David H. Blakey

The in vivo micronucleus assay in mammalian bone marrow and peripheral blood. A report of the U.S. environmental protection agency gene-tox program

The protocol recommended for the micronucleus assay in mammalian bone marrow has been revised and simplified. The number of sample times has been reduced to one or two, depending upon the dosing protocol. The minimum number of cells to be scored per treatment group has been increased to 20,000 to increase the ability of the assay to detect a doubling of the control micronucleus frequency. Use of both male and female animals is recommended. Scoring of micronuclei in polychromatic erythrocytes of peripheral blood is included as a variation of the bone marrow assay. Published data on chemicals tested by the micronucleus assay have been reviewed and are summarized.

In vivo rodent erythrocyte micronucleus assay

The following summary represents a consensus of the working group except where noted. The items discussed are listed in the order in which they appear in the OECD guideline (474) for easy reference. Introduction, purpose, scope, relevance, application and limits of test. The analysis of immature erythrocytes in either bone marrow or peripheral blood is equally acceptable for those species in which the spleen does not remove micronucleated erythrocytes. In the mouse, mature erythrocytes are also an acceptable cell population for micronucleus analysis when the exposure duration exceeds 4 weeks. Test substances. Organic solvents such as DMSO are not recommended. Freshly prepared solutions or suspensions should be used unless stability data demonstrate the acceptability of storage. Vegetable oils are acceptable as solvents or vehicles. Suspension of the test chemicals is acceptable for p.o. or i.p. administration but not for i.v. injection. The use of any unusual solvent should be justified. Selection of species. Any commonly used laboratory rodent species is acceptable. There is no strain preference. Number and sex. The size of experiment (i.e., number of cells per animal, number of animals per group) should be finalized based on statistical considerations. Although a consensus was not achieved, operationally it was agreed that 2000 cells per animal and four animals per group was a minimum requirement. In general, the available database suggests that the use of one gender is adequate for screening. However, if there is evidence indicating a significant difference in the toxicity between male and female, then both sexes should be used. Treatment schedule. No unique treatment schedule can be recommended. Results from extended dose regimens are acceptable as long as positive. For negative studies, toxicity should be demonstrated or the limit dose should be used, and dosing continued until sampling. Dose levels. At least three dose levels separated by a factor between 2 and square root of 10 should be used. The highest dose tested should be the maximum tolerated dose based on mortality, bone marrow cell toxicity, or clinical symptoms of toxicity. The limit dose is 2 g/kg/day for treatment periods of 14 days or less and 1 g/kg/day for treatment periods greater than 14 days. A single dose level (the limit dose) is acceptable if there is no evidence of toxicity. Controls. Concurrent solvent (vehicle) controls should be included at all sampling times. A pretreatment sample, however, may also be acceptable only in the short treatment period peripheral blood studies. A concurrent positive control group should be included for each experiment.(ABSTRACT TRUNCATED AT 400 WORDS)

In vivo rodent erythrocyte micronucleus assay. II. Some aspects of protocol design including repeated treatments, integration with toxicity testing, and automated scoring.

