Mutation Research, 259 (1991) 111-121

111

Elsevier MUTGEN 01626

Genotoxicity of 1-nitronaphthalene in Chinese hamster V79 cells B . G . B o y e s a, C . G . R o g e r s a a n d R . S t a p l e y b a Toxicology Research Division and b Food Statistics and Operational Planning Division, Bureau of Chemical Safety, Food Directorate, Health Protection Branch, Health and Welfare Canada, Ottawa, Ont. K1A OL2 (Canada)

(Received 21 May 1990) (Revision received 31 July 1990) (Accepted 31 July 1990)

Keywords: Sister-chromatidexchange; Thioguanine resistance; Short-term tests; 1-Nitronaphthalene

Summary 1-Nitronaphthalene (1-NN) has been identified in the U.S. National Toxicology Program as a noncarcinogen showing some evidence of in vitro genotoxicity. We tested this compound in Chinese hamster V79 cells at 2 0 - 8 0 / ~ g / m l with two endpoints: sister-chromatid exchange (SCE) and thioguanine resistance (TGR), with 5 repeat experiments. The SCE values in the presence of rat or hamster hepatocytes were consistently above the 95% and usually the 99% upper confidence limits for the corresponding control. Without hepatocyte activation, the control upper confidence limits were not exceeded except in one experiment in which the control SCE value was unusually low. T G R was scored both as proportion of plates with mutant colonies and as number of mutant colonies per plate. In 2 of 5 experiments, these values exceeded control 95% or 99% upper confidence limits; on the other hand, these values were substantially lower than those of the positive controls, dimethylbenz[a]anthracene (2.6 /~g/ml) with activation and ethyl methanesulfonate (155/~g/ml), which is direct-acting. For T G R, activation of 1-NN by either rat or hamster hepatocytes produced inconsistent results. Overall we would consider this compound to be a weak genotoxin, to which a cancer bioassay would be expected to be relatively insensitive.

Genotoxic non-carcinogens are of importance in assessing the power of short-term tests to predict carcinogenicity (Ashby and Purchase, 1985; Ashby and Tennant, 1988; Heddle, 1988; Ray et al., 1987; Shelby, 1988; Shelby and Stasiewicz, 1984; Tennant et al., 1987; Zeiger, 1987). 1Nitronaphthalene (1-NN) has been identified by

Correspondence: Dr. B.G. Boyes, Toxicology Research Division, Bureau of Chemical Safety, Food Directorate, Health Protection Branch, Health and Welfare Canada, Ottawa, Ont. K1A 0L2 (Canada).

Shelby and Stasiewicz (1984) as a non-carcinogen with in vitro genetic activity. Ennever and Rosenkranz (1986) interpret the same genotoxicity data to be inconclusive for prediction of carcinogenicity of this compound in both the National Toxicology Program test battery and in their own proposed test battery. 1 - N N is mutagenic in Salmonella, particularly in TA100, in the absence of $9 and in nitroreductase-producing strains (Benkendorf, 1978; Dunkel et al., 1985; E1-Bayoumy and Hecht, 1982; Massaro et al., 1983; Mortelmans et al., 1986; Tokiwa et al., 1981; Vance and Levin, 1984). In m a m -

0165-1218/91/$03.50 © 1991 ElsevierScience Publishers B.V. (Biomedical Division)

112

malian cell culture, chromosome aberrations were produced but sister-chromatid exchanges (SCE) were not (Shelby and Stasiewicz, 1984). 1-NN is non-mutagenic in Drosophila (Valencia et al., 1985). No evidence of carcinogenicity was demonstrated in the National Cancer Institute bioassay in Fischer 344 rats or B6C3F1 mice (NCI 1978). The tendency has been to interpret difficult-toclassify compounds as 'equivocal' or 'indeterminate' and to include these arbitrarily with either positive or negative genotoxins with consequent implications as to the predictive power of short-term tests. Since the chromosome aberration and SCE activity results for 1-NN were discordant and so far as we can determine not yet published in full, we have further characterised the genotoxic activity of this compound in mammalian cells in vitro. We assessed the genotoxicity of 1-NN at 2 0 - 8 0 / ~ g / m l in Chinese hamster V79 cell cultures in 5 separate experiments, using a screening procedure (Boyes et al., 1990) involving a cytogenetic endpoint (SCE) and a mutation endpoint (thioguanine resistance, T G R). Materials and methods

