Genotoxic effects of glycidyltrimethylammonium chloride

Mutation Research, 189 (1987) 387-394

387

Elsevier MTR 01232

Genotoxic effects of glycidyltrimethylammonium chloride C. Vleminckx, J. Arany, B. Hendrickx and W. Moens Department of Toxicology, Institute of Hygiene and Epidemiology, B-1050 Brussels (Belgium) (Received 20 February 1987) (Revision received 18 June 1987) (Accepted 24 June 1987)

Keywords: Epoxides; Salmonella/microsome test; Mitotic recombination; In vitro cytogenetics; DNA amplification.

Summary Evaluation of the genotoxicity of epoxides is best carried out on a case by case basis. Although glycidyltrimethylammonium chloride (GTAC) is widely used in several industrial applications, its genotoxicity is poorly documented. Therefore, we have evaluated GTAC in a battery of 4 in vitro short-term tests for genotoxicity. We report here that GTAC mediates the induction of base-pair substitutions in S. typhimurium, gene conversion in S. cerevisiae (D7), chromosomal aberrations in CHO cells and viral DNA amplification in Chinese hamster CO60 cells. In view of these results, it is advisable to consider GTAC a potential carcinogen.

Glycidyltrimethylammonium chloride (GTAC) is used in the textile industry as an enhancer of colouring efficiency for the treatment of polyamide and cellulosic fibers (Kawamoto et al., 1979; Rudkin et al., 1979; Bell et al., 1980; Chavannes et al., 1980; Rusznak et al., 1980; Balland, 1981). It is also an essential intermediate in the manufacture of cellulosic cationic ether derivatives and lignocellulosic material with enhanced draining properties (Fischer et al., 1981; Kao Soap Co., 1981; Soremark et al., 1981). In the electronic industry, GTAC is used in the manufacture of cationic polymer emulsions (Moritani et al., 1981), ferromagnetic polymer grains (ICI Australia Ltd., 1980), coagulated substances (Lion Corp., 1980a) and upper layers of electric conductors on paper Correspondence: C. Vleminckx, Department of Toxicology, Institute of Hygiene and Epidemiology, Rue J. Wytsman 14, B-1050 Brussels (Belgium).

support for electronic recording (Fuji Chemical Co., 1980). In the cosmetic industry, GTAC is a component in the preparation of some shampoos (Ajinomoto Co., 1980), capillary products (Yanagawa et al., 1980) and cosmetic formulae containing cellulosic powders (Arnold, 1978; Hoffmann-La Roche Co., 1978; Lion Corp., 1980b; Ajinomoto Co., 1981). In the cleaning industry, GTAC is used as a colour stabiliser (Weber et al., 1980; Claiborne, 1981). Voogd et al. (1981) have shown that potent mutagenic substances could be found among aliphatic and cyclic epoxides. Some of them were known carcinogens, whereas others were neither mutagenic nor carcinogenic. Due to this heterogeneity, the genotoxicity of each epoxide should be examined independently. Since GTAC has only recently been placed on the market, few toxicological data are yet available on this chemical. This substance was shown to be toxic for red fish

0165-1218/87/$03.50 © 1987 Elsevier Science Pubfishers B.V. (Biomedical Division)

388 (Bridi6 et al., 1979) and to increase the mutation rate of Klebsiella pneumoniae for streptomycin resistance (Voogd et al., 1981). The objective of this study was to investigate the genotoxic properties of GTAC at different biological endpoints in a battery of 4 in vitro tests: induction of mutations in the Salmonella/microsome test, mitotic recombination in S. cereoisiae (D7), chromosomal aberrations in CHO cells and viral D N A amplification in C O 6 0 cells.

(1983) by using dilutions of GTAC in water. BaP and AFB a were used as positive controls. Yeast test system Strain. Diploid strain D7 of Saccharomyces cereuisiae (a gift of Prof. F.K. Zimmermann, Institut fiir Mikrobiologie, Darmstadt, F.R.G.) was used to detect mitotic gene conversion at the 'trp 5' locus by means of production of nontryptophan-requiring colonies on selective media.

