GEORGE R. DOUGLAS, EARLE R. NESTMANN, C.E. GRANT, R.D.L. BELL,. J.M. WYTSMA and ])AVID J. KOWBEL. Mutagenesis Section, Environmental and ...
45
Mutation Research, 85 (1981) 45--56 Elsevier/North-Holland Biomedical Press
MUTAGENIC ACTIVITY OF DIALLATE AND TRIALLATE DETERMINED BY A BATTERY OF IN VITRO MAMMALIAN AND MICROBIAL TESTS
GEORGE R. DOUGLAS, EARLE R. NESTMANN, C.E. GRANT, R.D.L. BELL, J.M. WYTSMA and ])AVID J. KOWBEL
Mutagenesis Section, Environmental and Occupational Toxicology Division, Department of National Health and Welfare. Environmental Health Centre, Tunney's Pasture, Ottawa, Ontario, KIA OL2 (Canada) (Received 25 February 1980) (Revision received 17 September 1980)
(Accepted 16 October 1980)
Summary Diallate and TriaUate are carbamate herbicides used mainly for the pre-emergence control of wild oats in various crops. The genetic activity of these compounds was studied using a battery of microbial and mammalian in vitro tests. In the Salmonella/mammalian-microsome assay, Diallate and Triallate show dose-related increases without metabolic activation in strains TA1535, TA100 and TA98, indicating that these compounds cause both frameshift and basesubstitution mutations. Mutagenicity of both herbicides was enhanced greatly by incubation with Aroclor 1254 induced rat-liver $9. Genetic activity in mammalian cells was determined using a number of in vitro tests with Chinese hamster ovary (CHO) cells combined with metabolic activation as described above. Both Diallate and TriaUate caused dose-related decreases in colony-forming ability, with concomitant dose-related increases in the frequencies of cells with chromosome damage and in the number of sister-chromatid exchanges. However, only Diallate caused a reduction in DNA molecular weight as determined by alkaline sucrose gradient (ASG) sedimentation. DNA damage was negligible even at concentrations of Triallate that reduced colony-forming ability to zero. This suggests that the lesions in DNA detected by the ASG technique are not necessarily related to those that produce chromosomal damage. These data, taken together, strongly implicate both Diallate and TriaUate as capable of causing mutations in mammals. However the risk to man in terms of inherited disease or cancer remains to be established by appropriate in vivo methodology. Abbreviations: 9-AA. 9-aminoacridine hydrochloride: 2-AAF. 2-ecetylaminofluorene; B(a)P. benzola]pyrene; 2-AN, 2-anthramlne; DMN. dimethyln/trosamine; MNNG, N.methyl-N'-nitro-N.inltrosogtumidlne; 4-NQO, 4-rdtroqttino//ne oxide. 0165-1161/81/0000--0000/$02.50 © Elsevier/North-Holland Biomedical Press
46 DiaUate (2,3-dichloroalkyl diisopropylthiolcarbamate)and Triallate (2,3,3trichloroalkyl diisopropylthiolcarbamate) are herbicides used widely for the preemergence control of wild oats in cereal forage and vegetable crops. Statistics relating specifically to the use of these c o m p o u n d s are sparse. However, the following information is available and indicates that these chemicals are used extensively. Annual U.S. consumption of Diallate and Triallate was estimated in 1975 to be 0.14 and 0.59 million kg, resp. (Stanford Research Institute, 1976). In 1969, use of Triallate on the Canadian prairies amounted to as much as 510 290 kg on 303 520 hectares (Smith, 1969). Annual European production of Diallate was estimated to range from 1--5 million kg (IARC, 1976). Diallate has been reported to be carcinogenic in mice (Innes et al., 1969) and in rats (Ulland et al., 1973) after oral administration. Since most carcinogens are also mutagenic (McCann et al., 1975) it is not surprising that both Diallate and Triallate (which differs from Diallate by one chlorine atom) are mutagenic in Salmonella (De Lorenzo et al., 1978; Sikka and Florczyk, 1978; Carere et al., 1978). These studies have indicated that b o t h herbicides require in vitro metabolic activation by rat liver microsomal fraction ($9) before mutagenicity is observed. It is, thus, comprehensible that an earlier study (Andersen et al., 1972) not employing in vitro metabolic activation found no mutagenicity in Salmonella. Although they are used widely and may pose a potential hazard to human health in terms of cancer and germinal mutations inherited by future generations, little or no published data are available on the mutagenicity of Diallate and Triallate in mammalian cells. Consequently, the mutagenic activity of Diallate and Triallate was investigated in Chinese hamster ovary (CHO) cells using the following indicators: ( 1 ) c h r o m o s o m e aberrations, (2)sister-chromatid exchange, ( 3 ) D N A damage as determined b y