared with the solvent control. More specifically, for strain TA98, all arginine doses tested, i.e. from 0.4 to 8 mg/plate induced a statistically significant increase in the number of revertants. This increase was biologically significant from 1.2 to 8 mg/plate. For strain TA100, the five highest test doses, i.e., from 1.2 to 8 mg/plate, induced statistically and biologically significant increases in the number of revertants. A statistically significant increase in colony number was also observed in strain TA1535, but only at the maximal test dose of 8 mg/plate arginine. These increases were observed with arginine from two different sources, suggesting that the observed effect would not be due to the presence of potential impurities in the type of arginine used. Our findings show that a functional metabolic activation system was required to induce an increase in the number of colonies. The presence of vitamin C inhibited the arginine-induced increase in the number of revertant colonies in S. typhimurium strain TA98, suggesting a potential involvement of oxidative stress.

The existence of thresholds for indirect DNA-damaging agents has been widely accepted in the last few years. In contrast, DNA-reactive agents have been assumed to have a non-threshold mode of action, as they directly induce DNA lesions that have the potential to be converted into mutations. However, this does not take into account protective factors acting to reduce or repair genotoxic damage. Among the compounds acting through possible threshold-mechanisms, some of them induce DNA damage by oxidative stress. In this context, the aim of our study was to investigate the dose-response relationship of well-known DNA-oxidizing agents acting through different mechanisms of oxidative stress, viz. potassium bromate, bleomycin and hydrogen peroxide (by the action of glucose oxidase) by assessing the induction of chromosomal damage using the in vitro micronucleus test (MNT) on the human lymphoblastoid cell line TK6. In order to provide a first characterization of their genotoxic mechanism, two treatment schedules were applied. Cells received both short-term treatment followed by a recovery time (1 + 23 h, 2 + 22 h, 3 + 21 h or 6 + 18 h) and long-term treatment (24h continually). Our results show interesting non-linear dose-effect relationships starting with a range of non-mutagenic very low doses allowing the determination of a No-Observed-Effect Level (NOEL) and going step-wise up to higher doses. After a short exposure, three different plateaus were observed suggesting complex activations and interactions of different cellular mechanisms whose nature and efficiency were dose-dependent. In contrast, after a long treatment, the dose-response curves were different depending on the test compound investigated. Therefore, the in vitro MNT seems to be an appropriate predictive test to establish the NOEL(s) of DNA-oxidizing agents. In order to confirm and to determine the origin of the different cellular step-wise responses observed, additional mechanistic studies would be required, especially by means of other genotoxicity endpoints and gene-expression profiling.

Aloe-emodin (AE) and derivatives may be present as undesired components co-extracted during extraction of plants containing anthraquinonic derivatives for preparation of diacetylrhein. AE is a well-known in vitro mutagen, but up to now it failed to induce any clear in vivo genotoxic activity in the chromosome aberration assay in rat bone marrow or the in vivo/in vitro UDS test in liver. However, the two target organs noted during rodent carcinogenicity studies with danthron and emodin, two other well-known anthraquinone derivatives, are the colon and the kidney. Therefore, the choice of the organs for testing the genotoxicity of AE, i.e. bone marrow and liver, may be considered inadequate to demonstrate a possible in vivo genotoxic activity. In this context, the in vivo mouse comet assay was performed on both isolated kidney and colon cells in order to demonstrate a possible organospecific genotoxicity after oral administration of AE. Concurrently, the Ames test and the in vitro micronucleus assay with TK6 human lymphoblastoid cells were performed in their microscale version both with S9 from Aroclor 1254-induced liver or kidney, and without S9. AE induced primary DNA damage in the liver and in the kidney as observed between 3 and 6h after two oral administrations at 500, 1000 and 2000mg/kg bw, underlining an in vivo genotoxic mechanism of action. Furthermore, AE induced a clear genotoxic activity both in the Salmonella typhimurium strains TA1537 and TA98 and in the in vitro micronucleus assay in the absence as well as in the presence of metabolic activation. As no significant variation in the genotoxic activity of AE was noted when using either liver or kidney S9-mix, it seems that no quantitatively and/or qualitatively specific renal metabolism occurs. The kidney may be a target organ of AE as it is the major route of excretion. Under such conditions the separation of AE components should take place and the residual content of undesired AE derivatives should be made as low as reasonably achievable. AE present in plant extracts should be considered as an in vivo genotoxin and this property should be taken into account in the risk assessment for human exposure.

The purpose of the present work was to assess the mutagenic potency of soil samples presumably not contaminated by industrial wastes and discharges. A set of 51 soil samples was collected from areas considered as not contaminated by a known industrial activity: 11 urban samples (collected in cities), 15 suburban samples (collected in villages), 7 agricultural samples, and 18 forest or natural samples. Each soil sample was collected at the surface (0-5cm deep), dried, sieved (2mm), homogenized before organic extraction (dichloromethane/acetone 1/1 (v/v), 37 degrees C, 4h, soil/solvent ratio 1/2, m/v), solvent exchange to DMSO and sterilizing filtration. The micro-method adaptation of the standard bacterial mutagenicity test on Salmonella typhimurium strain TA98 was performed with and without a metabolic activation system (rat-liver homogenate S9), and thus detected the effect of pro-mutagens and direct mutagens, respectively. The use of a pre-incubation method increased the sensitivity of the assay. The results obtained showed a wide range of effect levels, from no effect to clear mutagenicity. In particular, the extract of all 11 urban soil samples demonstrated mutagenic activity, while the extracts of 10 of the 15 suburban samples showed mutagenicity. On the other hand, the extract of only one of the 7 agricultural samples studied induced mutations, and none of the 18 natural or forest-soil samples investigated produced mutagenic extracts. These findings seem to indicate the crucial influence of the diffuse pollution originating from different human activities on the mutagenic potency of urban soil samples. These findings make it possible to classify the soils according to their mutagenic potency. No clear correlation was found between the mutagenicity detected in soil extracts and the measured polycyclic aromatic hydrocarbon (PAH) content of the soils investigated.