Materials and methods

Chemicals The following chemicals were used in this study: glycidyltrimethylammonium chloride (GTAC, CAS No. 3033-77-0; Fluka, purum > 97% purity), mitomycin C (MMC; CAS No. 50-07-7; Sigma), ethyl methanesulphonate (EMS; CAS No. 62-50-0; Sigma), cyclophosphamide (END; CAS No. 605519-2; Aldrich), benzo[a]pyrene (BaP; CAS No. 50-32-8; Aldrich), aflatoxin B a (AFB1; CAS No. 1162-65-8; Calbiochem-Behring) and the nitronaphthofuran derivatives 7-methoxy-2-nitronaphtho[2,1-b]furan (R-7000) and 1-methyl-7-methoxy2-nitronaphtho[2,1-b]furan (R-7372; Dr. R. Royer, Institut Curie, Paris). Redistilled dimethyl sulphoxide (DMSO; Merck, spectrophotometrical grade) was used as the solvent for END, BaP, AFB 1, R-7000 and R-7372. GTAC, EMS and MMC were dissolved in distilled water. Salmonella / microsome test Strains of S. typhimurium TA1535, TA100, TA1537, TA97, TA98 were kindly provided by Professor B.N. Ames. The liver enzyme activation mixture ($9 mix) was prepared according to the method of Maron and Ames (1983) from liver post-mitochondrial fractions of rats pretreated with Aroclor 1254. All strains were first spread on Difco nutrient agar. For strains TA100, TA97 and TA98, 5 /~g ampicillin/ml were incorporated into the nutrient agar to select bacteria still possessing the R factor. A single colony was then used to inoculate the nutrient broth which, after overnight incubation, was used for the test. Plate incorporation assays were performed according to Maron and Ames

Media. The composition of synthetic and complete growth media used in mutagenicity testing with yeast have been described by Zimmermann (1975). Cell treatment with carcinogens. Culture cells at the stationary phase of growth selected for their adequate spontaneous gene conversion frequency were centrifuged and resuspended in a 5 times diluted complete medium at 7.5 × 107 cells/ml. Incubation mixtures were prepared by adding to 0.4 ml of this suspension 0.01 ml of the test or control substance solution and 0.6 ml of 5 times diluted complete medium or $9 mix. The suspensions were incubated for 1 h at 37 ° C followed by 16 h at 2 9 ° C with shaking. The treatment was stopped by insertion of the tubes into an ice bath. Five plates of selective medium were spread with 0.1 ml of each culture and 3 synthetic complete agar plates with 0.1 ml of an appropriate dilution in distilled water in order to spread approximately 200 cells/plate. These plates were then incubated for 5 days at 2 9 ° C before counting the colonies. Conversion frequencies were then expressed as convertant colonies per plating unit as determined from colony numbers on complete plates. Microsomal actwation mix for the yeast study. The rat-liver supernatant ($9 fraction) was prepared as above. The $9 mix is constituted of (for 3 ml): $9 fraction (1 ml), 0.4 M MgC12 (0.1 ml), 1.65 M KC1 (0.1 ml), G-6-P (200 mg) and N A D P (50 mg) pooled in 0.8 ml water and 3 times diluted complete growth medium (1 ml). This solution is freshly prepared and kept at 4°C. 0.6 ml is used per ml of treatment mixture.

389 Chromosome aberration test Cell line. Chinese hamster ovary (CHO) cells are a nearly diploid cell line with a chromosome modal number of 21 _+ 2. The karyogram was found similar to the one published in the ATCC catalogue (1979). These cells were originally obtained from Prof. P. Lohman, TNO-MBL, Rijswijk, The Netherlands. CHO cells were cultivated below confluency in Eagle minimum essential medium (Flow), supplemented with 10% foetal calf serum (Gibco), 100 /~g/ml streptomycin and 100 U / m l penicillin. The cells were incubated at 3 7 ° C in a CO 2 incubator with 5% CO 2 and 98% humidity. Cell treatment with carcinogens. One-day-old sparse cultures were exposed for 18 h to a freshly dissolved compound. Triplicate cultures were performed for each experimental point. The solvent was used as negative control. MMC dissolved in water was used as the positive control. At the end of the treatment, the cultures were used for cytotoxicity assay and cytogenetics. Cytotoxicity measurement. The cytotoxicity of the compounds was estimated by the survival index (SI). In practice, the surviving cells were trypsinized at the end of the treatment and resuspended in phosphate-buffered saline (PBS). The SI was defined as 100 times the ratio of the number of living cells in treated cultures to the number of living cells in the control culture as measured under a phase-contrast microscope with an haemocytometer (SD - 3%). The nuclei of the living cells are gold-red whereas those of dead cells are either absent or grey. Cytogenetics and data collection. Metaphase spreads were prepared according to Wahl et al. (1982) with slight modifications, and stained with 10% Giemsa. Metaphases were examined under a Leitz microscope equipped with an X63 PLAPO objective. For the determination of the mitotic index (% of cells in mitosis), 1000-2000 cells were scored. Numerical aberrations were analysed in the first 50 randomly selected metaphases. The percentage of aneuploid + polyploid metaphases for each dose was compared to control values