alkaline sucrose gradient (ASG) sedimentation, ( 4 ) c y t o t o x i c i t y as determined by survival of colony-forming ability. In addition, experiments were performed to provide confirmation of the mutagenicity of Diallate and Triallate in the Salmonella/mammalian microsome test (Ames et al., 1973). This battery of tests was chosen to reflect a variety of possible mutagenic events. The Salmonella/microsome test and chromosome aberration tests were included because they detect the ability of a chemical to cause types of mutations, namely gene and chromosome. In addition to these basic tests, the sister-chromatid exchange and DNA-damage assays serve as auxiliary tests which detect events that may be associated with the induction of mutation and therefore provide complementary data. Cytotoxicity was determined to form a basis for comparison of any possible effects in mammalian cells caused b y the test chemicals and to assist in the selection of the range of concentrations to be tested. Methods and materials
Cell culture. Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100, and media for bacterial testing were as described by Ames et al. (1975). Monolayer cultures of CHO cells were maintained as described previously (Nestmann et al., 1979) in minimal essential medium with non-essential amino
47 acids and sodium pyruvate (MEM, Gibco). MEM was supplemented with 10% fetal calf serum (FCS). Cells were grown at 37 ° , 5% C02 and high relative humidity. Cultures were routinely screened for the presence of mycoplasma (Ho and Quinn, 1977)and found to be free of contamination. All cell manipulations were carried out under illumination from gold fluorescent lamps (General Electric, F40GO). Chemicals. Diallate (98%) and Triallate (99+%) (Chem Services) were dissolved in ethyl alcohol for the bacterial and mammalian tests. Concentrated stock solutions of the test chemicals were prepared so that a 100 × dilution produced the desired concentration when treating CHO cells. Final concentration of ethyl alcohol in tissue culture medium did not exceed 1%. This concentration does not measurably affect the levels of the parameters assayed here in CHO cells (G.R. Douglas, unpublished data). N-Methyl-N'-nitro-N.nitrosoguanidine (MNNG; Aldrich), 4-nitroquinoline oxide (4-NQO; ICN K and K), 9-aminoacridine hydrochloride (9-AA; Matheson, Coleman and Bell), 2-acetylaminofluorene (2-AAF; ICN K and K), benzo[a]pyrene (B(a)P; Aldrich) and 2-anthramine (2-AN; Aldrich) served as positive controls in the bacterial tests. Dimethylnitrosamine (DMN, Aldrich) was the positive control in experiments with CHO cells. Bacterial testing procedures. The procedures were those described previously (Ames et al., 1975; Nestmann and Kowbel, 1979). Aroclor 1254 (Analabs)induced liver homogenate ($9) was prepared from Sprague--Dawley rats following the method of Ames et al. (1973). Experiments were performed with duplicate plates at each treatment. Dilutions of all test materials were prepared to allow addition of 0.1 ml of solution per plate. Each herbicide was tested in 3 separate experiments. Alkaline sucrose gradient sedimentation. CHO cells, treated essentially as described elsewhere (Nestmann et al., 1979), were labelled overnight in 60-ram tissue culture dishes containing 8 X 10 s cells in MEM supplemented with 10% newborn calf serum and 0.5 uCi [3H]TdR/ml (46 Ci/mM) or 1.0 uCi [14C]TdR/ ml (50 mCi/mM). The cells were chased with non-radioactive medium for 2 h prior to treatment. The treatment mixture contained $9 (7.1%), 1 mM HEPES buffer (pH 7.2), 1.2 mM MgC12, 7.8 mM KC1, 1.2 mM glucose-6-phosphate, 1.0 mM NADP, distilled H20 (6.67%), MEM with 2.5% FCS (75.2%), and 1% test chemical in solvent. Cells were treated with 1.4 ml of the treatment mix for 1 h. After removal of the mixture, the cells were washed 3 times in ice-cold, calcium- and magnesium-free, phosphate-buffered saline containing 10 mM EDTA (PBS). Cells were removed from the culture dishes by gentle scraping and resuspended in 1 ml ice-cold PBS. The method of Palcic and Skarsgard (1972) as modified by Nestmann et al. (1979) was used for alkaline sucrose sedimentation. Each gradient contained 4.8 ml composed of 4.5 ml 5--20% alkaline sucrose and a 0.3 ml lysis layer. Approx. 8 × 103 ['4C]TdR-labelled control cells and 8 × 103 [3H]TdR-labelled treated cells were lysed on each gradient for 10.5 h at 20°C and spun at 15 000 rpm for 6 h in a Beckman SW50.1 rotor. Gradients were fractionated from the top into 0.2-ml fractions, collected in mini-scintillation vials, and counted in 5 ml aquasol (New England Nuclear) after neutralization with 0.5 N HC1.