Cells The V79 Chinese hamster lung cells used throughout this study were derived from a culture kindly provided by Dr. E. Elmore, NSI Technology Services Corp., Research Triangle Park, NC (U.S.A.). The cells were grown and maintained in Williams' medium E (WE) (Gibco Canada, Burlington, Ont., Canada) containing 10% fetal bovine serum (Dextran Products, Toronto, Ont.), L-glutamine (2 mM) (Flow Laboratories, Mississauga, Ont.) and gentamicin (10/~g/ml) (Schering Corp., Kenilworth, N J, U.S.A.). At regular intervals, cultures were pregrown in antibiotic-free medium and found to be free of Mycoplasma as determined by the Hoechst 33258 fluorochrome stain technique (Chen, 1977).

contaminant, samples of the supplied material were examined by Fourier transform infrared ( F T - I R ) spectroscopy, capillary gas chromatographic-mass spectrometric ( G C - M S ) analysis and by high-resolution (400 MHz) 1H-NMR spectroscopy. The F T - I R analysis ruled out the possibility of any appreciable 2-NN content in the 1-NN material, except to the extent that a minor peak for 2-NN could remain undetected in the base line envelope of the 1-NN IR spectrum. The G C - M S demonstrated a single component indicative of chromatographically pure material, which very closely matched the NBS mass spectral reference for 1-NN and 2-NN, which are not distinguished by this test. Moreover, high-resolution 1H-NMR analysis, which would distinguish 1-NN from 2-NN, revealed a clean spectrum consistent with 1-NN with no indication of the presence of the 2-NN isomer. These results are consistent with purity of the 1-NN material of at least 99.9%. Ethyl methanesulfonate (EMS) was from Eastman Kodak Company, Rochester, NY; 6-thioguanine (6-TG) and 7 , 1 2 - d i m e t h y l b e n z [ a ] a n t h r a c e n e (DMBA) were from Sigma Chemical Co., St. Louis, MO; colcemid and 5-bromo-2'-deoxyuridine (BrdUrd) were from Boehringer-Mannheim Canada, Montreal, Que.; dimethyl sulfoxide (DMSO) (gold label, > 99%) was from Aldrich Chemical Co., Milwaukee, WI. All chemical solutions were prepared immediately before use. EMS was dissolved and diluted in WE medium. DMBA was first dissolved in DMSO, then diluted in WE medium so that the final DMSO concentration did not exceed 0.2% v / v .

Preparation of hepatoeytes Primary hepatocytes were freshly prepared by in vivo collagenase perfusion of the fiver of a male Wistar rat or golden Syrian hamster, using the methods of Williams et al. (1977) and Maslanski and Williams (1982) with modifications as described by Rogers and Hrroux-Metcalf (1983) and by Rogers et al. (1985).

Chemicals 1-Nitronaphthalene (reagent grade, 99%) was from Aldrich Chemical Company, Milwaukee, WI, and was used without further purification. To exclude the possibility that the 1-NN could contain minute quantities of a possibly genotoxic

Cytotoxicity The cytotoxicity of 1-NN to V79 cells was determined in the absence of hepatocytes by measurement of the effect on plating efficiency, using the method of Rogers and Hrroux-Metcalf (1983).

113

1-NN was tested at dose levels as high as 200 /~g/ml of medium.

Mutagenicity Mutagenicity was evaluated in a hepatocytemediated assay according to protocols of Langenbach et al. (1978, 1981) with modifications as reported by Rogers et al. (1985, 1988). The period of exposure to chemicals, with or without hepatocyte activation, was 48 h. 20 plates per treatment group were examined. Sister-chromatid exchange V79 cells, with and without test chemical, were incubated 24 h in the presence and absence of rat or hamster hepatocytes. The test cultures were then trypsinized, reseeded at 5 × 105 cells in 9 ml of medium (75-cm 2 flasks) and allowed to attach for 1 h. Following the addition of BrdUrd (6.0 t~g/ml), incubation was continued in darkness for 2 cycles of cell division (ca. 23 h). Colcemid (1.0 / t g / m l ) was then added. After 3 h, the cells were collected by gentle scraping, resuspended in hypotonic solution, fixed, air-dried and stained with Hoechst 33258 (Boehringer-Mannheim, Montreal, Que.) followed by Giemsa (Gibco, Burlington, Ont.) (Perry and Wolff, 1974). Slides were scored blind. In most cases, 30 metaphases (19-22 chromosomes per metaphase) were examined in each treatment group and scored for SCE by standard cytogenetic procedures (Latt et al., 1981). Statistical methodology For each experiment, separate analyses were carried out for each 'group' of treatments, where a 'group' is defined as a set of treatments without hepatocytes or with hepatocytes. Expt. 33-88 contains 3 groups of treatments since 2 types of hepatocytes were used. For each analysis, treatments were compared with the corresponding control for that group. Details of these analyses are provided below. The SCE data were analyzed on the natural log scale as this was found to be the most appropriate scale (based on previous analyses of this type of data). For each group of treatments within an experiment, a one-way Analysis of Variance (AOV) was performed on the Iog(SCE) levels. The residuals from this analysis were tested for normality

