Genotoxicity risk assessment: a proposed classification strategy

Recent advances in genetic toxicity (mutagenicity) testing methods and in approaches to performing risk assessment are prompting a renewed effort to harmonize genotoxicity risk assessment across the world. The US Environmental Protection Agency (EPA) first published Guidelines for Mutagenicity Risk Assessment in 1986 that focused mainly on transmissible germ cell genetic risk. Somatic cell genetic risk has also been a risk consideration, usually in support of carcinogenicity assessments. EPA and other international regulatory bodies have published mutagenicity testing requirements for agents (pesticides, pharmaceuticals, etc.) to generate data for use in genotoxicity risk assessments. The scheme that follows provides a proposed harmonization approach in which genotoxicity assessments are fully developed within the risk assessment paradigm used by EPA, and sets out a process that integrates newer thinking in testing battery design with the risk assessment process. A classification strategy for agents based on inherent genotoxicity, dose-responses observed in the data, and an exposure analysis is proposed. The classification leads to an initial level of concern for genotoxic risk to humans. A total risk characterization is performed using all relevant toxicity data and a comprehensive exposure evaluation in association with the genotoxicity data. The result of this characterization is ultimately used to generate a final level of concern for genotoxic risk to humans. The final level of concern and characterized genotoxicity risk assessment are communicated to decision makers for possible regulatory action(s) and to the public.     

Relevance and follow-up of positive results in in vitro genetic toxicity assays: an ILSI-HESI initiative

In vitro genotoxicity assays are often used to screen and predict whether chemicals might represent mutagenic and carcinogenic risks for humans. Recent discussions have focused on the high rate of positive results in in vitro tests, especially in those assays performed in mammalian cells that are not confirmed in vivo. Currently, there is no general consensus in the scientific community on the interpretation of the significance of positive results from the in vitro genotoxicity assays. To address this issue, the Health and Environmental Sciences Institute (HESI), held an international workshop in June 2006 to discuss the relevance and follow-up of positive results in in vitro genetic toxicity assays. The goals of the meeting were to examine ways to advance the scientific basis for the interpretation of positive findings in in vitro assays, to facilitate the development of follow-up testing strategies and to define criteria for determining the relevance to human health. The workshop identified specific needs in two general categories, i.e., improved testing and improved data interpretation and risk assessment. Recommendations to improve testing included: (1) re-examine the maximum level of cytotoxicity currently required for in vitro tests; (2) re-examine the upper limit concentration for in vitro mammalian studies; (3) develop improved testing strategies using current in vitro assays; (4) define criteria to guide selection of the appropriate follow-up in vivo studies; (5) develop new and more predictive in vitro and in vivo tests. Recommendations for improving interpretation and assessment included: (1) examine the suitability of applying the threshold of toxicological concern concepts to genotoxicity data; (2) develop a structured weight of evidence approach for assessing genotoxic/carcinogenic hazard; and (3) re-examine in vitro and in vivo correlations qualitatively and quantitatively. Conclusions from the workshop highlighted a willingness of scientists from various sectors to change and improve the current paradigm and move from a hazard identification approach to a "realistic" risk-based approach that incorporates information on mechanism of action, kinetics, and human exposure..     

Assessment of reproductive risks

In the regulatory process, the hazards posed by potentially toxic agents to the female and male reproductive systems and to developing young are evaluated by risk assessment procedures. In this paper, toxicity testing and the regulatory process are discussed, with emphasis on risk assessment. The suggested testing protocols of the Pesticide Assessment Guidelines (U.S. EPA) are presented as an example of testing that might be done to produce toxicity data for an agent. Protocols and end points that are utilized in testing for reproductive effects are described. Included are acute, subchronic, chronic, and short-term tests. The four components of reproductive risk assessment (hazard identification, dose-response assessment, exposure assessment, and risk characterization) are examined. Effects of dibromochloropropane on rabbit testicular parameters are used to demonstrate approaches that could be taken in doing a reproductive risk assessment. Research needs for screening methods, adequate dose-response testing, toxicokinetics, end point development, and extrapolation methods are identified. Finally, this paper discusses selected areas in which changes in reproductive risk assessment are anticipated, as well as the mechanism for influencing the nature and extent of those changes.     