7-Hydroxy-5-methyl-3-phenyl-6,7,8,9-tetrahydropyrido[3',2':4,5]imidazo[1,2-a]pyrimidin-5-ium chloride (PhIP-M1) is a newly identified intestinal microbial metabolite from the food carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Although the mutagenic potential of the endogenous N-hydroxy PhIP derivate has been reported, the risks associated with PhIP-M1 have not yet been explored. In this work, the cytotoxic and genotoxic effects originating from PhIP-M1 were assessed in the epithelial intestinal Caco-2 cell line. PhIP-M1 significantly decreased in a time- and dose-dependent manner mitochondrial dehydrogenase activity and protein synthesis, with IC50 values of, respectively, 180+/-39.4 and 173+/-20.3 microM after 24h, and 33.8+/-3.5 and 37.3+/-10.9 microM after 72 h. Apoptosis within the concentration ranges of cytotoxicity was confirmed by morphological examination, DAPI nuclear staining and annexin V staining. PhIP-M1 provoked cell cycle arrest, characterized by a significant increase in the number of nucleoids in the G2/M phase. A dose-dependent increase in DNA damage, as quantified by the alkaline comet assay, was observed after 3h in the 50-200 microM range. Because these PhIP-M1-induced genomic and cellular events may contribute to the carcinogenicity of PhIP, the potency of the colon microbiota to bioactivate PhIP must be included in future risk assessments.

The objective of this study was to determine the ability of the alkaline in vivo Comet assay (pH>13) to distinguish genotoxic carcinogens from epigenetic carcinogens when performed on freshly isolated kidney cells and to determine the possible interference of cytotoxicity by assessing DNA damage induced by renal genotoxic, epigenetic or toxic compounds after enzymatic isolation of kidney cells from OFA Sprague-Dawley male rats. The ability of the Comet assay to distinguish (1) genotoxicity versus cytotoxicity and (2) genotoxic versus non-genotoxic (epigenetic) carcinogens, was thus investigated by studying five known genotoxic renal carcinogens acting through diverse mechanisms of action, i.e. streptozotocin, aristolochic acids, 2-nitroanisole, potassium bromate and cisplatin, two rodent renal epigenetic carcinogens: d-limonene and ciclosporine and two nephrotoxic compounds: streptomycin and indomethacin. Animals were treated once with the test compound by the appropriate route of administration and genotoxic effects were measured at the two sampling times of 3-6 and 22-26h after treatment. Regarding the tissue processing, the limited background level of DNA migration observed in the negative control groups throughout all experiments demonstrated that the enzymatic isolation method implemented in the current study is appropriate. On the other hand, streptozotocin, 20mg/kg, used as positive reference control concurrently to each assay, caused a clear increase in the mean Olive Tail Moment median value, which allows validating the current methodology. Under these experimental conditions, the in vivo rodent Comet assay demonstrated good sensitivity and good specificity: all the five renal genotoxic carcinogens were clearly detected in at least one expression period either directly or indirectly, as in the case of cisplatin: for this cross-linking agent, the significant decrease in DNA migration observed under standard electrophoresis conditions was clearly amplified when the duration of electrophoresis was increased up to 40min. In contrast, epigenetic and nephrotoxic compounds failed to induce any signifcant increase in DNA migration. In conclusion, the in vivo rodent Comet assay performed on isolated kidney cells could be used as a tool to investigate the genotoxic potential of a test compound if neoplasic/preneoplasic changes occur after subchronic or chronic treatments, in order to determine the role of genotoxicity in tumor induction. Moreover, the epigenetic carcinogens and cytotoxic compounds displayed clearly negative responses in this study. These results allow excluding a DNA direct-acting mechanism of action and can thus suggest that a threshold exists. Therefore, the current in vivo rodent Comet assay could contribute to elucidate an epigenetic mechanism and thus, to undertake a risk assessment associated with human use, depending on the exposure level.

This report summarizes the proceedings of the September 9-10, 2005 meeting of the Expert Working Group on Hazard Identification and Risk Assessment in Relation to In Vitro Testing, part of an initiative on genetic toxicology. The objective of the Working Group was to develop recommendations for interpretation of results from tests commonly included in regulatory genetic toxicology test batteries, and to propose an appropriate strategy for follow-up testing when positive in vitro results were obtained in these assays. The Group noted the high frequency of positive in vitro findings in the genotoxicity test batteries with agents found not to be carcinogenic and thought not to pose a carcinogenic health hazard to humans. The Group agreed that a set of consensus principles for appropriate interpretation and follow-up testing when initial in vitro tests are positive was needed. Current differences in emphasis and policy among different regulatory agencies were recognized as a basis of this need. Using a consensus process among a balanced group of recognized international authorities from industry, government, and academia, it was agreed that a strategy based on these principles should include guidance on: (1) interpretation of initial results in the "core" test battery; (2) criteria for determining when follow-up testing is needed; (3) criteria for selecting appropriate follow-up tests; (4) definition of when the evidence is sufficient to define the mode of action and the relevance to human exposure; and (5) definition of approaches to evaluate the degree of health risk under conditions of exposure of the species of concern (generally the human). A framework for addressing these issues was discussed, and a general "decision tree" was developed that included criteria for assessing the need for further testing, selecting appropriate follow-up tests, and determining a sufficient weight of evidence to attribute a level of risk and stop testing. The discussion included case studies based on actual test results that illustrated common situations encountered, and consensus opinions were developed based on group analysis of these cases. The Working Group defined circumstances in which the pattern and magnitude of positive results was such that there was very low or no concern (e.g., non-reproducible or marginal responses), and no further testing would be needed. This included a discussion of the importance of the use of historical control data. The criteria for determining when follow-up testing is needed included factors, such as evidence of reproducibility, level of cytotoxicity at which an increased DNA damage or mutation frequency is observed, relationship of results to the historical control range of values, and total weight of evidence across assays. When the initial battery is negative, further testing might be required based on information from the published literature, structure activity considerations, or the potential for significant human metabolites not generated in the test systems. Additional testing might also be needed retrospectively when increase in tumors or evidence of pre-neoplastic change is seen. When follow-up testing is needed, it should be based on knowledge about the mode of action, based on reports in the literature or learned from the nature of the responses observed in the initial tests. The initial findings, and available information about the biochemical and pharmacological nature of the agent, are generally sufficient to conclude that the responses observed are consistent with certain molecular mechanisms and inconsistent with others. Follow-up tests should be sensitive to the types of genetic damage known to be capable of inducing the response observed initially. It was recognized that genotoxic events might arise from processes other than direct reactivity with DNA, that these mechanisms may have a non-linear, or threshold, dose-response relationship, and that in such cases it may be possible to determine an exposure level below which there is negligible concern about an effect due to human exposures. When a test result is clearly positive, consideration of relevance to human health includes whether other assays for the same endpoint support the results observed, whether the mode or mechanism of action is relevant to the human, and - most importantly - whether the effect observed is likely to occur in vivo at concentrations expected as a result of human exposure. Although general principles were agreed upon, time did not permit the development of recommendations for the selection of specific tests beyond those commonly employed in initial test batteries.

