Evaluation of the rodent micronucleus assay by a 28-day treatment protocol: Summary of the 13th Collaborative Study by the Collaborative Study Group for the Micronucleus Test (CSGMT)/Environmental Mutagen Society of Japan (JEMS)-Mammalian Mutagenicity Study Group (MMS).

To examine whether micronucleus tests can be incorporated into general toxicology assays, we performed micronucleus tests applying the treatment protocols typically used in such assays. In this 13th Collaborative Study of the CSGMT, both rats and mice were tested, although rats were used in the majority of the studies. Fifteen mutagens were tested in rats, mainly by oral (p.o.) administration. Micronucleus induction was evaluated 2, 3, and 4 days, and 1, 2, 3, and 28 days after the beginning of the treatment in the peripheral blood, and at 28 days in the bone marrow. Of the 15 chemicals that induced micronuclei in rats in short-term assays, two chemicals (1,2-dimethylhydrazine.2HCl and mitomycin C) were negative in all our experiments, possibly because of insufficient dose levels. The remaining 13 were positive within the estimated dose range of a general toxicology assay, suggesting the possibility of integrating the micronucleus assay into general toxicology assays. Three patterns were observed in micronucleus induction during the period of repeated treatment: (1) gradual increases in micronucleus frequency with sequential doses, (2) a peak at 3-5 days followed by gradual decreases in micronucleus frequency with sequential doses, and (3) a rapid increase in micronucleus frequency followed by a plateau. We evaluated factors that might have been involved in those patterns, such as the spleen function, target organ exposure, extramedullary hematopoiesis, hypothermia, and hypoxia. Another factor we considered was dosage. Because the dosages employed in a general toxicity assay are usually lower than those used in short-term micronucleus assays, this discrepancy was considered the greatest potential problem for integrating the micronucleus assay into general toxicology assays. Our results indicate that the integration of the micronucleus assay into a 28-day toxicological assay is feasible. To serve this purpose, blood samples collected 4 days after the beginning of treatment and blood and bone marrow samples collected at autopsy should be examined. Furthermore, although it is recognized that mice may be suitable for performing independent micronucleus assays, we propose that rats can provide biologically important and relevant information regarding potential chemical mutagens that can be evaluated under conditions used in the conduct of general toxicology studies.

ICH-harmonised guidances on genotoxicity testing of pharmaceuticals: evolution, reasoning and impact.

The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) has convened an expert working group which consisted of the authors of this paper and their respective committees, consulting groups and task forces. Two ICH guidances regarding genotoxicity testing have been issued: S2A, 'Guidance on Specific Aspects of Regulatory Genotoxicity Tests' and S2B, 'Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals.' Together, these guidance documents now form the regulatory backbone for genotoxicity testing and assessment of pharmaceuticals in the European Union, Japan, and the USA. These guidances do not constitute a revolutionary new approach to genotoxicity testing and assessment, instead they are an evolution from preexisting regional guidelines, guidances and technical approaches. Both guidances describe a number of specific criteria as well as a general test philosophy in genotoxicity testing. Although these guidances were previously released within the participating regions in their respective regulatory communiqués, to ensure their wider distribution and better understanding, the texts of the guidances are reproduced here in their entirety (see Appendix A) and the background for the recommendations are described. The establishment of a standard battery for genotoxicity testing of pharmaceuticals was one of the most important issues of the harmonisation effort. This battery currently consists of: (i) a test for gene mutation in bacteria, (ii) an in vitro test with cytogenetic evaluation of chromosomal damage with mammalian cells or an in vitro mouse lymphoma tk assay, (iii) an in vivo test for chromosomal damage using rodent hematopoietic cells. A major change in testing philosophy is the acceptance of the interchangeability of testing for chromosomal aberrations in mammalian cells and the mouse lymphoma tk assay. This agreement was reached on the basis of the extensive review of databases and newly generated experimental data which are in part described in this publication. The authors are fully aware of the fact that some of the recommendations given in these ICH guidances are transient in nature and that the dynamic qualities and ongoing evolution of genetic toxicology makes necessary a continuous maintenance process that would serve to update the guidance as necessary.

Synthesis of high-molecular-weight poly([R]-(-)-3-hydroxybutyrate) in transgenic Arabidopsis thaliana plant cells.

