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Lack of mutagenicity of chlormequat chloride in Drosophila and in bacteria
Mutation Research, 31 (1975) 65-68 0 Elsevier Scientific Publishing Company,
Amsterdam-Printed
in The Netherlands
65
Lack of mutagenicity of chlormequat chloride in Drosophila and in bacteria The growth regulator (z-chloroethyl)-trimethylammonium chloride (CCC, chlormequat chloride) has a widespread application. In the Netherlands it is used in agriculture, to prevent lodging of wheat, and in fruit and flower culture. Conflicting data have been presented on the degradation of CCC in whkat1~5~6~8. In two independent studies it was shown that in wheat grains the compound was still present in measurable amounts three months after spraying (1.4 ppm), but had disappeared I year after harvestinga9s. Assuming a daily intake of dry bread wheat of 200 g, this amount of 1.4 ppm would result in a daily exposure of 0.28 mg CCC, or approximately 4 ,ug/kg/day. In the wheat plant CCC or its metabolites appear not to be bound to nucleic acidsc. CCC seems to be excreted rather quickly in rats, a relatively large fraction of the remaining compound being lett in the gonads l. Chronic toxicity experiments in rats were negative, but in their testes the growth of abnormally big cells was demonstrated3. In male as well as in female locusts meiosis was almost completely inhibited after feeding CCC at a total dosage of 5 mg/g body weight, or after injection at dosages of 0.2 or 2 mg/g body weight; in the cotton stainer (Dysdercus cardinal&) CCC caused a decrease in fertility2y3. Based on these data and on the suspicion that CCC, since it is a quaternary ammonium compound, could have alkylating properties, it was considered worthwhile to test the compound for mutagenicity. Mutagenicity testing was carried out with Drosophila melanogaster (P. G. N. KRAMERS AND A. G. A. C. KNAAP) and with two species of bacteria : Klebsiella pneunzoniae and Citrobacter freundii (C. E. VOOGD). Drosophila tests
Two types of genetic damage were measured in male germ cells, namely (1) sexlinked recessive lethal mutations and (2) dominant lethal mutations. CCC was administered by injection (approx. 0.2 ,ul) into the abdomen of 4-day-old males, in concentrations of I and IO mg/ml in saline, to which 0.5% DMSO was added. In the sex-linked recessive lethal experiments the flies were of the following genetic constitution: R(l)2 y B/yfY(JJ) and Inscy; bw; st pp($?$?). Atter treatment the males were individually mated every two days to three virgins, in order to sample different germ cell stages. Assuming no delay in spermatogenesis following treatment the first three broods were considered to represent post-meiotic stages and the last three broods meiotic and pre-meiotic ones. The frequencies of sex-linked recessive lethal mutations were scored in the F,, according to standard procedures. The results of these tests are given in Table I. Since no significant differences existed between the broods, the data of the postmeiotic broods (A-C) as well as those pertaining to the meiotic and premeiotic broods (D-F) respectively, were pooled. It is evident, that in these experiments the injection of CCC did not increase the mutation rate over the control frequency. Actually, with the numbers of germ cells tested, a z.5-fold increase of the mutation frequency in the CCC series over the control rate would have been detected as a significant increase at the 5% level’,lo. Abbreviations:CCC, (z-chloroethyl)trimethylammonium
dimethyl sulphoxide.
chloride (chlormequat
chloride)
; DMSO,
SHORT COMMUNICATIONS
66
TABLE I FREQUENcIEs OF SEX-LINKED RECESSIVE L.ETH.4L.S AFTERINJECTIoNOFccc Cont. (mg/ml)
Broods A-C Days o-6
Broods D-F Days 7-12
Number of yO lethals chrom. Control (2 expts.) I (I expt.) IO (2 expts.)
3374 2479 5396
0.24 0.08 0.07
95% conf.& limits
Number of yO lethals chrom.