This study, coordinated by the SFTG (French branch of European Environmental Mutagen Society), included 38 participants from Europe, Japan and America. Clastogens (bleomycin, urethane), including base and nucleoside analogs (5-fluorouracil and cytosine arabinoside), aneugens and/or polyploidy inducers (colchicine, diethylstilboestrol, griseofulvin and thiabendazole), as well as non-genotoxic compounds (mannitol and clofibrate), were tested. Four cell types were used, i.e. human lymphocytes in the presence of cytochalasin B and CHO, CHL and L5178Y cell lines, in the presence or absence of cytochalasin B, with various treatment-recovery schedules. Mitomycin C was used as a positive control for all cell types. Mannitol and clofibrate were consistently negative in all cell types and with all treatment-recovery conditions. Urethane, known to induce questionable clastogenicity, was not found as positive. Bleomycin and mitomycin C were found positive in all treatment-recovery conditions. The base and nucleoside analogs were less easy to detect, especially 5-fluorouracil due to the interference with cytotoxicity, while cytosine arabinoside was detected in all cell types depending on the treatment-recovery schedule. Aneugens (colchicine, diethylstilboestrol and griseofulvin) were all detected in all cell types. In this study, the optimal detection was ensured when a short treatment followed by a long recovery was associated with a long continuous treatment without recovery. There was no impact of the presence or absence of cytochalasin B on the detection of micronucleated cells on cell lines. Scoring micronucleated cells in both mononucleated and binucleated cells when using cytochalasin B was confirmed to be useful for the detection and the identification of aneugens. In conclusion, these results, together with previously published validation studies, provide a useful contribution to the optimisation of a study protocol for the detection of both clastogens and aneugens in the in vitro micronucleus test.

This study on the in vitro micronucleus assay, comprising 11 laboratories using human lymphocytes, was coordinated by an organizing committee supported by the SFTG (the French branch of the European Environmental Mutagen Society). Nine coded substances were assessed for their ability to induce micronuclei in human lymphocytes in vitro, mitomycin C being used as a positive control. Cultures were exposed to the test substances for a short (early or late) time or for a long time, followed by a short or long recovery period, in the presence of cytochalasin B. Each chemical was evaluated, generally in two laboratories, using three treatment schedules at least twice. The data were assessed for acceptability, and then classified as negative, positive or equivocal. Two of seven genotoxic compounds, namely colchicine and bleomycin, clearly induced micronuclei. Reproducible results were difficult to obtain for some substances, which tended to be those acting at specific stages of the cell cycle. Cytosine arabinoside, diethylstilboestrol and 5-fluorouracil were classified as equivocal. Urethane and thiabendazole were classified as negative. The two presumed non-genotoxic compounds, mannitol and clofibrate, did not induce micronuclei. Repeat testing, exposing cells at both an early and late time after mitogenic stimulation, was needed to detect substances classified as equivocal. These results show the importance of achieving sufficient inhibition of nuclear division to avoid the possibility of missing an effect. The evaluation of micronuclei in mononucleated as well as binucleated cells was particularly useful to detect aneugens. There were no false positive results using lymphocytes, indicating a high specificity. It is concluded that the clastogenic or aneugenic potential in vitro of the substances tested was correctly identified in this study, but that refining the protocol to take into account factors such as the stages of the cell cycle exposed to the compound, or the duration of recovery would be likely to improve the sensitivity of detection using lymphocytes.

In this report, results are presented from an international study of the in vitro micronucleus assay using mouse lymphoma L5178Y cells. This study was coordinated by an organizing committee supported by the SFTG (the French branch of the European Environmental Mutagen Society). Test chemicals included mannitol, bleomycin, 5-fluorouracil, colchicine and griseofulvin. Mitomycin C was used as a positive control. Each chemical was evaluated in at least two laboratories following a variety of different protocols (short and long exposures, varying recovery times, with and without cytochalasin B) in order to help determine a standard protocol for routine testing in mouse lymphoma L5178Y cells. Mannitol was the only exception, being tested in only one laboratory. Mannitol was negative, while bleomycin induced a concentration-dependent increase in micronucleated cells. Equivocal results were obtained for 5-fluorouracil, colchicine and griseofulvin. High levels of cytotoxicity interfered with the assessment of aneuploidy for colchicine and griseofulvin, preventing the ability to obtain clear results in all the treatment schedules. Experiments with 5-fluorouracil, colchicine and griseofulvin showed that both short and long treatment times are required as each compound was detected using one or more treatment protocol. No clear differences were seen in the sensitivity or accuracy of the responses in the presence of absence of cytochalasin B. It was also found that a recovery period may help to detect compounds which induce a genotoxicity associated to a reduction in cell number or cell proliferation. Overall, the results of the present study show that mouse lymphoma L5178Y cells are suitable for the in vitro micronucleus assay.