Strategies and testing methods for identifying mutagenic risks

The evolution of testing strategies and methods for identification of mutagenic agents is discussed, beginning with the concern over potential health and population effects of chemical mutagens in the late 1940s that led to the development of regulatory guidelines for mutagenicity testing in the 1970s and 1980s. Efforts to achieve international harmonization of mutagenicity testing guidelines are summarized, and current issues and needs in the field are discussed, including the need for quantitative methods of mutagenic risk assessment, dose-response thresholds, indirect mechanisms of mutagenicity, and the predictivity of mutagenicity assays for carcinogenicity in vivo. Speculation is offered about the future of mutagenicity testing, including possible near-term changes in standard test batteries and the longer-term roles of expression profiling of damage-response genes, in vivo mutagenicity testing methods, and models that better account for differences in metabolism between humans and laboratory model systems.     

Use of in vivo genetic toxicity data for risk assessment

Mutagenicity studies have been used to identify specific agents as potential carconogens or other human health hazards; however, they have been used minimally for risk assessment or in determining permissible levels of human exposure. The poor predictive value of in vitro mutagenesis tests for carcinogenic activity and a lack of mechanistic understanding of the roles of mutagens in the induction of specific cancers have made these tests unattractive for the purpose of risk assessment. However, the limited resources available for carcinogen testing and large number of chemicals which need to be evaluated necessitate the incorporation of more efficient methods into the evaluation process. In vivo genetic toxicity testing can be recommended for this purpose because in vivo assays incorporate the metabolic activation pathways that are relevant to humans. We propose the use of a multiple end-point in vivo comprehensive testing protocol (CTP) using rodents. Studies using sub-acute exposure to low levels of test agents by routes consistent with human exposure can be a useful adjunct to methods currently used to provide data for risk assessment. Evaluations can include metabolic and pharmacokinetic endpoints, in addition to genetic toxicity studies, in order to provide a comprehensive examination of the mechanism of toxicity of the agent. A parallelogram approach can be used to estimate effects in non-accessible human tissues by using data from accessible human tissues and analogous tissues in animals. A categorical risk assessment procedure can be used which would consider, in order of priority, genetic damage in man, genetic damage in animals that is highly relevant to disease outcome (mutation, chromosome damage), and data from animals that is of less certain relevance to disease. Action levels of environmental exposure would be determined based on the lowest observed effect levels or the highest observed no effect levels, using sub-acute low level exposure studies in rodents. As an example, the known genotoxic effects of benzene exposure at low levels in man and animals are discussed. The lowest observed genotoxic effects were observed at about 1–10 parts per million for man and 0.04–0.1 parts per million in subacute animal studies. If genetic toxicity is to achieve a prominent role in evaluating carcinogens and characterizing germ-cell mutagens, minimal testing requirements must be established to ascertain the risk associated with environmental mutagen exposure. The use of the in vivo approach described here should provide the information needed to meet this goal. In addition, it should allow truly epigenetic or non-genotoxic carcinogens to be distinguished from the genotoxic carcinogens that are not detected by in vitro methods.     

Update on genotoxicity and carcinogenicity testing of 472 marketed pharmaceuticals

This survey is a compendium of genotoxicity and carcinogenicity information of 838 marketed drugs, whose expected clinical use is continuous for at least 6 months or intermittent over an extended period of time. Of these 838 drugs, 366 (43.7%) do not have retrievable genotoxicity or carcinogenicity data. The remaining 472 (56.3%) have at least one genotoxicity or carcinogenicity test result. Of the 449 drugs with at least one genotoxicity test result, 183 (40.8%) have at least one positive finding. Of the 338 drugs with at least one carcinogenicity test result, 160 (47.3%) have at least one positive result. Concerning the predictivity of genetic toxicology findings for long-term carcinogenesis assays, of the 315 drugs which have both genotoxicity and carcinogenicity data 116 (36.8%) are neither genotoxic nor carcinogenic, 50 (15.9%) are non-carcinogens which test positive in at least one genotoxicity assay, 75 (23.8%) are carcinogenic in at least one sex of mice or rats but test negative in genotoxicity assays, and 74 (23.5%) are both genotoxic and carcinogenic. Only 208 (24.8%) of the 838 drugs considered have all data required by current guidelines for testing of pharmaceuticals. However, it should be noted that a large fraction of the drugs considered were developed and marketed prior to the present regulatory climate. Although the laws do not require re-testing based on revised standards, in the absence of epidemiological studies excluding a carcinogenic risk to humans, a re-evalutation would be appropriate.     