0.1-0.5 0.01-0.3
1579 1149 4364
0.02-0.2
a Confidence limits are calculated according to STEVENS
0.38 0.0 0.16
95% conf.” limits 0.1-0.8 0.0-0.3 0.06-0.3
(ref.IO)
TABLE II AMOUNTS Cont.
(mglml)
OFCCC
FOUND
Time between injection and fixation
IN THE FLIES AFTERINJECTION
Number of flies
Total amount”
ofCCCinWed
(h) 0
0
50
0
IO
0
IO0
10
3
50
75
150
Cd
OfCCC
Total amountb
Percentage of the injected quantity determined
8
_
z
59
determined (Mls)
70
& Based on an average injected volume of
0.2 ~1 per fly. b With this method amounts below IO ,ug cannot be measured accurately.
The ability of CCC to induce dominant lethal mutations was measured according to the method described by SANKARANARAYANAN~,using wild type flies (Oregon-K). In two experiments a total of 5233 eggs were counted in the CCC series and 2630 in the control. The proportion of unhatched eggs in the CCC series was equal to that in the control series (11.8%, confidence limits of the control group: 10.6-13.1%). This result provides no evidence for the induction of dominant lethal mutations by CCC. In order to investigate whether this lack of mutagenicity in Drosophila was due to CCC being rapidly metabolized, a quantitative determination of the compound was carried out in the flies at two different times after the treatment. These analyses were performed by Dr. DEKHUIJZEN (Wageningen), according to the method described in ref. 4. The results given in Table II show that most of the injected compound was still present 3 h after injection; this finding excludes the possibility that the lack of mutagenic effect ot CCC was due to a rapid metabolic breakdown of the compound. The dosages used in our experiments are in the same range--at a mg/g body weight basis-as those inhibiting meiosis in grasshoppers, as shown by CARLISLE et a1.293. If CCC had a similar effect in Drosophila, this should have caused severe sterility in broods E and F, which, however, was not found. Bacterial tests To test for the mutagenic
potentials OI CCC in bacteria a fluctuation test was carried out as described by VOOGD et al. Il. As test organisms we used Klebsiella pneumoniae, requiring uracil and proline for growth, and Citrobncter freundii 425. This test was performed by dividing nutrient broth containing the substance under test and seeded with IOO bacteria/ml into rag portions of equal volume in culture tubes. After incubation for 20 h at 37”, the total number of streptomycin-
SHORTCOMMUNICATIONS
67
resistant and -dependent bacteria was determined in IOO portions by the pour-plate technique using nutrient agar supplemented with streptomycin (IOO pg/ml) and 3 days of incubation at 37“. The number of bacteria present in the 5 remaining tubes was determined by using nutrient agar, without streptomycin. From the number of portions without streptomycin-resistant or -dependent mutants the mutation rate was calculated by the Poisson distribution. As it is essential to the test to obtain a sufficient number of portions without mutants, the volumes per tube (portion) were 2.5 ml using Klebsiella pneumoniae and 0.5 m.l using Citrobacter freundii. The compound was applied in concentrations up to 5 mg/ml in the culture medium. Table III shows the results of the experiments. As in the Drosophila tests, there is no difference in mutation rate between the treated and the control series. The absence of a mutagenic effect of acridine orange in this test suggests the system’s inability to detect frameshift mutagens. Therefore, the negative results obtained with CCC are likely to apply only to base-pair substitutions. TABLE III MUTAGENICACTION OF CCC ON Klebsiella pneumoniae AND ON Citrobacter freundii
(mslml)
Portions without mutants
Kl. pneumoniae
5 2 O&
C. freundii
56 49 50
5 0”
::
Test organism
Average a 0.1676
Cone.
