Diana Anderson

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)

Saccharin: An epigenetic carcinogen/mutagen?

Saccharin, together with four of the impurities found in the commercial material and a further nine functional analogues gave negative results in the Salmonella reverse mutation assay of Ames et al. (Mutation Res. 1975, 31, 347) and the cell-transformation assay of Styles (Br. J. Cancer 1977, 36, 558). The significance of these and related in vitro results is evaluated within the context of the ability of commercial saccharin to cause bladder cancer in rats and dominant lethal effects in mice. In particular, evidence that the carcinogenicity and the dominant lethal effect associated with saccharin may be mediated by an epigenetic mechanism is evaluated, as is the possible role played by impurities in the generation of these and other biological effects. The case for separating carcinogens into two classes, namely those that operate by a genotypic mechanism and those that elicit their effects via an epigenetic mechanism, is discussed. The compounds evaluated in the current study were saccharin base, sodium saccharin, o-toluenesulphonamide, o-sulphonamidobenzoic acid, 5-chlorosaccharin, 3-iminosaccharin, 6-chlorosaccharin, 5-chlorobenzisothiazoline-1,1-dioxide, 6-aminosaccharin. 6-nitrosaccharin, 3-chloro-ψ-saccharin, 3,6-dichloro-ψ-saccharin, 3-dimethylamino-ψ-saccharin, 3-piperidinyl-ψ-saccharin and 3-pyrrolidino-ψ-saccharin.

Issues relevant to the assessment of chemically induced chromosome damage in vivo and their relationship to chemical mutagenesis

Rats have been exposed by different routes of administration (inhalation, orally and intraperitoneally) to known mutagens and their bone-marrow cells sampled at different times to determine the extent of chromosome damage. The mutagens investigated were ethyl methanesulphonate, mitomycin C, trimethylphosphate, benzene and vinyl chloride, at single and/or multiple doses (5 consecutive daily). Various categories of chromosome damage were observed in all cases. However, the extent of damage due to chromosome and chromatid gaps was greater than, and generally increased in parallel with, other categories of damage. It has been suggested tht chromosome and chromatid gaps are indicative of toxic phenomena but this study suggests that such aberrations could be useful and sensitive indicators of chemically induced genetic damage. In addition the study has also confirmed that single exposure is as effective as multiple exposure in producing chromosome damage and that the correct sampling time is necessary to detect this damage. Therefore for screening purposes a time course sampling after a single treatment regime would be suitable for detecting the mutagenic potential of a chemical.

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)

Chromosomal analyses in vinyl chloride exposed workers. Results from analysis 18 and 42 months after an initial sampling.

In a previous study (Purchase et al., Mutation Res., 57 (1978) 325-334) it was reported that 57 workers occupationally exposed to vinyl chloride had an increase in the incidence of chromosomal abnormalities in their lymphocytes by comparison with 24 control workers. Since that time (July 1974) threshold limit values for vinyl chloride and plant exposure levels have been reduced. In the present study, 2 further samples from the same population of workers have been analysed for chromosomal aberrations 18 and 42 months after the initial sampling. At 18 months, 21 VC workers and 6 on-site controls were investigated as were 23 workers and 8 on-site controls at 42 months. In the second sampling there was a tendency to an increase in chromosomal abnormalities of VC-exposed workers except in those people who had changed occupation. By the third sampling, however, there was a decrease by comparison with previous samplings and the levels of abnormalities had returned to values similar to those of the controls. Thus, reduction in exposure to vinyl chloride is accompanied by a reduction in the chromosomal abnormalities to levels indistinguishabe from those of controls.

Vinyl chloride: Dominant lethal studies in male CD-1 mice

The mutagenic activity of vinyl chloride (VC) at three exposure levels was assessed in fertile male CD-1 mice with the dominant lethal test. Male mice were exposured by inhalation to VC at 3000, 10,000 and 30,000 ppm for 6 h a day for 5 days. By comparison with control males exposed to air, no mutagenic effects on any maturation stage of spermatogeneisis in treated males were detected. There was no significant increase in the number of post-implantational early foetal deaths as shown by the number of females with one or more early deaths or number of early deaths/pregnancy or the number of early deaths/total implants/pregnancy. There was no evidence of pre-implantational egg losses as indicated by the total implants/pregnant female. There was also no reduction in fertility. The lack of effect was not due to the insensitivity of the system used since a dominant lethal effect was clearly demonstated in male mice dosed i.p. with cyclophosphamide (CTX) at 200 mg/kg body weight and ethyl methanesulphonate (EMS) orally at 200 mg/kg body weight once a day for 5 days. During dosing these animals were housed under dimilar exposure conditions to those animals exposed to the test substances but with a flow of air through the exposure chambers. Thus vinyl chloride is not mutagenic in the mouse at the stated exposure levels as measured by the dominant lethal test.