Threshold effects in genetic toxicity: perspective of chemicals regulation in Germany

In the regulation of chemical substances, it is generally agreed that there are no thresholds for genotoxic effects of chemicals, i.e. , that there are no doses without genotoxic effects. When classifying and labelling chemicals, dangerous properties of chemicals are to be identified. In this context, in general, the mode of action (threshold or not) is not considered for genotoxic substances. In the process of quantitative risk assessment, however, determination of the type of dose-effect relationships is decisive for the outcome and the type of risk management. The presence of a threshold must be justified specifically in each individual case. Inter alia, the following aspects may be discussed in this respect: aneugenic activity, indirect modes of action, extremely steep dose-effect relationships in combination with strong toxicity, specific toxicokinetic conditions which may lead to 'metabolic protection' prior to an attack of DNA. In the practice of the regulation of chemical substances with respect to their genotoxic effects, the discussion of thresholds has played a minor role. For notified new substances, there are, in general, no data available that would allow a reasonable discussion. Concerning substances out of the European programme on existing substances, so far 29 have been assessed in our institute with respect to genetic toxicity. Eight out of these have shown considerable evidence for genotoxicity. For two of them, a possible threshold is discussed: one substance is an aneugen, the other one is metabolised to an endogenic compound with genotoxic potential. In the practice of risk assessment of genotoxic substances, the discussion of the mode of action for genotoxicity is frequently associated with the evaluation of potential carcinogenic effects. Here, tissue-specific genotoxic effects in target organs for carcinogenicity are to be discussed. Moreover, the contribution of genotoxicity to the multifactorial process of tumour development should be assessed.     

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.     

Synergy between systemic toxicity and genotoxicity: relevance to human cancer risk

The health risk manager and policy analyst must frequently make recommendations based upon incomplete toxicity data. This is a situation which is encountered in the evaluation of human carcinogenic risks as animal cancer bioassay results are often not available. In this study, in order to assess the relevance of other possible indicators of carcinogenic risks, we used the "chemical diversity approach" to estimate the magnitude of the human carcinogenic risk based upon Salmonella mutagenicity and systemic toxicity data of the "universe of chemicals" to which humans have the potential to be exposed. Analyses of the properties of 10,000 agents representative of the "universe of chemicals" suggest that chemicals that have genotoxic potentials as well as exhibiting greater systemic toxicity are more likely to be carcinogens than non-genotoxicants or agents that exhibit lesser toxicity. Since "genotoxic" carcinogenicity is a hallmark of recognized human carcinogens, these findings are relevant to human cancer risk assessment.     

Development of a highthroughput yeast-based assay for detection of metabolically activated genotoxins

