Henry E. Holden

A direct comparison of mouse and rat bone marrow and blood as target tissues in the micronucleus assay

Rats and mice were treated concurrently with mitomycin C at a dose of 1 mg/kg/day i.p. for 3 days, a regimen known to induce micronuclei in polychromatic erythrocytes (MN-PCE) in the bone marrow of rats and mice and the peripheral blood of mice. The incidence of micronuclei was evaluated in the peripheral blood and the bone marrow of both species. Early reports suggested that the efficiency of the rat spleen in removing micronuclei from the circulation precluded the use of rat peripheral blood in the detection of chemically-induced micronuclei. The data in the present study demonstrate that the induction of micronuclei in polychromatic erythrocytes as the result of treatment with a clastogen can be demonstrated equally well in the bone marrow or the peripheral blood of both rats and mice.

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)

Hemizygous Tg.AC transgenic mouse as a potential alternative to the two-year mouse carcinogenicity bioassay: evaluation of husbandry and housing factors

The dermal Tg.AC transgenic mouse model has been proposed as a potential alternative to the conventional (e.g. oral, dermal, parenteral, inhalation, etc.) 2‐year rodent bioassay for detecting chemical carcinogenicity. The present study was designed to address a number of technical aspects of this model as well as to augment the database being developed with the Tg.AC system at the NIEHS. Hemizygous Tg.AC mice were implanted s.c. with microchips for identification and housed individually in polycarbonate (i.e. ‘plastic’) or suspended stainless‐steel wire‐bottom (i.e. ‘metal’) cages. Treatment consisted of dermal application of the test or control material in treatment volumes of 200 μl of acetone. Groups of 10 males and 10 females were treated as follows: G1—shaved, no treatment; G2—acetone control seven times a week; G3—100 μl of benzene three times a week; G4—150 μl of benzene three times a week; G5—1.25 μg of phorbol ester (PMA) twice a week. The G1–G5 mice were housed in plastic caging with Alpha‐dri® bedding. Three additional groups were housed in stainless‐steel wire‐bottom caging: G6—shaved, no treatment; G7—acetone control seven times a week; G8—1.25 μg of PMA twice a week. The PMA‐treated mice (G5 and G8) served as the positive controls. Mice were treated for 20 weeks followed by a 6‐week recovery period prior to necropsy. The incidence of dermal papillomas in the shaved area was recorded weekly. There were no spontaneous papillomas in the target area of any of the untreated (G1) or vehicle control (G2) animals in the polycarbonate cages. One papilloma was observed in the untreated mice (G6) and one in the vehicle control group (G7) in the steel cages. This suggests that the type of caging, the shaving process, microchip implantation and daily acetone treatment for 20 weeks are all consistent with a very low background incidence of papillomas in this model. Papillomas were observed in the positive control groups as early as 4 weeks of treatment and increased both in number per mouse and number of mice affected up to a maximum average of 3.5 papillomas per mouse and 55% (11/20) mice with papillomas in G5 and 2.7 and 80% (16/20) in G8. A plateau was reached at about week 13 and the numbers of papillomas remained stable through the rest of the treatment and recovery phases. The low dose of benzene (100 μl) showed no significant effect, whereas the higher dose (150 μl) produced a moderate number of papillomas beginning at about week 11. The results of this study are comparable with earlier studies at the NIEHS and indicate reproducibility between laboratories and that the Tg.AC transgenic mouse model is suitable for use in an industrial pre‐clinical safety evaluation context.

Oxymetholone: I. Evaluation in a Comprehensive Battery of Genetic Toxicology and In Vitro Transformation Assays:

Oxymetholone is generally assumed to be a nongenotoxic carcinogen. This assumption is based primarily on the results of an Ames test, existing data in repeat-dose toxicology studies, and the predicted results of a 2-yr National Toxicology Program (NTP) rat carcinogenicity bioassay. To provide a comprehensive assessment of its genotoxicity in a standard battery of mutagenicity assays, oxymetholone was tested in microbial and mammalian cell gene mutation assays, in an in vitro cytogenetics assay (human lymphocytes), and in an in vivo micronucleus assay. Oxymetholone was also tested in an in vitro morphologic transformation model using Syrian hamster embryo (SHE) cells. These studies were initiated and completed prior to the disclosure of the results of the NTP bioassay. Oxymetholone was tested at doses up to 5,000 μg/plate in the bacterial plate incorporation assay using 4 Salmonella strains and the WP2 uvrA (pKM101) strain of Escherichia coli. There was no induction of revertants up to the highest dose levels, which were insoluble as well as toxic. In the L5178Y tk+/- mouse lymphoma assay, doses up to 30 μg/ml reduced relative survival to ∼30% with no increase in mutants. Male or female human lymphocytes were exposed in vitro to oxymetholone for 24 hr without S9 or 3 hr with S9 and evaluated for the induction of chromosomal aberrations. There was no increase in aberration frequency over control levels and no difference between male and female cells. Peripheral blood from Tg.AC transgenic mice treated dermally for 20 wk with 0, 1.2, 6.0, or 12.0 mg/day of oxymetholone and from p53 transgenic mice treated orally by gavage for 26 wk with 125, 625, or 1,250 mg/kg/day of oxymetholone was evaluated for micronuclei in polychromatic and normochromatic erythrocytes. There was no difference in micronuclei frequency between control and treated animals. These results confirm that oxymetholone is not genotoxic in a comprehensive battery of mutagenicity assays. In the SHE assay, oxymetholone produced a significant increase in morphologically transformed colonies at dose levels of 13-18 μg/ml. The lack of genotoxicity of oxymetholone, the positive response in the in vitro transformation assay, and the results of transgenic mouse carcinogenicity assays will provide an interesting perspective on the results of an on-going NTP rat carcinogenicity bioassay.