High-molecular-weight poly([R]-(-)-3-hydroxybutyrate) (PHB), a biodegradable thermoplastic, was produced from a suspension culture of transgenic Arabidopsis thaliana plant cells expressing two genes from the bacterium Alcaligenes eutrophus involved in the synthesis of PHB. The molecular structure of the plant-produced polymer was analysed by gas chromatography, mass spectrometry, proton nuclear magnetic resonance spectroscopy, infra-red spectroscopy, spectropolarimetry, differential scanning calorimetry, X-ray diffraction and size exclusion chromatography. The results indicate that the polymer from transgenic plants appears to have a chemical structure identical to that of PHB produced by bacteria. However, the molecular weight distribution of the plant-produced PHB was much broader than that of typical bacterial PHB.

Induction of mutagenesis and transformation in BALB/c-3T3 clone A31-1 cells by diverse chemical carcinogens.

BALB/c-3T3 cells were employed to examine the genotoxic potential of a variety of known chemical carcinogens. BALB/c-3T3 cells displayed a dose-dependent transformation response to a variety of carcinogens (polycyclic hydrocarbons, methylating agents, ethylating agents, aflatoxin B1 [AFB1], and 4-nitroquinoline-N-oxide [4-NQO]). When the ability of these compounds to induce mutagenesis to resistance to the cardiac glycoside ouabain (OUAR) was examined, we found the short chain alkylating agents to be particularly effective mutagens, causing biologic effects at doses below those necessary to induce a transformation response. In contrast, the polycyclic hydrocarbons which were potent transforming agents were weaker, albeit significant, mutagens for the OUAR locus in this system, while AFB1 was quite weak. Further studies were performed with 5-azacytidine (5-AZA) and the nongenotoxic carcinogen cinnamyl anthranilate (ClN). 5-AZA was a potent transforming agent, but failed to cause mutagenesis. ClN similarly caused in vitro transformation. When a series of eight structurally diverse compounds were examined in both the BALB/c-3T3 and C3H10T1/2 mouse fibroblast transformation systems, the BALB/c-3T3 system was shown to be sensitive to a wide variety of potential carcinogens, whereas the C3H10T1/2 system proved routinely sensitive only to the polycyclic hydrocarbons.

Morphological transformation of BALB/3T3 cells by various procarcinogens in the presence of a rat liver S-9 activation system.

An Aroclor-induced rat hepatic S-9 metabolic activation system was incorporated into the BALB/3T3 cell transformation assay to increase its sensitivity to a wide range of procarcinogens. S-9 was prepared from Aroclor 1254-induced (500 mg/kg) Fischer 344 rats. Cyclophosphamide, dimethylnitrosamine, 2-aminofluorene, and 2-naphthylamine were metabolized to reactive forms capable of inducing both dose-dependent toxicity and morphological transformation of BALB/3T3 cells. Treatments without an exogenous metabolic activation system were nontoxic and nontransforming. Adaptation of this commonly used exogenous metabolic activation system to BALB/3T3 cells will allow detection of the transforming potential of procarcinogens which test negative in a standard assay.

Interlaboratory evaluation of the C3H/10T1/2 cell transformation assay.

The C3H/10T1/2 transformation assay was evaluated for its responsiveness and interlaboratory reproducibility. Two laboratories participated in this study and tested a series of 46 chemicals. The majority of these chemicals were tested under code. Of the 46 chemicals tested, seven were determined to be active in both laboratories, and 14 were determined to be inactive. When the total number of chemicals is adjusted for assays considered "no test" in either one or both laboratories as well as for tests of chemicals yielding positive results in only one laboratory, reproducible responses were obtained for 21/35, or 60%, of the chemicals tested.

A method for the amplification of chemically induced transformation in C3H/10T1/2 clone 8 cells: its use as a potential screening assay.