The potential genotoxicity of drug candidates is a serious concern during drug development. Therefore, it is important to assess the potential genotoxicity and mutagenicity of a compound early in the discovery phase of drug development. AMES Salmonella assay is the most widely used assay for the assessment of mutagenicity and genotoxicity. However, the AMES assay is not readily adaptable to highthroughput screening and several strains of Salmonella must be employed to ensure that different types of DNA damage can be studied. Therefore, an additional robust highthroughput genotoxicity screen would be of significant value in the early detection and elimination of genotoxicity. The complexity of DNA damage requires numerous cellular pathways, thus using single model organism to predict genotoxicity in early stage is challenging. Another critical component of such screens is that they incorporate the capability of metabolic activation to ensure that no genotoxic metabolites are generated. We have developed a novel highthroughput reporter assay for DNA repair that detects genotoxicity, and which incorporates metabolic activation. The assay has a low compound requirement as compared to Ames, and relies upon two different reporter genes cotransfected into a yeast strain. The gene encoding Renilla luciferase is fused to the constitutive 3-phosphoglycerate kinase (PGK1) promoter and integrated into the yeast genome to provide a control for cell numbers. The firefly luciferase gene is fused to the RAD51 (bacterial RecA homolog) promoter and used to report an increase in DNA repair activity. A dual luciferase assay is performed by measuring the firefly and Renilla luciferase activities in the same sample. The result is expressed as the ratio of the two luciferase activities; changes from the base level (control) are interpreted as induction of the RAD51 promoter and evidence of DNA repair activity in eukaryote cells due to DNA damage. The yeast dual luciferase reporter has been characterized with and without S-9 activation using positive and negative control agents. This assay is efficient, requires little time and low amounts of compound. The assay is compatible with metabolic activation, adaptable to a highthroughput platform, and yields data that accurately and reproducibly detects DNA damage. Whereas the normal yeast cell wall, plasma membrane composition and the presence of active transporters can prevent the entry or persistence of some compounds internally in yeast cells, our assay did show concordance with regulatory mutagenicity assays, many of which require metabolic activation and are poorly detected by bacterial mutagenicity assays. Although there were false negative results, in our hands this assay performs as well as or better than other commercially available genetox assays. Furthermore, the RAD51 gene is strongly inducible by homologous intrachromosomal recombination; thus this assay may provide a means to detect clastogens. The RAD51 promoter fused dual luciferase assay represents a valuable addition to the armamentarium for the early detection of genotoxic compounds.     

Ecotoxicological Evaluation of a Bench-Scale Bioslurry Treating Explosives-Spiked Soil

The ecotoxicological effects of four bioslurry reactors treating 2,4,6-trinitotoluene (TNT)- and 1,3,5-trinitro-1,3,5-triazacyclohexane (RDX)-spiked soil were evaluated. A control bioslurry reactor was used to assess the endogenous toxicity of the bioslurry operation conditions. A battery of ecotoxicity tests was used: Microtox, green algae growth inhibition, bacterial genotoxicity and mutagenicity, and earthworm mortality and growth inhibition. Bioslurry soluble and solid phases were separated by centrifugation in order to identify toxicity and possible toxicants associated with each phase. Microtox toxicity values were initially very high in both bioslurry reactors spiked with TNT, in relation with TNT concentration. Initial toxicity was also detected by algal growth inhibition, earthworm lethality, genotoxicity and mutagenicity tests. An endogenous toxicity was detected in the control bioreactor using the Microtox and the SOS Chromotest. The soluble phase of the control bioslurry was genotoxic, suggesting that some potentially genotoxic agents were induced in the bioslurry samples. At the end of the bioremediation treatment, data showed that toxicity was reduced using all of the bioassays, except for earthworm lethality and growth inhibition tests in both RDX-spiked bioslurries. This study demonstrates the usefulness of a battery of toxicity tests to monitor bioremediation processes.     

The genotoxicity of priority polycyclic aromatic hydrocarbons in complex mixtures