using Fisher's exact probability test (Siegel, 1956). For each experimental point, 50-100 well-spread metaphases were analysed for structural aberrations. The selected metaphases were those containing +_2 centromeres of the modal number (21 chromosomes for CHO cells). Scoring of chromosomal aberrations was done according to UKEMS (Dean, 1983) and OECD guidelines (1983). Metaphases with more than 10 aberrations were classified as having multiple aberrations (MA). Their maximal number was fixed to a value of 10 in the calculation of the frequency of aberrations/ metaphase. For the analysis of the number of metaphases with chromosome aberrations, Fisher's exact probability test (Siegel, 1956) was used. For the analysis of chromosomal aberration frequencies, the significance of frequency variations was estimated using the Mann-Whitney test (U test) (Siegel, 1956). D N A amplification test (Vanhorick and Moens, 1983) Cell line. The C O 6 0 cell line is a clonal derivative of an agar colony of SV40-transformed Chinese hamster embryo secondary cultures (Lavi, 1981). The cells were grown in Dulbecco's modification of Eagle's medium (Gibco) supplemented with 10% foetal calf serum (Gibco) and penicillin-streptomycin (Sigma) in the watersaturated atmosphere of a water-jacketted CO 2 incubator (Format). Cell treatment with carcinogens. One-day-old sparse cultures were exposed to a freshly dissolved test compound. After 24 h, the medium was removed and cells were further incubated in a fresh test compound-free medium. Thereafter, cells were collected 4 days later for the dispersed cell assay. R-7000 and R-7372 were respectively used as positive and negative direct-acting standard controls (Royer et al., 1985; Vleminckx and Moens, 1986). Dispersed cell assay. The extent of SV40 D N A amplification (SDA) was measured by filter hybridisation of alkali-denatured cells using [32p]sv40-DNA as the probe. The radioactive probe was prepared by nick translation (Maniatis et al., 1975)

390 o f SV40 D N A

(prepared

(Mukhopadhyay

and

from plasmid pSV40

Mandal,

1983))

and AF variations being normally distributed (data

using

not

shown), the

t

test m a y b e a p p l i e d to the

A m e r s h a m ' s n i c k t r a n s l a t i o n kit (cat. N o . N 5 0 0 0 ) .

r e s u l t s . I n o u r t e s t c o n d i t i o n s , a d o u b l i n g o f A F is

The

s i g n i f i c a n t at P < 0.001 f o r at l e a s t 15 d e g r e e s o f freedom.

assay was performed

e x a c t l y as d e s c r i b e d

p r e v i o u s l y ( V a n h o r i c k a n d M o e n s , 1983). T h e A m p l i f i c a t i o n F a c t o r ( A F ) w a s d e f i n e d as the ratio of SV40-specific radioactivity b o u n d to t h e filter c a r r y i n g a c e r t a i n n u m b e r o f t r e a t e d cells

Results

to the SV40-specific radioactivity b o u n d

Mutagenic activity of GTAC in the Salmonella/ microsome test

to the

filter c a r r y i n g t h e s a m e n u m b e r o f u n t r e a t e d cells, radioactivities being

corrected

for

backgrounds

A s s h o w n i n T a b l e 1, G T A C i n d u c e d h i s t i d i n e reversion m u t a n t s in S a l m o n e l l a strains TA1535

( S D ~< 10%). T h e SI w a s d e f i n e d a b o v e . B o t h SI TABLE 1

EFFECT OF GTAC ON 5 S. typhimurium TESTER STRAINS WITH AND WITHOUT METABOLIC ACTIVATION ($9) All plates were made in triplicate and the mean values are given. Treatment (mg/plate)

TA100 - $9

+ $9

- $9

+ $9

- $9

+ $9

- $9

+ $9

- $9

+ $9

GTAC

106 168 222 611 936

114 184 235 588 1042

16 86 155 643 1107

12 104 159 618 979

4 2 0 3 1

4 4 3 3 6

87 113 96 116 121

121 125 125 137 151

18 24 19 26 20

30 33 35 31 28

. .

