Reproductive toxicity, mutagenicity and antigenicity of pamiteplase (genetical recombination).

Pamiteplase (genetical recombination), YM866, is a novel recombinant modified human tissue-type plasminogen activator developed by Yamanouchi Pharmaceutical Co. Ltd., Tokyo, Japan. An intended route of administration in the clinical use of this drug is intravenous administration. We conducted an intravenous fertility and general reproduction studies of this drug in male and female rats and teratology study of this drug in rabbits at the dose levels of 0 (vehicle control), 0.1, 0.3 or 1 mg/kg/day. In the rat, no treatment-related abnormalities were observed up to the maximum dose in parental animals and their offspring. In the teratology study in rabbits, prolonged coagulation time at the injection site was observed at 0.3 mg/kg or more. One death and one abortion occurred at 1 mg/kg on days 22 and 23 of pregnancy, respectively. No toxic effects on the litters were observed up to the maximum dose. Results of evaluation of the mutagenicity of YM866 and its ability to induce chromosome aberrations using the L5178Y TK+/- mouse lymphoma assay, human lymphocyte chromosome aberration assay and the micronucleus assay in mice were negative. Evaluation of the immunogenicity of YM866 by repeated intravenous injection in chimpanzees elicited no confirmed antibody titers.

Evaluation of spontaneous and chemical-induced lacI mutations in germ cells from lambda/lacI transgenic mice.

The spontaneous mutant frequency in germ cells isolated from seminiferous tubules of two lambda/lacI transgenic mouse strains, C57BL/6 and B6C3F1 was evaluated. At least 500 000 phage were screened for mutation at lacI for each animal using standardized assay procedures. The germ cell spontaneous lacI mutant frequency was 17.8 +/- 8.1 x 10(-6) in C57BL/6 mice and 17.0 +/- 10.0 x 10(-6) in B6C3F1 mice. The induction of germ cell mutations by three well characterized alkylating agents were also evaluated in C57BL/6 mice on day 3 after a single dose administration. The lacI mutant frequencies were significantly elevated in transgenic mice dosed with ENU at 150 mg/kg (2-fold increase above control) and iPMS at 200 mg/kg (3-fold increase above control) but not in those receiving MMS at 40 mg/kg. These findings suggest that single dose studies using the lambda/lacI transgenic system may be capable of detecting germ mutations induced by chemicals characterized either by point mutations or small, intragenic deletions but not those characterized by a predominance of multi-locus deletions.

Evaluation of potential genotoxicity of pulsed electric and electromagnetic fields used for bone growth stimulation.

Medical devices emitting pulsed electric and electromagnetic fields have been found to be effective for a number of clinical applications including stimulation of bone and tissue growth. To determine whether pulsed fields of the type used in these clinical applications present a mutagenic hazard, electric and electromagnetic fields at two exposure levels were tested in the Ames test, CHO cell chromosomal aberration assay, BALB/3T3 cell transformation assay and unscheduled DNA synthesis assay in primary rat hepatocytes. For both field types, initial and independent repeat studies were performed for each assay at both clinical and supra clinical doses. In all assays, the results show a lack of cytotoxic, transforming and mutagenic activity. The data suggest that pulsed electric and electromagnetic fields of the type and dose levels used in bone growth stimulation lack mutagenic and transforming activity.

Multilaboratory comparison of in vitro tests for chromosome aberrations in CHO and CHL cells tested under the same protocols.

