Regulatory aspects of genotoxicity testing: from hazard identification to risk assessment.

This symposium focused on the use of tests for chromosomal damage, and other genotoxicity measures, for detection of potentially harmful chemicals. The speakers discussed the information that has been gained over the last three decades about the use of "short-term tests" for genotoxicity in cultured cells and in animals (mainly rodents), and the ongoing debates about the rational use of data from such experimental systems in trying to extrapolate to an understanding of potential human risk. The overall theme was that the field of regulatory toxicology currently is over-reliant on qualitative outcomes of in vitro hazard-screening tests, generally conducted at the maximum achievable exposures, and needs a more realistic approach that incorporates in vivo exposure levels and dose-response information.

Strategy for genotoxicity testing: hazard identification and risk assessment in relation to in vitro testing.

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.

Association of marginal folate depletion with increased human chromosomal damage in vivo: demonstration by analysis of micronucleated erythrocytes.

Recent studies have demonstrated that in the absence of spleen function, frequencies of micronuclei (Howell-Jolly bodies) in peripheral blood rbcs can be used to measure in vivo cytogenetic damage. Among 20 subjects studied greater than or equal to 6 months after splenectomy, 1 had a frequency of micronucleated rbcs more than an order of magnitude higher than rates for the others. Initial data suggested that this subject was mildly folate-depleted, and a therapeutic trial with folate rapidly reduced the frequency of micronucleated rbcs to normal values. These observations suggest a need to evaluate further the contribution of mild levels of folate depletion to spontaneous chromosomal damage. The approach used here provides a sensitive index of clastogenic damage and offers unique opportunities for investigating the determinants of cytogenetic damage in humans.

Assessment of cytogenetic damage by quantitation of micronuclei in human peripheral blood erythrocytes.

The incidence of micronuclei (Howell-Jolly bodies) in peripheral blood erythrocytes of splenectomized and nonsplenectomized humans was evaluated as an index of genotoxic exposure. Subjects with intact spleens had very low frequencies of micronucleated cells among circulating erythrocytes, even when these individuals were exposed to known clastogenic agents used in cancer therapy (no micronuclei were seen in 100,000 cells). After splenectomy, the frequency of micronuclei among erythrocytes of untreated subjects rose slowly and after 4 mo established a steady-state level of approximately 2.0/1000, a value similar to that reported for human bone marrow (Goetz et al. Relationship between experimental results in mammals and man: cytogenetic analysis of bone marrow injury induced by a single dose of cyclophosphamine. Mutat. Res., 31: 247-254, 1985; and Hogstedt et al. Micronuclei and chromosome aberrations in bone marrow cells and lymphocytes of humans exposed mainly to petroleum vapors. Hereditas, 94: 179-187, 1981. Chemotherapy increased these levels, with individual samples from patients on daily treatment often having values greater than 5 times higher than control levels. The frequency of micronucleated erythrocytes rose as the duration of clastogenic exposure increased and returned to near base-line levels approximately 4 mo after treatment was discontinued. These findings suggest that it will be possible to use analyses of circulating erythrocytes to assess genotoxic exposures among splenectomized human populations. The ease of sample preparation and scoring should make it possible to monitor individuals with greater statistical power than is feasible with conventional cytogenetic techniques.

Micronucleated erythrocytes as an index of cytogenetic damage in humans: demographic and dietary factors associated with micronucleated erythrocytes in splenectomized subjects.

Erythrocytes containing micronuclei serve as an indicator of genotoxic exposure in splenectomized individuals. Micronucleated erythrocytes, derived from cytogenetically damaged RBC precursors, are not selectively removed from peripheral blood in individuals who lack splenic function. The relationship between micronucleated cell frequencies and demographic, environmental, and dietary factors was examined in 44 subjects with previous splenectomy due to trauma. Their micronucleated cell counts fit a log-normal distribution, with geometric means of 3.3 micronucleus-containing cells/1000 reticulocytes and 2.7/1000 normochromatic erythrocytes. A multiple regression analysis showed that drinking five cups of coffee or tea/day (relative to none) was associated with an approximately 2-fold higher frequency of micronucleated cells. Weaker statistical associations were also noted with micronucleus frequency and the consumption of calcium supplements (associated with a higher frequency) and vitamins A, C, or E (lower frequency). An apparent trend of higher micronucleus counts with age was attenuated when other factors were considered in the regression. Cigarette smoking and decaffeinated coffee consumption were among the factors not associated with elevated micronucleated cell frequencies. Because the occurrence of micronuclei in reticulocytes reflects cytotoxic exposures within the past 3-8 days, it may be possible to test directly the relationship of these factors to micronucleus formation through intervention studies.