. .

0 5 10 50 100

TA1535

BaP a AFB1 a

517 663

. .

TA1537

TA97

. .

. .

TA98

. .

. .

a BaP (5 ~g/plate) and AFB a (0.2 ~g/plate) were used as positive controls.

TABLE 2 INDUCTION OF GENE CONVERSION IN S. cerevisiae (D7) BY GTAC 3 × 107 cells/ml were treated with increasing concentrations of GTAC in the presence or absence of metabolic activation for 1 h at 37 ° C followed by 16 h at 29 ° C. Appropriate dilutions of the treated cultures were spread on complete or selective media for the estimation of respectively the survival and the gene conversion induction. Results shown are means derived from 2 independent experiments. Treatment

- $9

(mg/ml)

Survival (%)

Gene conversion (trp+/105 survivors)

Survival (%)

+ $9 Gene conversion (trp+/105 survivors)

GTAC 0.00 0.01 0.05 0.10 0.50 1.00 5.00

100 83 86 124 118 84 82

1.02 (288) 1.91 (223) 2.59 (314) 2.37 (413) 8.47 (1402) 21.01 (2481) 97.82 (11282)

100 78 108 100 81 110 102

1.05 (126) * 1.51 (126) 1.39 (181) 1.83 (218) 4.80 (466) 4.82 (632) 27.32 (3 337)

DMSO EMS a END "

89 95 110

124 71

0.97 (129) * 20.06 (2120)

1.08 3.76 2.18

(121) * (503) (269) *

a EMS (0.1 /zl/ml) and END (4 mg/ml) were used as positive controls. Numbers in parentheses are the actual total number of colonies on 10 plates ( * on 9 plates).

391

Genotoxic activity of GTAC in the yeast D7 system G T A C dose-dependently increased the frequency of gene conversion at the ' t r p 5' locus. The presence of a metabolic activation mixture slightly reduced the mutagenic activity of the molecule (Table 2).

and TA100. On the other hand, G T A C was not mutagenic in strains TA1537, TA97 and TA98. This indicates that G T A C is an inducer of basepair substitutions but not an inducer of frameshift mutations in this test. G T A C was equally active in the presence or absence of $9 mix. TABLE 3

ABERRATION LEVELS IN CHO CELLS F O L L O W I N G EXPOSURE TO GTAC A: Survival index (SI), mitotic index (MI) and numerical aberrations. B: Frequency of aberrations per cell. C: Percentage of cells with aberrations. 106 CHO cells were exposed for 18 h to MMC (0.02 # g / m l and 0.1 ~ g / m l ) or GTAC dissolved in water. Cells were collected at the end of treatment. MI and SI are defined in ' Materials and methods'. After GTAC or MMC treatment, the increase of the percentage of cells with numerical aberrations (aneuploid + polyploid cells) was statistically not significant. For the analysis of the structural aberrations 100 metaphases were examined in the case of GTAC and 50 in the case of MMC. A: Treatment (~g/~)

SI (%)

MI (%)

Aneuploid metaphases (%) Polyploid metaphases (%)

GTAC

100 87 86 70 57 56

10.4 7.7 8.3 6.5 6.0 3.1

0 4 0 0 0 2

2 2 4 6 4 4

8 4 6 6 12 4

0 2 0 0 4 6

78 62

5.5 2.8

0 0

6 10

16 12

2 2

MMC

0 5 10 20 50 100 0.02 0.10

B: Treatment (/~g/ml) GTAC

MMC

0 5 10 20 50 100 0.02 0.10

C: Treatment (~g/ml) GTAC

MMC

0 5 10 20 50 100 0.02 0.10

< 19

> 23

PCC or PM (%)

