The purpose of these guidelines is to provide concise guidance on the planning, performing and interpretation of studies to monitor groups or individuals exposed to genotoxic agents. Most human carcinogens are genotoxic but not all genotoxic agents have been shown to be carcinogenic in humans. Although the main interest in these studies is due to the association of genotoxicity with carcinogenicity, there is also an inherent interest in monitoring human genotoxicity independently of cancer as an endpoint. The most often studied genotoxicity endpoints have been selected for inclusion in this document and they are structural and numerical chromosomal aberrations assessed using cytogenetic methods (classical chromosomal aberration analysis (CA), fluorescence in situ hybridisation (FISH), micronuclei (MN)); DNA damage (adducts, strand breaks, crosslinking, alkali-labile sites) assessed using bio-chemical/electrophoretic assays or sister chromatid exchanges (SCE); protein adducts; and hypoxanthine-guanine phosphoribosyltransferase (HPRT) mutations. The document does not consider germ cells or gene mutation assays other than HPRT or markers of oxidative stress, which have been applied on a more limited scale.

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IPCS guidelines for the monitoring of genotoxic effects of carcinogens in humans

Safety Evaluation and Risk Assessment of the Herbicide Roundup and Its Active Ingredient, Glyphosate, for Humans

The International Collaborative Project on Micronucleus Frequency in Human Populations (HUMN) was organized to collect data on micronucleus (MN) frequencies in different human populations and different cell types. The test procedures considered by this project are assays using human lymphocytes (cytokinesis-block method), exfoliated epithelial cells, and other cell types. Data (including descriptions of the populations monitored, detailed test protocols, and test results) are being obtained from a large number of laboratories throughout the world and are being entered into a unified database. The information will be used to: (1) determine the extent of variation of 'normal' values for different laboratories and the influence of other factors potentially affecting baseline MN frequency, e.g., age, gender and life-style; (2) provide information on the effect of experimental protocol variations on MN frequency measurements; (3) design and test optimal protocols for the different cell types; and (4) determine the extent to which MN frequency is a valid biomarker of ageing and risk for diseases such as cancer.

The HUman MicroNucleus Project--An international collaborative study on the use of the micronucleus technique for measuring DNA damage in humans.

Polymorphisms in several DNA repair genes have recently been identified, but little is known about their phenotypic significance. To determine whether variation in DNA repair genes is related to host DNA damage, we studied the association between polymorphisms in XRCC1 (codon 399) and ERCC2 (codon 751) and two markers of DNA damage, sister chromatid exchange (SCE) frequencies (n = 76) and polyphenol DNA adducts (n = 61). SCE frequencies were determined using a modified fluorescence-Giemsa method and polyphenol DNA adducts were determined using a P1-enhanced 32 P-post-labeling procedure. XRCC1 and ERCC2 genotypes were identified using PCR-RFLP. Mean SCE frequencies among current smokers who were homozygous carriers of the 399Gln allele in XRCC1 were greater than those in 399Arg/Arg current smokers. We also observed a possible gene-dosage effect for XRCC1 399Gln and detectable DNA adducts, and significantly more adducts among older subjects who were carriers of the 399Gln allele than in younger subjects with the 399Arg/Arg genotype. The polymorphism in ERCC2 was unrelated to SCE frequency or DNA adduct level. Our results suggest that carriers of the polymorphic XRCC1 399Gln allele may be at greater risk for tobacco- and age-related DNA damage.

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Polymorphisms in the DNA repair genes XRCC1 and ERCC2 and biomarkers of DNA damage in human blood mononuclear cells

The human relevance framework (HRF) outlines a four-part process, beginning with data on the mode of action (MOA) in laboratory animals, for evaluating the human relevance of animal tumors. Drawing on U.S. EPA and IPCS proposals for animal MOA analysis, the HRF expands those analyses to include a systematic evaluation of comparability, or lack of comparability, between the postulated animal MOA and related information from human data sources. The HRF evolved through a series of case studies representing several different MOAs. HRF analyses produced divergent outcomes, some leading to complete risk assessment and others discontinuing the process, according to the data available from animal and human sources. Two case examples call for complete risk assessments. One is the default: When data are insufficient to confidently postulate a MOA for test animals, the animal tumor data are presumed to be relevant for risk assessment and a complete risk assessment is necessary. The other is the product of a data-based finding that the animal MOA is relevant to humans. For the specific MOA and endpoint combinations studied for this article, full risk assessments are necessary for potentially relevant MOAs involving cytotoxicity and cell proliferation in animals and humans (Case Study 6, chloroform) and formation of urinary-tract calculi (Case Study 7, melamine). In other circumstances, when data-based findings for the chemical and endpoint combination studied indicate that the tumor-related animal MOA is unlikely to have a human counterpart, there is little reason to continue the risk assessment for that combination. Similarly, when qualitative considerations identify MOAs specific to the test species or quantitative considerations indicate that the animal MOA is unlikely to occur in humans, such hazard findings are generally conclusive and further risk assessment is not necessary for the endpoint-MOA combination under study. Case examples include a tumor-related protein specific to test animals (Case Study 3, d-limonene), the tumor consequences of hormone suppression typical of laboratory animals but not humans (Case Study 4, atrazine), and chemical-related enhanced hormone clearance rates in animals relative to humans (Case Study 5, phenobarbital). The human relevance analysis is highly specific for the chemical-MOA-tissue-endpoint combination under analysis in any particular case: different tissues, different endpoints, or alternative MOAs for a given chemical may result in different human relevance findings. By providing a systematic approach to using MOA data, the HRF offers a new tool for the scientific community's overall effort to enhance the predictive power, reliability and transparency of cancer risk assessment.