TABLE
1
37
.
0.010
0.150
0.01
2-AN
9-AA
B(a)P
.
.
.
.
.
19
c
457
--
--
--
--
--
93 c
.
107
557
388
176
125
44
.
.
.
.
.
.
372
227
227
166
145
93
96
.
448
.
.
1690
.
.
84
74
97
108
103
c
c
c
c
c
.
.
. 1450
.
.
.
.
.
339
1317
1080
986
612
484
237
135
+S9
c
c
.
.
.
.
.
.
2
2
2
7
4
5
9
3
5
4
.
.
1046
.
--$9
TA1537
a Values are averages of duplicate plates from 1 of 3 experiments showing similar results. b Results for these strains from a separate experiment. c Reduction in His- background lawn.
.
.
.
0.050
80 c
10.0
0.0005
57 c
2-AAF
36 c
2.0
4.0
4oNQO
30
1.0
1818
38
0.50
0.005
.
0.25
c
9
14
0.050
.
0.025
0.10
8
11
14
0.005
0.020
12
0.001
0.010
12
0
--$9
--S9
+S9
TA100
TA1535
a
Revertants/plate
IN SALMONELLA
Amount
DIALLATE
per plate (rag)
OF
MNNG
Dianate
Substance
MUTAGENICITY
.
.
.
.
b
.
. . 116
.
.
.
.
.
.
3 c
2 c
6
7
8
5
6
5
6
+S9
.
.
.
.
.
.
.
.
.
10
71
II
6
11
9
.
9
9
5
8
9
--S9
.
TA1538
.
.
b
.
.
20
21
25
. .
-1464
.
58 c
21
19
16
22
18
+$9
. . .
10
136
54 c
46 c
35 c
27 c
25
__
20
18
--
21
18
11
16
22
--$9
TA98
903
--
--
__
_
__
143
_
188
128
--
65
48
41
35
29
+$9
c
c
00
49 Calculation of molecular weights was carried out using the method described by Palcic and Skarsgard (1972), employing a sedimentation constant derived from the sedimentation characteristics of bacteriophage T4 DNA. The cosedimentation of control and treated DNA greatly increases the precision of the breakage estimate. Cell survival. Cell survival was estimated by determining colony-forming ability at different concentrations of Diallate and Triallate. Approx. 200 cells were plated in 60-mm tissue culture dishes (4 dishes per concentration)in MEM, 10% FCS, and allowed to attach for 4--5 h. The cells were then treated as above for I h, washed gently with Earle's balanced salt solution 3 times, and incubated in MEM containing 10% FCS for 6--7 days. Colonies were fixed in ethanol : glacial acetic acid (3 : 1), stained with Giemsa, and counted. Survival was expressed as the mean colony-forming ability relative to the solvent-alone control treatment. Chromosome aberrations. Approx. 2 × l 0 s cells were seeded in 60-mm tissue culture dishes in MEM, 10% FCS. After 24 h incubation at 37°C, cells were treated with the test chemical for 1 h as above. Cells were then washed in fresh medium and cultured for 24 h. Colcemid (0.08 ug/ml)was added to the cultures for the final 2 h. After hypotonic treatment in 0.075 M KC1 for 15 min, cells were fixed in 3 : 1 ethyl alcohol : glacial acetic acid and washed 3 times in fresh fixative. Chromosome preparations were made by dropping the cell suspension onto clean distilled water-wetted microscope slides and air dried. The slides were subsequently stained with Giemsa (3%, Gurr R66) for 7 min and mounted in Eukitt (Otto C. Watzka). Chromosome damage was enumerated by classifying all aberrations according to defined criteria (Savage, 1975). Approx. 200 cells per treatment were scored (approx. 50 per slide). Sister-chromatid exchange. The method used was essentially as described elsewhere (Perry and Wolff, 1974). 5 × 104 cells were seeded in 60-mm tissue culture dishes in MEM, 10% FCS and incubated for 48 h. Cultures were treated with the test chemical exactly as described above. Cells were washed in Earle's salt solution (Gibco) and incubated in MEM, 10% FCS containing 10 uM 5-bromodeoxyuridine (BUdR) until harvested 24 h later. Slides were prepared and stained in Hoechst 33258 (0.5 ug/ml) for 15 min, rinsed in distilled H20, and coverslips were mounted in distilled H20 and sealed with rubber cement. Slides were exposed to fluorescent ceiling lights for 22 h. After removal of coverslips, slides were immersed in 2 × SSC at 60°C for 1 h, and stained in Giemsa (Gurr R66, 3%)for 7 min. 25 cells per treatment were scored for the number of sister-chromatid exchanges. Results