Chromatid aberrations

Chromosome aberrations

G

B

IC

G

B

R

D/PC

m

All aberrations including G

All aberrations excluding G

0.05 0.09 0.30 0.24 0.65 0.30

0.05 0.02 0.05 0.05 0.09 0.13

0.00 0.00 0.00 0.07 0.12 0.20

0.02 0.01 0.07 0.03 0.07 0.07

0.03 0.12 0.11 0.11 0.21 0.32

0.00 0.00 0.00 0.01 0.02 0.00

0.01 0.02 0.01 0.01 0.01 0.03

0.00 0.01 0.05 0.04 0.04 0.02

0.16 0.27 0.59 0.56 1.31 1.07

0.09 0.17 0.22 0.29 0.59 d 0.70 d

0.22 0.54

0.00 0.12

0.10 0.62

0.08 0.04

0.28 0.74

0.00 0.02

0.00 0.04

0.02 0.02

0.70 2.34

0.40 c

MA

All aberrations including G

All aberrations excluding G

Chromatid aberrations Chromosome aberrations

1.76 d

G

B

IC

G

B

R

D/PC

m

4 9 21 19 40 19

4 2 5 4 6 12

0 0 0 6 10 14

2 1 5 3 7 7

3 5 9 9 18 24

0 0 0 1 2 0

1 2 1 1 1 3

0 1 1 4 3 2

0 0 0 0 1 0

12 18 31 0 37 d 63 d 53 d

8 9 15 20 b 35 d 42 d

16 28

0 12

10 42

8 2

22 48

0 2

0 2

2 2

0 2

50 d 80 d

30 d 74 ~

PCC, premature chromosome condensations; PM, pulverized metaphases; G," gaps; B, breaks; IC, interchanges (including complex rearrangements); R, rings; D / P C , di/polycentrics; m, minutes; MA, metaphases containing multiple aberrations. a p = 0.025-0.05; b p = 0.01-0.025; c p = 0.001-0.01 and d p < 0.001.

392

Clastogenic activity of GTA C in CHO cells As illustrated in Table 3, both the frequency of aberrations (including and excluding gaps) per cell and the percentage of cells with all aberrations increased in a dose-dependent manner after exposure to GTAC, while the survival index (SI) and the mitotic index (MI) decreased. These effects were highly significant ( P < 0.001) at a GTAC concentration of 50 /~g/ml. Chromatid gaps, interchanges and chromosome breaks are the predominant types of induced chromosome aberrations. GTAC also induced premature chromosome condensation (PCC) and shattered metaphases. No significant increase of the percentage of cells with numerical aberrations (aneuploid + polyploid cells) was observed. This compound is about 1000 times less cytotoxic and clastogenic than MMC used as the positive standard.

TABLE 4 CYTOTOXICITY A N D ABILITY OF GTAC TO I N D U C E SDA IN C O 6 0 CELLS Cultures containing 5 x 105 C O 6 0 cells were treated for 24 h with R-7000 (0.1 ~tg/ml), R-7372 (0.1 ktg/ml), DMSO (0.5%, the solvent of R-7000 and R-7372) or various concentrations of GTAC (3-150 ttg/ml) dissolved in water. Three days after the end of treatment, the cells were dispersed with trypsin, samples of 1 million cells were trapped on nitrocellulose filters. After alkali denaturation, the cellular DNA was hybridised against [32p]sv40_DNA (1.5-2.0 X 108 c.p.m.//xg, 5 X 105 c.p.m./filter). The filter-bound radioactivity was measured by scintillation counting. Three background filters without cells were processed as the other filters. Their radioactivities ranged from 120 to 200 c.p.m. The values presented for each dose were mean values from triplicate cultures. Treatment

SI (%)

AF

(~g/n~) GTAC

DMSA R-7000 R-7372

0 3 7.5 15 75 150 0.5 a 0.1 0.1

100 100 100 102 34 8

1.0 0.9 0.7 1.2 5.4 b 34.4 b

100 46 100

1.0 7.4 b 1.0

a DMSO, 0.5% solution. AF, amplification factor; SI, survival index. b p < 0.001.

Induction of DNA amplification by GTAC GTAC is an inducer of SV40-DNA amplification in C O 6 0 cells in the absence of an exogenous microsomal bioactivation system. This is illustrated in Table 4 by the significant increase of the amplification factor for GTAC at concentrations around 50 /~g/ml. As compared to previously tested chemicals (Vanhorick and Moens, 1983; Vleminckx and Moens, 1986) and to R-7000, GTAC is a weak direct inducer of D N A amplification.