using the Shapiro-Wilk statistic (Shapiro and Wilk, 1965). In addition, the residuals were screened for outliers (Grubbs, 1969). Significant outliers ( p < 0.01) (a total of 3 values overall) were deleted from analyses. For each of the experiments and treatments considered here, the assumption that the natural logarithms of the SCE levels were normally distributed was reasonable ( p > 0.05 in all cases). Treatment means which exceeded the 95% or 99% upper confidence limits (u.c.l.'s) for the controls were identified. Assuming variance homogeneity among the treatments and control for the group, the pooled variance (a weighted sum of the individual variances within the group) was used as the variance estimate for the calculation of these u.c.l.'s. A test for variance homogeneity (Bartlett, 1937) was conducted initially. If there was evidence of variance heterogeneity ( p < 0.05), the variance estimate was based on the individual variances for the treatment-control pair. A twotailed F-test was used to test for the homogeneity of variances of the treatment-control pair. If this test indicated variance heterogeneity ( p < 0.05), then individual variances, using Satterthwaite's (1946) approximation for the degrees of freedom of the t-statistic, were used. For the thioguanine resistance data, two variables were analyzed; namely, the proportion of the 20 dishes with at least one mutant colony and the average number of mutant colonies/dish. For the analysis of the former variable, the proportion of the 20 dishes with at least one mutant colony was calculated for each control and treatment within a group. Based on a normal approximation to the binomial distribution, treatment proportions which exceeded the 95% or 99% u.c.l.'s for the controls were identified. The standard deviation which was used in comparing control and t r e a t m e n t p r o p o r t i o n s was e s t i m a t e d b y ~/p(1 - p ) ( 1 / n c + 1 / n t ) , where p was the pooled proportion estimate, i.e. p = (ncp c + n t P t ) / ( n c q - n t ) , n c and n t were the number of dishes examined for the control and treatment groups respectively (n c = n t = 20 in most cases), and Pc and Pt were the proportions of dishes with at least one mutant colony for the control and treatment groups respectively. The analysis of mutant colonies per dish proceeded under the assumption that the number of mutant colonies/dish for treatment or control

114

groups had a Poisson distribution. Plots of the average number of mutant colonies/dish versus the variance of the number of mutant colonies/ dish revealed this assumption to be reasonable. After screening the data for outliers (Barnett and Lewis, 1978), the total number of mutant colonies observed on the control dishes was calculated. Based on this total, 95% and 99% u.c.l.'s (Pearson and Hartley, 1970) for the expected number of mutant colonies/dish for the control group were determined. Treatment groups for which the average number of mutant colonies/dish exceeded these 95% or 99% u.c.l.'s were identified. In addition to the analyses described above in which SCE and T G R treatment levels were compared with concurrent control levels, a screening procedure using the decision rule and historical limits given in Tables 3 and 4 of Boyes et al. (1990) was applied to the data. This strategy was developed to take advantage of the reproducibility and accuracy characteristics of these two assays, as derived from multiple replications on the known genotoxic chemicals dimethylbenzanthracene with hepatocytes and ethyl methanesulfonate, along with corresponding non-genotoxic treatments (Boyes et al., 1990). In this procedure, SCE results were screened first, and if negative the treatment was declared negative. If SCE results were positive, then T G R results were screened. If T G R results were positive, then the treatment was declared positive. If T G R results were negative, then further testing was required. The above screening procedure was not applied to treatment groups for which hamster hepatocytes were used (Expts. 25-88 and 33-88) as no historical limits had been established.

Results Cytotoxicity is shown in Fig. 1 as plating efficiency after treatment with 1-NN in the absence of hepatocytes. On this basis, treatment doses (20, 40, 8 0 / z g / m l ) were chosen that showed extensive toxicity while permitting the preparation of technically adequate slides for SCE studies. Summaries of the treatment control comparisons for SCE and T G R are contained in Tables 1 5. Each experiment is presented individually. For each table, the geometric means of the

100( 90807060150% 50I-NITRONAPHTHALENE NO2 ..I 40-

C1oH7NO2 FW 173.17

I'- 30Z ZU I.IJ O. 20-

10

5'0

1()0

150

200

I-NN (p.g/ml)

Fig. 1. Percent survival (plating efficiency) of V79 cells treated with 1-NN (inset) in the absence of hepatocytes.