Diallate and Triallate induce dose-related increases in mutant frequencies in strains TA1535 and TA100 in the presence of $9 (Tables 1 and 2). Higher doses result in mutation induction also in strain TA98 with $9. The highest doses of both herbicides, causing bacterial lethality, lead to low levels of reversion without $9 in strains TA1535 and TA100, as well as in strain TA98 with Diallate. No mutagenic effect was found in strain TA1537 or TA1538 for either compound (Tables 1 and 2). Diallate appears to be a more active muta-
TABLE
2
Triallate
Triallate
Triallate
MNNG 4-NQO 9-AA 2-AN B(a)P 2-AAF
1
2
2
1--3
0.005 0.0005 0.15 O.010 0.01 0.05
0 0.5 1.0 2.0 5.0
0 0.125 0.25 0.50 1.25
0 0.005 0.010 0.020 0.050
Amount per plate (nag)
2040 . . 52 . .
16 28 52 67 b 0 b
9 11 16 26 46
11 22 21 17 18
.
.
308
--
.
.
.
.
1904 . . 345 . .
110 162 222 285 b 302 b
100 91 120 148 237
108 118 117 97 102
.
. 1697
.
.
.
113 1538 1914 1526 b 0 b
92 863 1601 1523 1374
116 118 136 204 497
+$9
882 . 14
.
6 6 8 7 6
8 6 4 6 4
9 10 10 5 6
--$9
TA1537
.
. 116
.
12 12 16 0 b 0 b
5 3 4 7 6
12 11 8 8 11
+S9
for the positive controls which are averages of 6 plates.
.
.
26 521 509 462 b 0 b
14 134 348 284 442
21 16 18 34 79
--$9
--$9
+S9
TA100
per plate a
TA1535
Revertants
IN SALMONELLA
a Values are averages of duplicate plates except b Reduction in His- background lawn.
Substance
OF TRIALLATE
Expt.
MUTAGENICITY
.
.
. . 12
. 78 .
10 12 10 10 b 12 b
10 i0 6 6 5
6 6 8 6 8
--89
.
TA1538
.
.
1042
.
.
--
.
19 20 22 34 b 0 b
31 18 10 13 18
13 20 17 19 16
+$9
.
.
. . 24
140
16 30 22 28 b 22 b
21 17 21 23 27
28 27 26 24 32
--$9
TA98
1244
--
34 225 278 282 b 0 b
21 77 168 240 246
43 36 41 40 46
+$9
C~ O
51
3"0 I00
*
• ~
,
#, , , ,,i
'~2.5
I00 2"0
8O >
o
=>
~
I'0
,4
8o
08
6o
0'6 %-
40
0.4
g
1"5
60
~
t~ c~
l,u
I'0
.o
~ m
2O
0
ff . 10_5
. 6
.
. 8
. . 10_4
2
DIALLATE CONCENTRATION(M)
m
0"5
20
0"0
0
02 #l
,
10_5
6
i , , I 8
10_4
2
4 I0.0
TRIALLATE CONCENTRATION(M)
Fig. I . E f f e c t o f D i a l l a t e a f t e r in v i t r o m e t a b o l i c a c t i v a t i o n o n cell curvival in C H O cells as d e t e c t e d b y c o l o n y - f o r m i n g a b i l i t y (A), a n d o n D N A d a m a g e as d e t e c t e d b y A S G s e d i m e n t a t i o n ( e ) . T h e survival a n d DNA-damage curves are, resp., the pooled results from 4 and 3 separate experiments involving overlapping concentration ranges. Fig. 2. E f f e c t o f T r i a l l a t e a f t e r in v i t r o m e t a b o l i c a c t i v a t i o n o n cell survival in C H O cells as d e t e c t e d b y c o l o n y - f o r m i n g a b i l i t y (A), a n d o n D N A d a m a g e as d e t e c t e d b y A S G s e d i m e n t a t i o n (e). T h e survival a n d D N A - d a m a g e c u r v e s a r e , zesp., t h e p o o l e d r e s u l t s f r o m 2 a n d 3 s e p a r a t e e x p e r i m e n t s i n v o l v i n g o v e r l a p p i n g concentration ranges.
