Mutation Research, 260 (1991) 187-193
187
© 1991 ElsevierSciencePublishers B.V. 0165-1218/91/$03.50 ADONIS 016512189100095X MUTGEN 01658
Genotoxicity studies on the preemergence herbicide trifluralin Michael L. Garriott, Elizabeth R. Adams, Gregory S. Probst, John L. Emmerson, Thomas J. Oberly, Delinda E.F. Kindig, Steven B. Neal, Bernard J. Bewsey and Marcia A. Rexroat Toxicology Division, Lilly Research Laboratories, A Division of Eli Lilly and Company, Greenfield, IN 46140 (U.S.A.)
(Received23 July 1990) (Revision received11 November 1990) (Accepted 25 November 1990)
Keywords: Trifluralin; Herbicide; Short-termassays; Genotoxicity
Trifluralin, a dinitroaniline (Fig. 1), is a preemergence soil-incorporated herbicide that is registered with the U.S. Environmental Protection Agency and in countries worldwide for use on more than 50 crops. Trifluralin has been subjected to a variety of short-term tests designed to measure the potential genotoxic activity of the compound. These studies have been conducted to satisfy regulatory requirements in various countries and include a Salmonella/microsome mutagenicity (Ames) test, an L5178Y mouse lymphoma N (CH2C~2C~3) 2
O2N
NO~
C~ 3
Fig. 1. Trifluralin, 2,6-dinitro-N,N-dipropyl-4-(trifluoromethyl)benzenamine.CAS No. 1582-09-08.
Correspondence: Dr. Michael L. Garriott, Toxicology Division, Lilly Research Laboratories, A Division of Eli Lilly and Company, Greenfield,IN 46140 (U.S.A.)
assay, an in vitro chromosome aberration assay, and an in vivo test for sister-chromatid exchange induction in Chinese hamsters. This paper reports the results of studies conducted within the Lilly Research Laboratories and discusses additional studies with trifluralin that have been reported in the literature. Materials and methods Chemicals
All chemicals/reagents used in these studies were reagent-grade and were not further analyzed. Trifluralin (CAS 1582-09-08) was synthesized at Eli Lilly and Company. The material for the Ames, mouse lymphoma, and sister-chromatid exchange (SCE) assays was > 95% pure, while the material for the in vitro chromosome aberration assay was > 99% pure. The following positive control compounds were used in the studies reported herein: N - m e t h y l - N '-nitro-N-nitrosoguanidine (MNNG), 2-nitrofluorene (2-NF), 9-aminoacridine (9-AmAc), 2-aminoanthracene (2-AA) (Aldrich Chemical Co.), ethyl methanesulfonate (EMS), 3-methylcholanthrene (3-MC) (Eastman Kodak), cyclophosphamide (CP), and mitomycin C (MC) (Sigma Chemical Co.). In addition, bromodeoxyuridine (BrdU) (Calbiochem) and Velban ® (Eli
188 Lilly and Company) were used for sister-chromatid differentiation and as the mitotic arrestant, respectively, in the SCE assay.
Salmonella / microsome mutagenicity test The Salmonella/microsome mutagenicity test was conducted according to the method of Ames et al. (1975). All Salmonella typhimurium strains (see Table 1) employed in this assay were obtained from Dr. Bruce Ames (Berkeley, CA) and were tested prior to use for appropriate genetic markers. Trifluralin was evaluated at the exposure levels shown (Table 1), either with or without added metabolic activation by Aroclor 1254-induced, Fischer 344 rat-liver $9. The plates were scored for revertant colony formation using a New Brunswick Biotran II Automated Colony Counter (New Brunswick Scientific, New Brunswick, N J). L5178Y mouse lymphoma assay The L5178Y T K +/- assay was conducted essentially by the method of Clive et al. (1975, 1979) with minor modification by Amacher et al. (1979, 1980) and Oberly et al. (1982). Ceils in RPMI 1640 media were exposed to trifluralin for 4 h, both with and without metabolic activation at the concentrations shown in Table 2. Colonies were counted in the same manner and with the same instrumentation used for the Salmonella/microsome test. Sister-chromatid exchange assay The sister-chromatid exchange assay was conducted using the BudR tablet method as adapted from King et al. (1982) and with some modification of the method reported by Neal and Probst (1983). Doses tested and results appear in Table 3. In vitro chromosome aberration assay CHO-WB L cells were obtained from Hazleton Laboratories America, Inc. (Kensington, MD) and were maintained in McCoy's 5A medium supplemented with 25 rag/1 gentamicin sulfate (Gibco Laboratories, Grand Island, NY) and 10% fetal calf serum (Gibco Laboratories). Concentrations of trifluralin used in the assay were selected based on results of preliminary toxicity studies. Exponentially growing cells were plated in 75-cm2 plastic tissue culture flasks at an