In this report, are presented the results of an international collaborative study on the in vitro micronucleus assay, using CHL cells. Fourteen laboratories participated in this study which was coordinated by an organizing committee supported by the SFTG (the French branch of the European Environmental Mutagen Society). Nine coded substances, having different modes of action and at different levels were assessed in the in vitro micronucleus test, using a common protocol. Mitomycin C was used as a positive control. In order to help to define a standard protocol on CHL cells, short and long treatment periods followed by various recovery times, with or without cytochalasin B, were compared. After an evaluation of the acceptability of the assays, the tested chemicals were classified as negative, positive or equivocal. Mannitol and clofibrate were judged as negative in all treatment schedules. Bleomycin was positive in all the treatment schedules, with an increase in the number of micronucleated cells in both mononucleate and binucleate cells when using cytochalasin B. This was also shown for the aneugens colchicine, diethylstilboestrol and griseofulvin, as expected. Urethane was judged as equivocal only after long treatment with cytochalasin B, and negative in all other treatment schedules. In any case, no genotoxic compound would have been missed with schedules including a short and a long treatment time, whether the treatment was followed by a recovery period or not and whether cytochalasin B was used or not. Thus, these results show that CHL cells were suitable for accurately detecting clastogenic and aneugenic compounds of various types in the in vitro micronucleus test.

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

Aneugenic compounds act on non-DNA targets to exert genotoxicity via an indirect mechanism. In contrast to DNA-binding agents, these compounds are expected to possess threshold levels of activity. Therefore, the risk for adverse effects following human exposure to an aneugen could be minimal, if the threshold of activity has been clearly determined in vivo and in vitro and providing the human exposure level is below this threshold. Thus, the development of a single-cell model to allow comparisons between in vitro and in vivo threshold values for aneugenic compounds is of importance. The in vivo micronucleus test is one of the main assays used in genetic toxicology, and is often performed in the mouse. Thus, an extensive database is available in the literature. However, there are only few data concerning the in vitro micronucleus assay using mouse cells, as the majority of in vitro micronucleus assays have been performed using human lymphocytes. In addition, there is a lack of data concerning thresholds for any compound using this model. First, we evaluated whether the use of mouse splenocytes would be an acceptable alternative to that of human lymphocytes to identify aneugens. To allow valid comparisons, the two protocols were first harmonized. Thus, phytohemagglutinin (PHA) and concanavalin A were used as specific mitogens for human lymphocytes and mouse splenocytes, respectively, in order to achieve similar cell-proliferation rates. To achieve similar and sufficient numbers of binucleated cells, cytochalasin B was added 44 and 56 h after culture initiation of the human and mouse cells, respectively. Second, we compared the sensitivity of the mouse protocol with that of the human protocol by exposing the cells to the aneugens nocodazole and paclitaxel. There was good reproducibility of the cytotoxic/genotoxic responses of the two cell models following exposure to the aneugens. The sensitivity of the mouse splenocytes to paclitaxel was higher than that of the human lymphocytes. The two cell types were equally sensitive to nocodazole.

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 aim of this study was to investigate the influence of four extraction parameters (type of solvent, temperature, duration of extraction, and soil mass/solvent volume ratio) on the mutagenicity of soil extracts. Four urban soil samples were submitted to the micro-method adaptation of the Ames test on Salmonella typhimurium according to the following sequence: identification of the most sensitive strain (TA98 or TA100), the best solvent(s), the optimum extraction temperature and extraction time, and finally the optimal soil/solvent ratio. Extraction was thus performed using eight different solvents (distilled water, dichloromethane, acetonitrile, acetone, cyclohexane, methanol, hexane, or ethanol), two temperatures (room temperature or 37 degrees C), two durations (4 or 24 h), and two soil mass/solvent volume ratios (1:2 or 1:10). The results show that strain TA98 was more sensitive than strain TA100, and the observed mutagenicity was expressed as number of TA98 revertants per mg of soil equivalent. No mutagenicity was induced by the distilled water extracts, whereas most of the organic solvent extracts induced a significant mutagenic response. A dichloromethane/acetone mixture appeared to be the best compromise for extraction of mutagens from the urban soils tested. Moreover, the present study showed that a higher mutagenic activity was generally obtained with a temperature of 37 degrees C (compared to room temperature), with an extraction time of 24 h (compared to 4 h), and with a soil mass/solvent volume ratio of 1:10 (compared to 1:2).

Okadaic acid (OA) is a marine toxin produced by dinoflagellates and responsible for human intoxications. OA is a specific inhibitor of serine/threonine protein phosphatases PP1 and PP2A and a potent tumor promoter in mouse skin and rat glandular stomach. In a previous study, we demonstrated that OA induced aneuploidy in CHO-K1 cells using the cytokinesis-block micronucleus (CBMN) assay coupled to FISH and concluded that OA was not a direct mutagen. As some previous in vitro mutagenicity studies had given positive results with OA, we decided to perform two additional in vitro mutagenicity assays in accordance with the OECD guidelines: (i) the CHO/Hprt test, which provides end points about locus-specific gene mutation; (ii) the in vitro unscheduled DNA synthesis (UDS) assay in rat hepatocytes, which measures [(3)H]thymidine incorporation into DNA undergoing excision repair. In the CHO/Hprt assay, there was no significant increase in the number of mutants for doses ranging from 5 to 5000 nM in the presence or absence of rat liver S9 fraction. In the in vitro UDS assay, OA did not induce primary DNA damages in rat hepatocytes following 18 h exposure at concentrations between 1.32 and 100 nM. As OA could affect the DNA repair systems via the inhibition of protein phosphatases, its effects on the repair kinetic of 2AAF-induced DNA damage were also investigated with the UDS assay. The results showed that OA did not interact with the DNA-repair process involved in in vitro UDS in rat hepatocytes. We concluded that OA failed to induce direct DNA damage but acted principally by altering the chromosome number, which could contribute to its carcinogenic effect.