Oxymetholone: III. Evaluation in the p53+/- Transgenic Mouse Model

Oxymetholone has been identified as a suspected nongenotoxic carcinogen and has recently completed testing in a conventional National Toxicology Program (NTP) 2-yr rodent bioassay program. As a synthetic androgen with a limited historical database in toxicology, oxymetholone is an ideal candidate for prospective examination of the performance of short-term transgenic mouse models in the detection of carcinogenic activity. In the present series of 3 articles, studies are described where oxymetholone was evaluated prior to disclosure of the results of the NTP 2-yr bioassay. The accompanying articles provide evidence showing that oxymetholone is devoid of mutagenic activity yet elicits a positive carcinogenic response in the Tg.AC transgenic mouse model. In the present study, oxymetholone was administered by oral gavage to p53 heterozygous male and female mice for 26 wk at doses of 125, 625, and 1,250 mg/kg/day. The vehicle was 0.5% aqueous methylcellulose. Positive controls consisted of mice treated daily by oral gavage with 200 or 400 mg/kg/day of p-cresidine in corn oil. The oxymetholone-treated females showed significantly increased body weight gain and clitoral enlargement attributable to drug treatment. In addition, significant alterations in kidney, liver, and testis weights were attributable to oxymetholone. However, there were no neoplastic lesions that were attributable to oxymetholone in either sex. p-Cresidine produced unequivocal bladder neoplasms in both sexes at the high dose and in males at the lower dose. The absence of a neoplastic response with oxymetholone is consistent with the selectivity of the p53+/- mouse model for detecting carcinogens that act by genotoxic mechanisms.

Oxymetholone: II. Evaluation in the Tg-AC transgenic mouse model for detection of carcinogens.

Several rodent models are under examination as possible alternatives to the classical 2-yr carcinogenicity bioassay. The Tg.AC transgenic mouse has been proposed as a shorter term model offering the possibility of detecting nongenotoxic and genotoxic carcinogenic agents. Retrospective studies of chemicals with established carcinogenic potential have revealed a close correlation between classical bioassay results and the production of skin tumors in the Tg.AC mouse model. Oxymetholone is a synthetic testosterone derivative that is a suspected carcinogen but has shown no evidence of genotoxic activity in a comprehensive battery of genetic toxicity assays. It currently is being tested by the National Toxicology Program (NTP) in a 2-yr rat carcinogenicity bioassay. Because of its nongenotoxicity and the ongoing chronic bioassay, oxymetholone was considered an ideal candidate for a prospective evaluation of the predictive validity of the Tg.AC dermal carcinogenicity model. Consequently, a 6-mo dermal study with oxymetholone in the Tg.AC mouse model was initiated and completed prior to disclosure of the NTP rat bioassay results. In this study, male and female hemizygous Tg.AC mice, 7-8 wk old, were housed individually in suspended plastic cages. An area of dorsal skin was shaved to accommodate dermal applications of 200-μl doses of vehicle control (acetone), drug (1.2, 6.0, or 12 mg oxymetholone in dimethylsulfoxide: acetone, 20:80), or positive control (1.25 μg 12- o-tetradecanoyl-phorbol-13-acetate [TPA]) solutions. Mice received oxymetholone or acetone daily or TPA twice weekly for 20 wk followed by a 6-wk recovery period. The acetone control groups exhibited low spontaneous incidences of papillomas, whereas dermal application of oxymetholone produced dose-related increases in the numbers of papilloma-bearing mice and the numbers of papillomas per animal. Females showed a somewhat greater response to the androgen than did the males. TPA caused an unequivocal increase in papillomas, with males exhibiting a greater response than females. The results of this study indicate that this nongenotoxic androgenic compound possesses proliferative properties. The results predict that chronic systemic administration of oxymetholone will most likely be associated with increased incidences of neoplasms.

Selection of agar for use in Salmonella typhimurium and Escherichia coli mutation assays

The spontaneous and induced revertant frequency of four Salmonella typhimurium strains (TA1535, TA1537, TA98, and TA100) and Escherichia coli [WP2 uvrA (pKM101)] was evaluated using Vogel Bonner minimal plates prepared with ten different agars. In addition to the Difco Bacto agar originally recommended by Ames, Difco Noble, granulated and Bitek agars; BD grade A, BBL granulated and purified agars; Oxoid purified and No. 1 agars; and GIBCO select agar were tested. Several of these agars have been reported as acceptable alternatives for these Salmonella strains, but comparable studies with E. coli have not been done. The bacteria were treated with DMSO or an appropriate positive control in the presence or absence of an Aroclor 1254‐induced rat liver activation system. With the exception of Noble agar in the presence of S9, there was little difference among the responses of the Salmonella strains on any of the agars. However, with E. coli the responses include either a reduction or an increase in spontaneous revertants numbers as well as a reduction in absolute and relative induced revertant frequency. Difco Bacto agar appears to be the most consistent agar for use with these strains. As an alternative, only BBL purified agar resulted in consistent results for all of these strains under all testing conditions. These results emphasize the need to evaluate the components of the standard mutation assay when incorporating additional bacterial strains. Suboptimal responses related to the agar or other components could compromise the detection of weak mutagens. Environ. Mol. Mutagen. 32:192–196, 1998