initial density of about I × 106 cells in 10 ml of medium and incubated for 24 h. Medium was then replaced with serum-free medium and cells exposed to trifluralin for 4 h, both with and without metabolic activation. Following exposure, the cells were washed, the medium changed to growth medium, and the cultures incubated for an additional 16-18 h. At approximately 21 h post treatment, cells were harvested by mitotic shakeoff, centrifuged, diluted, and counted on a hemacytometer (Newman et al., 1987). Chemical precipitate was observed at concentrations >t 30 /xg/ml without metabolic activation and >/300 /~g/rnl with metabolic activation. For the aberration assays, initial plating, dosing, and exposure proceeded as described above. At approximately 19 h after dosing, colcemid (0.1/~g/ml) was added to 2 of 3 replicate cultures at each test concentration. The third culture served as a concurrent cytotoxicity test and was processed as described previously. Metaphase cells were collected from the remaining 2 cultures per treatment group approximately 2 h after administration of colcemid. At least 2 slides were prepared per culture, stained in 4% Giemsa in p H 6.8 phosphate buffer for 10 rain, air dried, and coded prior to evaluation to control for bias. 100 ceUs/treatment (50/culture) were evaluated for chromosome aberrations from the 3 highest concentrations yielding approximately 50% cell survival a n d / o r little compound precipitate. The results are shown in Table 4. R ~
Results of this testing were uniformly negative. No reverse mutations were noted in S. typhimurium strains TA98, TA100, TA1535, TA1537, or TA1538 when treated with trifluralin at concentrations ranging from 25 to 400 /xg/plate in the nonactivated test and from 50 to 800/~g/plate in the test with metabolic activation (Table 1). In both tests, concentrations of trifluralin were limited by precipitation and not by toxicity. In contrast to the bacterial system, trifluralin was found to be severely toxic to L5178Y mouse lymphoma cells at concentrations >/100 ~tg/rnl and 250 # g / m l in preliminary toxicity tests without and with activation, respectively (data not shown). However, no increase in forward muta-
189 t i o n s was o b s e r v e d w i t h o r w i t h o u t m e t a b o l i c a c t i v a t i o n at t r i f l u r a l i n c o n c e n t r a t i o n s r a n g i n g b e t w e e n 0.5 a n d 20 / x g / m l , d e s p i t e o b s e r v e d c y t o t o x i c i t y ( T a b l e 2). I n the in v i v o s i s t e r - c h r o m a t i d e x c h a n g e assay, c y t o t o x i c i t y , c h a r a c t e r i z e d b y a n i n c r e a s e in the n u m b e r o f f i r s t - d i v i s i o n m e t a p h a s e cells, was evid e n t in all a n i m a l s r e c e i v i n g b o t h 500 a n d 400 m g / k g t r e a t m e n t s a n d 2 o f the 3 a n i m a l s r e c e i v i n g t h e 3 0 0 - m g / k g t r e a t m e n t w i t h trifluralin. H o w ever, n o i n c r e a s e in 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 s was o b s e r v e d i n C h i n e s e h a m sters t r e a t e d w i t h 2 0 0 - 5 0 0 m g / k g o f t h e test a r t i c l e ( T a b l e 3).
F i n a l l y , n o i n c r e a s e in c h r o m o s o m e a b e r r a t i o n s w a s n o t e d in C H O cells t r e a t e d in v i t r o w i t h t r i f l u r a l i n at c o n c e n t r a t i o n s r a n g i n g f r o m 3 to 30 # g / m l o r 25 to 100 / ~ g / r n l in t h e tests w i t h o u t a n d w i t h a c t i v a t i o n , r e s p e c t i v e l y ( T a b l e 4).
Discussion I n a d d i t i o n to t h e s t a n d a r d g e n e t i c t o x i c o l o g y tests r e p o r t e d h e r e i n , n e g a t i v e results w e r e also o b t a i n e d in a d o m i n a n t l e t h a l a s s a y w h e r e i n m a l e W i s t a r r a t s r e c e i v e d 5 d a i l y o r a l d o s e s o f 100 o r 1000 m g / k g / d a y o f trifluralin. N o t r e a t m e n t - r e l a t e d e f f e c t s w e r e o b s e r v e d in the s t a n d a r d p a r a m -
TABLE 1 AN EVALUATION OF TRIFLURALIN FOR THE INDUCTION OF BACTERIAL MUTATION USING THE AMES TEST Treatment
/xg/plate
Revertant colony counts (mean_+ S.D.) a TA1535
TA1537
TA1538
TA98
TA100
Test without metabolic activation DMSO b 0.05 ml DMSO c 0.05ml
26_+ 8 334- 7
13-~ 4 12+ 3
174194-
1 3
204- 5 204- 2
1704186_+
Triflnralin Trifluralin Trifluralin Trifluralin Trifluralin
25 50 100 200 400
31426-+ 32+ 32-+ 37_+
646-+ 841258_+
17519425+ 27-+ 23_+
4 4 7 4 3
22420419418-+ 254-
1 5 5 2 9
124+103 188+ 34 188-+ 17 181_+ 14 1954- 6
MNNG d MNNG 9AmAc a 9AmAc 2NF d 2NF
1 2 50 100 0.5 5
NT NT NT NT 121_+30 800 4- 57
1316 _+ 20 1734 5 : 9 8 NT NT NT NT
7 6 8 6 1
1 1 2 6 2
1 318 _+73 2 073 5:89 NT e NT NT NT
NT NT 67 4-11 8014- 33 NT NT
Test with metabolic activation DMSO b 0.051111 DMSO c 0.05ml
164- 3 194- 1
94- 2 64- 1
30+ 384-
0 4
254- 6 304-11
1694- 7 167± 18
Trifluralin Trifluralin Trifluralin Trifluralin Trifluralin
19418512419426+
4± 5-t644444-
25428_+ 26428_+ 384-
3 9 4 3 6
29-121_+ 24_+ 28_+ 384-
7 4 8 6 8
178± 16 1984- 12 213_+ 6 1954- 6 1744- 14
747_+22 11395:25
1197-t- 6 1504 4- 72
2AA d 2AA a b c d
50 100 200 400 800 2.5 5
1 3 3 8 6
143_+23 243 4- 27
2 3 3 1 2
1094-15 350 4-11
NT NT NT NT 198_+ 3 1353_+ 10
5 7
10565-120 1725 -+ 57
Mean + standard deviation of counts from triplicate plates. Values represent corrected counts for 100% of the plate area. Dimethyl sulfoxide control value for the tester strain plated at the initiation of plating. Dimethyl sulfoxide control value for the tester strain plated at the termination of plating. Positive control compounds: MNNG (N-methyi-N'-rfitro-N-nitrosoguanidine), 9AmAc (9-aminoacridine), 2NF (2-nitrofluorene), 2AA (2-aminoanthracene). * NT, not tested.