Since epidemiological studies have implicated the co-exposition of iron oxides and polycyclic aromatic hydrocarbons as potential etiological factors involved in the excess of mortality from lung cancer in miners, experimental studies have been performed to investigate the role of iron on benzo[a]pyrene (B[a]P)-induced lung pathogenesis. We demonstrated previously that in vivo damage was higher when B[a]P was coated onto hematite than when B[a]P was administered alone. In order to determine the role of (i) different cell types and (ii) adsorption of hematite in this potentiation, in vitro studies were developed. The Comet assay was first used to measure DNA damage in four isolated cell types from Sprague-Dawley rats at 1, 2, 4, 8 and 24h after in vitro treatment with hematite (Fe2O3) or B[a]P or B[a]P coated onto hematite. For the two treatments with B[a]P, no damage was observed in alveolar macrophages, but significant increases in damage were seen in lymphocytes, hepatocytes and lung cells (where the effects of B[a]P coated onto hematite were stronger than those of B[a]P alone). In a second part of the study, the Comet assay was conducted with lung cells to measure the in vitro effect of (i) the coating and (ii) the role of the physical properties of Fe2O3. A statistically significant increase in damage was observed for the coating of B[a]P onto Fe2O3 compared (i) with their simple addition and (ii) with the coating of B[a]P onto graphite used as an inert compound. This study showed that (i) Fe2O3/B[a]P acts essentially in lung cells, (ii) the coating is a primordial step and (iii) the physical properties of Fe2O3 play a very minor role, which suggests another mechanism of action to explain the higher toxicity. Hence, our data may contribute to explain the excess of mortality in epidemiological studies and overall why exposures to B[a]P coated onto Fe2O3 resulted in higher toxicity in rodents compared to exposure to B[a]P alone.

With the re-emergence of Human African Trypanosomiasis (HAT) on the one hand, which are increasingly resistant to current therapies, and the stage-dependent effectiveness or even the prohibitive cost of these therapies on the other hand, megazol, a 5-nitroimidazole thiadiazole highly active against various trypanosomal species, was assessed for its genotoxic potential. Very little information has become available until now. Two batches of megazol were provided by two different suppliers: Far-Manguinhos, a part of the Fiocruz foundation, under the Brazilian Minister of Health, and Delphia, a French company. These two batches, obtained by different synthetic routes, were studied by means of the in vitro micronucleus assay on L5178Y mouse lymphoma cells, in its microscale version. Both batches of magazol displayed a strong genotoxic activity in this screening assay. A second batch from Delphia was then investigated by use of two tests, i.e. the in vitro metaphase analysis with human lymphocytes and the in vivo micronucleus test in rat bone-marrow. Megazol was shown to be a potent inducer of in vitro and in vivo chromosomal aberrations. Although megazol is a potent trypanocidal agent and is orally bio-available, its toxicity dictates that it should not be developed further for the treatment of HAT and Chagas disease. All development work has therefore been discontinued.

Conditions in which clastogens produce positive responses have been increasingly challenged, and several situations have been described in which clastogenic responses would be considered not to be relevant. For example, extreme culture conditions lead to high variations of pH, osmolality or ionic strength. Apoptosis is induced in extreme culture conditions and contributes to false-positive results in the in vitro micronucleus test performed with CTLL-2 cells. These cells can enter apoptosis when exposed to apoptosis stimuli or after IL-2 deprivation, whereas the CTLL-2 Bcl2 cell line is protected from apoptosis due to the over-expression of the apoptosis inhibitor Bcl2 in bcl2-transfected CTLL-2 cells. The two cell lines were treated in extreme culture conditions of either pH or osmolality or were submitted to high ionic strength. The apoptosis level was measured in parallel with the in vitro micronucleus test using the annexin V-FITC method. Data obtained in the two cell lines suggested that apoptosis caused by extreme culture condition induces the formation of micronucleated cells, which leads to false-positive results in the in vitro micronucleus test.

2-Hydroxy-1,4-naphthoquinone (HNQ) has been found positive in a previous chromosome aberration test in Chinese hamster ovary (CHO) cells and in a mouse bone marrow micronucleus test at 72h after oral administration (vehicle: DMSO). However it was negative at 24 and 48h sampling times, and in subsequent micronucleus tests that used 0.5% aqueous methyl cellulose (MC) as vehicle. We performed a bone marrow micronucleus test in male and female NMRI BRL/BR mice at oral doses of 75, 150 and 300mg/kg in two vehicles (DMSO and 0.5% aqueous MC), evaluated micronuclei at 24, 48 and 72h, plasma levels of HNQ at 0.5, 1 and 4h, and haematology parameters at 72h after administration. The mechanism of in vitro clastogenic activity of HNQ was investigated by evaluation of the potential of HNQ to produce oxidative DNA damage after treatment of CHO with 10mM HNQ, followed by quantification of DNA fragments using the comet assay. In the micronucleus test, HNQ at 300mg/kg produced mortality and clinical signs at similar incidence and severity for both vehicles. Levels of HNQ in the plasma of treated mice were dose-related, of similar magnitude for both vehicles, but higher in females than in males. Maximum concentrations were found at 0.5 or 1h. At 300mg/kg, HNQ slightly affected RBC parameters suggesting haematotoxicity. No increase in the frequency of micronuclei was observed for any dose, vehicle or time point, whereas the positive control substance (CPA) produced a clear positive response. No evidence of HNQ-induced oxidative DNA damage was found at clastogenic concentrations in vitro, whereas the positive control substance (H(2)O(2)) produced a clear increase. In conclusion, HNQ was negative for induction of bone marrow micronuclei in mice up to 72h after administration in two different vehicles, and its in vitro clastogenicity was not due to oxidative damage. These results confirm that HNQ poses no or negligible genotoxic risk.