A framework for human relevance analysis of information on carcinogenic modes of action.

This paper describes the four cytogenetic endpoints most frequently used in hazard identification assays as the first step in the risk assessment process. These are structural chromosome aberrations, micronuclei, aneuploidy, and sister chromatid exchanges. The biological mechanisms involved in the formation of the alterations observed in each assay are briefly discussed. Variations in and recent improvements to each assay are described, with an emphasis on the use of molecular techniques to improve the sensitivity of the assay, and to allow for detection of specific alterations that are, or could be, associated with cancer induction. This, in turn, will make the data obtained in the cytogenetic assays more useful in cancer and genetic risk assessment. Thus, the aim of this paper is to encourage cytogeneticists to design their experiments in such a way that the data obtained will be of maximum possible benefit for characterizing and quantifying adverse human health effects, particularly cancer.

Chromosome aberrations, micronuclei, aneuploidy, sister chromatid exchanges, and cancer risk assessment.

Results from new chromosome studies in laboratory animals, comparative investigations of styrene metabolism and pharmacokinetics in humans and animals, and several recent cytogenetic surveys of styrene-exposed workers have necessitated a comprehensive re-evaluation of the chromosome-damaging effects of this chemical. Both styrene and its genotoxic metabolite, styrene oxide, can induce chromosome aberrations (CA) and sister chromatid exchanges (SCE) in vitro, but the chromosome-damaging ability of styrene is only manifested if test conditions favour its metabolic activation over inactivation. There is no convincing evidence of styrene clastogenicity in experimental animals. Styrene oxide is clastogenic only at lethal concentrations via i.p. injection in Chinese hamsters (but not via inhalation) or after oral treatment of mice, a route considered inappropriate for investigating the chromosome-damaging potential of inhaled styrene in man. Styrene and styrene oxide can induce SCE in animals at very high concentrations. Eighteen of 52 cytogenetic studies (CA, micronuclei, SCE) on peripheral blood lymphocytes of styrene workers have reported increases in chromosome damage. The positive findings are not compatible with the conclusion that styrene is responsible for the cytogenetic effects for the following reasons. 1. (a) The positive or negative outcome of the various investigations bears no relationship to the degree of exposure of the workers. 2. (b) There is no convincing evidence of a positive dose response relationship. 3. (c) The relative induction of CA and SCE in worker studies are the opposite of observations of styrene effects in cultured lymphocytes and in laboratory animals. 4. (d) The reports of chromosome-type exchanges in some studies of styrene workers is inconsistent with observations of styrene clastogenicity in cultured lymphocytes. 5. (e) Reports of SCE induction in workers exposed to low concentrations of styrene are not compatible with results of animal inhalation studies, particularly in view of the differences in styrene metabolism and pharmacokinetics between humans and rodents. The increases in cytogenetic effects reported in some studies on styrene workers are probably attributable to the presence of other chromosome-damaging agents in the workplace and/or to inadequate investigations.

A re-evaluation of the cytogenetic effects of styrene

Cytogenetic assays are an integral component of the battery of short-term assays that are used for the hazard identification component of a cancer risk assessment. The protocol for the conduct of such assays for maximal sensitivity for detecting clastogenicity has to be attendant to the mechanism of induction of the endpoint being assessed and the fact that several aberration types are cell lethal necessitates that analysis be for cells at their first posttreatment metaphase. Cytogenetic assays for human populating monitoring have been used for predicting potential for carcinogenicity in humans. However, the assays as typically conducted are not appropriate for chronic exposures because nontransmissible alterations are assessed. The use of fluorescent in situ hybridization (FISH) techniques for the assessment of transmissible changes such as reciprocal translocations are required to make population monitoring studies interpretable, and for removing some of the concern over the influence of confounders on outcome. The database for the cytogenetic effects of ethylene oxide in vitro and in vivo, with an emphasis on human population monitoring, has been critically reviewed. Based on the endpoints studied, the size of the study groups, the information on exposure, the nature of any exposure response data, and the possible influence of confounders (i.e., control matching), it is concluded that acute, high exposures to ethylene oxide with sampling shortly (a few days) after exposure can be detected by increases in chromosome aberrations or SCE in peripheral lymphocytes. Such increases are indicators of exposure to a genotoxic chemical and not predictors of subsequent adverse health effects to individuals. The effect of chronic and/or low level (less than about 25 ppm) exposures cannot be reliably evaluated using current methods. The use of FISH, for example, for assessing reciprocal translocation frequencies (as a measure of transmissible events) will greatly improve the ability to detect chronic exposures to clastogenic chemicals.

R J Preston

Papers

Chromosome aberrations, micronuclei, aneuploidy, sister chromatid exchanges, and cancer risk assessment.

A re-evaluation of the cytogenetic effects of styrene

Cytogenetic effects of ethylene oxide, with an emphasis on population monitoring.

A Critical Review of the Cytogenetic Effects of Styrene with an Emphasis on Human Population Monitoring: A Synopsis