190 TABLE 2 S U M M A R Y OF RESULTS F O R T H E M O U S E L Y M P H O M A F O R W A R D M U T A T I O N ASSAY W I T H T R I F L U R A L I N Treatment
Concentration (/~g/ml)
Percent cloning efficiency
Percent total survival
N u m b e r of m u t a n t colonies
Mutation frequency
Mutation index
100 100 47
100 100 20
18 17 192
2.3 2.4 54.9
23.4
20 15 10 /.5 5.0 2.5 1.0 0.5
106 94 95 102 101 116 109 99
18 17 24 38 47 50 110 103
19 18 13 15 15 18 17 18
2.4 2.6 1.8 2.0 2.0 2.1 2.1 2.4
DMSO a DMSO a 3-MC b
(1%) (1%) 5
100 100 51
100 100 4
17 15 81
3.4 3.0 31.2
Trifluralin Trifluralin Trifluralin Trifluralin Trifluralin Trifluralin Trifluralin Trifluralin
20 15 10 7.5 5.0 2.5 1.0 0.5
92 87 94 97 95 95 127 128
3 5 19 28 42 67 146 104
28 32 20 19 20 16 16 18
6.0 7.3 4.2 3.9 4.2 3.3 2.5 2.8
Nonactivated test
DMSO a DMSO a EMS b Trifluralin Trifluralin Trifluralin Trifluralin Trifluralin Trifluralin Trifluralin Trifluralin
(1%) (1%)
620
(2.35) c
(1.o)
1.0 1.1 0.8 0.9 0.9 0.9 0.9 1.0
A ctivated test
(3.2) c
(1.o) 9.8 1.9 2.3 1.3 1.2 1.3 1.0 0.8 0.9
a Solvent control, dimethyl sulfoxide. b Positive control, ethyl methane sulfonate (EMS), 3-methylcholanthrene (3-MC). c Mean of solvent control.
TABLE 3 S U M M A R Y VALUES F O R T H E IN VIVO I N D U C T I O N OF S I S T E R - C H R O M A T I D E X C H A N G E IN BONE M A R R O W OF CHINESE HAMSTERS TREATED ORALLY WITH TRIFLURALIN Chemical treatment Solvent control (10% aqueous a c a c i a / D M S O ) Cyclophosphamide Trifluralin Trifluralin Trifluralin Trifluralin a b c a ¢ t
Dose (mg/kg) 10 m g / k g 50 500 400 300 200
Total metaphases scored
Percent first-division cells d (mean + S.D.)
50 a 25 b
26.5 5 : 3 . 5 60
75 75 75 75
46.7 + 6.7 ~ 44.3 + 4.7 ~ 30.7 5:13.6 32.7 + 2.5
~ ¢ c ¢
Negative control, 2 a n i m a l s / d o s e group, compound solubilized in D M S O and diluted in 10% acacia. Positive control, 1 a n i m a l / d o s e group. 3 animals/group, 25 metaphases/animal. 100 metaphases counted per animal. Cytotoxic response, distribution of metaphase figures shifted in favor of first-division staining pattern. Significantly different from control as determined by Durmett's t-test.
SCE/metaphase (mean 5: S.D.) 3.6 + 1.6 29.8 + 10.2 t 3.5 + 3.1 + 3.2 + 3.5 +
1.6 1.4 1.5 1.6
Concentration (/tg/ml)
3 15 30 d
25 50 100 d
86
105 136
100
92
100 100
100
25
81 77
94
100 87
100
100 100 100
100 25
I
I
6
2
1
SG
ctb
I
9
8
TB
--
cte
2
2
TR
I
6
1
QR
I
CR
I
2
ID
1 1
2
9
4
1 2
SB
csb D
cse R
CI
other
1
DM
PU
GT
0.01 0.01
0.05
1.12
0
0.04
0 0
0.01 0.6
of aberrations/cell
TG
g
Cells scored
Percent cell survival
Number
Number and type of aberration a
1 1
4
60 c
0
3
0 0
1 40 c
with aberrations
% Cells
0 0
1
28
0
1
0 0
0 12
with > 1 aberrations
% Cells
a Abbreviations: TG, chromatid gap; SG, chromosome gap; TB, chromatid break; SB, chromosome break; DM, 'double minute' fragment; ID, interstitial deletion; TR, triradial; QR, quadriradial; CR, complex rearrangement; D, dicentric; R, ring chromosome; CI, chromosome intrachange; PU, pulverized chromosome; GT, greater than 10 aberrations. b DMSO, dimethyl sulfoxide; MC, mitomycin C; CP, cyclophospharmde. c Significantly greater than the solvent controls, P < 0.01. d Precipitate observed.