SCEs/metaphase, the means and standard errors of SCEs/metaphase on the natural log scale, the proportion of dishes with at least one mutant colony and the average number of mutant colonies/dish are provided. In addition, the 95% and 99% upper confidence limits (u.c.l.'s) for the expected number of mutant colonies/dish for the control groups are shown. Treatment levels which exceeded the 95% or 99% u.c.l.'s for the controls are indicated. The levels of SCE and T G R for the positive controls (EMS, D M B A with either rat or hamster hepatocytes) exceeded the 99% u.c.l.'s for the controls in all cases (Tables 1-5). The levels of SCE and T G R for the negative control, D M B A without hepatocytes, did not exceed the 95% u.c.1, for the controls except for SCE in Table 1, where the control level was low relative to the other 4 Expts. For the test chemical 1-nitronaphthalene, the most consistent experiment to experiment results occurred for the SCE data. Here, for treatments

115 TABLE 1 TREATMENT-CONTROL COMPARISONS FOR SCE AND TGR: Expt. 13-87 Treatment

SCE

TG R

Geometric mean a

Natural log scale b Mean ± S.E.

Proportion of dishes with mutant colonies

Average number of mutant colonies/ dish

2.017 ± 0.061

0.250

0.25 (0.53 c, 0.66 d)

3.253 _+0.060 2.228 ± 0.082 2.378 ± 0.069 2.201 ± 0.068

1.000 * * 0.050 0.400 0.000

12.15 * * 0.05 0.60 * 0.00

2.101 ± 0.057

0.100

0.10 (0.31 c, 0.42 d)

2.939±0.071 2.358 ± 0.064 2.854±0.083

1.000 ** 0.300 0.250

30.40 ** 0.40 * 0.35 *

Screening decision e

(i) Without hepatocytes Control EMS (155/tg/ml) DMBA (2.6 #g/ml) 1-Nitro (20 #tg/ml) 1-Nitro (80/tg/ml) (ii) With rat hepatocytes Control DMBA (2.6 ~g/ml) 1-Nitro (20/zg/ml) 1-Nitro (80 ~tg/ml)

7.51 25.88 9.28 10.78 9.04

** * ** *

8.17 18.89 ** 10.57 * * 17.36 * *

+ ? + ?

+ + ?

a Geometric means of sister-chromatid exchanges/metaphase. b Means and standard errors of sister-chromatid exchanges/metaphase are presented on the natural log scale as they were analyzed in this form. c 95% upper confidence limit for the expected number of mutant colonies/dish. d 99% upper confidence limit for the expected number of mutant colonies/dish. e Results obtained with the screening procedure using historical controls described in Boyes et al. (1990). Treatments which were declared as positive, negative or those for which further testing was required are indicated as ' + ', ' - ' and "?' respectively. * Exceeded the 95% upper confidence limit for the controls. * * Exceeded the 99% upper confidence limit for the controls.

w i t h o u t h e p a t o c y t e s , t h e levels o f S C E d i d n o t e x c e e d t h e 95% u.c.l.'s f o r t h e c o n t r o l s f o r a n y o f t h e c o n c e n t r a t i o n levels, w i t h t h e e x c e p t i o n o f T a b l e s 1 a n d 5 w h e r e t h e S C E levels f o r t h e c o n t r o l g r o u p s w e r e low. F o r t r e a t m e n t s w i t h h e p a t o c y t e s , t h e t r e a t m e n t levels f o r S C E exc e e d e d t h e 99% u.c.l.'s f o r t h e c o n t r o l s f o r all 3 c o n c e n t r a t i o n l e v e l s e x c e p t i n T a b l e 2, w h e r e t h e level o f S C E f o r t h e 2 0 / ~ g / m l c o n c e n t r a t i o n g r o u p e x c e e d e d t h e 95% u.c.1, f o r t h e c o n t r o l . C o m p a r a ble values were observed when either rat or hamster hepatocytes were used for activation. A c r o s s e x p e r i m e n t s , a n a l y s e s s h o w e d less c o n sistency for TG R data than for the SCE data. The effects of activation by either rat or hamster hepatocytes, while comparable, were also incon-