Determination of tissue distribution of an intramuscular plasmid vaccine using PCR and in situ DNA hybridization.

The increasing use of nucleic acid-based therapeutics has created a need for new methods of determining tissue distribution and levels. Radiolabel methods may not always be appropriate because nucleic acids are easily degraded. Quantitation using the polymerase chain reaction (PCR) has the advantage that only continuous stretches of DNA will be amplified. In situ hybridization allows detection of specific sequences in histological preparations. We have used quantitative PCR and in situ hybridization techniques to study the pharmacokinetics and distribution of PGagPol (a potential anti-HIV plasmid vaccine) in rabbits. Samples were obtained 4 hr, 24 hr, 7 days, and 28 days after intramuscular injection of 100 micrograms or 400 micrograms of plasmid. A simplified procedure for collecting and processing tissues for PCR that minimizes the risk of contamination was developed. Using PCR, plasmid was found principally in the skin and muscle of the injection site and in blood plasma. At 4 hr after dosing with 400 micrograms, the plasmid was detected at the injection site with mean copy numbers of 10(6) (in muscle) and 4 x 10(4) (in skin) per microgram of tissue. Plasmid copy number declined rapidly in muscle during the first 24 hr and was undetectable at 7 and 28 days after injection. The decline was slower in the skin, and the plasmid was still detectable at 28 days. With in situ hybridization, plasmid was detected in muscle, mainly in the perimysium and to a lesser degree in the endomysium and within the muscle fibers. These data indicate that quantitative PCR and in situ hybridization are sensitive methods for examining tissue distribution of DNA used for gene therapy.

Pesticide use in agriculture.

During the last three decades, the use of modern organic synthetic pesticides has increased about 40-fold. Total U.S. production, for domestic and expert use, in 1976 was about 1.4 million pounds. Crops receiving the most intensive application of various pesticides were cotton for insecticides, corn for herbicides, and fruits and vegetables for fungicides. Examination of use trends of pesticides indicates that the volume in pounds of herbicides used on crops is increasing, whereas the quantities of insecticides and fungicides remain stable. New chemical classes of compounds such as the synthetic pyrethroid insecticides are being introduced, but are not yet significant in terms of their share of the market. The increased usage of pesticides, together with knowledge of some of their adverse effects, has alerted the public to the need for regulation. To assist in the regulatory decision-making process, emphasis is being placed on benefit-cost analyses. Additional and improved biological inputs and methodologies are needed to provide accurate analyses.

Oral treatment with trimethoprim-sulfamethoxazole and zidovudine suppresses murine accessory cell-dependent immune responses.

Trimethoprim-sulfamethoxazole (TMP-SMX), commonly used for prophylaxis of Pneumocystis carinii pneumonia (PCP) in AIDS patients, often produces a high incidence of treatment-limiting reactions. We investigated the effect of oral administration of TMP-SMX alone or in combination with the antiretroviral drug zidovudine (ZDV) on hematopoiesis and cellular immunity in BALB/c mice. Daily treatment for 28 days with TMP-SMX (160:800 mg/kg) had no effect on hematopoiesis or the ex vivo proliferative response of splenic T lymphocytes to allogeneic tumor cells (EL-4) or to concanavalin A (ConA), or that of splenic B cells to lipopolysaccharide (LPS). ZDV at 240 mg/kg/day was not immunosuppressive but caused a mild macrocytic anemia. Combined treatment produced severe pancytopenia, a significant drop in splenic cellularity, and a 61% decrease in the percentage of splenic macrophages. The percentage of splenic CD3+ lymphocytes increased 150% in the TMP-SMX + ZDV group, but the ratios of T-cell subsets and the frequency of B cells remained unchanged. Combined drug treatment did not impair the proliferative response of B cells to LPS or that of T cells to EL-4 cells. In concert with the reduction in the percentage of macrophages, the proliferative response of T lymphocytes to ConA decreased significantly. Optimal ConA-induced T-cell proliferation requires the participation of accessory cells (AC) (e.g., macrophages); EL-4 cells are able to function as AC. These data indicate that ZDV synergizes with TMP-SMX, causing severe hematotoxicity and suppressing AC-dependent immune function, and suggest that this therapeutic regimen may contribute to the immune deterioration in AIDS patients.