Since epidemiological studies have firmly implied the co-exposition between iron oxides and polycyclic aromatic hydrocarbons (PAH) as potential etiological factor involved in the excess of mortality by lung cancer in miners, experimental studies have been performed to investigate the role of iron particles on benzo[a]pyrene (B[a]P)-induced lung pathogenesis. In the present study, the alkaline single-cell gel electrophoresis (SCGE; Comet Assay) was used to measure DNA single-strand breaks in four cell types (alveolar macrophages, lung cells, peripheral lymphocytes and hepatocytes) of OFA Sprague-Dawley rats 24h after endotracheal administration of a single dose of an iron oxide (hematite; Fe(2)O(3)) (0.75mg) or B[a]P (0.75mg) or B[a]P (0.75mg) coated onto hematite particles (0.75mg). No damage was observed in cell from the four investigated organs in rats treated with iron oxide alone, while a statistically significant increase in DNA damage was observed compared with control animals in all tested cell types of rats treated with B[a]P alone or in association with hematite. The highest levels of damage were observed in lung cells and peripheral lymphocytes; the levels of damage in alveolar macrophages and hepatocytes were increased, but to a lesser extent compared with the first two cell types. The main finding was to notice a statistically significant increase of the damage in all organs of rats treated with B[a]P coated onto hematite (approximately two-fold increases; P<0.001), versus B[a]P alone. The current study shows that iron particles increase the genotoxic properties of B[a]P in the respiratory tract of endotracheally treated OFA Sprague-Dawley rats. Hence, our data may contribute to explain the excess mortality by lung cancer in epidemiological studies and overall why exposures to B[a]P coated onto Fe(2)O(3) particles resulted in higher toxicity in rodents compared with exposure to B[a]P alone.

2-Hydroxy-1,4-naphthoquinone (HNQ; Lawsone; CAS 83-72-7) is the principal natural dye ingredient contained in the leaves of Henna (Lawsonia inermis). Published genotoxicity studies on HNQ suggested it was a weak bacterial mutagen for Salmonella typhimurium strain TA98 or was more clearly mutagenic for strain TA 2637, both in the presence of metabolic activation. HNQ was unable to induce sex-linked recessive lethal mutations in Drosophila melanogaster. However, a small increase in micronucleus frequency was reported in the bone marrow of mice at a single mid-range dose level, 24h after intraperitoneal injection. In view of the wide use of Henna hair dyes it was deemed necessary to conduct a thorough investigation, under Good Laboratory Practice conditions, of the genotoxicity of HNQ. HNQ was non-mutagenic in bacterial (Ames test) or mammalian (V79 hprt) assays. It was borderline positive in a mouse lymphoma tk mutation assay and a chromosome aberration test (CHO cells), results that may reflect a similar clastogenic mechanism. Negative in vivo genotoxicity results were noted in the rat hepatocyte in vivo/in vitro UDS test, in peripheral lymphocytes (chromosome aberrations) of rats receiving repeated oral doses of HNQ at the MTD for 28 days, and in mouse and hamster bone marrow chromosome aberration tests. However small, but statistically significant increases in the incidence of bone marrow micronuclei were observed in two out of five tests at 72 h after dosing, but not at 24 or 48 h. There was evidence of haematotoxicity at 72 h, which may have been enhanced by the vehicle (DMSO) used in the positive tests. As erythropoiesis and administration of haematotoxic agents are known to induce small increases in the frequency of bone marrow micronuclei, typically at delayed sampling times, the data suggest that the positive 72 h response produced by HNQ is consistent with stimulation of haematopoiesis subsequent to haematological toxicity of HNQ, and not due to a DNA-reactive mechanism. Overall, the weight of evidence suggests that Henna and HNQ pose no genotoxic risk to the consumer.

Although benzo[a]pyrene (B[a]P) is a well-known genotoxic agent, little is known about the extent of DNA effects induced by B[a]P in rat tissues after pulmonary exposure. The alkaline single-cell gel electrophoresis (comet assay) was used to measure DNA single-strand breaks in alveolar macrophages, lung cells, peripheral lymphocytes and hepatocytes of OFA Sprague-Dawley rats exposed to a single dose of B[a]P by endotracheal administration. Statistically significant damage was observed in all organs tested after 3, 24 and 48h of pulmonary exposure to 3mg of B[a]P per animal, with a time-dependent relationship. The maximum damage was observed in the four cell types 24h after exposure. The higher level of damage was observed both in lung cells and peripheral lymphocytes; in alveolar macrophages and hepatocytes the level of damage was increased, but at a lower level than in the two other cell types. Furthermore, B[a]P demonstrated a clear dose-related genotoxic activity in the lung cells when tested at doses of 0.75, 1.5 and 3mg. The current study shows that B[a]P caused DNA single-strand breaks in the respiratory tract of endotracheally treated OFA Sprague-Dawley rats. The study also suggests that pulmonary exposure to B[a]P can induce a high level of DNA damage in peripheral lymphocytes. The clear relationship between lung exposure to B[a]P and consequences observed in lymphocytes suggests that the comet assay in peripheral lymphocytes can be used as a sensitive marker in human monitoring studies.

The potential mutagenic properties (micronucleus and the Ames tests) of fluoro-A-85380 (2-fluoro-3-[2(S)-2-azetidinylmethoxy]pyridine) were evaluated as a mandatory pre-clinical step. No statistically significant increase in the frequency of micronucleated polychromatic erythrocytes was found in animals treated at any dose tested. No biologically significant increase in the mean number of revertants was noted in all the Salmonella typhimurium strains tested with fluoro-A-85380. Therefore, fluoro-A-85380 demonstrated no mutagenic properties using these two tests.