An expert working group on the in vivo micronucleus assay, formed as part of the International Workshop on Genotoxicity Test Procedures (IWGTP), discussed protocols for the conduct of established and proposed micronucleus assays at a meeting held March 25–26, 1999 in Washington, DC, in conjunction with the annual meeting of the Environmental Mutagen Society. The working group reached consensus on a number issues, including: (1) protocols using repeated dosing in mice and rats; (2) integration of the (rodent erythrocyte) micronucleus assay into general toxicology studies; (3) the possible omission of concurrently‐treated positive control animals from the assay; (4) automation of micronucleus scoring by flow cytometry or image analysis; (5) criteria for regulatory acceptance; (6) detection of aneuploidy induction in the micronucleus assay; and (7) micronucleus assays in tissues (germ cells, other organs, neonatal tissue) other than bone marrow. This report summarizes the discussions and recommendations of this working group. In the classic rodent erythrocyte assay, treatment schedules using repeated dosing of mice or rats, and integration of assays using such schedules into short‐term toxicology studies, were considered acceptable as long as certain study criteria were met. When the micronucleus assay is integrated into ongoing toxicology studies, relatively short‐term repeated‐dose studies should be used preferentially because there is not yet sufficient data to demonstrate that conservative dose selection in longer term studies (longer than 1 month) does not reduce the sensitivity of the assay. Additional validation data are needed to resolve this point. In studies with mice, either bone marrow or blood was considered acceptable as the tissue for assessing micronucleus induction, provided that the absence of spleen function has been verified in the animal strains used. In studies with rats, the principal endpoint should be the frequency of micronucleated immature erythrocytes in bone marrow, although scoring of peripheral blood samples gives important supplementary data about the time course of micronucleus induction. When dose concentration and stability are verified appropriately, concurrent treatment with a positive control agent is not necessary. Control of staining and scoring procedures can be obtained by including appropriate reference samples that have been obtained from a separate experiment. For studies in rats or mice, treatment/sampling regimens should include treatment at intervals of no more than 24 hr (unless the test article has a half‐life of more than 24 hr) with sampling of bone marrow or blood, respectively, within 24 or 40 hr after the last treatment. The use of a DNA specific stain is recommended for the identification of micronuclei, especially for studies in the rat. In the case of a negative assay result with a non‐toxic test article, it is desirable that systemic exposure to the test article is demonstrated. The group concluded that successful application of automated scoring by both flow cytometry and image analysis had been achieved, and defined criteria that should be met if automated scoring is employed. It was not felt appropriate to attempt to define specific recommended protocols for automated scoring at the present time. Other issues reviewed and discussed by the working group included micronucleus assays that have been developed in a number of tissues other than bone marrow. The group felt that these assays were useful research tools that could also be used to elucidate mechanisms in certain regulatory situations, but that these assays had not yet been standardized and validated for routine regulatory application. Environ. Mol. Mutagen. 35:234–252, 2000

Meeting report: Validation of toxicogenomics-based test systems: ECVAM-ICCVAM/NICEATM considerations for regulatory use

This is the report of the first workshop “Validation of Toxicogenomics-Based Test Systems” held 11–12 December 2003 in Ispra, Italy. The workshop was hosted by the European Centre for the Validation of Alternative Methods (ECVAM) and organized jointly by ECVAM, the U.S. Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), and the National Toxicology Program (NTP) Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM). The primary aim of the workshop was for participants to discuss and define principles applicable to the validation of toxicogenomics platforms as well as validation of specific toxicologic test methods that incorporate toxicogenomics technologies. The workshop was viewed as an opportunity for initiating a dialogue between technologic experts, regulators, and the principal validation bodies and for identifying those factors to which the validation process would be applicable. It was felt that to do so now, as the technology is evolving and associated challenges are identified, would be a basis for the future validation of the technology when it reaches the appropriate stage. Because of the complexity of the issue, different aspects of the validation of toxicogenomics-based test methods were covered. The three focus areas include a) biologic validation of toxicogenomics-based test methods for regulatory decision making, b) technical and bioinformatics aspects related to validation, and c) validation issues as they relate to regulatory acceptance and use of toxicogenomics-based test methods. In this report we summarize the discussions and describe in detail the recommendations for future direction and priorities.

Report from the working group on the in vivo mammalian bone marrow chromosomal aberration test