Conclusions GTAC is a chemical intermediate widely used in various manufacturing procedures. To our knowledge, there is only one report on the mutagenicity of G T A C in the literature (Voogd et al., 1981). In this study, we have investigated the genotoxic properties of GTAC in a battery of 4 short-term tests. These tests have been selected on the basis of their complementary biological properties and genetic endpoints. Such an approach can provide strong indications of potential carcinogenicity; short-term tests considered separately (Lave and Omenn, 1986; Quinto et al., in press) or a single long-term bioassay (National Toxicology Program, 1980) are of less accuracy. We report that GTAC is positive in all the tests carried out. GTAC behaves as a base-pair substitution inducer in S. typhimurium with and without metabolic activation. It is able to induce gene conversion in S. cerevisiae (D7). GTAC is clearly a clastogenic agent and is also able to mediate the induction of viral DNA amplification in Chinese hamster cells. A long-term animal study is presently unavailable. Nevertheless, in view of our in vitro test results, it is advisable to consider G T A C a potential carcinogen.

Acknowledgements The authors thank Dr. R. Royer (Institut Curie, Paris) for the supply of the nitronaphthofurans and Dr. E. Bonnyns for statistical support. The technical assistance of C. Bagyary, Y. Willems and J. Henrotte is gratefully acknowledged. We are also grateful to M.J. Deroisy for typing the manuscript.

393

References Ajinomoto Co. (1980) Shampoos, Chem. Abstr., 93 (22), 210095f. Ajinomoto Co. (1981) Ionized polymers and nonionic surfacrants in hair-rinse preparations, Chem. Abstr., 95 (2), 12570K. Arnold, H.A. (1978) Cosmetic preparation containing colored powdered cellulose, Chem. Abstr., 89 (6), 48803d. Balland, J. (1981a) Use of quaternary epoxyalkylammonium compounds for pretreating polyamide fibers, Chem. Abstr., 95 (22), 188598q. Balland, J. (1981b) Use of quaternary epoxyalkylammonium compounds in short bath dyeing of cellulose fibers, Chem. Abstr., 95 (22), 188597p. Bell, V.A., D.M. Lewis, P.R. Brady, P.G. Cookson and K.W. Fincher (1980) Pretreatment of textiles prior to transfer printing, Chem. Abstr., 93 (4), 27684h. Bridie, A.L., J.M. Wolff and M. Winter (1979) The acute toxicity of some petrochemicals to goldfish, Water Res., 13, 623-626. Chavannes, J.P., R. Fischer, F. Palacin and J.P. Vierling (1980) Dyeing or printing of textiles containing cellulose fibers, Chem. Abstr., 93 (8), 73681c. Claiborne, J.L. (1981) Discoloration preventing agent for textile materials and its use in laundry systems, Chem. Abstr., 95 (22), 188992p. Dean, B.J. (Ed.) (1983) Report of the UKEMS Sub-Committee on Guidelines for Mutagenicity Testing, Part 1, UKEMS, London. Eschwey, A., H. Fischer, H.F. Pfeiffer and R. Schmid (1980) Enzyme-stabilizing agent, Chem. Abstr., 93 (1), 3079p. Fischer, W., M. Langer and G. Pohl (1981) Cationic starch ethers, Chem. Abstr., 95 (10), 82755y. Fleche, G., S. Gosset and M. Huchette (1980) Cationized starch and its use, Chem. Abstr., 92 (24), 200132f. Fuji Chemical Co. (1980) Electrically conductive coating compositions for electro-recording paper supports, Chem. Abstr., 93 (24), 228588d. Hatt, H.D., and M.J. Gantt (Eds.) (1979) The American Type Culture Collection, Catalogue of Strains II (2nd edn.), Library of Congress, Washington, DC. Hoffmann-La Roche Co. (1978) Cosmetics, Chem. Abstr., 89 (18), 152575s. ICI Australia Ltd. Commonwealth Scientific and Industrial Research Organization (1980) Production of ferromagnetic polymer particles, Chem. Abstr., 94 (26), 209696s. Kao Soap Co. (1981) Cellulose ether cationic derivatives, Chem. Abstr., 95 (16), 134651h. Kawamoto, N. (1979) Quaternary ammonium derivatives of cellulose, Chem. Abstr., 92 (6), 43522p. Lave, L.B., and G.S. Omenn (1986) Cost-effectiveness of short-term tests for carcinogenicity, Nature (London), 324, 29-34. Lavi, S. (1981) Carcinogen-mediated amplification of viral DNA sequences in simian virus 40-transformed Chinese hamster embryo cells, Proc. Natl. Acad. Sci. (U.S.A.), 78, 6144-6148.