Pyrimethamine impairs host resistance to infection with Listeria monocytogenes in BALB/c mice.

Increased mortality has been observed when HIV-infected patients were treated with pyrimethamine (Pyr) as prophylaxis for toxoplasmic encephalitis, suggesting that Pyr might possess immunosuppressive activity. To analyze this in an animal model, immune function was assessed in BALB/c mice using a battery of in vivo and ex vivo assays and an in vivo model of host resistance to Listeria monocytogenes infection. Treatment for 30 days with 60 mg/kg Pyr decreased circulating white blood cell and lymphocyte counts but not neutrophil, red blood cell, or platelet counts or hemoglobin levels. Splenic B cell percentages and lipopolysaccharide-induced B cell proliferation decreased significantly after treatment with 60 mg/kg Pyr, as did levels of anti-keyhole limpet hemocyanin (KLH) IgM in serum 7 days after immunization with KLH. Anti-KLH IgG levels 14 days after immunization were not affected. Percentages of splenic T cells and macrophages and T cell proliferation in the presence of concanavalin A or allogeneic cells were not decreased by Pyr treatment. An ex vivo assay of T-cell-mediated cytotoxicity was also unaffected. When host resistance to L. monocytogenes infection was assessed, dramatic increases in mortality were observed in Pyr-treated compared to control mice. Increased numbers of L. monocytogenes organisms were observed in liver and spleen of Pyr-treated mice, compared to controls. The reduction in Listeria resistance, which is T cell mediated, contrasts with the fact that no significant changes in T-cell-mediated immunity were observed. It is possible that Pyr affects parameters of innate immunity, which were not monitored in this study.

In vitro human tissue models in risk assessment: report of a consensus-building workshop.

Advances in the technology of human cell and tissue culture and the increasing availability of human tissue for laboratory studies have led to the increased use of in vitro human tissue models in toxicology and pharmacodynamics studies and in quantitative modeling of metabolism, pharmacokinetic behavior, and transport. In recognition of the potential importance of such models in toxicological risk assessment, the Society of Toxicology sponsored a workshop to evaluate the current status of human cell and tissue models and to develop consensus recommendations on the use of such models to improve the scientific basis of risk assessment. This report summarizes the evaluation by invited experts and workshop attendees of the current status of such models for prediction of human metabolism and identification of drug-drug interactions, prediction of human toxicities, and quantitative modeling of pharmacokinetic and pharmaco-toxicodynamic behavior. Consensus recommendations for the application and improvement of current models are presented.

Environmental mutagenesis: past and future directions.

On the occasion of the 25th anniversary of the Environmental Mutagen Society, this article presents a personal reflection on the development of environmental mutagenesis as a unique scientific discipline. The events following the demonstration of chemical mutagenesis in the 1940s and leading to the formation of the Environmental Mutagen Society in 1969 are reviewed. Scientific progress and regulatory practices in the field of environmental mutagenesis during the twenty-five years since the founding of the Society are discussed, and speculation on the likely future of the field is presented.

Flavone mutagenicity in Salmonella typhimurium: dependence on the pKM101 plasmid and excision-repair deficiency.

Flavones mutagenic in Salmonella typhimurium fall into two distinct classes, characterized by different structural and metabolic activation requirements and by different strain responses. The mutagenic potencies of a prototype agent of each class, quercetin (3,3',4',5,7-hydroxyflavone) and norwogonin (5,7,8-hydroxyflavone), were determined in tester strains differing in excision-repair capability and in the presence or absence of plasmid pKM101. Two series of strains were used, one with the hisD3052 frameshift mutation and one with the hisG46 missense mutation. With both agents and for both series of strains, the mutagenic response was markedly dependent on the absence of excision repair and the presence of the pKM101 plasmid.

Assays of three carcinogen/non-carcinogen chemical pairs for in vivo induction of chromosome aberrations, sister chromatid exchanges and micronuclei.