The following summary represents a consensus of the working group, except where noted. The goal of this working group was to identify the minimal requirements needed to conduct a scientifically valid and practical in vivo chromosomal aberration assay. For easy reference, the items discussed are listed in the order in which they appear in OECD guideline 475. Specific disagreement with the current and/or proposed OECD guideline is presented in the text. Introduction, purpose, scope, relevance, application, and limits of test: This test would not be appropriate in situations where there was sufficient evidence to indicate that the test article or reactive metabolites could not reach the bone marrow. Test substances: Solid and liquid test substances should be dissolved, if possible, in water or isotonic saline. If insoluble in water/saline, the test substance should be dissolved or homogeneously suspended in an appropriate vehicle (e.g., vegetable oil). A suspension was not considered suitable for an intravenous injection. The use of dimethyl sulfoxide as an organic solvent was not recommended. The use of any uncommonly used solvent/vehicle should be justified. Freshly prepared solutions or suspensions of the test substance should be employed unless stability data demonstrate the acceptability of storage. Selection of species: Any commonly used rodent species was deemed acceptable but rats or mice were preferred, with no strain preference. Number and sex: A consensus could not be reached as to the requirement for both sexes versus one sex in this assay. It was suggested that a single sex should be used unless pharmacokinetic and/or toxicity data indicated a difference in metabolism and/or sensitivity between males and females. The size of the experiment (i.e., number of cells per animal, number of animals per treatment group) should be based on statistical considerations. Lacking a formal analysis, it was agreed that at least 100 metaphase cells should be scored per animal while at least five animals of any one sex should be evaluated per treatment group. Recently, a formal analysis of the numbers of cells to score per animal and numbers of animals to score per treatment group was conducted at a workshop on statistics for in vivo mutagenicity tests (Adler et al., 1994). The conclusion of this workshop was that, based on a type I error of 0.05 and a power of 80% to detect at least a doubling in the control frequency, the minimal number of cells to score per animal was 200 and the minimal number of animals to score per sex per treatment group was four.(ABSTRACT TRUNCATED AT 400 WORDS)

Genotoxicity tests as predictors of carcinogens: An analysis

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

The role of excision repair in the removal of transient benzo[a]pyrene-induced DNA lesions in Chinese hamster ovary cells

In Chinese hamster ovary (CHO) cells, benzo[a]pyrene induces both persistent and transient lesions that are detected by alkaline sucrose gradient sedimentation analysis (ASG sites). The transient lesions disappear within 15 min while the persistent lesions can be detected for several hours following treatment. Although the persistent ASG sites are believed to be repaired by excision repair, the process responsible for the disappearance of the transient ASG sites is unknown. To determine the contribution of excision repair to the removal of these transient lesions, CHO cells were treated with benzo[a]pyrene (B(a)P) in the presence of the inhibitors of excision repair, araC and novobiocin. The results indicate that: (1) araC inhibits the removal of persistent, but not the transient B(a)P-induced ASG sites; (2) novobiocin, a putative inhibitor of the incision step of DNA excision repair, reduced the number of lesions detected immediately following treatment, indicating that many of these lesions may represent single-strand discontinuities generated during repair; and (3) the lesions detected in the presence of novobiocin disappear rapidly following treatment. Based on these results, we concluded that B(a)P-induced transient ASG sites are repaired by a process other than excision repair.

Induction of chromosomal aberrations in Chinese hamster ovary cells by triethyllead acetate

Organolead compounds enter the environment primarily through the combustion of leaded gasoline and industrial discharge. Lead and lead-containing compounds have been shown to induce a broad spectrum of toxic effects, including hematopoietic, renal, neurologic, and carcinogenic effects. In this study, the mutagenic activity of triethyllead acetate (Et3PbAc) was determined by measuring the induction of chromosomal aberrations in Chinese hamster ovary cells. The results indicate that Et3PbAc is very cytotoxic and a potent clastogen. In preliminary cytotoxicity studies used to determine appropriate test concentrations for chromosomal aberration analysis, the LC50 of Et3PbAc was approximately 10 microM in the absence of metabolic activation, and 80 microM in the presence of metabolic activation. The maximal response was greater with metabolic activation than without. However, a much higher dose was required to elicit a significant response in the presence of metabolic activation than in its absence.

A screening tool to prioritize public health risk associated with accidental or deliberate release of chemicals into the atmosphere

The Chemical Events Working Group of the Global Health Security Initiative has developed a flexible screening tool for chemicals that present a risk when accidentally or deliberately released into the atmosphere. The tool is generic, semi-quantitative, independent of site, situation and scenario, encompasses all chemical hazards (toxicity, flammability and reactivity), and can be easily and quickly implemented by non-subject matter experts using freely available, authoritative information. Public health practitioners and planners can use the screening tool to assist them in directing their activities in each of the five stages of the disaster management cycle.