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Mutagenicity in v79 cells does not correlate with carcinogenity in small rodents for 12 aromatic amines a

b

Gianfranco Fassina , Angelo Abbondandolo , Laura Mariani d

, Maurizio Taningher & Silvio Parodi

c

d

a

Istituto Nazionale per la Ricerca sul Cancro, Genoa—Centro di Studio per la Neurofisiologia Cerebrale , C.N.R. , Viale Benedetto XV, 10, Genova, 16132, Italy b

Istituto Nazionale per la Ricerca sul Cancro, Genoa—Department of Genetics , University of Genoa , Italy c

Istituto di Mutagenesi e Differenziamento , C.N.R. , Pisa, Italy d

Istituto Nazionale per la Ricerca sul Cancro, Genoa—Department of Oncology , University of Genoa , Italy Published online: 20 Oct 2009.

To cite this article: Gianfranco Fassina , Angelo Abbondandolo , Laura Mariani , Maurizio Taningher & Silvio Parodi (1990) Mutagenicity in v79 cells does not correlate with carcinogenity in small rodents for 12 aromatic amines, Journal of Toxicology and Environmental Health: Current Issues, 29:1, 109-130, DOI: 10.1080/15287399009531376 To link to this article: http://dx.doi.org/10.1080/15287399009531376

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MUTAGENICITY IN V79 CELLS DOES NOT CORRELATE WITH CARCINOGENITY IN SMALL RODENTS FOR 12 AROMATIC AMINES Gianfranco Fassina Istituto Nazionale per la Ricerca sul Cancro, Genoa—Centro di Studio per la Neurofisiologia Cerebrale, C.N.R., Genoa, Italy

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Angelo Abbondandolo Istituto Nazionale per la Ricerca sul Cancro, Genoa—Department of Genetics, University of Genoa, Italy Laura Mariani Istituto di Mutagenesi e Differenziamento, C.N.R., Pisa, Italy Maurizio Taningher Istituto Nazionale per la Ricerca sul Cancro, Genoa—Department of Oncology, University of Genoa, Italy Silvio Parodi Istituto Nazionale per la Ricerca sul Cancro, Genoa—Department of Oncology, University of Genoa, Italy

The aim of this investigation was to study the correlation between carcinogenicity in small rodents and mutagenic potency of aromatic amines, as measured by the induction of 6-thioguanine resistance in V79 Chinese hamster cells. It has been previously shown that the carcinogenic potency of these compounds is not correlated to their ability to induce DNA breakage, SCEs, or point mutations in bacteria, but a correlation exists with autoradiographic DNA repair test (in primary hepatocyte cultures).

This work was supported by CNR, Special Project "Oncology," contracts 88.00797.44 and 88.00485.44; by EEC, contract EV-0036-I (A); and by a grant from Regione Liguria. We are grateful to Drs. Adriana Albini and Douglas Noonan for revising the manuscript and to Miss Cabriella Frigerio for typing it. Requests for reprints should be sent to Gianfranco Fassina, Istituto Nazionale per la Ricerca sul Cancro, Viale Benedetto XV, 10, 16132 Genova, Italy. 109 Journal of Toxicology and Environmental Health, 29:109-130, 1990 Copyright © 1990 by Hemisphere Publishing Corporation

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Twelve aromatic amines were tested and the rat liver S9 fraction was routinely incorporated in the mutation assay; mouse liver and hamster liver S9 fractions were a/so used as metabolizing systems. The comparison of the ranks of mutagenic and oncogenic potencies by means of the Spearman test shows no correlation between carcinogenicity and V79 cell mutagenicity of the tested aromatic amines. There was a generally low mutagenicity seen for aromatic amines in V79 cells. In some cases this could be attributed to an insufficient metabolic activation by rat S9. For example, benzidine, which was inactive when assayed in the presence of rat S9, became mutagenic when in the presence of mouse S9. On the other hand, hamster S9, which has been shown to be the best activating system for 2-acetylaminofluorene in the Ames test, did not activate this compound in V79 cells. Inadequate metabolic activation of the standard system (rat S9) used in this work could explain the low mutagenicity and the lack of correlation observed between mutagenicity and carcinogenicity. A second possibility is that point mutation is not the essential end point for the initiating activity of aromatic amines during the carcinogenic process. A third possibility is that the activity of some aromatic amines is not restricted to the initiation step in carcinogenesis. Chronic treatments with the sublethal doses often result in significant promoting activities, which could mask efficiently the initiating potential of the same chemicals.