Dominant-lethal test results with known mutagens in two laboratories.

Abstract During investigations of the potential dominant lethal activity of various chemicals, concurrent controls were run to check the sensitivity and the reproducibility of the test systems. These experiments were carried out over an 18-month period in two laboratories using similar protocols. Negative control substances used were maize oil, dispersol, tween 80 and water. Positive control substance used were cyclophosphamide, ethyl methane-sulphonate and mechlorethamine hydrochloride (nitrogen mustard). The substances were administered either intraperitoneally or by gavage. The results were analysed using, principally, a hierarchical analysis of variance of the total implants per pregnancy and the transformed early deaths per pregnancy data. Pregnancy frequency generally did not fall below 80%. The total number of implantations per pregnancy was usually about 11.8 in negative controls and was variably reduced by the mutagens used. With cyclophosphamide or ethyl methanesulphonate (EMS), there were some quantitative variations in the results obtained but qualitative agreement was good in the results both between experiments in the same laboratory and between the two laboratories. It was demonstrated that EMS is a useful positive control substance in experiments with orally administered materials. Sensitivity of the system was indicated by positive effects with a relatively low dose of EMS and consistent positive results with mechlorethamine for which a dominant lethal effect has not been demonstrated previously. It is concluded that the dominant lethal test gives reproducible data and it is, thus, possible to have confidence in the results and to compare findings in different laboratories using similar protocols.

Dominant lethal studies with paraquat and diquat in male CD-1 mice

The herbicides paraquat and diquat were tested for dominant lethal activity in male CD-1 mice. No mutagenic effects were detected on any stage of spermatogenesis in treated males if these compounds were administered orally up to 4 mg paraquat/kg/body weight/day of 10 mg diquat/kg/body weight/day for 5 days. There was no increase in the number of post-implantation losses as shown by the number of females with one or more early deaths or number of early deaths/pregnancy or the number of early deaths/total implants/pragnancy. There was no evidence of pre-implantation losses as indicated by the total implants/pregnant female. There was no anti-fertility effect on the treated males as measured by the pregnancy frequency or successful mating frequency. The lack of dominant lethal effect was not due to particular insensitivity of the animals used since such effects were amply demonstrated in mice doses orally on 5 days with ethyl methanesulphonate at 100 mg/kg/body weight/day, or intraperitoneally on one day with cyclophosphamide at 200 mg/kg/body weight.

Irradiated laboratory animal diets: Dominant lethal studies in the mouse

In 4 separate dominant lethal experiments groups of mice of either Charles River CD1 or Alderley Park strains were fed laboratory diets (Oakes, 41B, PRD, BP nutrition rat and mouse maintenance diet No. 1). The diets were either untreated (negative control diets) or irradiated at 1, 2.5 and 5 megarad and were freshly irradiated, or stored. The animals were fed their test diets for a period of 3 weeks prior to mating. Groups of mice given a single intraperitoneal injection of 200 mg cyclophosphamide per kg body weight served as the positive controls. Freshly irradiated PRD diet fed to male mice of both strains caused an increase in early deaths in females mated to the males in week 7 and to a lesser extent in week 4. The increase due to irradiation was small by comparison with that produced by the positive control compound. The responses for the other irradiated diets showed no significant increases in early deaths although some values for Oakes diet were high. The effect of storage was examined with PRD and BPN diet on one occasion and produced conflicting results. Thus there was some evidence that irradiated PRD diet has weak mutagenic activity in the meiotic and/or pre-meiotic phase of the spermatogenic cycle which appeared to be lessened on storage; the inclusion of such a diet in toxicological studies would therefore need to be carefully considered.

The parallelogram approach in studies of genotoxic effects.

Over the past two decades mutagenicity tests have been used for the identification of potential human mutagens and have had an ancillary role, as supportive evidence in the assessment of human carcinogens. The demonstration of human germinal mutagens has been beyond the main scope of short-term testing strategies. However, just as mutagenicity tests have been useful in detecting potential carcinogens so should carcinogenicity tests assist the identification of presumptive germ cell mutagens. Cancer is an easily observable phenotype of mutation for genotoxic carcinogens and multi-site carcinogens or gonadal carcinogens logically could be germ cell mutagens. Thus carcinogenicity and mutagenicity data for a given genotoxic chemical should be considered together in the identification of putative germinal mutagens. Clearly, most classified human carcinogens are genotoxic thus helping to build the case for human germ cell mutagenicity. This paper describes the issues involved in such thinking and suggests an enhanced parallelogram approach incorporating the cancer endpoint. The enhanced parallelogram is explored using 1,3-butadiene and ethylene oxide as examples. The obvious lack of data for extrapolations using the parallelogram method suggests the need for targeted studies specifically designed for use in this approach.