gen in Salmonella than Triallate in that significant mutagenesis and lethality are induced at lower doses. All assays involving CHO cells were done in the presence of S9 only, because the mutagenic activity of both herbicides in Salmonella is greatly enhanced by metabolic activation. In CHO cells, both Diallate and Triallate cause doserelated cytotoxicity expressed in terms of reduced colony-forming ability (Figs. 1 and 2). However, Triallate is a more p o t e n t c y t o t o x i c agent in CHO cells in that the Do (concentration required to reduce survival to 37%) for Diallate is approx. 2.8 × 10 -4 M, whereas the Do for Triallate is approx. 2.1 × 10 -4 M. Data showing the effect of Diallate and Triallate on chromosome aberrations are summarized in Tables 3 and 4, resp., and also are represented graphically in Fig. 3. Both herbicides cause very rapid dose-related increases in the frequency of cells with damaged chromosomes. Virtually all chromosome damage observed after 24 h w a s of the chromatid-type. These effects, as with cytotoxicity, were observed over a narrow range of concentrations. Although Triallate causes chromosome damage at lower concentrations than Diallate, when chromosome aberration yield at equitoxic concentrations is compared, both compounds produce similar responses. For example, at the Do for both chemicals, approx. 10% of cells have damaged chromosomes. Diallate causes a dose-related increase in DNA damage as detected by the ASG technique (Fig. 1). The effective dose range overlaps the range for cyto-
t~
TABLE 3 E F F E C T O F D I A L L A T E O N C H R O M O S O M E A B E R R A T I O N S IN C H O C E L L S Diallate concentration (M)
Number of cells
Number of cells w i t h aberrations a
0 b
260
2
0.8
0
2.0 X 10 --4 2.25 X 10 -4
240 230
6 9
2.5 3.9
0.42 0
2.5 X 10 -4 2 . 7 5 X 10-4
210 230
15 14
7.1 6.1
3.0 X 10 -4 3.25 × 10-4
210 290
40 35
5.0 X 10 -2 DMN c
130
65
a Excluding achromatic lesions. b Solvent alone, c Positive control, dime~ylnitrosamine.
Percentage of cells w i t h aberrations a
Aberrations/l O 0 cells
Chromatid exchanges
Chromatid breaks
Chromosome exchanges
Chromosome breaks
Achromatic lesions
0.38
0
0.77
0
1.6 3.9
0 0
0.42 0.87
0.42 0
8.6 1.3
7.1 6.5
0.48 0.87
2.4 0.43
0 0.43
19.0 12.1
16.2 7.9
12.4 6.6
3.3 1.7
1.9 1.7
1.9 0
50.0
49.2
36.9
0.77
0
0.77
56 55
200 200
230 171
167
2.0 X 1 0 - 4 2.33 X 10 -4
2.66 × 1 0 - 4
5.0 X 10 -2 D M N c
75
5 4
• E x c l u d i n g a c h r o m a t i c lesions. b Solvent alone. c Positive c o n t r o l , d i m e t h y l n / t r o s a m l n e .