Trifluralin Trifluralin Trifluralin
With activation DMSO b (1%) CP b 10
Trifluralin Tdfluralin Trifluralin
Without activation DMSO b (1%) MC b 0.5
Treatment
SUMMARY RESULTS FROM THE INDUCTION OF CHROMOSOME ABERRATIONS IN CHINESE HAMSTER OVARY (CHO) CELLS BY TRIFLURALIN
TABLE 4
192
eters measured in this test (Markham and Hoyt, unpublished). The mutagenic potential of trifluralin has also been extensively investigated by researchers outside of Lilly Research Laboratories. These studies have included additional tests for gene mutations, chromosomal effects, DNA damage, and genetic damage in germinal cells. Although the majority of these tests have produced negative results (Shirasu et al., 1976; Wates et al., 1982; Moriya et al., 1983; Galloway et al., 1985), some tests have shown evidence of genetic activity. Murnik (1978) and Bryant and Murnik (1979) reported an increase in the rate of X and Y chromosome loss in larval-fed Drosophila melanogaster males while Foureman (1981) reported an increase in XXY nondisjunction in progeny of similarly treated males. The significance of these results is not clear since the rat dominant lethal assay, cited above, would have detected these types of chromosomal effects and none were observed in the mammalian system. Nehez et al. (1980, 1981) reported that trifluralin induced chromosome abnormalities in mouse bone marrow cells as well as mutagenic responses in the mouse spermatocyte and dominant lethal tests. However, the material tested was product formulated for agricultural use and contained only 26% trifiuralin. The purity of the trifluralin used in the formulation is not known and the formulation was an emulsifiable concentrate containing solvents and surfactants. Although the influence of the other ingredients is unclear, uniformly negative results have repeatedly been obtained in tests employing technical trifluralin (>/95% pure). The chromosome aberration findings in mouse bone marrow are particularly suspect since trifluralin has been found negative for chromosome aberration induction in vitro (study reported herein; Galloway et al., 1985) and since in vitro cytogenetic assays are more sensitive than in vivo cytogenetic assays (Thompson, 1986). In summary, the large number of negative findings in a variety of short-term tests designed to measure intrinsic genotoxic activity, and particularly the negative responses obtained in the mammalian assays, indicate that trifluralin does not pose a genotoxic hazard to man.
References Amacher, D.E., S. Paillet and V.A. Ray (1979) Point mutations at the thymidine kinase locus in L5178Y mouse lymphoma ceils, I. Application to genetic toxicology testing, Mutation Res., 64, 391-406. Amacher, D.E., S.C. PaiUet, G.N. Turner, V.A. Ray and D.S. Salsbury (1980) Point mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells, II. Test validation and interpretation, Mutation Res., 72, 447-474. Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/ mammalian microsome test, Mutation Res., 31, 347-364. Bryant, M.L., and M.R. Mumik (1979) Mutagenicity of the herbicide trifluralin in Drosophila melanogaster, Genetics, 91, Suppl. 2. Clive, D., and J.F.S. Spector (1975) Laboratory procedures for assessing specific locus mutations at the TK locus in cultured L5178Y mouse lyrnplioma cells, Mutation Res., 31, 17-29. Clive, D., K.O. Johnson, J.F.S. Spector, A.G. Batson and M.M.M. Brown (1979) Validation and characterization of the L5178Y TK +/- mouse lymphoma mutagen/carcinogen assay system, Mutation Res., 59, 61-108. Foureman, P.A. (1981) Identification of aneuploidy inducing chemicals in Drosophila, Environ. Mutagen., 3, 319. Galloway, S.M., A.D. Bloom, M. Resnick, B.H. Margolin, F. Nakamura, P. Archer and E. Zeiger (1985) Development of a standard protocol for in vitro cytogenetic testing with Chinese hamster ovary ceils: Comparison of results for 22 compounds in two laboratories, Environ. Mutagen., 7, 1-51. King, M.T., D. Wild, E. Gocke and K. Eckhardt (1982) 5Bromodeoxyuridine tablets with improved depot effect for analysis of in vivo sister chromatid exchanges in bone marrow and spermatogonial cells, Mutation Res., 97, 117129. Markham, J.K., and J.A. Hoyt (1983) A dominant lethal study with technical trifluralin in the Wistar rat, Lilly Research Laboratories, unpublished. Moriya, M.T., T. Ohta, K. Watanabe, T. Miyazawa, K. Kato and Y. Shirasu (1983) Further mutagenicity studies on pesticides in bacterial reversion assay systems, Mutation Res., 116, 185-216. Murnik, M.R. (1978) Mutagenicity of the herbicide trifluralin in Drosophilamelanogaster, Mutation Res., 53, 235-236. Neal, S.B., and G.S. Probst (1983) Chemically-induced sister chromatid exchange in vivo in bone marrow of Chinese hamsters. An evaluation of 24 compounds, Mutation Res., 113, 33-43. Nehez, M., A. Paldy, A. Selypes and G. Berencsi (1980) Experiments on the mutagenic effects of two pesticides, DNOC and trifluralin, Mutation Res., 74, 202-203. Nehez, M., A. Selypes, A. Paldy, E. Mazzag, G. Berencsi and R. Jarmay (1981) Histopathological and cytogenetical picture of microtoxicological effects of 2 nitrophenol-strnctured pesticides in mouse experiments on germ cells, Mutation Res., 85, 256.
193 Newman, D., D.D. Sedor, R.J. Preston and J.W. Oldham (1987) A comparison of cytotoxicity indicators for in vitro cytogenetics, Environ. Mutagen., 9, 78. Oberly, T.J., C.E. Piper and D.S. McDonald (1982) Mutagenicity of metal salts in the L5178Y mouse lymphoma assay, J. Toxicol. Environ. Health 9, 367-376. Shirasu, Y., M. Moriya, K. Kato, A. Furuhashi and T. Kada (1976) Mutagenicity screening of pesticides in the microbial system, Mutation Res., 40, 19-30.
Thompson, E.D. (1986) Comparison of in vivo and in vitro cytogenetic assay results, Environ. Mutagen. 8, 753-767. Waters, M.D., S.S. Sandhu, V.F. Sirnmon, K.E. Mortelmans, A.D. Mitchell, T.A. Jorgenson, D.C. Jones, R. Valencia and N.E. Garret (1982) Study of pesticide genotoxicity, Basic Life Sci., 21,275-326.