s i s t e n t . T r e a t m e n t p r o p o r t i o n s w h i c h w e r e less t h a n t h e 95% u.c.1, f o r t h e c o n t r o l s w o u l d b e c o n s i d e r e d s u f f i c i e n t e v i d e n c e , i n m o s t cases, t o c l a s s i f y t h e t e s t c h e m i c a l as n o n m u t a g e n i c . H o w ever, f o r c a s e s i n w h i c h t h e p r o p o r t i o n o f d i s h e s with at least one mutant colony for the control g r o u p is h i g h ( T a b l e 4), t h e a v e r a g e n u m b e r o f mutant colonies/dish should be assessed. There was at least one treatment group in each of Expts. 2 5 - 8 7 a n d 3 3 - 8 8 ( T a b l e s 3 a n d 5) f o r w h i c h b o t h proportion and number of colonies indicated positive m u t a g e n i c a c t i v i t y . H o w e v e r , n o t o n l y w a s t h e expected number of mutant colonies/dish less than one for both of these treatment groups, but these expected numbers were substantially lower t h a n t h o s e o f t h e p o s i t i v e c o n t r o l s . F o r t h e s e cases,

116 TABLE 2 TREATMENT-CONTROL COMPARISONS FOR SCE AND TGR: Expt. 19-87 Treatment

(i)

TG R

SCE Geometric mean a

Natural log scale b Mean + S.E.

Proportion of dishes with mutant colonies

Average number of mutant colonies/ dish

Without hepatocytes Control

11.05

2.402 _+0.071

0.050

0.05 (0.24 c, 0.33 d)

EMS (old) (155/~g/ml) EMS (new) (155/~g/ml) DMBA (2.6 #g/ml) 1-Nitro (20/~g/ml) l-Nitro (40/Lg/ml) 1-Nitro (80/Lg/ml)

38.57 ** 29.09 ** 11.34 9.71 12.10 11.57

3.652_+0.048 3.371_+0.053 2.428 _+0.061 2.274_+0.085 2.494 _+0.077 2.449 -+0.077

1.000 ** 1.000 ** 0.100 0.100 0.100 0.050

9.40 ** 8.90 ** 0.15 0.10 0.15 0.05

2.177 _+0.086

0.100

0.10 (0.31 c, 0.42 ~)

3.025_+0.066 2.380_+0.071 2.696 _+0.098 2.877 _+0.117

1.000 ** 0.050 0.050 0.250

12.95 ** 0.05 0.05 0.25

(ii) With rat hepatocytes Control DMBA (2.6/~g/ml) 1-Nitro (20 #g/ml) 1-Nitro (40 ~g/ml) 1-Nitro (80 t~g/ml)

8.82 20.60 10.81 14.82 17.77

** * ** **

Screening decision e

+ +

-

+ 9 9

a Geometric means of sister-chromatid exchanges/metaphase. b Means and standard errors of sister-chromatid exchanges/metaphase are presented on the natural log scale as they were analyzed in this form. c 95% upper confidence limit for the expected number of mutant colonies/dish. d 99% upper confidence limit for the expected number of mutant colonies/dish. e Results obtained with the screening procedure using historical controls described in Boyes et al. (1990). Treatments which were declared as positive, negative or those for which further testing was required are indicated as "+', ' - ' and '?' respectively. * Exceeded the 95% upper confidence limit for the controls. * * Exceeded the 99% upper confidence limit for the controls.

t h e t e s t c h e m i c a l w o u l d b e c o n s i d e r e d , a t b e s t , to be weakly mutagenic. The results which were obtained by applying t h e d e c i s i o n r u l e o f B o y e s et al. ( 1 9 9 0 ) a r e i n f a i r l y good agreement, particularly for the SCE data (Tables 1-5). At the concentration levels considered here, 1-nitronaphthalene (with rat or hamster hepatocytes) tested positive for SCE and questiona b l e f o r T G R. W i t h o u t h e p a t o c y t e s , t h i s c h e m i c a l generally tested negative for SCE but results were i n c o n s i s t e n t f o r T G R. Discussion