INTRODUCTION In previous studies we have shown that a quantitative approach to correlation between short-term tests and carcinogenicity data in small rodents seems feasible (Parodi et al., 1982a, 1982b, 1984a, 1984b; Bolognesi et al., 1986; Taningher et al., in press). This possibility is supported by two weeks of Peto, Ames, and coworkers for carcinogenicity data (Gold et al., 1984; Peto et al., 1984) and a work of Waters (IARC, 1987) for short-term test data, in which the authors have made an attempt not only to give the qualitative result but also to measure the potency of the compounds tested. In the present study we have investigated a group of 12 aromatic amines. These compounds are very important in chemical industry and have a widespread utilization in the modern world. Previous works with chemicals of this class have shown that it is apparently difficult to find a short-term test with an accurate predictivity of their carcinogenic potential. No predictivity whatsoever was found for the SCEs induction in bone marrow cells (Parodi et al., 1983) and for the alkaline DNA fragmentation in vivo (Parodi et al., 1981). For the Ames test no predictivity was found utilizing the response obtained in the most sensitive of four different strains. A low degree of predictivity (correlation coefficient between test results and oncogenic potencies: r = .4) was found selecting a posteriori the response obtained only in the TA98 strain (Parodi et al., 1981). On the contrary, a reasonable predictivity (r = .54; p < .05) was shown by autoradiographic DNA repair in rat liver cells (Bolognesi et al., 1986).

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Mutagenicity in mammalian cells is usually considered a relevant end point for prediction of carcinogenic potential. Some authors (Clive et al v 1979; Kuroki et al., 1980) have shown a good correlation between the data obtained in these systems and carcinogenicity for compounds belonging to different chemical classes. However, contrasting results have been obtained testing aromatic amines for mutagenicity with different test systems (Knaap et al., 1981; Carver et al., 1981; Jotz and Mitchell, 1981; Oglesby et al., 1983; Coppinger et al., 1984). For these reasons and to investigate whether a mutagenicity assay in mammalian cells could give a better predictivity of carcinogenicity than the tests already examined, a representative group of 12 aromatic amines has been tested in homogeneous conditions. We detected the 6thioguanine (6-TG) resistant mutants in the hypoxanthine guanine phosphoribosyltransferase (HGPRT) system in V79 Chinese hamster cells. MATERIALS AND METHODS Chemicals

Chemicals were obtained as follow: 2-acetylaminofluorene (AAF) and 1-naphthylamine from Sigma, St. Louis, Mo.; aniline, auramine O, and rhodamine B from Merck, Darmstadt, Federal Republic of Germany; pdimethylaminoazobenzene (DAB) from Fluka, Bucks, Switzerland; 2nephthylamine from Serva, Heidelberg, Federal Republic of Germany; phenacetin from Carlo Erba, Milan, italy; benzidine either from Sigma or Imperial Chemical Industries (ICI), Macclesfield, United Kingdom; and 4, 4'-diaminoterphenyl (DAT) from ICI. Benzidine and DAT were supplied by Dr. J. Ashby, Central Toxicology Laboratory of ICI, in the context of the II UKEMS (United Kingdom Environmental Mutagen Society) Collaborative Study. Benzidine from Sigma and 2-naphthylamine were research grade; auramine O, DAB, and rhodamine B were standard grade; and the chemical purity of the other compounds was as follows: 1naphthylamine, 90%; AAF, 96%; phenacetin, 98%; aniline, 99.5%; DAT and benzidine from Dr. Ashby, 99%. Dulbecco's modified Eagle's medium (D-MEM), Leibovitz L-15 medium (L-15), fetal calf serum (FC), HEPES, Dulbecco's phosphate-buffered saline (PBS), and trypsin solution were purchased from Flow Laboratories, Irvine, United Kingdom; flasks and petri dishes from Corning, Corning, N.Y.; 6-thioguanine (6-TG), ethylenglycol-bis-(2-aminoethylether)N,/V'-tetraacetic acid (EGTA), collagenase I, and |8-naphthoflavone from Sigma; phenobarbital and dimethylnitrosamine (DMN) from Merck; Aroclor from Monsanto, St. Louis, Mo.; benzo[a]pyrene (BaP) from Fluka; dimethyl sulfoxide (DMSO) from Carlo Erba; /3-nicotinamide adenine dinucleotide phosphate (NADP) and glucose 6-phosphate (G6P) from Boehringer, Mannheim, Federal Republic of Germany; penicillin and