Ochratoxin A (OTA) was, up to now, believed to be non-mutagenic in the classical Salmonella typhimurium reverse mutation test. This was confirmed using rat liver microsomal fractions with the strains, TA1535, TA1538 and TA98, and up to 1210 micrograms/plate, utilizing an Ames microtest. However, using mice kidney microsomal fractions as metabolic activators, reverse mutations were obtained with the three strains used, in the presence of either NADP or arachidonic acid as cofactors. The mutagenicity was higher with arachidonic acid than with NADP using the TA1535 strain. This lends support to the results concerning the DNA or dGMP adducts obtained in vitro which were also higher in the presence of arachidonic acid, and indicate that several metabolic pathways of OTA can lead to genotoxic compounds. In addition, both base pair substitutions and frameshift mutations can be caused by OTA after metabolic activation.

Radiopharmaceuticals should be tested in a battery of assays to evaluate human risk assessment, as for every drug. The battery of tests used for this evaluation should be adapted taking into account the specificity of radiopharmaceuticals and particularly the short duration of treatment, the toxicity associated both to the unlabelled part of the molecule and to the radioactivity itself, but also to a specific type of impurities due to the radiolytic degradation products.

The in vitro micronucleus test is available to demonstrate the clastogenic and aneugenic potencies of chemicals. This test is easier to evaluate than metaphase analysis and, although used routinely by some laboratories as a prescreening test for the determination of chromosomal mutation induction potency, it has not been recognised by regulatory authorities as a test to be included in the battery of assays for human risk assessment. This limitation is due to the lack of a fixed protocol, a limited data bank, particularly in the case of clastogens, and to the lack of sufficient robust interlaboratory validation studies. If we aim to recognize this test in the form of an OECD guideline and to introduce it as an alternative to in vitro metaphase analysis in cell culture, it is necessary to begin a collaborative study in order to determine the best protocol which is valid for the detection of all clastogens as well as to determine clearly the limits of the test. The use of this in vitro test as a prescreening assay could be very useful as it takes structural and numerical chromosomal abnormalities into account and is simpler to perform than in vitro metaphase analysis.

Two in vitro tests (Ames test and SOS chromotest), one for bacterial mutagenicity and one for primary DNA damage, were assayed to determine the genotoxic activity of 6 pesticides (atrazine, captafol, Captan, chlorpyrifosmethyl, molinate and tetrachlorvinphos). Assays were carried out both in the absence and presence of S9 fractions of liver homogenate from rat (Sprague-Dawley) pretreated with Aroclor 1254. Captan and captafol were genotoxic on both the Ames test and the SOS chromotest. Comparisons with mutagenesis data in Salmonella indicated that the SOS assay detected as genotoxic the pesticides that were mutagenic on the Salmonella test. Non-genotoxic effects were not detected in vitro either in the Salmonella/microsome assay nor in the SOS chromotest when bacterial tester strains were exposed to atrazine, molinate, chlorpyrifosmethyl and tetrachlorvinphos in the absence or presence of S9 mix.

The chlorination by-products chloral hydrate and chloropicrine were assayed for genotoxicity in three short-term tests. Chloropicrine was 100-fold more potent than chloral in inducing mutations in strain TA100 of S. typhimurium (fluctuation test) and, at variance with chloral, was positive in the SOS chromotest using strain PQ37 of E. coli. On the other hand, only chloral caused a significant increase in the frequency of micronucleated erythrocytes following in vivo exposure of the amphibian Pleurodeles waltl newt larvae.

Three short-term assays (the SOS chromotest, the Ames-fluctuation test and the newt micronucleus test) were carried out to evaluate the genotoxicity of six halogenated acetonitriles identified in chlorinated waters (monochloro-, dichloro-, trichloro-, monobromo-, dibromo- and bromochloroacetonitrile). With the SOS chromotest, three of the chemicals studied (dichloro-, dibromo- and bromochloroacetonitrile) were found to induce primary DNA damage in Escherichia coli PQ37. In the Ames-fluctuation test, all the compounds except dibromoacetonitrile showed mutagenic activity on Salmonella typhimurium strain TA100. The newt micronucleus assay detected a clastogenic effect on the peripheral blood erythrocytes of Pleurodeles waltl larvae for all the six haloacetonitriles studied. Moreover, two structure-activity relationships were noted: (1) the genotoxic activity of haloacetonitriles containing bromine substituents appeared higher than the corresponding chlorinated acetonitriles and (2) the clastogenic activity of the chlorinated acetonitriles increased with the number of chlorine substituents.

Three short-term assays (the SOS chromotest, the Ames-fluctuation test and the newt micronucleus test) were carried out to evaluate the genotoxicity of five chlorinated propanones identified in several chlorinated waters (monochloropropanone, 1,1-dichloropropanone, 1,3-dichloropropanone, 1,1,1-trichloropropanone and 1,1,3-trichloropropanone). In the SOS chromotest, all the compounds except monochloropropanone were found to induce primary DNA damage in Escherichia coli. With the fluctuation test, all five chloropropanones showed mutagenic activity on Salmonella typhimurium strain TA100. The newt micronucleus assay detected a clastogenic effect on the peripheral blood erythrocytes of Pleurodeles waltl larvae only for 1,3-dichloropropanone and 1,1,3-trichloropropanone. Moreover, two structure-activity relationships are noticeable: (1) chloropropanones with chlorine substituents on both carbon positions (1,3-DCP and 1,1,3-TCP) are by far more genotoxic than chloropropanones substituted only on one carbon position (1,1-DCP and 1,1,1-TCP); (2) the increase of the number of chlorine substituents decreases the mutagenic activity (fluctuation test) of the chlorinated propanones studied.

Three short-term assays (the SOS chromotest, the Ames fluctuation test and the newt micronucleus test) were used to evaluate the genotoxicity of seven chemicals (4-nitroquinoline 1-oxide, potassium dichromate, formaldehyde, sodium hypochlorite, benzo[a]pyrene, cyclophosphamide and 2-naphthylamine). In the SOS chromotest, all seven compounds except sodium hypochlorite and cyclophosphamide were found to induce primary DNA damage in E. coli. With the Ames fluctuation test, all seven chemicals except sodium hypochlorite showed mutagenic activity on Salmonella typhimurium TA100, TA102 or TA98. The newt micronucleus assay detected a clastogenic effect of all seven compounds except formaldehyde on the peripheral blood erythrocytes of Pleurodeles waltl larvae. For each compound, the sensitivity of the tests was compared; (1) the SOS chromotest is the least sensitive assay in every case, (2) the Ames fluctuation test is the most sensitive assay for studied chemicals with direct genotoxic effect and (3) the newt micronucleus assay is the most sensitive test for tested compounds with indirect genotoxic activity. The potential contribution of these three tests in the monitoring of water genotoxicity is discussed.