Three pairs of structurally similar carcinogenic/non-carcinogenic chemicals were tested for in vivo genotoxic activity in B6C3F1 mice. The carcinogenic/non-carcinogenic pairs, respectively, were o-toluidine hydrochloride/o-anthranilic acid, 4-chloro-o-phenylenediamine/4-nitro-o-phenylenediamine, and 3-(chloromethyl)pyridine hydrochloride/2-(chloromethyl)pyridine hydrochloride. Bone marrow cells from mice given intraperitoneal injections of up to the maximum tolerated dose were evaluated for chromosomal aberration, sister chromatid exchange, and micronucleus induction, o-anthranilic acid and o-toluidine hydrochloride did not increase the frequency of chromosomal aberrations or micronuclei. o-Toluidine hydrochloride increased the frequency of sister chromatid exchanges in two successive trials, while o-anthranilic acid had a positive effect on sister chromatid exchanges in two of three trials. Both 2-(chloromethyl) and 3-(chloromethyl)pyridine hydrochloride were negative for all three endpoints. Assays for chromosomal aberrations and micronuclei each distinguished between 4-chloro-o-phenylenediamine and its non-carcinogenic companion, 4-nitro-o-phenylenediamine. In the aberration test, 4-chloro-o-phenylenediamine produced a few cells with very large numbers of aberrations rather than an even distribution of damage among cells.

Micronucleated erythrocyte frequency in peripheral blood of B6C3F(1) mice from short-term, prechronic, and chronic studies of the NTP carcinogenesis bioassay program.

The mouse peripheral blood micronucleus (MN) test was performed on samples collected from 20 short-term, 67 subchronic, and 5 chronic toxicity and carcinogenicity studies conducted by the National Toxicology Program (NTP). Data are presented for studies not previously published. Aspects of protocol that distinguish this test from conventional short-term bone marrow MN tests are duration of exposure, and absence of repeat tests and concurrent positive controls. Furthermore, in contrast to short-term bone marrow MN tests where scoring is limited to polychromatic erythrocytes (PCE), longer term studies using peripheral blood may evaluate MN in both, or either, the normochromatic (NCE) or PCE populations. The incidence of MN-PCE provides an index of damage induced within 72 hr of sampling, whereas the incidence of MN in the NCE population at steady state provides an index of average damage during the 30-day period preceding sampling. The mouse peripheral blood MN test has been proposed as a useful adjunct to rodent toxicity tests and has been effectively incorporated as a routine part of overall toxicity testing by the NTP. Data derived from peripheral blood MN analyses of dosed animals provide a useful indication of the in vivo potential for induced genetic damage and supply an important piece of evidence to be considered in the overall assessment of toxicity and health risk of a particular chemical. Although results indicate that the test has low sensitivity for prediction of carcinogenicity, a convincingly positive result in this assay appears to be highly predictive of rodent carcinogenicity.

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.

Micronuclei as an index of cytogenetic damage: past, present, and future.

The workshop was designed to present what is known about the production of micronuclei, what protocols are now accepted or proposed internationally, what new results have been obtained, and what new methods and protocols are likely to be forthcoming. This report is designed to convey the flavour of the workshop and to provide the essence of the new information. After the workshop an effort was made to determine what single protocol would satisfy the requirements set for the micronucleus test by as many regulatory agencies as possible. The result, reported here, includes the requirements of six regulatory authorities in Canada, the European Economic Community, the Organization for Economic Co-operation and Development, Japan, and the United States.

Immunohematotoxicity studies with combinations of dapsone and zidovudine.

We investigated the immunohematoxicities of the antiparasitic drug dapsone (DDS) and the antiretroviral drug zidovudine (ZDV, AZT) given alone or in combination in BALB/c mice. DDS is used for prophylaxis and treatment of Pneumocystis carinii infection in AIDS patients. We examined the impact of concurrent administration of these drugs on the immune and hematopoietic systems because DDS causes hematotoxicity and ZDV therapy results in bone marrow toxicity. Daily oral administration of DDS at 25 and 50 mg/kg for 28 days caused a slight anemia, marked methemoglobinemia, reticulocytosis, and a moderate leukopenia (P < 0.01 for all parameters) but had no discernible effect on platelet count. In DDS-treated mice, the proliferative response of splenic T cells to concanavalin A was > or = 35% higher than that manifested by splenocytes from vehicle-treated control mice. ZDV at 240 and 480 mg/kg was not immunosuppressive but caused low-grade macrocytic anemia, thrombocytosis, and neutropenia; these effects were drug dose-dependent and statistically significant (P < 0.01). Concurrent administration of DDS and ZDV augmented the severity of ZDV-mediated macrocytic anemia, and 7 of 12 (58%) mice did not survive treatment with the high doses of DDS and ZDV (50 and 480 mg/kg, respectively). On the other hand, co-administration of ZDV mitigated DDS-induced methemoglobinemia and the DDS-associated elevation in lymphoproliferative response. These data suggest interaction between DDS and ZDV in mice and indicate a need for caution in using DDS as long-term therapy in AIDS patients receiving ZDV.