187
© 1991 ElsevierSciencePublishers B.V. 0165-1218/91/$03.50 ADONIS 016512189100095X MUTGEN 01658
Genotoxicity studies on the preemergence herbicide trifluralin Michael L. Garriott, Elizabeth R. Adams, Gregory S. Probst, John L. Emmerson, Thomas J. Oberly, Delinda E.F. Kindig, Steven B. Neal, Bernard J. Bewsey and Marcia A. Rexroat Toxicology Division, Lilly Research Laboratories, A Division of Eli Lilly and Company, Greenfield, IN 46140 (U.S.A.)
(Received23 July 1990) (Revision received11 November 1990) (Accepted 25 November 1990)
Keywords: Trifluralin; Herbicide; Short-termassays; Genotoxicity
Trifluralin, a dinitroaniline (Fig. 1), is a preemergence soil-incorporated herbicide that is registered with the U.S. Environmental Protection Agency and in countries worldwide for use on more than 50 crops. Trifluralin has been subjected to a variety of short-term tests designed to measure the potential genotoxic activity of the compound. These studies have been conducted to satisfy regulatory requirements in various countries and include a Salmonella/microsome mutagenicity (Ames) test, an L5178Y mouse lymphoma N (CH2C~2C~3) 2
O2N
NO~
C~ 3
Fig. 1. Trifluralin, 2,6-dinitro-N,N-dipropyl-4-(trifluoromethyl)benzenamine.CAS No. 1582-09-08.
Correspondence: Dr. Michael L. Garriott, Toxicology Division, Lilly Research Laboratories, A Division of Eli Lilly and Company, Greenfield,IN 46140 (U.S.A.)
assay, an in vitro chromosome aberration assay, and an in vivo test for sister-chromatid exchange induction in Chinese hamsters. This paper reports the results of studies conducted within the Lilly Research Laboratories and discusses additional studies with trifluralin that have been reported in the literature. Materials and methods Chemicals
All chemicals/reagents used in these studies were reagent-grade and were not further analyzed. Trifluralin (CAS 1582-09-08) was synthesized at Eli Lilly and Company. The material for the Ames, mouse lymphoma, and sister-chromatid exchange (SCE) assays was > 95% pure, while the material for the in vitro chromosome aberration assay was > 99% pure. The following positive control compounds were used in the studies reported herein: N - m e t h y l - N '-nitro-N-nitrosoguanidine (MNNG), 2-nitrofluorene (2-NF), 9-aminoacridine (9-AmAc), 2-aminoanthracene (2-AA) (Aldrich Chemical Co.), ethyl methanesulfonate (EMS), 3-methylcholanthrene (3-MC) (Eastman Kodak), cyclophosphamide (CP), and mitomycin C (MC) (Sigma Chemical Co.). In addition, bromodeoxyuridine (BrdU) (Calbiochem) and Velban ® (Eli
188 Lilly and Company) were used for sister-chromatid differentiation and as the mitotic arrestant, respectively, in the SCE assay.
Salmonella / microsome mutagenicity test The Salmonella/microsome mutagenicity test was conducted according to the method of Ames et al. (1975). All Salmonella typhimurium strains (see Table 1) employed in this assay were obtained from Dr. Bruce Ames (Berkeley, CA) and were tested prior to use for appropriate genetic markers. Trifluralin was evaluated at the exposure levels shown (Table 1), either with or without added metabolic activation by Aroclor 1254-induced, Fischer 344 rat-liver $9. The plates were scored for revertant colony formation using a New Brunswick Biotran II Automated Colony Counter (New Brunswick Scientific, New Brunswick, N J). L5178Y mouse lymphoma assay The L5178Y T K +/- assay was conducted essentially by the method of Clive et al. (1975, 1979) with minor modification by Amacher et al. (1979, 1980) and Oberly et al. (1982). Ceils in RPMI 1640 media were exposed to trifluralin for 4 h, both with and without metabolic activation at the concentrations shown in Table 2. Colonies were counted in the same manner and with the same instrumentation used for the Salmonella/microsome test. Sister-chromatid exchange assay The sister-chromatid exchange assay was conducted using the BudR tablet method as adapted from King et al. (1982) and with some modification of the method reported by Neal and Probst (1983). Doses tested and results appear in Table 3. In vitro chromosome aberration assay CHO-WB L cells were obtained from Hazleton Laboratories America, Inc. (Kensington, MD) and were maintained in McCoy's 5A medium supplemented with 25 rag/1 gentamicin sulfate (Gibco Laboratories, Grand Island, NY) and 10% fetal calf serum (Gibco Laboratories). Concentrations of trifluralin used in the assay were selected based on results of preliminary toxicity studies. Exponentially growing cells were plated in 75-cm2 plastic tissue culture flasks at an