Different test results have been reported for the same chemicals in two in vitro chromosome aberration test systems, CHL cells tested by a Japanese protocol and CHO cells tested by the US National Toxicology Program [Sofuni et al., Mutat Res 241:173-213,1990]. Here, laboratories in Japan, the US and the UK tested 9 such chemicals in CHL and CHO cells using the same protocols and found all 9 positive in both cell types; differences in earlier conclusions with these chemicals were due mainly to test protocol, not to different sensitivities of the cells. The most important protocol difference is sampling time. Chemicals that were negative in the NTP series using a sampling time of 10 to 13 hours often produced positive results when retested here with a 20- to 24-hour sampling time. While positive results were obtained in both cell types, CHL cells sometimes had higher aberration levels and survived at higher doses than CHO cells would tolerate. This may reflect some intrinsic difference in sensitivity but may also be affected by factors such as cell cycle length and culture media (e.g., oxygen scavenging capacity). The collaboration reported here also contributed to a better understanding of scoring aberrations, especially "gaps"; there was good agreement on what types of aberrations should be included in the totals when scoring criteria were clearly defined, for example, many changes classified as "gaps" by the Japanese system were classified as "breaks" in the scoring systems used in the United States and the United Kingdom, and were appropriately included in total aberration counts.

Interlaboratory comparison: liver spontaneous mutant frequency from lambda/lacI transgenic mice (Big Blue) (II).

Spontaneous mutant frequency in livers of two transgenic mouse strains, each carrying identical lambda shuttle vectors with a lacI target gene, was evaluated by two laboratories. These studies investigated variability in spontaneous mutant frequency between animals and as a function of the number of phage screened. Liver DNA was independently isolated from 7-11 week old C57BL/6 and B6C3F1 Big Blue transgenic mice. At least 500,000 phage were screened for mutation at lacI for each animal using standardized assay procedures. In the two labs, the C57BL/6 liver spontaneous mutant frequency was 45 +/- 9 x 10(-6) and 41 +/- 7 x 10(-6). The B6C3F1 liver spontaneous mutant frequency was 42 +/- 10 x 10(-6) at one lab and 43 +/- 12 x 10(-6) and 41 +/- 8 x 10(-6) in two trials at the second lab. Mean mutant frequency data from both labs, calculated in increments of 100,000 plaque forming units (pfu) scored for each mouse strain, show stabilized mean mutant frequency and standard deviation after approximately 200,000-300,000 pfu screened. The frequency of spontaneous lacI mutants was reproducible both within and between labs and was comparable between the two transgenic mouse strains.

Intralaboratory optimization and standardization of mutant screening conditions used for a lambda/lacI transgenic mouse mutagenesis assay (I).

A lambda/lacI shuttle vector transgenic mouse mutagenesis assay has been optimized and standardized for reproducible mutant detection. The mutagenic endpoints are blue lacI- phage plaques on a bacterial lawn resulting from the de-repression of beta-galactosidase activity acting on the chromogenic substrate X-gal. Non-mutant lacI phage plaques remain colorless. Factors demonstrated to affect mutant detection include X-gal concentration per assay tray, plaque density per assay tray, pH of plating agar, incubation time at 37 degrees C and the use of a red translucent screening filter over a light source to enhance mutant plaque visibility. In vivo mutant frequencies for liver in untreated animals using standard protocols and internal controls were repeatable in separate experiments using lambda/lacI B6C3F1 mice (4.3 +/- 1.2 x 10(-5) and 4.1 +/- 0.8 x 10(-5)). These studies analyze the use of internal controls to monitor the level of mutant phage plaque detection in a given experiment and evaluate the repeatability of observed mutant frequencies obtained when using standardized procedures.

Cytogenetic studies on commercial hexane solvent.

Commercial hexane is a solvent mixture of six-carbon isomers, consisting principally of n-hexane, 3-methylpentane, methylcyclopentane and 2-methylpentane. The potential of commercial hexane to produce chromosome aberrations was evaluated in both an in vitro assay using Chinese hamster ovary (CHO) cells and an in vivo cytogenetics assay using Sprague-Dawley rats. The CHO cells were exposed to media containing commercial hexane at concentrations of 0.014-0.42 microliters ml-1 in the presence and absence of an S-9 activation mixture. Cellular toxicity was observed at the higher dose levels, but no increase in chromosome aberrations was observed in either the non-activated or S-9-activated systems. For the in vivo cytogenetics assay, rats were exposed nose-only for 6 h per day for 5 consecutive days to commercial hexane vapor at target concentrations of 900, 3000 and 9000 ppm. Bone marrow cells were collected at 6 and 24 h after the midpoint of the last exposure. Metaphase cells were examined microscopically for chromosome aberrations. No statistically significant increases in aberrant cells were observed in the commercial hexane-exposed animals of any dose group at either of the bone marrow harvest times. In conclusion, commercial hexane did not produce chromosomal mutations under the conditions of these studies.