Lewis, R.H., I. Holder and M.L. Finney (1981) Multicolor coating system, using it to achieve a multicolor pattern on a surface and articles having such multicolor patterns, Chem. Abstr., 95 (8), 63627s. Lion Corp. (1980a) Coagulants, Chem. Abstr., 93 (26), 244887f. Lion Corp. (1980b) Weakly acidic cosmetic emulsions, Chem. Abstr., 94 (10) 71218d. Maniatis, T., A. Jeffrey and D.G. Kleid (1975) Nucleotide sequence of the rightward operator of phage, Proc. Natl. Acad. Sci. (U.S.A.), 72, 1184-1188. Maron, D.M., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. Moritani, T., T. Moritani, J. Yamauchi, Y. Tanaka and M. Shivaishy (1981) Cationic polymer emulsions, Chem. Abstr., 95 (2), 8227m. Mukhopadhyay, M., and N.C. Mandal (1983) A simple procedure for large-scale preparations of pure plasmid DNA free from chromosomal DNA from bacteria, Anal. Biochem., 133, 265-270. National Toxicology Program (1980) Tech. Bull., 1(3), 2. OECD (1981, 1983), Guidelines for Testing Chemicals, OECD, Paris. Purchase, I.F.H. (1980) Procedures for screening chemicals for carcinogenicity, Br. J. Ind. Med., 37, 1-10. Quinto, I., L. Tenenbaum and M. Radman, Genotoxicity profile of monofunctional alkylating agents in E. coli: quantitative correlation with carcinogenic potency in rodents, Proc. Natl. Acad. Sci. (U.S.A.), in press. Royer, R., J. Einhorn, P. Demerseman, W. Moens and P. Gayral (1985) Synth+se et premi+re ~tude biologique d'un d~riv~ dichlor~thylanin~ du nitro-2 naphto[2,1-b]furanne, Eur. J. Med. Chem.-Chim. Ther., 20, 429-432. Rudkin, A.L., J.H. Clint and K. Young (1979) Textile-treating agent, Chem. Abstr., 92 (24), 200145n. Rusznak, I., T. Kuszman, J. Leyer and J. Zobor Frankl (1980) Theoretical aspects and practical application of reactive auxiliaries in the dyeing of cellulosic fibers with direct dyes, Chem. Abstr., 92 (18), 148357s. Siegel, S. (Ed.) (1956) Non Parametric Statistics, International Student Edition, McGraw-Hill, New York. Soremark, C., H. Mats-Olov, B. Samuelsson, C.J. Alfthan, E. Soremark and Z. Lindh-Nomm (1981) Lignocelhilosic material with improved strength, drainability and heating capacity, Chem. Abstr., 95 (6), 45050u. Vanhorick, M., and W. Moens (1983) Carcinogen-mediated induction of SV40 DNA amplification is enhanced by acrylamide in Chinese hamster CO60 cells, Carcinogenesis, 4, 1459-1463. Vleminckx, C. and W. Moens (1986) Induction of microchromosomes by chemical carcinogens correlates with SV40-DNA amplification in SV40-transformed Chinese hamster cells, Carcinogenesis, 7, 229-234. C.E. Voogd, J.J. Van der Stel and J.J.J.A.A. Jacobs (1981) The mutagenic action of aliphatic epoxides, Mutation Res., 89, 269-282. Wahl, G.M., L. Vitro, R.A. Padgett and G.R. Stark (1982) Single-copy and amplified CAD genes in Syrian hamster

394 chromosomes localized by a highly sensitive method for in situ hybridization, Mol. Cell. Biol., 2, 308-319. Weber, R., H. Andree and G. Wetzel (1980) Textile washing composition with finishing action, Chem. Abstr., 93 (23) 206523b. Yanagawa, T., K. Nakayama and D. Saiga (1980) Hair preparations, Chem. Abstr., 93 (16) 155708f.

Zimmermann, F.K., R. Kern and H. Rasenberger (1975) A yeast strain for simultaneous detection of induced mitotic crossing-over, mitotic gene conversion and reverse mutation, Mutation Res., 28, 381-388.