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streptomycin from Flow; and rats and hamsters from Charles River, Como, Italy. Cell Culture

V79 cells were cultured under standard conditions of 5% CO2 in air at 37°C in an incubator humidified to 100%. They were routinely maintained as monolayers on plastic culture tissue dishes in D-MEM supplemented with 5% FC, penicillin G (100 U/ml), and streptomycin sulfate (100 Mg/ml). For subculturing, a trypsin-EDTA solution (0.05% trypsin 1:250 and 0.02% EDTA) was used. Population doubling time was 11-12 h and cloning efficiency normally 80-100%.

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Mutagenicity Assay

Resistance to 6-TG was used as a marker for measuring mutation induction in V79 Chinese hamster cells in the HGPRT system (Kuroki et al., 1980). Cells (2.5 x 106 were plated in 25-cm2 flasks and incubated overnight. After removing the medium, they were incubated at 37°C for 1 h with the tested chemical dissolved in DMSO and diluted in serumfree D-MEM with 20 mM HEPES buffer. About 5 x 106 cells were present at the time of the mutagenic treatment, corresponding to a density of about 2 x 105 cells/cm2. Treated cells were washed twice with prewarmed PBS, trypsinized, and resuspended in D-MEM with 5% FCS. They were then seeded in three 90-mm petri dishes at 5 x 105 cells/dish. 6-TG (5 jug/ml) was added after cell attachment. Three 60-mm dishes were plated with 100 cells/dish for evaluation of cell survival. At the same time five 90mm dishes, with 3 x 105 cells each, were incubated for 5 d (with an intermediate subcultivation) to obtain a new set of plates for the evaluation of the mutation frequency and cell survival, as done at zero time (114 h expression time). An identical procedure was repeated 162 h after treatment. Mutation and survival plates were stained with methylene blue for colony counting after 10 and 7 d of incubation, respectively. Mutation frequencies were corrected for the number of viable cells at the corresponding expression time and expressed as mutants/105 cells. When mean mutation frequencies and SD were calculated, data were not given a different relevance (weight) according to the initial population size, but negative results based on populatoins 24 mutants/106 cells, a new stock was unfrozen and utilized. No experiment, however, was discarded. No significant differences in mutation frequency were observed among the different expression times for untreated samples and 0 h expression time for treated samples Because of the length of time (about 18 mo) needed to test all the aromatic amines considered in this study, particular care was taken to check positive and negative controls that were performed during the entire time of experimentation. The results regarding positive controls for each new batch S9 (cells treated with DMN and BaP) are shown in Table 2, whereas the global situation of negative controls (historical controls including those matched to test samples) is illustrated in Fig. 1. Each S9 batch was used within 5 mo after preparation. In our experience S9 preparations are stable under liquid nitrogen for this span of time as tested with DMN (3-10 mM) or BaP (10-100 fiM) every two or three experiments (data not shown). Positive standards and relative dosages are reported in Table 2. The performance of different batches of rat liver S9 was sufficiently invariant for our purposes. We were unable to find suitable strongly positive aromatic amines to be tested as positive standards in our system. The results shown in Table 1 indicate that there was no strong muta-

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TABLE 1 . Mutagenicity of the 12 Aromatic Amines Tested as Evaluated with the V79/HGPRT System