Mutagenic activity of 1-NN has been studied m a i n l y i n S a l m o n e l l a , w h e r e 1 - N N is d e s c r i b e d as

a w e a k m u t a g e n ( E 1 - B a y o u m y et al., 1 9 8 2 ; F u et al., 1 9 8 5 ; M a s s a r o et al., 1 9 8 3 ; M e r m e l s t e i n et al., 1985), p r o d u c i n g f r a m e s h i f t e r r o r s ( T o k i w a e t al., 1981; Vance and Levin, 1984) and base-substitution errors; the latter are dependent on plasmid pKM101, which codes for an error-prone DNA r e p a i r e n z y m e ( V a n c e a n d L e v i n , 1984). 1 - N N is inactivated by ring hydroxylation and activated by n i t r o r e d u c t i o n ( B e l a n d et al., 1985, B e n k e n d o r f , 1 9 7 8 ; E 1 - B a y o u m y a n d H e c h t , 1 9 8 2 ; K a r p i n s k y et al., 1 9 8 2 ; R o s e n k r a n z a n d M e r m e l s t e i n , 1 9 8 3 ; Vance and Levin, 1984) to form the hydroxylamine which reacts with the DNA bases (Mermels t e i n et al., 1 9 8 5 ) f o r m i n g a d d u c t s p r e f e r e n t i a l l y a t 0 6 ( M c C o y et al., 1 9 8 1 ; R o s e n k r a n z et al., 1985). Genotoxic activity may occur in the presence or

117 TABLE 3 TREATMENT-CONTROL COMPARISONS FOR SCE AND TG R: Expt. 25-87 Treatment

SCE

TG R

Geometric mean a (i)

Without hepatocytes Control EMS (155 #g/ml) DMBA (2.6 #g/ml) 1-Nitro (20 #g/ml) 1-Nitro (40 #g/ml) 1-Nitro (80 #g/ml)

(ii) With rat hepatocytes Control DMBA (2.6/tg/ml) 1-Nitro (20 #g/ml) 1-Nitro (40 #g/ml) 1-Nitro (80 #g/ml)

9.45 25.56 * * 9.41 8.50 10.10 9.35 8.81 21.16 11.69 13.90 12.08

** ** ** **

Natural log scale b Mean _+S.E.

Proportion of dishes with mutant colonies

Average number of mutant colonies/ dish

2.246 + 0.061

0.050

0.05 (0.24 c, 0.33 d)

3.241 +0.052 2.242 + 0.045 2.140+0.056 2.313 _+0.063 2.236 _+0.067

1.000 * * 0.050 0.500 * * 0.211 0.150

5.40 * * 0.05 0.60 * * 0.26 * 0.15

2.176 + 0.078

0.350

0.40 (0.72 c, 0.87 d)

3.052+0.082 2.459+0.071 2.632 + 0.074 2.492 _+0.067

1.000 ** 0.211 0.579 0.150

18.25 ** 0.21 0.68 0.25

Screening decision e

+ ? ? ?

a Geometric means of sister-chromatid exchanges/metaphase. b Means and standard errors of sister-chromatid exchanges/metaphase are presented on the natural log scale as they were analyzed in this form. 95% upper confidence limit for the expected number of mutant colonies/dish. 99% upper confidence limit for the expected number of mutant colonies/dish. Results obtained with the screening procedure using historical controls described in Boyes et al. (1990). Treatments which were declared as positive, negative or those for which further testing was required are indicated as ' + ', ' - " and '?' respectively. * Exceeded the 95% upper confidence limit for the controls. * * Exceeded the 99% upper confidence limit for the controls.

absence of exogenous activation (Benkendorf, 1978; D u n k e l et al., 1985; T o k i w a et al., 1981), a n d is s e n s i t i v e to t h e g r o w t h s t a g e ( R o s e n k r a n z a n d M e r m e l s t e i n , 1983). O u r f i n d i n g o f s p o r a d i c w e a k a c t i v i t y for T G R in V79 is c o n s i s t e n t w i t h the a b o v e r e p o r t s of w e a k a c t i v i t y in S a l m o n e l l a . W h i l e we f o u n d little o r n o S C E a c t i v i t y in t h e a b s e n c e o f h e p a t o c y t e s e x c e p t in 1 (of 5) E x p t s . ( T a b l e 1) w h e r e t h e c o n t r o l v a l u e was u n u s u a l l y low, S C E f r e q u e n c i e s in t h e p r e s e n c e o f h e p a t o c y t e s w e r e a l w a y s signific a n t l y o r h i g h l y s i g n i f i c a n t l y e l e v a t e d . T h e s e positive f i n d i n g s are in c o n t r a s t to the n e g a t i v e S C E results r e p o r t e d in S h e l b y a n d S t a s i e w i c z (1984).