A method has been developed by which to amplify expression of phenotypic transformation of C3H/10T1/2 clone 8 mouse embryo cells not otherwise observed in the standard transformation assay. The expression of transformed foci was amplified by subcultivating chemically treated target cells after they had reached confluence and replating them at subconfluent cell densities. Conditions leading to the expression of the highest numbers of transformed foci include a) a cell seeding density for chemical treatment of 1 X 10(4) cells/dish, b) subculture 4 weeks after treatment, and c) replating cells at a density of 2 X 10(5) cells/-dish. Agents capable of inducing transformation in the standard assay (e.g., 4,4'-bis(dimethylamino)benzophenone, benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, and others) also yielded transformation in the replating assay. The more marginal transforming activities of chemicals such as ethyl methanesulfonate, 7-(bromomethyl)-12-methylbenz[a]anthracene, and N-methyl-N'-nitro-N-nitrosoguanidine were enhanced by the amplification procedure. Compounds that failed to elicit focal transformation in the standard assay (e.g., dibenz[a,h]anthracene, Tris(2,3-dibromopropyl) phosphate, lead acetate, benzidine, propyleneimine, N-hydroxy-2-fluorenylacetamide, and numerous other compounds of various chemical classes) induced significant levels of phenotypic transformation upon amplification. Noncarcinogens (e.g., phenanthrene, anthracene, 2-aminobiphenyl, cycloheximide, and others) failed to cause significant phenotypic transformation even when cells were replated. To further enhance the applicability of this new replating system, an exogenous source of metabolic activation was added: a 9,000 X g supernatant from Aroclor 1254-induced rat hepatic S-9. This activation system was found a) to be only minimally cytotoxic by itself and b) to be able to mediate NADPH-dependent, dose-dependent toxicity, and transformation by activating the procarcinogens dimethylnitrosamine, 2-naphthylamine, 2-aminoanthracene, and aflatoxin B1. With the use of this revised assay, 14 coded and 23 model compounds were tested. Agreement with in vivo results was observed to be over 85%. The marked sensitivity and discriminatory ability of this revised assay procedure suggest its usefulness as a screen for potential carcinogens of diverse chemical structure.

Analysis of the interlaboratory and intralaboratory reproducibility of the enhancement of simian adenovirus SA7 transformation of Syrian hamster embryo cells by model carcinogenic and noncarcinogenic compounds.

The intralaboratory and interlaboratory reproducibility of a DNA virus (SA7) transformation enhancement assay was investigated using nine carcinogenic and noncarcinogenic compounds representing a variety of chemical classes. By the use of standardized procedures designed to limit assay variables, replicate assay data were collected in two independent laboratories and analyzed for concurrence. The carcinogens, 7,12-dimethylbenz(a)anthracene, benzo(a)pyrene, and N-methyl-N'-nitro-N-nitrosoguanidine yielded reproducible dose-dependent cytotoxicity and positive transformation effects (defined as statistically significant [p less than or equal to 0.05] enhancement of virus transformation at two or more consecutive dose levels) in all experiments in both laboratories. The carcinogens lead chromate, diethylnitrosamine, 4-nitroquinoline-N-oxide, and 2-acetylaminofluorene demonstrated enhancement of SA7 transformation at two or more dose levels in 40-50% of the assays. The noncarcinogenic structural analogs anthracene and pyrene consistently did not produce positive assay responses when tested at dose levels up to the limits of solubility. Good interlaboratory concurrence was demonstrated for these model compounds in the Syrian hamster embryo cell-SA7 assay.

Influence of various parameters on benzo(a)pyrene enhancement of adenovirus SA7 transformation of Syrian hamster embryo cells.

Several of the major variable factors in the Syrian hamster embryo/simian adenovirus SA7 (SHE/SA7) viral enhancement assay were identified and the effects of these parameters on assay sensitivity were assessed. The extent of dose-dependent cytotoxicity and enhancement of SA7 transformation of primary SHE target cells by benzo(a)pyrene was examined through analysis of data obtained from 37 assays performed over a 2-year period. The variables analyzed for contribution to assay sensitivity included the number of SA7-induced transformed SHE cell foci enumerated in ten replicate dishes in the negative control condition (background focus count) (range: 26-139); the age of the SHE cell cultures at the time of exposure to benzo(a)pyrene (range: 72-144 hr postseeding); and the source of the pregnant hamsters used to prepare the primary SHE cells (Wilmington colony vs Lakeview colony, Charles River Laboratories, Inc., Wilmington, MA). The benzo(a)pyrene-induced cytotoxicity and enhancement of SA7 transformation responses were found to be independent of each of these variables, within the range of values tested.