Evaluation of the genotoxic potential of zinc pyrithione in the Salmonella mutagenicity (Ames) assay, CHO/HGPRT gene mutation assay and mouse micronucleus assay.

The mutagenic potential of zinc pyrithione (Znpt) was evaluated in vitro in the Salmonella/mammalian microsome plate incorporation mutagenicity (Ames) assay and the CHO/HGPRT gene mutation assay. The clastogenic potential of Znpt was evaluated in vivo using the mouse micronucleus test. Znpt was negative in the Ames test in five tester strains in the presence and absence of rat liver microsomal enzymes when assayed at concentrations ranging between 10 and 333 micrograms per plate and between 0.03 and 33 micrograms per plate, respectively. Znpt also produced negative results in the CHO/HGPRT assay. No significant increases in mutant frequencies were seen in the presence and absence of rat liver microsomal enzymes. In each case, the highest concentrations reduced cellular viability by 83% and 85%, respectively. Znpt also did not induce increased frequencies of micronuclei in mouse bone marrow cells when tested at the maximally tolerated dose (MTD) (44 mg kg-1). These data support the conclusion that Znpt lacks genotoxic activity under the conditions of these tests.

Genetic toxicology of acrylic acid.

Acrylic acid was tested for gene mutations in the in vitro CHO/HGPRT assay, for chromosome aberrations in CHO cells in culture, and for potential to induce unscheduled DNA synthesis in rat hepatocytes in culture. In vivo assays performed included the Drosophila sex-linked recessive lethal assay by both the feeding and injection routes, the in vivo cytogenetic assay in rat bone marrow cells after both a 1-day and 5-day oral dosing regimen, and a dominant lethal assay in mice by both an acute and 5-day dosing regimen. All results were negative (non-mutagenic) except for the in vitro chromosome aberration assay. This latter result is consistent with the previously reported possible clastogenic activity suggested by the results of the mouse lymphoma L5178Y TK locus assay in which a predominance of small-colony mutants was observed (Moore et al., Environmental and Molecular Mutagenesis 1988, 11, 49-63). The rapid clearance of acrylic acid in animals and the weight of evidence of genetic toxicity testing, including negative in vivo data in both somatic and germ cells, indicate a lack of genetic toxicity of acrylic acid in vivo.

Spectra of spontaneous and mutagen-induced mutations in the lacI gene in transgenic mice.