Risk assessment of complex environmental samples suffers from difficulty in identifying toxic components, inadequacy of available toxicity data, and a paucity of knowledge about the behavior of geno(toxic) substances in complex mixtures. Lack of information about the behavior of toxic substances in complex mixtures is often avoided by assuming that the toxicity of a mixture is simply the sum of the expected effects from each mixture component, i.e. no synergistic or antagonistic interactions. Although this assumption is supported by research investigating non-genotoxic end-points, the literature describing the behavior of genotoxic substances in complex mixtures is sparse and, occasionally, contradictory. In this study, the results of polycyclic aromatic hydrocarbon (PAH) analyses on freshwater bivalves were used to prepare realistic mixtures containing up to 16 PAHs. The SOS genotoxicity of the mixtures and each component were then assessed in an effort to evaluate the additivity of PAH genotoxicity. At nominal PAH concentrations above 1 microg/ml, observed genotoxic responses were far lower than those predicted under the assumption of additivity. At nominal concentrations below 0.75 microg/ml, differences are smaller and occasionally negligible, indicating that the genotoxicity of unsubstituted homocyclic PAHs is additive or slightly less than additive. Other researchers who have investigated the mutagenicity, carcinogenicity, and DNA binding activity of mixtures containing unsubstituted homocyclic PAHs have also reported additive effects. Therefore, the mutagenic risk posed by simple, well-characterized mixtures of priority PAHs can reasonably be estimated as the sum of the risks posed by the mixture components. Current data indicate that less-than-additive effects likely result from saturation of metabolic pathways needed to activate mutagenic PAHs.     

Assessment of the potential germ cell mutagenicity of industrial and plant protection chemicals as part of an integrated study of genotoxicity in vitro and in vivo

An approach is described that enables the germ cell mutagenicity of chemicals to be assessed as part of an integrated assessment of genotoxic potential. It is recommended, first, that the genotoxicity of a chemical be defined by appropriate studies in vitro. This should involve use of the Salmonella mutation assay and an assay for the induction of chromosomal aberrations, but supplementary assays may be indicated in specific instances. If negative results are obtained from these 2 tests there is no need for the conduct of additional tests. Agents considered to be genotoxic in vitro should then be assessed for genotoxicity to rodents. This will usually involve the conduct of a bone marrow cytogenetic assay, and in the case of negative results, a genotoxicity test in an independent tissue. Agents found to be non-genotoxic in vivo are regarded as having no potential for germ cell mutagenicity. Agents found to be genotoxic in vivo may either be assumed to have potential as germ cell mutagens, or their status in this respect may be defined by appropriate germ cell mutagenicity studies. The basis of the approach, which is supported by the available experimental data, is that germ cell mutagens will be evident as somatic cell genotoxins in vivo, and that these will be detected as genotoxins in vitro given appropriate experimentation. The conduct of appropriate and adequate studies is suggested to be of more value than the conduct of a rigid set of prescribed tests.     

Thresholds in genotoxicity responses

It has been commonly accepted that risk assessments of genotoxic chemicals are based on linear extrapolation methods. However, there is substantial evidence that some chemicals may be genotoxic only at high doses by mechanisms that do not occur at low doses, or only under specific conditions in genotoxicity assays, but are inactive at concentrations within the range of human exposure levels. There are a variety of possible mechanisms of thresholded genotoxicity, including disruption of cell division and chromosome segregation, inhibition of DNA synthesis, overloading of oxidative defence mechanisms, metabolism or plasma binding capacity, disturbances of metal homeostasis, cytotoxicity and physiological perturbations in in vivo assays. The degrees of evidence supporting the proposed mechanisms are variable and not all are sufficiently robust to be universally accepted as yet by the scientific community. However, a survey of industrial companies indicated that data have been accepted by some regulatory authorities indicating thresholds contributing to genotoxicity responses.     

Acrylamide: a review of its genotoxicity and an assessment of heritable genetic risk

An updated review of the genotoxicity studies with acrylamide is provided. Then, using data from the studies generating quantitative information concerning heritability of genetic effects, an assessment of the heritable genetic risk presented by acrylamide is presented. The review offers a discussion of the reactions and possible mechanisms of genotoxic action by acrylamide and its epoxide metabolite glycidamide. Several genetic risk approaches are discussed, including the parallelogram, direct (actually a modified direct), and doubling dose approaches. Using data from the specific-locus and heritable translocation assays, the modified direct and doubling dose approaches are utilized to quantitate genetic risk. Exposures of male parents to acrylamide via inhalation, ingestion, and dermal routes are also quantitated. With these approaches and measurements and their underlying assumptions concerning extrapolation factors (including germ cell stage specificity, DNA repair variability, locus specificity), number of human loci associated with dominant disease alleles, and spontaneous mutation rates, an assessment of heritable genetic risk for humans is calculated for the three exposure scenarios. The calculated estimates for offspring from fathers exposed to acrylamide via drinking water are up to three offspring potentially affected with induced genetic disease per 10⁸ offspring. Estimates for inhalation or dermal exposures suggest higher risks for induced genetic disease in offspring from fathers exposed in occupational settings.     