Chemical

Metabolic activation

AAF

None

Rat S9e

Hamster S9^

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Aniline

None

Rat S9e

Rat hepatocytes

Auramine O

None

Rat S9e

Benzidine

None

Rat S9e

RatS9S

Mouse S9*

Concentration 0% positive in control experiments. ± , 20% positive treated experiments. +, >20% Positive treated experiments and 0% positive control experiments. + +; >40% Positive treated experiments and 0% positive control experiments. 'NS, p > .05 at each dose tested. g The probability that r - 0 is given in parentheses. h \n this column a numerical synthetic view of the results obtained with the three statistical approaches is shown. To the various symbols the following values are attributed: ± , 0.5; + , 1 ; + +, 2; p < .05,1; p < .02, 2. 'S9 from rats induced with phenobarbital and /3-naphthoflavone. 'S9 from hamster induced with Aroclor. ^Significant only at the concentration of 60 mM. 'Significant only at the concentration of 4 mM. m S9 from rats induced with Aroclor. n S9 from uninduced mice. "According to the concentration tested; 3 mM not significant. ''Calculated from the ascendent part of the curve (0-0.75 mM). 'Significant only at the concentration of 0.025 mM. 'Data from Loprieno et al. (1982). s Data from N. Loprieno, personal communication. 'All doses were pooled. "For each treatment. Significant for 2- and 4-h treatments. In this case in the equation of the regression line y — a + bx, x was not simply the concentration Cof the chemical, but the parameter (Ct) where r is the exposure time. "'Significant only at the concentration of 7.5 mM. Significant at all doses tested.

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should also be considered as positive on the basis of this analysis. Unequivocal negative responses were obtained with AAF, DAB, 2,4diaminotoluene, rhodamine B, and with most substances when assayed without activation or with inefficient metabolicy systems, as in the case of benzidine. If we look at the cumulative statistical evaluation for the most active treatment (Table 3), a relatively clear discrimination between positive and negative results seems possible because no positivity lower than 2.5 and no negativity higher than zero was observed. The low mutagenicity and the discrepancies in the statistical evaluation of the responses that have been found in this study are not surprising in light of previous studies analyzing aromatic amines in different systems [see Prog. Mutat. Res. 1: (1981) and Le et al. (1985)]. A modest increase in the mutation frequency of the positive compounds is exactly what one would expect from weak mutagens, whose demonstration of activity requires optimal conditions with reference to dosage, type of metabolic activation, choice of cells and markers, and other features of the detection system. Predictive Capability of the V79/HGPRT System for Aromatic Amines

To evaluate the correlation existing between mutagenicity in the V79/HGPRT system and carcinogenicity elicted by the 12 aromatic amines considered in this study, two different approaches were followed. The first is a quantitative one and compares each to the carcinogenic and mutagenic potency parameters. The second approach is derived from the first one but is more qualitative in nature. With this approach chemicals were classified for mutagenicity and carcinogenicity by ranks of potency and then subdivided in two subsets: chemicals in the first half of the classification and chemicals in the second half. The number of correct couplings for subset was then evaluated. The aim of these computations was to investigate the capability of the V79/HGPRT system to predict the carcinogenic potency of compounds belonging to a well-defined chemical class. The qualitative correlation between short-term test results and carcinogenicity has been evaluated to be about 90% in the first studies (McCann et al., 1975; McCann and Ames, 1976; Sugimura et al., 1976). More recent investigations have steadily reduced the correlation to a level of about 60% (ICPEMC, 1984; Tennant et al., 1987). It has been noted by other authors that when the fraction of nongenotoxic chemicals in a data base goes up, correspondingly the correlation between carcinogenicity and short-term tests results goes down (ICPEMC, 1988; Ramel, 1988; Rosenkranz, 1988). The predictivity of carcinogenic potency obtainable with the V79/HGPRT system for the examined aromatic amines is reported in Tables 4 and 5.

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TABLE 4. Mutagenic and Oncogenic Potencies of the 12 Aromatic Amines Testeda Ranks

Compounds

OPI b

Ranks

Compounds

MPl

1 2

DAT AAF 2,4-Diaminotoluene

2080c 265 123

1 2

3

Benzidine DAB Auramine 0 2,4-Diaminoanisole 2-Napthylamine Phenacetin

88.5 68.9 29.2 12.3 8.2 0.284

DAT Benzidine 2,4-Diaminotoanisole 2-Naphthylamine 1-Naphthylamine Phenacetin Auramine 0

440 61.1 20.8 14.7 13.2 2.6 2.5

1-Naphthylamined Aniline Rhodamine Bd

iae ia ia

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4 5

6

3

4 5

Aniline AAF DAB Rhodamine B 2,4-Diaminotoluene

0.5 iae ia ia ia

a The subsets span a 10 times interval. The last MPl and OPl subsets include the inactive compounds. MPl (mutagenic potency index) and OPl (oncogenic potency index) are defined in the discussion section. b OPI data were obtained from Gold et al. (1984), except data marked with c and d. c Obtained from Eldridge et al. (1985). d Bonser et al. (1956).

e

ia., inactive.