A rapid screen for cumulative chromosomal damage in mice. Accumulation of circulating micronucleated erythrocytes.

The frequency of micronuclei in normochromatic peripheral blood erythrocytes was found to be a useful index of cumulative chromosomal damage in repeatedly exposed mice. Although approximately 5 weeks of continuous treatment is required to reach the maximum steady state level, significant elevations in this index were achieved after only 5 daily exposures to non-lethal doses of all six genotoxins tested. The frequencies of micronucleated cells per 1000 normochromatic erythrocytes in weanling mice after 5 daily intraperitoneal (i.p.) injections of each test agent were: triethylenemelamine (0.5 mg/kg), 13.7; mitomycin C (2 mg/kg), 7.1; cyclophosphamide (22 mg/kg), 6.6; colchicine (1 mg/kg), 4.0; nitrogen mustard (0.4 mg/kg), 3.6; 7,12-dimethylbenz[a]anthracene (5 mg/kg), 6.6. Controls averaged 1.2 per 1000. During extended exposure to nitrogen mustard (5 i.p. injections of 0.4 mg/kg per week), the incidence of micronucleated erythrocytes rose steadily to a value of 12.0 per 1000, approaching the steady state after about 5 weeks of treatment. These results indicate that the measurement of micronuclei in peripheral blood erythrocytes is an effective and rapid method for estimating chromosomal damage in subchronically or chronically exposed animals. In practical application, routine blood smears taken during 90-day subchronic toxicity tests could be scored for micronuclei in less than 1 day, providing an economical estimate of in vivo chromosomal damage.

Mutagenicity of plant flavonoids: structural requirements for mutagenic activity in Salmonella typhimurium.

40 compounds structurally related to the plant flavonol quercetin were tested for mutagenic activity in Salmonella typhimurium strain TA98. 10 flavonols, quercetin, myricetin, rhamnetin, galangin, kaempferol, tamarixetin, morin, 3'-O-methylquercetin, 7,4'-di-O-methylquercetin and 5,7-di-O-methyl-quercetin, exhibited unequivocal mutagenic activity. 4 compounds, quercetin, myricetin, rhamnetin and 5,7-di-O-methylquercetin, were active without metabolic activation, although metabolic activation markedly enhanced their activity. All 4 have free hydroxyl groups at the 3' and 4' positions of the B ring. The other active compounds required an in vitro rat-liver metabolizing system for significant activity. Structural features which appear essential for mutagenic activity in this strain are a basic flavanoid ring structure with (1) a free hydroxyl group at the 3 position, (2) a double bond at the 2, 3 position, (3) a keto group at the 4 position, and (4) a structure which permits the proton of the 3-hydroxyl group to tautomerise to a 3-keto compound. The data are consistent with the requirement for a B ring structure that permits oxidation to quininoid intermediates. Free hydroxyl groups in the B ring are not essential for activity if a rat-liver metabolic activating system is employed. Data from 12 compounds which differ only at the essential sites described above indicate that the structural requirements for mutagenicity in strain TA100 are the same as those for activity in strain TA98. Based on the above structural requirements, a metabolic pathway for flavonol activation to DNA-reactive species is proposed.

The micronucleus test: statistical design and analysis.

Alternative statistical procedures are discussed which may be employed to compare the incidences among treatment groups of micronucleated polychromatic and normochromatic erythrocytes and their ratios. Comparison of incidences of micronucleated polychromatic erythrocytes using a sequential sampling strategy based on the negative binomial distribution is shown to require fewer animals for the same sensitivity of test than a similar procedure based on the binomial distribution. The sequential test is superior, both in power and number of animals required, to an alternative 1-stage test based on the same distribution. The procedure described permits the investigator to optimize the number of animals in each test group and the number of cells counted per animal to detect a predetermined increase in the incidence of micronucleated cells over that observed in the control population within chosen limits of type I and type II error. An alternative sequential approach based on the binomial distribution is presented, which is applicable when the number of cells analyzed per animal is variable.