T h e y e x p o s e d for 2 h in t h e p r e s e n c e a n d 1 0 - 1 2 h in the a b s e n c e o f A r o c l o r - i n d u c e d rat $9 (M. Shelby, p e r s o n a l c o m m u n i c a t i o n ) , w h i l e w e u s e d 24-h e x p o s u r e in the p r e s e n c e a n d a b s e n c e o f u n i n d u c e d rat o r h a m s t e r h e p a t o c y t e s . L i k e l y t h e longer exposure time we used, along with the d i f f e r e n t a c t i v a t i o n system, a c c o u n t s for t h e diff e r e n c e in t h e S C E results. T h e S C E levels for t r e a t m e n t s i n v o l v i n g 1 - N N w i t h h e p a t o c y t e s r a r e l y r o s e as h i g h as the p o s i t i v e c o n t r o l s ( D M B A w i t h rat o r h a m s t e r h e p a t o c y t e s , E M S ) , a l t h o u g h t h e y w e r e c l o s e r t h a n w a s seen using the TG R endpoint. T h e p u r i t y o f t h e 1 - N N m a t e r i a l w e used, w h i c h

118 TABLE 4 TREATMENT-CONTROL COMPARISONS FOR SCE AND TG R: Expt. 25-88 Treatment

SCE Geometric mean a

(i) Without hepatocytes Control

EMS (155/~g/ml) DMBA (2.6/Lg/ml) 1-Nitro (40 ~g/ml) 1-Nitro (80 ~g/ml) (ii) With hamster hepatocytes f Control DMBA (2.6 ~g/ml) 1-Nitro (40/Lg/rnl) 1-Nitro (80/xg/ml)

9.99 25.67 ** 8.36 9.79 9.21 9.03 14.77 * * 13.23 * * 16.54 * *

TG R Natural log scale b Mean _+S.E.

Proportion of dishes with mutant colonies

Average number of mutant colonies/ dish

2.302 ± 0.074

0.850

2.15 (2.77 c, 3.04 d)

3.245_+0.058 2.124 _+0.066 2.281 ± 0.081 2.221 ± 0.076

1.000 * 0.800 0.650 0.950

15.10 * * 1.50 1.05 3.85 * *

2.201 ± 0.065

0.850

2.00 (2.60 c, 2.87 J)

2.692 ±0.082 2.582 _+0.069 2.806 ± 0.058

1.000 * 0.850 NA

10.10 2.25 NA

Screening decision e

a Geometric means of sister-chromatid exchanges/metaphase. b Means and standard errors of sister-chromatid exchanges/metaphase are presented on the natural log scale as they were analyzed in this form. 95% upper confidence limit for the expected number of mutant colonies/dish. 99% upper confidence limit for the expected number of mutant colonies/dish. '~ Results obtained with the screening procedure using historical controls described in Boyes et al. (1990). Treatments which were declared as positive, negative or those for which further testing was required are indicated as ' + ' , ' - ' and '?' respectively. r Historical limits have not been established. NA, No data available. * Exceeded the 95% upper confidence limit for the controls. '** Exceeded the 99% upper confidence limit for the controls.

was at least 99.9% b y o u r c h e m i c a l analyses, m a k e s it v e r y u n l i k e l y t h a t the o b s e r v e d effects c o u l d b e d u e to a c o n t a m i n a n t . R a t h e r t h a n b e i n g ' i n d e t e r m i n a t e ' or ' e q u i v o c a l ' , the e v i d e n c e s u g g e s t s t h a t 1 - N N is a w e a k g e n o t o x i n . It is p o s i t i v e in S a l m o n e l l a as d i s c u s s e d earlier, p o s i t i v e in t h e N C I / N T P test p r o g r a m m e for a b e r r a t i o n s a l t h o u g h n o t for S C E , a n d p o s i t i v e in o u r test s y s t e m f o r S C E w i t h a c t i v a t i o n ; e v e n for T G R s p o r a d i c w e a k p o s i t i v e results o c c u r . O v e r a l l t h e s e p o s i t i v e results, w h i l e s t a t i s t i c a l l y s i g n i f i c a n t , are n o t large. D u e to t h e r e l a t i v e l y s m a l l n u m b e r s o f a n i m a l s (50 m a l e a n d 50 f e m a l e , t w o c o n c e n t r a t i o n s , t w o species) u s e d in t h e N C I / N T P b i o a s s a y , this test