For the quantitative evaluation (Table 4) we adopted the data base elaborated by Gold et al. (1984), for potencies of carcinogenicity, whose approach is sufficiently similar to ours (Parodi et al., 1982a, 1984a, 1984b). In that data base carcinogenic potency is defined, according to Peto et al. (1984), as TD50. For each chemical the experimental dose giving the highest oncogenic potency was selected. The oncogenic potency indices (OPl values) reported in Table 4 were computed by us as 1/TD50 (with TD50 expressed in mmol/kg-d). The mutagenic potency indices (MPl values) were calculated by dividing the effect observed by the concentration administered in the V79/HGPRT system. A correction was also introduced to compensate for the length of treatment: MPl +

number of resistant cells (above control) x 10~ (millimoles) x (hours of treatment)

Also for MPl values the dosage giving the highest potency was utilized. In order to take into account only relatively major differences, OPl

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TABLE 5. Subsets of Aromatic Amines above or below Average Ranking for Carcinogenicity and Mutagenicity

OPI,A

OP1,B

DAT AAF 2,4-Diaminotoluene Benzidine DAB Auramine 0

2,4-Diaminoanisole 2-Naphthylamine Phenacetin 1-Napthylamine Rhodamine B Aniline

OPI,A

OPI, B

DAT Benzidine 2,4-Diaminoanisole 1-Naphthylamine 2-Naph thylamine Phenacetin

Auramine O Aniline AAF DAB Rhodamine B 2,4-Diaminotoluene

a

Above average ranking, A and C; below average ranking, B and D.

and MPI values were classified in ranks of potency spanning a 10 times interval, as shown in Table 4. For both values, in active compounds were classified in the last rank of potency. Since the difference in potency of chemicals belonging to different consecutive ranks is 10 times on average, it seems evident that differences in computation methods that could have given a different result by two or even three times are minor and relatively unimportant. Examining the variability of our positive controls, it appears very unlikely that our mutagenic responses could be wrong for more than three times. This tolerance ensures a higher degree of objectivity to the computations. For statistical analysis we considered appropriate the Spearman test for rank correlation (Siegel, 1956). Using the couples of MPI and OPI values relative to each chemical reported in Table 4, we obtained a correlation coefficient rs •= .10. In other words, absolutely no correlation was found between mutagenic and carcinogenic potencies. With the qualitative approach the chemicals were divided in four subsets (Table 5): subset A, chemicals in the first half of rank classifications for carcinogenic potency; subset B, chemicals in the second half of the above classification; subset C, chemicals in the first half of rank classification for mutagenic potency; and subset D, chemicals in the second half of the above classification. If no correlation exists between carcinogenicity and mutagenicity the chemicals of set A will have a 50% chance of belonging also to set C, and a similar reasoning holds for set B and set D. As shown in Table 5, sets A

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and C have only two compounds in common and the same is true for set B and set D, so the response is slightly worse than an average random distribution. This special type of qualitative approach is probably better than a subdivision in positive and negative compounds, because we have absolutely no guarantee that the threshold of sensitivity of the two tests would be similar. On the other hand, it is perhaps worthwhile to underline that the difference between the quantitative and qualitative approach is only a difference of degree. For each test we go down from five or six ranks to only two ranks, but the two approaches remain formally homogeneous. Some caution is needed in accepting the complete negativity of these results because, the sample being rather small from a statistical point of view, it is subjected to very large confidence limits. However, even with this caution in mind, the negative result found remains the most likely. A further reason for caution in the interpretation of the results is related to the fact that the purity of the compounds tested is not extreme. A similar argument holds in general for the chemicals used in the carcinogenicity studies. If the methods of chemical synthesis were different, which is not unlikely, different impurities could be present in different amounts. This possibility can add further statistical noise (difficult to evaluate) to the correlation analysis. CONCLUSIONS Up to now the group of aromatic amines considered in this study has been investigated in several short-term tests: alkaline DNA fragmentation (Parodi et a!., 1981), Ames test (Parodi et al., 1981), SCE induction (Parodi et al., 1983), and autoradiographic repair test (Bolognesi et al., 1986). In all cases except DNA repair we have always found essentially a complete lack of correlation between short-term test results and carcinogenic potencies. For the Ames test some correlation was found, but only selecting arbitrarily a posteriori the results obtained in a single strain. With the present work we show now that even the test of mutagenicity in mammalian cells (6-thioguanine resistance) is not predictive for this class of compounds. This trend is more the exception than the rule: in fact, the global predictivity of carcinogenicity for all the shor-term tests we examined was statistically significant although not very high (Parodi et al., 1988). The majority of the aromatic amines we investigated are not very potent carcinogens (on a molar basis) and our results become not very surprising in view of the recent findings of Tennant et al. (1987). Perhaps a better correlation could have been found using other aromatic amines. However, the results we have obtained up to now do not justify the use of the HGPRT mutation test in mammalian cells for the prediction of the