Acrylamide: its metabolism, developmental and reproductive effects, genotoxicity, and carcinogenicity

Monomeric acrylamide is an important industrial chemical primarily used in the production of polymers and copolymers. It is also used for producing grouts and soil stabilizers. Acrylamide's neurotoxic properties have been well documented. This review will focus on pertinent information concerning other, non-neurotoxic, effects observed after exposure to acrylamide, including: its genotoxic, carcinogenic, reproductive, and developmental effects. It will also cover its absorption, metabolism, and distribution. The data show that acrylamide is capable of inducing genotoxic, carcinogenic, developmental, and reproductive effects in tested organisms. Thus, acrylamide may pose more than a neurotoxic health hazard to exposed humans. Acrylamide is a small organic molecule with very high water solubility. These properties probably facilitate its rapid absorption and distribution throughout the body. After absorption, acrylamide is rapidly metabolized, primarily by glutathione conjugation, and the majority of applied material is excreted within 24 h. Preferential bioconcentration of acrylamide and/or its metabolites is not observed although it appears to persist in tests and skin. Acrylamide can bind to DNA, presumably via a Michael addition-type reaction, which has implications for its genotoxic and carcinogenic potential. The available evidence suggests that acrylamide does not produce detectable gene mutations, but that the major concern for its genotoxicity is its clastogenic activity. This clastogenic activity has been observed in germinal tissues which suggest the possible heritability of acrylamide-induced DNA alterations. Since there is 'sufficient evidence' of carcinogenicity in experimental animals as outlined under the U.S. EPA proposed guidelines for carcinogen risk assessment, acrylamide should be categorized as a 'B2' carcinogen and therefore be considered a 'probable human carcinogen.' The very limited human epidemiological data do not provide sufficient evidence to enable one to judge the actual carcinogenic risk to humans. Acrylamide is able to cross the placenta, reach significant concentrations in the conceptus and produce direct developmental and post-natal effects in rodent offspring. It appears that acrylamide may produce neurotoxic effects in neonates from exposures not overtly toxic to the mothers. Acrylamide has an adverse effect on reproduction as evidenced by dominant lethal effects, degeneration of testicular epithelial tissue, and sperm-head abnormalities.     

Considerations in the U.S. Environmental Protection Agency's testing approach for mutagenicity.

OPP: This paper provides the rationale and support for the decisions the OPP will make in requiring and reviewing mutagenicity information. The regulatory requirement for mutagenicity testing to support a pesticide registration is found in the 40 CFR Part 158. The guidance as to the specific mutagenicity testing to be performed is found in the OPP's Pesticide Assessment Guidelines, Subdivision F, Hazard Evaluation: Human and Domestic Animals (referred to as the Subdivision F guideline). A revised Subdivision F guideline has been presented that becomes the current guidance for submitters of mutagenicity data to the OPP. The decision to revise the guideline was the result of close examination of the version published in 1982 and the desire to update the guidance based on developments since then and current state-of-the-science. After undergoing Agency and public scrutiny, the revised guideline is to be published in 1991. The revised guideline consists of an initial battery of tests (the Salmonella assay, an in vitro mammalian gene mutation assay and an in vivo cytogenetics assay which may be either a bone marrow assay for chromosomal aberrations or for micronuclei formation) that should provide an adequate initial assessment of the potential mutagenicity of a chemical. Follow-up testing to clarify results from the initial testing may be necessary. After this information as well as all other relevant information is obtained, a weight-of-evidence decision will be made about the possible mutagenicity concern a chemical may present. Testing to pursue qualitative and/or quantitative evidence for assessing heritable risk in relation to human beings will then be considered if a mutagenicity concern exists. This testing may range from tests for evidence of gonadal exposure to dominant lethal testing to quantitative tests such as the specific locus and heritable translocation assays. The mutagenicity assessment will be performed in accordance with the Agency's Mutagenicity Risk Assessment Guidelines. The mutagenicity data would also be used in the weight-of-evidence consideration for the potential carcinogenicity of a chemical in accordance with the Agency's Carcinogen Risk Assessment Guidelines. In instances where there are triggers for carcinogenicity testing, mutagenicity data may be used as one of the triggers after a consideration of available information. It is felt that the revised Subdivision F guideline will provide appropriate, and more specific, guidance concerning the OPP approach to mutagenicity testing for the registration of a pesticide. It also provides a clearer understanding of how the OPP will proceed with its evaluation and decision making concerning the potential heritable effects of a test chemical.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mutagens in contaminated soil: a review