2.6 × 1 0 - 4
11 34
200 200
1.37 X 10 -4 1.75 × 1 0 - 4
0 3
200 200
0 b 1.0 X 10 -4
Number of cell- w i t h aberrations a
Number o f ceils
TriaLlate concentration
58.3 77.8 43.7
44.9
8.6 31.0
0 0
0 0
Chromatid exchanges
42.5
31.6 35.7
4.5 26.0
2.5 2.5
0 1.0
Chromatid breaks
AberratlocLsl 1 0 0 cells
24.4 32.2
5.5 17.0
2.5 2.0
0 1.5
Percentage of cells w i t h aberrations •
E F F E C T OF T R I A L L A T E ON C H R O M O S O M E A B E R R A T I O N S IN CHO CELLS
TABLE 4
0.6
0.5 0
1.0 0.5
0.5 0
0 0.5
Chromosome exchanges
0
0 1.8
0 0
0 0
0 0
Chromosome breaks
0
0 0.58
0 0.5
0 0
0 0
Achromatic lesions
r.O
54
35 "//i
'
'
,
'
'
,
,
,i
3O
o
25
8 2o
2O
3 bJ ~D
03
I0
I
0
I
2 3 CONCENTRATION I M x l O 4 )
0 10-5
10-4 CONCENTRATION
(M)
Fig. 3. E f f e c t o f Diallate (o) a n d Triallate (o) o n t h e p e r c e n t a g e o f cells w i t h c h r o m o s o m e a b e r r a t i o n s in C H O cells a f t e r in v i t r o m e t a b o l i c a c t i v a t i o n . Bars r e p r e s e n t s t a n d a r d errors. O t h e r d a t a f r o m t h e s e e x p e r i m e n t s are s h o w n in T a b l e s 2 a n d 3. Fig. 4. E f f e c t of Diallate ( e ) a n d Triallate (o) o n t h e f r e q u e n c y o f s i s t e r - c h r o m a t i d e x c h a n g e in CHO cells a f t e r in v i t r o m e t a b o l i c a c t i v a t i o n . P o i n t s r e p r e s e n t m e a n s b a s e d o n 25 cells p e r c o n c e n t r a t i o n . Bars r e p r e s e n t s t a n d a r d e r r o r s . T h e z e r o c o n c e n t r a t i o n t r e a t m e n t is t h e s o l v e n t w i t h o u t t e s t c h e m i c a l .
toxicity (Fig. 1) and chromosome aberrations (Fig. 3). However, in 3 separate experiments no effect of Triallate on DNA sedimentation was seen even at lethal concentrations (Fig. 2). Dose-related increases in the frequency of sister-chromatid exchanges were observed after treatment of CHO cells with both Diallate and Triallate (Fig. 4). Data for 2 X 10 -4 M and 3 X 10 -4 M Triallate could n o t be obtained apparently because inhibition of cell division delayed the occurrence of second-division metaphases and hence cells with sister~hromatid differentiation. It is perhaps for this reason that the maximum yield was much greater with Diallate, even though the initial responses are similar. Discussion
The results of the bacterial mutagenicity tests presented here indicate that Diallate and Triallate cause base-substitution mutations in Salmonella strains TA1535 and T A 1 0 0 in the presence of a mammalian activation system ($9). These findings are in agreement with the results of 2 earlier studies (De Lorenzo et al., 1978; Sikka and Florczyk, 1978). The present results also indicate that both herbicides at higher doses are mutagenic with $9 activation in strain TA98, which reverts b y frame-shift mutation. The reason for the failure of the earlier reports to find the mutagenic responses in strain TA98 is n o t
55 clear. Possible explanations could be based on the sources of herbicides or the types of $9 in each study. In addition, comparison of our data with those from the literature is hampered by the absence of data for positive controls and for strain TA98 (De Lorenzo et al., 1978), or by the use of different control compounds (Sikka and Florczyk, 1978). Our data differ further from these published reports in that we find Diallate and Triallate to be mutagenic in strains TA1535 and TA100 at high doses without metabolic activation. Because the highest concentrations in the present study were 5--40 times higher, it is possible that the weak mutagenic activity observed without $9 activation is caused by the presence of impurities, by breakdown products produced spontaneously or by bacterial metabolism. Alternatively, it is possible that these chemicals are weak direct acting mutagens. In accordance with the results of bacterial tests, the mammalian tests performed show that both Diallate and TriaUate possess mutagenic activity in mammalian cells with metabolic activation. Both herbicides cause chromosome aberrations in CHO cells (Fig. 3). The dose response for both compounds occurs over intervals of approximately 1 X 10-4M between minimal and maximal response. This abrupt and steep response suggests the occurrence of a threshold where active metabolites cannot initially reach the DNA target due to factors such as permeability restrictions or cellular deactivation mechanisms. A similar narrow effective dose range has been reported previously by Rosin et al. (1980) for ascorbate mutagenesis. DiaUate, but not Triallate, causes an increase in DNA lesions recognized by the ASG technique. This finding is somewhat surprising as DiaUate and TriaUate have very similar chemical structures, differing only by one chlorine atom. However, Schuphan et al: (1979) have suggested that the ultimate mutagens for Diallate and Triallate are different. Accordingly, it would not be surprising if different DNA lesions, which have different forms of expression, are produced by each herbicide. Under the experimental conditions used here the data further suggest that, at least for Triallate, cytotoxicity. SCE and chromosome aberrations are not induced by DNA lesions detected by the ASG technique, but by other lesions not recognized by this assay. A similar lack of concordance for ASG