Transgenic mice with a lambda shuttle vector containing a lacI target gene were generated for use as a short-term, in vivo mutagenesis assay. The gene is recovered from the treated mice by exposing mouse genomic DNA to in vitro packaging extracts and plating the rescued phage on agar plates containing 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-Gal). Phage with mutations in the lacI gene form blue plaques, whereas phage with a nonmutated lacI form colorless plaques. Spontaneous background mutant rates using this system range from 0.6 x 10(-5) to 1.7 x 10(-5), depending upon tissue analyzed, with germ cells exhibiting less than one-third the background rate of somatic tissue. Treatment of the mice with N-ethyl-N-nitrosourea (EtNU), benzo[a]pyrene (B[a]P), or cyclophosphamide caused an induction of mutations over background. Recovery of the lacI target for sequence analysis was performed by genetic excision of a plasmid from the phage using partial filamentous phage origins. The predominant mutations identified from untreated and treated populations were base substitutions. Although it has been shown by others that 70% of all spontaneous mutations within the lacI gene, when replicated in Escherichia coli, occur at a hot spot located at bases 620-632, only 1 of 21 spontaneous mutations has been identified in this region in the transgenic mouse system. In addition, 5 of 9 spontaneous transitions analyzed occur at CpG dinucleotides, whereas no transition mutations were identified at the prokaryotic deamination hot spots occurring at dcm sites (CCA/TGG) within the lacI gene. For EtNU, approximately equal amounts of transitions and transversions were observed, contrasting with B[a]P-induced mutations, in which only transversions were obtained. In addition, B[a]P mutagenesis showed a predominance of mutations (81%) involving cytosines and/or guanines, consistent with its known mode of action. The discovery of a spontaneous mutation spectrum different from that of bacterial assays, coupled with the concordance of EtNU and B[a]P base mutations with the known mechanisms of activity for these mutagens, suggests that this transgenic system will be useful as a short-term, in vivo system for mutagen assessment and analysis of mechanisms leading to mutations.

Analysis of spontaneous and induced mutations in transgenic mice using a lambda ZAP/lacI shuttle vector.

A short term, in vivo mutagenesis assay has been developed utilizing a lacl target gene contained within a lambda ZAP shuttle vector which has been incorporated into transgenic mice. Following chemical exposure, the target gene was recovered from mouse genomic DNA by mixing the DNA with in vitro lambda phage packaging extract. Mutations within the lacl target were identified by infecting host E. coli with the packaged phage and plating on indicator plates containing Xgal. Phage plaques with mutations in the lacl appeared blue, while intact phage were colorless. The ratio of blue plaques to colorless plaques is a measure of the mutagenicity of the compound. This system was used to obtain significant induction (up to 74-fold) over background levels for a variety of compounds, including N-ethyl-N-nitrosourea, benzo(a)pyrene (BaP), cyclophosphamide, and methylnitrosourea. Sequence analysis of selected mutant clones derived from this system was accomplished through the use of partial filamentous phage origins which allow rapid transfer of the target gene from phage to plasmid. Sequence analysis of spontaneous mutants derived from the mice primarily found of base substitutions, differing markedly from the previous data for spontaneous mutations in lacl derived from E. coli, where the preponderance of mutations were found at a single site, a repeated tetramer sequence. Upon sequence analysis of BaP derived base substitutions, only transversions were obtained, consistent with the known mechanism of BaP mutagenesis. Use of the well-characterized lacl gene in transgenic mice should allow for extrapolation of the extensive pool of in vitro data to whole animals, as well as provide insight into the tissue specific effects of mutagenic compounds.

Genotoxicity evaluation of lithium hypochlorite.

Lithium hypochlorite (LiOCl), the pool and spa sanitizer/algicide, was evaluated for genotoxicity in a battery of studies designed to evaluate potential mutagenicity, DNA damage and chromosome aberrations. LiOCl was not mutagenic in the Ames test when tested in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, TA1538 or in the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) mutation assay in Chinese hamster ovary (CHO) cells without metabolic activation. LiOCl did not induce DNA damage in the unscheduled DNA synthesis assay using rat primary hepatocytes. Effects on metaphase chromosomes were evaluated in vitro in CHO cells at 12 and 18 h exposure without S9 and at 12 and 22 h following a 2 h exposure with S9. LiOCl induced a statistically significant increase in chromosome aberrations at the high dose only at both harvest times without S9 and at the late harvest time with S9. There were significant increases in chromosome aberrations at the low dose, low-mid and high doses, but not at the high mid-dose at the early harvest time with S9. However, LiOCl did not increase chromosome aberrations when tested orally in rats at maximally tolerated doses. Bone marrow cells, collected 6, 24 and 48 h after a single oral dose of LiOCl to rats (100, 500, 1000 mg/kg in males; 50, 250, 500 mg/kg in females) showed no increase in the incidence of aberrations. In general, the weight of the evidence indicates that LiOCl is not genotoxic.