w o u l d b e e x p e c t e d to b e i n s e n s i t i v e to a w e a k c a r c i n o g e n . A t least in S a l m o n e l l a , 1 - N N a c t i v i t y is s e n s i t i v e to the g r o w t h s t a g e o f t h e test p o p u l a t i o n ( R o s e n k r a n z a n d M e r m e l s t e i n , 1983). S i m i larly, the m e t a b o l i c f a t e of 1 - N N m i g h t b e d e p e n d e n t o n slight d i f f e r e n c e s b e t w e e n i n d i v i d u a l s o r b e t w e e n species. F u r t h e r m o r e , t h e c o n c e n t r a t i o n s o f 1 - N N u s e d in the p r e s e n t s t u d y ( m a x . 8 0 / ~ g / m l o f m e d i u m , i.e. 0.008%) a r e l o w e r t h a n t h o s e u s e d in t h e N C I b i o a s s a y ( u p to 0.18% o f feed, a s s u m i n g i n c o r p o r a t i o n p r o p o r t i o n a l to f e e d c o n c e n t r a tion). T h e d i f f i c u l t y of e s t a b l i s h i n g a n e g a t i v e result has b e e n e m p h a s i z e d b y C l a y s o n (1989). W e c o n s i d e r , t h e r e f o r e , t h a t 1 - N N is g e n o t o x i c ; w h e t h e r it m i g h t also b e c a r c i n o g e n i c u n d e r c o n d i -

119 TABLE 5 TREATMENT-CONTROL

C O M P A R I S O N S F O R SCE A N D T G R : Expt. 33-88

Treatment

SCE Geometric mean a

(i)

Without hepatocytes Control

E M S (155 ~ g / m l ) D M B A (2.6 b t g / m l ) 1-Nitro (40 b t g / m l ) 1-Nitro ( 8 0 / ~ g / m l ) (ii) W i t h rat h e p a t o c y t e s Control

D M B A (2.6 p g / m l ) 1-Nitro (40 # g / m l ) 1-Nitro (80 # g / m l ) (iii) W i t h h a m s t e r h e p a t o c y t e s t Control

D M B A (2.6 p g / m l ) 1-Nitro (40 ~ g / m l ) 1-Nitro (80 ~ g / m l )

7.87

27.25 * * 8.44 9.14 9.95 *

7.74

21.83 * * 16.14 * * 13.34 * *

8.03

19.06 * * 12.76 * * 12.16 * *

T G l~ N a t u r a l log scale b M e a n _+S.E.

P r o p o r t i o n of dishes with m u t a n t colonies

A v e r a g e n u m b e r of mutant colonies/ dish

2.063 __.0.086

0.200

0.20 (0.46 c, 0.58 d)

3.305-1-0.044 2.133 _+0.064 2.212_+0.081 2.298 + 0.070

1.000 * * 0.400 0.600 * * 0.550 *

14.65 * * 0.40 0.95 * * 0.90 * *

2.046 + 0.080

0.300

0.35 (0.66 c, 0.80 d)

3.083+0.071 2.781 + 0.066 2.590 + 0.045

1.000 * * 0.450 0.450

24.15 * * 0.60 0.50

2.083 + 0.079

0.200

0.20 (0.46 c, 0.58 d)

2.947+0.083 2.546 + 0.084 2.498 ___0.055

1.000 * * 0.350 0.450 *

11.95 * * 0.70 * * 0.60 * *

Screening decision e

+ + +

+ ? ?

a G e o m e t r i c m e a n s of s i s t e r - c h r o m a t i d e x c h a n g e s / m e t a p h a s e . b M e a n s a n d s t a n d a r d errors of sister-chromatid e x c h a n g e s / m e t a p h a s e are p r e s e n t e d o n the n a t u r a l tog scale as they were a n a l y z e d in this form. c 95% u p p e r c o n f i d e n c e l i m i t for the expected n u m b e r of m u t a n t c o l o n i e s / d i s h . d 99% u p p e r c o n f i d e n c e limit for the expected n u m b e r of m u t a n t c o l o n i e s / d i s h . e Results o b t a i n e d with the screening p r o c e d u r e using historical controls described in Boyes et al. (1990). T r e a t m e n t s w h i c h were declared as positive, negative or those for which further testing was required are i n d i c a t e d as ' + ', ' - ' a n d '?' respectively. f H i s t o r i c a l limits have n o t b e e n established.

tions different from those used in the N C I / N T P bioassay is open to question.

analyses and GLP certification on our sample of 1-NN.

Acknowledgements References We thank Irene Langlois, Claudette H6rouxMetcalf and Leigh Martin for excellent technical assistance. We also especially thank Dr. George Neville of the Drug Identification Division, Bureau of Drug Research, Drugs Directorate, Health and Welfare Canada, for performing the purity

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Genotoxicity of 1-nitronaphthalene in Chinese hamster V79 cells.

1-Nitronaphthalene (1-NN) has been identified in the U.S. National Toxicology Program as a non-carcinogen showing some evidence of in vitro genotoxici...
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