initial density of about I × 106 cells in 10 ml of medium and incubated for 24 h. Medium was then replaced with serum-free medium and cells exposed to trifluralin for 4 h, both with and without metabolic activation. Following exposure, the cells were washed, the medium changed to growth medium, and the cultures incubated for an additional 16-18 h. At approximately 21 h post treatment, cells were harvested by mitotic shakeoff, centrifuged, diluted, and counted on a hemacytometer (Newman et al., 1987). Chemical precipitate was observed at concentrations >t 30 /xg/ml without metabolic activation and >/300 /~g/rnl with metabolic activation. For the aberration assays, initial plating, dosing, and exposure proceeded as described above. At approximately 19 h after dosing, colcemid (0.1/~g/ml) was added to 2 of 3 replicate cultures at each test concentration. The third culture served as a concurrent cytotoxicity test and was processed as described previously. Metaphase cells were collected from the remaining 2 cultures per treatment group approximately 2 h after administration of colcemid. At least 2 slides were prepared per culture, stained in 4% Giemsa in p H 6.8 phosphate buffer for 10 rain, air dried, and coded prior to evaluation to control for bias. 100 ceUs/treatment (50/culture) were evaluated for chromosome aberrations from the 3 highest concentrations yielding approximately 50% cell survival a n d / o r little compound precipitate. The results are shown in Table 4. R ~
Results of this testing were uniformly negative. No reverse mutations were noted in S. typhimurium strains TA98, TA100, TA1535, TA1537, or TA1538 when treated with trifluralin at concentrations ranging from 25 to 400 /xg/plate in the nonactivated test and from 50 to 800/~g/plate in the test with metabolic activation (Table 1). In both tests, concentrations of trifluralin were limited by precipitation and not by toxicity. In contrast to the bacterial system, trifluralin was found to be severely toxic to L5178Y mouse lymphoma cells at concentrations >/100 ~tg/rnl and 250 # g / m l in preliminary toxicity tests without and with activation, respectively (data not shown). However, no increase in forward muta-
189 t i o n s was o b s e r v e d w i t h o r w i t h o u t m e t a b o l i c a c t i v a t i o n at t r i f l u r a l i n c o n c e n t r a t i o n s r a n g i n g b e t w e e n 0.5 a n d 20 / x g / m l , d e s p i t e o b s e r v e d c y t o t o x i c i t y ( T a b l e 2). I n the in v i v o s i s t e r - c h r o m a t i d e x c h a n g e assay, c y t o t o x i c i t y , c h a r a c t e r i z e d b y a n i n c r e a s e in the n u m b e r o f f i r s t - d i v i s i o n m e t a p h a s e cells, was evid e n t in all a n i m a l s r e c e i v i n g b o t h 500 a n d 400 m g / k g t r e a t m e n t s a n d 2 o f the 3 a n i m a l s r e c e i v i n g t h e 3 0 0 - m g / k g t r e a t m e n t w i t h trifluralin. H o w ever, n o i n c r e a s e in 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 s was o b s e r v e d i n C h i n e s e h a m sters t r e a t e d w i t h 2 0 0 - 5 0 0 m g / k g o f t h e test a r t i c l e ( T a b l e 3).
F i n a l l y , n o i n c r e a s e in c h r o m o s o m e a b e r r a t i o n s w a s n o t e d in C H O cells t r e a t e d in v i t r o w i t h t r i f l u r a l i n at c o n c e n t r a t i o n s r a n g i n g f r o m 3 to 30 # g / m l o r 25 to 100 / ~ g / r n l in t h e tests w i t h o u t a n d w i t h a c t i v a t i o n , r e s p e c t i v e l y ( T a b l e 4).
Discussion I n a d d i t i o n to t h e s t a n d a r d g e n e t i c t o x i c o l o g y tests r e p o r t e d h e r e i n , n e g a t i v e results w e r e also o b t a i n e d in a d o m i n a n t l e t h a l a s s a y w h e r e i n m a l e W i s t a r r a t s r e c e i v e d 5 d a i l y o r a l d o s e s o f 100 o r 1000 m g / k g / d a y o f trifluralin. N o t r e a t m e n t - r e l a t e d e f f e c t s w e r e o b s e r v e d in the s t a n d a r d p a r a m -
TABLE 1 AN EVALUATION OF TRIFLURALIN FOR THE INDUCTION OF BACTERIAL MUTATION USING THE AMES TEST Treatment
/xg/plate
Revertant colony counts (mean_+ S.D.) a TA1535
TA1537
TA1538
TA98
TA100
Test without metabolic activation DMSO b 0.05 ml DMSO c 0.05ml
26_+ 8 334- 7
13-~ 4 12+ 3
174194-
1 3
204- 5 204- 2
1704186_+
Triflnralin Trifluralin Trifluralin Trifluralin Trifluralin
25 50 100 200 400
31426-+ 32+ 32-+ 37_+
646-+ 841258_+
17519425+ 27-+ 23_+
4 4 7 4 3
22420419418-+ 254-
1 5 5 2 9
124+103 188+ 34 188-+ 17 181_+ 14 1954- 6
MNNG d MNNG 9AmAc a 9AmAc 2NF d 2NF
1 2 50 100 0.5 5
NT NT NT NT 121_+30 800 4- 57
1316 _+ 20 1734 5 : 9 8 NT NT NT NT
7 6 8 6 1
1 1 2 6 2
1 318 _+73 2 073 5:89 NT e NT NT NT
NT NT 67 4-11 8014- 33 NT NT
Test with metabolic activation DMSO b 0.051111 DMSO c 0.05ml
164- 3 194- 1
94- 2 64- 1
30+ 384-
0 4
254- 6 304-11
1694- 7 167± 18
Trifluralin Trifluralin Trifluralin Trifluralin Trifluralin
19418512419426+
4± 5-t644444-
25428_+ 26428_+ 384-
3 9 4 3 6
29-121_+ 24_+ 28_+ 384-
7 4 8 6 8
178± 16 1984- 12 213_+ 6 1954- 6 1744- 14
747_+22 11395:25
1197-t- 6 1504 4- 72
2AA d 2AA a b c d
50 100 200 400 800 2.5 5
1 3 3 8 6
143_+23 243 4- 27
2 3 3 1 2
1094-15 350 4-11
NT NT NT NT 198_+ 3 1353_+ 10
5 7
10565-120 1725 -+ 57
Mean + standard deviation of counts from triplicate plates. Values represent corrected counts for 100% of the plate area. Dimethyl sulfoxide control value for the tester strain plated at the initiation of plating. Dimethyl sulfoxide control value for the tester strain plated at the termination of plating. Positive control compounds: MNNG (N-methyi-N'-rfitro-N-nitrosoguanidine), 9AmAc (9-aminoacridine), 2NF (2-nitrofluorene), 2AA (2-aminoanthracene). * NT, not tested.