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.

In vitro and in vivo evaluation of the genotoxic potential of 2-ethyl-1,3-hexanediol.

2-Ethyl-1,3-hexanediol (EHD) has intentional human exposure because of its application to skin as an insect repellent and its use in various skin care products. Genotoxicity studies on EHD were conducted to determine mutagenic and clastogenic potential using in vitro and in vivo test systems. In vitro tests were conducted both with and without an Aroclor-induced, rat-liver S9 metabolic activation system and within a range of cytotoxic to non-cytotoxic doses. EHD did not produce dose-related positive increases in gene mutations in the Salmonella (Ames) test or in the CHO/HGPRT forward mutation test. No statistically significant or dose-related increases in sister chromatid exchanges indicative of DNA damage were produced by EHD in CHO cells. Small but statistically significant increases in chromosome aberrations were produced in CHO cells only in tests with S9 activation. However, no evidence of clastogenicity of EHD was obtained in vivo in a mouse peripheral blood micronucleus test or in 2 rat bone marrow chromosome aberration studies using single or repeated dosing procedures. The overall negative pattern of mutagenic and clastogenic results in the majority of tests conducted suggests that EHD is unlikely to pose significant hazard as a genotoxic agent or to possess carcinogenic initiating activity in animals.

Genotoxicity of fecapentaene-12 in bacterial and mammalian cell assay systems.

Fecapentaene-12 (Fec-12), a compound thought to be responsible for much of the mutagenicity in fecal extracts from groups at high risk for colon cancer, was assayed for genotoxic potential in a battery of bacterial and mammalian cell short-term assays. This compound demonstrated significant mutagenic activity with Salmonella typhimurium tester strains TA104, TA100 and TA98, inducing approximately 1400, 700 and 100 revertants/micrograms, respectively. Fec-12 caused dose-dependent increases in unscheduled DNA synthesis in both rat hepatocytes and human fibroblasts, indicating its potential genotoxicity to mammalian as well as bacterial cells. Finally, Fec-12 had the ability to induce neoplastic transformation in mouse BALB/c 3T3 cells in the absence of exogenous metabolic activation.

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.

Chemical enhancement of SA7 virus transformation of hamster embryo cells: evaluation by interlaboratory testing of diverse chemicals.

Twelve chemicals from diverse structural classes were tested under code for their capacity to enhance the transformation of Syrian hamster embryo cells by simian adenovirus SA7 in two independent laboratories. Pretreatment of hamster cells with eight of those chemicals (reserpine, dichlorvos, methapyrilene hydrochloride, benzidine dihydrochloride, diphenylhydantoin, cinnamyl anthranilate, 11-aminoundecanoic acid, and 4,4'-oxydianiline) produced repeatable enhancement of SA7 transformation at two or more consecutive dose levels, which constitutes clear evidence of enhancing activity in this assay. Both toxic and nontoxic doses of each of these chemicals caused enhancement of virus transformation. Two chemicals (2,6-dichloro-p-phenylenediamine and cinnamaldehyde) produced some evidence of enhancing activity (repeatable transformation enhancement at one dose). Dose ranges for cytotoxicity and enhancement of SA7 transformation were similar in both laboratories for all chemicals producing activity. The final two chemicals, chloramphenicol sodium succinate and ethylene thiourea, failed to reproducibly demonstrate either significant cytotoxicity or enhancement of SA7 transformation at concentrations up to 10-20 mM. The test results for these 12 chemicals were combined with the test results for 9 known carcinogens and noncarcinogens in order to evaluate relationships between activity, dose response, and lowest effective enhancing concentration for these compounds, as well as to correlate them with rodent carcinogenesis classifications. The Syrian hamster embryo cell-SA7 system demonstrated reproducible test responses in both intra- and interlaboratory studies and detected 13 out of 15 known rodent carcinogens.

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.