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aromatic amines carcinogenic potential. Our judgment is of course referring only to the standard test: perhaps an ad hoc modified test could display a better predictivity of carcinogenicity. In addition, we have to take into account the possibility that in longterm experiments on carcinogenicity at subtoxic doses some aromatic amines act as promotors in the carcinogenetic process. Classical works of experimental pathologists, working for instance with the rat liver model or the skin model of carcinogenesis, have underlined the dramatic role played in carcinogenesis by effects of promotion. Recent papers on hazard evaluation (Tennant et al., 1987; Ashby and Tennant, 1988) have pointed out the possibility that chronic experiments of carcinogenesis in rodents could sometimes reflect to a larger extent promoting activities more than effects of initiation. As a consequence, our results should be more correctly correlated with a test for initiation (initiation with aromatic amines + standard promotion and evaluation of preneoplastic nodules or papillomas in different tissues) than with a global test of carcinogenicity. Because of this, for the moment we cannot discard the possibility that our test is indeed an acceptable predictor of the initiating potential for our class of chemicals. Correlation studies with initiation, instead of global carcinogenicity, could become the future trend for a better assessment of usefulness of short-term tests of genotoxicity. However, at present, the number of chemicals tested with initiation-promotion experiments is very limited (Tsuda et al., 1980; Ito et al, 1988). REFERENCES Ashby, J., and Tennant, R. W. 1988. Chemical structure. Salmonella mutagenicity and extent of carcinogenicity as indicators of genotoxic carcinogenesis among 222 chemicals tested in rodents by the U.S. NCI/NTP. Mutat. Res. 204:17-115. Bolognesi, C., Taningher, M., Parodi, S., and Santi, L. 1986. Quantitative predictivity of carcinogenicity of the autoradiographic repair test (primary hepatocyte cultures) for a group of 80 chemicals belonging to different chemical classes. Environ. Health Perspect. 70:247-253. Bonser, G. M., Clayson, D. B., and Jull, J. W. 1956. The induction of tumors of the subcutaneous tissues, liver and intestine in the mouse of certain dye stuffs and their intermediates. Br. J. Cancer 10:653-667. Bradley, M. O., Bhuyan, B., Francis, M. C., Langenbach, R., Peterson, A., and Huberman, E. 1981. Mutagenesis by chemical agents in V79 Chinese hamster cells: A review and analysis of the literature. A report of the Gene-Tox Program. Mutat. Res. 87:81-142. Carver, J. H., Salazar, E. P., Knize, M. G., and Wandres, D. L. 1981. Mutation induction at multiple loci in Chinese hamster ovary cells: The genetic activity of 15 coded carcinogens and noncarcinogens. Prog. Mutat. Res. 1:594-601. Clive, D., Johnson, K. O., Spector, J. F. S., Batson, A. G., and Brown, M. M. M. 1979. Validation and characterization of the L5178Y/TK mouse lymphoma mutagen assay system. Mutat. Res. 59:61108. Coppinger, W. J., Brennan, S. A., Carver, J. H., and Thompson, E. D. 1984. Locus specificity of mutagenicity of 2,4-diaminotoluene in both L5178Y mouse lymphoma and AT3-2 Chinese hamster ovary cells. Mutat. Res. 135:115-123.

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Mutagenicity in V79 cells does not correlate with carcinogenity in small rodents for 12 aromatic amines.

The aim of this investigation was to study the correlation between carcinogenicity in small rodents and mutagenic potency of aromatic amines, as measu...
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