190 TABLE 2 S U M M A R Y OF RESULTS F O R T H E M O U S E L Y M P H O M A F O R W A R D M U T A T I O N ASSAY W I T H T R I F L U R A L I N Treatment
Concentration (/~g/ml)
Percent cloning efficiency
Percent total survival
N u m b e r of m u t a n t colonies
Mutation frequency
Mutation index
100 100 47
100 100 20
18 17 192
2.3 2.4 54.9
23.4
20 15 10 /.5 5.0 2.5 1.0 0.5
106 94 95 102 101 116 109 99
18 17 24 38 47 50 110 103
19 18 13 15 15 18 17 18
2.4 2.6 1.8 2.0 2.0 2.1 2.1 2.4
DMSO a DMSO a 3-MC b
(1%) (1%) 5
100 100 51
100 100 4
17 15 81
3.4 3.0 31.2
Trifluralin Trifluralin Trifluralin Trifluralin Trifluralin Trifluralin Trifluralin Trifluralin
20 15 10 7.5 5.0 2.5 1.0 0.5
92 87 94 97 95 95 127 128
3 5 19 28 42 67 146 104
28 32 20 19 20 16 16 18
6.0 7.3 4.2 3.9 4.2 3.3 2.5 2.8
Nonactivated test
DMSO a DMSO a EMS b Trifluralin Trifluralin Trifluralin Trifluralin Trifluralin Trifluralin Trifluralin Trifluralin
(1%) (1%)
620
(2.35) c
(1.o)
1.0 1.1 0.8 0.9 0.9 0.9 0.9 1.0
A ctivated test
(3.2) c
(1.o) 9.8 1.9 2.3 1.3 1.2 1.3 1.0 0.8 0.9
a Solvent control, dimethyl sulfoxide. b Positive control, ethyl methane sulfonate (EMS), 3-methylcholanthrene (3-MC). c Mean of solvent control.
TABLE 3 S U M M A R Y VALUES F O R T H E IN VIVO I N D U C T I O N OF S I S T E R - C H R O M A T I D E X C H A N G E IN BONE M A R R O W OF CHINESE HAMSTERS TREATED ORALLY WITH TRIFLURALIN Chemical treatment Solvent control (10% aqueous a c a c i a / D M S O ) Cyclophosphamide Trifluralin Trifluralin Trifluralin Trifluralin a b c a ¢ t
Dose (mg/kg) 10 m g / k g 50 500 400 300 200
Total metaphases scored
Percent first-division cells d (mean + S.D.)
50 a 25 b
26.5 5 : 3 . 5 60
75 75 75 75
46.7 + 6.7 ~ 44.3 + 4.7 ~ 30.7 5:13.6 32.7 + 2.5
~ ¢ c ¢
Negative control, 2 a n i m a l s / d o s e group, compound solubilized in D M S O and diluted in 10% acacia. Positive control, 1 a n i m a l / d o s e group. 3 animals/group, 25 metaphases/animal. 100 metaphases counted per animal. Cytotoxic response, distribution of metaphase figures shifted in favor of first-division staining pattern. Significantly different from control as determined by Durmett's t-test.
SCE/metaphase (mean 5: S.D.) 3.6 + 1.6 29.8 + 10.2 t 3.5 + 3.1 + 3.2 + 3.5 +
1.6 1.4 1.5 1.6
Concentration (/tg/ml)
3 15 30 d
25 50 100 d
86
105 136
100
92
100 100
100
25
81 77
94
100 87
100
100 100 100
100 25
I
I
6
2
1
SG
ctb
I
9
8
TB
--
cte
2
2
TR
I
6
1
QR
I
CR
I
2
ID
1 1
2
9
4
1 2
SB
csb D
cse R
CI
other
1
DM
PU
GT
0.01 0.01
0.05
1.12
0
0.04
0 0
0.01 0.6
of aberrations/cell
TG
g
Cells scored
Percent cell survival
Number
Number and type of aberration a
1 1
4
60 c
0
3
0 0
1 40 c
with aberrations
% Cells
0 0
1
28
0
1
0 0
0 12
with > 1 aberrations
% Cells
a Abbreviations: TG, chromatid gap; SG, chromosome gap; TB, chromatid break; SB, chromosome break; DM, 'double minute' fragment; ID, interstitial deletion; TR, triradial; QR, quadriradial; CR, complex rearrangement; D, dicentric; R, ring chromosome; CI, chromosome intrachange; PU, pulverized chromosome; GT, greater than 10 aberrations. b DMSO, dimethyl sulfoxide; MC, mitomycin C; CP, cyclophospharmde. c Significantly greater than the solvent controls, P < 0.01. d Precipitate observed.