The genotoxicity of 5 compounds: 2 fulvic acids, a trade humic acid, a synthetic humic material (SHM), and 2,5-dihydroxybenzoic acid, was assessed after chlorination, by means of the SOS Chromotest for tester strain E. coli PQ 37 without metabolic activation. Chlorination was carried out for humic material concentration of 0.5 mg total organic carbon per liter, and chlorine concentrations in the range of 0.1-2.0 chlorine equivalents per mole of carbon. Among all the considered criteria that can account for potent toxicity: chemical degradation determined by the UV absorption decrease, chlorine consumption, average molecular weight, only the polymerization index (O.D. 665 nm/O.D. 465 nm) can be related to the genotoxicity of humic samples. This latter criterion appears a possible predictor of genotoxic potency, revealed subsequent to the aqueous chlorination of humic materials. Looking at the various genotoxic activities of the tested compounds, SHM can be considered a better model of natural humic materials than the trade humic acid.

The short term effects of fenofibrate (150 mg/kg per day), administered by gavage, on lipoprotein and fatty acid distribution have been investigated in an hypertriglyceridemic model, the Zucker rat. Lean rats were compared to control obese and treated obese rats, and control obese animals to treated obese littermates. Classically, plasma cholesterol and triacylglycerol increased by 1.8- and 7.9-fold, respectively, in control obese versus lean rats. Treatment of the Zucker obese rats with fenofibrate reduced their plasma cholesterol by 10% and raised triacylglycerol by 47% (P less than 0.001) in comparison to untreated control obese rats. These effects were accompanied by a change in the composition of all plasma lipoproteins. The cholesterol/triacylglycerol ratio in VLDL rose by 32% while that in LDL and HDL fell by 43 and 47%, respectively. Drug therapy altered the fatty acid profile in both plasma and liver; the percentage of polyunsaturated fatty acids fell while monounsaturated fatty acids increased. The increased proportion of monounsaturated fatty acids in plasma suggests that the fatty acid composition of circulating lipoproteins is modified, particularly in VLDL. This, in association with the altered lipid distribution in VLDL may reflect an abnormal metabolism of this lipoprotein. In view of these abnormalities, we conclude that this rat is not an appropriate model for the short-term study of clofibrate analogues.

Radioimmunoassay, rate nephelometric inhibition immunoassay and substrate-labelled fluorescent immunoassay were employed for the quantitative determination of dibekacin in serum. The cross-reactivity of the antibody provided with each assay allowed the use of tobramycin assay procedures for measuring dibekacin concentrations. With radioimmunoassay and nephelometric immunoassay, a dibekacin calibration curve was required, whereas fluorescent immunoassay was directly suitable for dibekacin assay, with cross-reactivity of nearly 100%. This allows the purchase of one assay kit for testing two antibiotics and thus reduces the cost to medical laboratories.

The genotoxic potential of 48 inorganic derivatives was studied using the bacterial colorimetric assay: the SOS Chromotest. Some of these compounds are known as carcinogens (As, CR(VI), Cd, Ni) or suspected carcinogens for human beings (Hg, Pb), others are known as non-carcinogens. Among these 48 derivatives, only the two Cr(VI) compounds and the Sn(II) compounds gave positive results.

These studies examine the effects of a hypercholesterolemic diet (with butter and cholesterol added), or a hypertriglyceridemic diet (30% sucrose (w/v) in drinking water), on murine plasma lipids and lipoproteins prepared either by sequential ultracentrifugation or gel exclusion chromatography. The hypercholesterolemic diet increased plasma cholesterol (186%), particularly that associated with very low (VLDL) and low (LDL) density lipoproteins. In addition, the cholesterol/triglyceride ratio in the VLDL fraction rose significantly from 0.10 to 4.0. The hypertriglyceridemic diet raised markedly plasma triglyceride levels (46%) by expanding the circulating VLDL pool (+39%). These dietary modifications were used to provide a model for the examination of 3 classical hypolipidemic drugs (fenofibrate, gemfibrozil and nicotinic acid). In animals receiving the standard diet, fenofibrate, gemfibrozil and nicotinic acid decreased the triglyceride (TG) level (-28%, -31%, and -38%) by lowering VLDL-TG (-37%, -42%, and -49%), fenofibrate and gemfibrozil increased HDL-cholesterol by 18% and 31%, respectively. In animals receiving the hypercholesterolemic diet fenofibrate lowered the total plasma cholesterol level by 40%, at the same time increasing HDL-cholesterol by 23%. In animals fed sucrose, on the other hand, fenofibrate (100 mg/kg per day) and nicotinic acid (900 and 600 mg/kg per day) reduced plasma triglyceride levels (-20%, -40% and -33%), and nicotinic acid (900 mg/kg per day) decreased VLDL-TG by 58%. These results are in good agreement with clinical data from studies in man and suggest that this animal model may provide a useful and rapid screening system for testing new lipid lowering drugs.

Troxerutin, a flavonoid derivative, used in vascular diseases was studied in 4 mutagenicity tests: the Ames test, the point mutation test (V79/HPRT), the in vitro metaphase analysis in human lymphocytes and the micronucleus test in mice. The aglycone trihydroxyethylquercetin (THEQ) and quercetin were studied too. Troxerutin was not mutagenic, whereas quercetin was positive in the Ames test, V79 cells and in vitro metaphase analysis. THEQ was negative in the Ames test. The substitution of quercetin with hydroxyethyl groups in 7,3' and 4' positions abolished mutagenic activity of quercetin.