Effects of 3-aminobenzamide on the induction of morphologic transformation by diverse compounds in Balb/3T3 cells in vitro.

When cells were exposed simultaneously for a 24-h period to the poly(ADP-ribose) synthetase inhibitor 3-aminobenzamide (3AB) (1 or 3 mM) plus aflatoxin B1 (AfB1), no increase in toxicity and limited enhancement of transformation frequency (less than 2 X) was observed. Similarly, simultaneous treatment of cell with 3AB plus methylcholanthrene (MCA) had limited effects, slightly decreasing both toxicity and transformation. In contrast, simultaneous treatment with non-toxic doses of 3AB together with the alkylating agents N-methyl-N'-N-nitro-nitrosoguanidine (MNNG) or ethyl methanesulfonate (EMS) resulted in substantial enhancement of the toxicity and transforming effects of both short-chain alkylating agents. When EMS and varying doses of 3AB (0.1-3 mM) were administered simultaneously for 24 h, increasing levels of 3AB were found to cause a dose-dependent enhancement in toxicity and transformation. To explore the relationship of MNNG- and 3AB-induced effects, two further experiments were performed. First, cells were treated with MNNG plus 3AB for varying lengths of time (4, 24, 72 h). Although exposure for as little as 4 h enhanced toxicity and transformation, these effects were even more profound following 24 or 72 h exposure. Second, cells were exposed to 3AB for varing times prior to or after MNNG exposure. Under these conditions the addition of 3AB up to 6 h post MNNG exposure caused profound enhancement of toxicity and transformation, whereas addition of 3AB 24 h post exposure had minimal effects. Thus the co-carcinogenic effect of 3AB is agent-specific, time-specific and dose-dependent.

Effect of 3-aminobenzamide on the induction of toxicity and transformation by ethyl methanesulfonate and methylcholanthrene in BALB/3T3 cells.

3-Aminobenzamide, a potent inhibitor of nuclear poly ADP-ribosyl synthetase, was tested for its ability to alter the toxic and/or transforming effects of ethyl methanesulfonate, methyl methanesulfonate and 3-methylcholanthrene in BALB/3T3 clone A31-1 cells. 3-Aminobenzamide enhanced the toxic effects of both ethyl methanesulfonate and methyl methanesulfonate in a dose dependent manner, but had minimal effects on 3-methylcholanthrene induced toxicity. Similarly, 3-aminobenzamide greatly enhanced ethyl methanesulfonate induced transformation while failing to enhance the transformation of BALB/3T3 clone A31-1 cells by 3-methylcholanthrene. These results stress the importance of poly ADP-ribosyl synthetase in repair of DNA damage and the chemical induction of transformation in vitro.

Induction of neoplastic transformation and DNA single-strand breaks in C3H/10T1/2 clone 8 cells by polycyclic hydrocarbons and alkylating agents.

The standard C3H/10T1/2 clone 8 (C3H/10T1/2 CL8) cell transformation assay was tested for its ability to identify a variety of polycyclic hydrocarbons and alkylating agents. Dose-dependent morphologic transformation occurred with benzo[a]pyrene (BaP), 3-methylcholanthrene (MCA), 7,12-dimethylbenz[a]anthracene, BaP-7,8-dihydroxy-7,8-dihydrodiol (BaP-7,8-diol), as well as with the relatively weak in vivo carcinogen benzo[e]pyrene. Dibenz[a,h]anthracene yielded a relatively weak response, whereas anthracene and phenanthrene were negative. In contrast, treatment of C3H/10T1/2 CL8 cells with two directly acting alkylating agents, N-nitroso-N-methylnitroguanidine (MNNG) and styrene oxide, gave no transformation, whereas a third alkylating agent, ethyl methanesulfonate (EMS), gave a weak response. Treatment with MCA (2.5 micrograms/ml) yielded a reproducible positive response and, therefore, served as a positive control for routine use of the C3H/10T1/2 CL8 assay. When cells treated with the hydrocarbons BaP, BaP-7,8-diol, or MCA were analyzed for nonspecific DNA damage (single-strand breaks or alkaline-labile sites) by alkaline elution techniques, little if any DNA damage was observed. In contrast, the alkylating agents MNNG, styrene oxide, and EMS yielded substantial numbers of single-strand breaks.