Trifluralin Trifluralin Trifluralin
With activation DMSO b (1%) CP b 10
Trifluralin Tdfluralin Trifluralin
Without activation DMSO b (1%) MC b 0.5
Treatment
SUMMARY RESULTS FROM THE INDUCTION OF CHROMOSOME ABERRATIONS IN CHINESE HAMSTER OVARY (CHO) CELLS BY TRIFLURALIN
TABLE 4
192
eters measured in this test (Markham and Hoyt, unpublished). The mutagenic potential of trifluralin has also been extensively investigated by researchers outside of Lilly Research Laboratories. These studies have included additional tests for gene mutations, chromosomal effects, DNA damage, and genetic damage in germinal cells. Although the majority of these tests have produced negative results (Shirasu et al., 1976; Wates et al., 1982; Moriya et al., 1983; Galloway et al., 1985), some tests have shown evidence of genetic activity. Murnik (1978) and Bryant and Murnik (1979) reported an increase in the rate of X and Y chromosome loss in larval-fed Drosophila melanogaster males while Foureman (1981) reported an increase in XXY nondisjunction in progeny of similarly treated males. The significance of these results is not clear since the rat dominant lethal assay, cited above, would have detected these types of chromosomal effects and none were observed in the mammalian system. Nehez et al. (1980, 1981) reported that trifluralin induced chromosome abnormalities in mouse bone marrow cells as well as mutagenic responses in the mouse spermatocyte and dominant lethal tests. However, the material tested was product formulated for agricultural use and contained only 26% trifiuralin. The purity of the trifluralin used in the formulation is not known and the formulation was an emulsifiable concentrate containing solvents and surfactants. Although the influence of the other ingredients is unclear, uniformly negative results have repeatedly been obtained in tests employing technical trifluralin (>/95% pure). The chromosome aberration findings in mouse bone marrow are particularly suspect since trifluralin has been found negative for chromosome aberration induction in vitro (study reported herein; Galloway et al., 1985) and since in vitro cytogenetic assays are more sensitive than in vivo cytogenetic assays (Thompson, 1986). In summary, the large number of negative findings in a variety of short-term tests designed to measure intrinsic genotoxic activity, and particularly the negative responses obtained in the mammalian assays, indicate that trifluralin does not pose a genotoxic hazard to man.
References Amacher, D.E., S. Paillet and V.A. Ray (1979) Point mutations at the thymidine kinase locus in L5178Y mouse lymphoma ceils, I. Application to genetic toxicology testing, Mutation Res., 64, 391-406. Amacher, D.E., S.C. PaiUet, G.N. Turner, V.A. Ray and D.S. Salsbury (1980) Point mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells, II. Test validation and interpretation, Mutation Res., 72, 447-474. Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/ mammalian microsome test, Mutation Res., 31, 347-364. Bryant, M.L., and M.R. Mumik (1979) Mutagenicity of the herbicide trifluralin in Drosophila melanogaster, Genetics, 91, Suppl. 2. Clive, D., and J.F.S. Spector (1975) Laboratory procedures for assessing specific locus mutations at the TK locus in cultured L5178Y mouse lyrnplioma cells, Mutation Res., 31, 17-29. Clive, D., K.O. Johnson, J.F.S. Spector, A.G. Batson and M.M.M. Brown (1979) Validation and characterization of the L5178Y TK +/- mouse lymphoma mutagen/carcinogen assay system, Mutation Res., 59, 61-108. Foureman, P.A. (1981) Identification of aneuploidy inducing chemicals in Drosophila, Environ. Mutagen., 3, 319. Galloway, S.M., A.D. Bloom, M. Resnick, B.H. Margolin, F. Nakamura, P. Archer and E. Zeiger (1985) Development of a standard protocol for in vitro cytogenetic testing with Chinese hamster ovary ceils: Comparison of results for 22 compounds in two laboratories, Environ. Mutagen., 7, 1-51. King, M.T., D. Wild, E. Gocke and K. Eckhardt (1982) 5Bromodeoxyuridine tablets with improved depot effect for analysis of in vivo sister chromatid exchanges in bone marrow and spermatogonial cells, Mutation Res., 97, 117129. Markham, J.K., and J.A. Hoyt (1983) A dominant lethal study with technical trifluralin in the Wistar rat, Lilly Research Laboratories, unpublished. Moriya, M.T., T. Ohta, K. Watanabe, T. Miyazawa, K. Kato and Y. Shirasu (1983) Further mutagenicity studies on pesticides in bacterial reversion assay systems, Mutation Res., 116, 185-216. Murnik, M.R. (1978) Mutagenicity of the herbicide trifluralin in Drosophilamelanogaster, Mutation Res., 53, 235-236. Neal, S.B., and G.S. Probst (1983) Chemically-induced sister chromatid exchange in vivo in bone marrow of Chinese hamsters. An evaluation of 24 compounds, Mutation Res., 113, 33-43. Nehez, M., A. Paldy, A. Selypes and G. Berencsi (1980) Experiments on the mutagenic effects of two pesticides, DNOC and trifluralin, Mutation Res., 74, 202-203. Nehez, M., A. Selypes, A. Paldy, E. Mazzag, G. Berencsi and R. Jarmay (1981) Histopathological and cytogenetical picture of microtoxicological effects of 2 nitrophenol-strnctured pesticides in mouse experiments on germ cells, Mutation Res., 85, 256.
193 Newman, D., D.D. Sedor, R.J. Preston and J.W. Oldham (1987) A comparison of cytotoxicity indicators for in vitro cytogenetics, Environ. Mutagen., 9, 78. Oberly, T.J., C.E. Piper and D.S. McDonald (1982) Mutagenicity of metal salts in the L5178Y mouse lymphoma assay, J. Toxicol. Environ. Health 9, 367-376. Shirasu, Y., M. Moriya, K. Kato, A. Furuhashi and T. Kada (1976) Mutagenicity screening of pesticides in the microbial system, Mutation Res., 40, 19-30.
Thompson, E.D. (1986) Comparison of in vivo and in vitro cytogenetic assay results, Environ. Mutagen. 8, 753-767. Waters, M.D., S.S. Sandhu, V.F. Sirnmon, K.E. Mortelmans, A.D. Mitchell, T.A. Jorgenson, D.C. Jones, R. Valencia and N.E. Garret (1982) Study of pesticide genotoxicity, Basic Life Sci., 21,275-326.