The intentional and accidental discharges of toxic pollutants into the lithosphere results in soil contamination. In some cases (e.g., wood preserving wastes, coal-tar, airborne combustion by-products), the contaminated soil constitutes a genotoxic hazard. This work is a comprehensive review of published information on soil mutagenicity. In total, 1312 assessments of genotoxic activity from 118 works were examined. The majority of the assessments (37.6%) employed the Salmonella mutagenicity test with strains TA98 and/or TA100. An additional 37.6% of the assessments employed a variety of plant species (e.g., Tradescantia clone 4430, Vicia faba, Zea mays, Allium cepa) to assess mutagenic activity. The compiled data on Salmonella mutagenicity indicates significant differences (p<0.0001) in mean potency (revertents per gram dry weight) between industrial, urban, and rural/agricultural sites. Additional analyses showed significant empirical relationships between S9-activated TA98 mutagenicity and soil polycyclic aromatic hydrocarbon (PAH) concentration (r2=0.19 to 0.25, p<0.0001), and between direct-acting TA98 mutagenicity and soil dinitropyrene (DNP) concentration (r2=0.87, p<0.0001). The plant assay data revealed excellent response ranges and significant differences between heavily contaminated, industrial, rural/agricultural, and reference sites, for the anaphase aberration in Allium cepa (direct soil contact) and the waxy locus mutation assay in Zea mays (direct soil contact). The Tradescantia assays appeared to be less responsive, particularly for exposures to aqueous soil leachates. Additional data analyses showed empirical relationships between anaphase aberrations in Allium, or mutations in Arabidopsis, and the 137Cs contamination of soils. Induction of micronuclei in Tradescantia is significantly related to the soil concentration of several metals (e.g., Sb, Cu, Cr, As, Pb, Cd, Ni, Zn). Review of published remediation exercises showed effective removal of genotoxic petrochemical wastes within one year. Remediation of more refractory genotoxic material (e.g., explosives, creosote) frequently showed increases in mutagenic hazard that remained for extended periods. Despite substantial contamination and mutagenic hazards, the risk of adverse effect (e.g., mutation, cancer) in humans or terrestrial biota is difficult to quantify.     

Toxicity and genotoxicity of surface water before and after various potabilization steps

Many studies have revealed the presence of compounds with genotoxic activity in drinking water by means of short-term mutagenicity tests. In this study, the influence of the different steps of surface water treatment on the mutagenicity of drinking water was evaluated. Four different types of samples were collected: raw lake water, water after pre-disinfection with chlorine dioxide, water after filtration on granular activated carbon, and tap water. Water extracts underwent a bacterial toxicity test (Microtox test) and different in vitro genotoxicity tests: a test with Salmonella typhimurium strains, a Saccharomyces cerevisiae test, the SOS Chromotest with Escherichia coli and the Mutatox test with Vibrio fischeri. The Microtox test revealed high toxicity in the treated water samples. The disinfection steps increased the toxicity: the Mutatox test confirmed these results and the Salmonella/microsome test at the highest doses showed toxicity that could conceal mutagenicity. The SOS Chromotest was positive in all treated water samples without metabolic activation. In the test with S. cerevisiae both toxicity and genotoxicity generally increased during the water treatment steps, especially in cells without induction of cytochrome P450.