Number of
Mutation frequency
bacteria/ml
1.18.109 1.27.10~ 1.03~10~ 1.97’109 2.89.10~
0.137.10-9 O.I55.10_9 0.187. IO-~ 0.231.10-9 o.200~Io-9
spontaneous mutation frequency currently obtained * 0.0304. IO-~; b 0.2616 + 0.0760. IO-~.
in control series with these strains:
Conclusion It is concluded that the present data provide no evidence for a mutagenic activity of CCC in Drosophila melanogaster, or in the bacteria Klebsiella pneumoniae and Citrobacter freundii. This work was supported by the Netherlands Ministry of Public Health and Environmental Hygiene. We thank Dr. H. M. DEKHUIJZENfor his suggestion to test CCC for mutagenicity. We are indebted to Prof. F. H. SOBELSfor his encouraging advice and to Dr. K. SANKARANARAYANAN for reading the manuscript. We want to acknowledge the technical assistance of RIA Los, NETTY PEX and WILMA SEGERS. Department
of Radiation Genetics and Chemical
Mutagenesis, State University of Leiden, Wassenaarseweg 62, Leiden and
PIETERG. N. KRAMERS ADA G. A. C. KNAAP “C. E. VOOGD
* National Institute of Public Health, Bilthoven (The Netherlands) I BLINN, R., Biochemical behaviour of z-chloroethyl-trimethylammonium chloride in wheat and in rats, J. Agr. Food Chem., 15 (1967) 984-988. 2 CARLISLE, D. B., AND P. E. ELLIS, z-Chloroethyl-trimethylammonium chloride inhibits gametogenesis in locusts, Nature, 220 (1968) 189.
SHORT
68
COMMUNICATIONS
3 CARLISLE, D. B., P. E. ELLIS, AND D. J. OSBORNE, Effects of plant growth regulators on locusts and cotton stainer bugs, J. Sci. Food Agr., 20 (1969) 391-393. and persistence of chlormequat 4 DEKHUIJZEN, H. M., AND K. B. A. BODLAENDER, Distribution in potato plants, Pesticide Sci., 4 (1973) 619-627. and degradation of chlormequat in 5 DEKHUIJZEN, H. M., AND C. R. VONK, The distribution wheat plants, Pesticide Biochem. Physiol., 4 (1974) in press. of chlorocholine in plants, Z. Pflanzennachr., 114 6 JUNG, J,, AND M.E. EL-FOULY, Degradation (1966) 128-134. the statistical significance of 7 KASTENBAUM, M. A., AND K. 0. BOWMAN, Tables for determining mutation frequencies, Mutation Res., g (1970) 527-549. of chlorocholinechloride residues in 8 MOONEY. R. P.. AND N. R. PASARELA, Determination wheat grain, straw, green wheat foliage,. J. Agr. Food Chem., 15 (1967) 989-995. K., The effect of nitrogen and oxygen treatments on the frequencies of 9 SANKARANARAYANAN, X-ray-induced dominant lethals and on the physiology of the sperm in Drosophila nzelano-
gaster, Mutation Res., 4 (1967) 641-661. 10 STEVENS, W. L., Accuracy of mutation rates, J. Genet., 43 (1942) 301-307. II VOOGD, C. E., J. J. J. A. A. JACOBS, AND J. J. VAN DER STEL, On the mutagenic chlorvos, Mutation Res., 16 (1972) 413-416.
Received March zgth, 1974 Revision received August zy-d, 1974
action
of di-
Amsterdam-Printed
in The Netherlands
65
Lack of mutagenicity of chlormequat chloride in Drosophila and in bacteria The growth regulator (z-chloroethyl)-trimethylammonium chloride (CCC, chlormequat chloride) has a widespread application. In the Netherlands it is used in agriculture, to prevent lodging of wheat, and in fruit and flower culture. Conflicting data have been presented on the degradation of CCC in whkat1~5~6~8. In two independent studies it was shown that in wheat grains the compound was still present in measurable amounts three months after spraying (1.4 ppm), but had disappeared I year after harvestinga9s. Assuming a daily intake of dry bread wheat of 200 g, this amount of 1.4 ppm would result in a daily exposure of 0.28 mg CCC, or approximately 4 ,ug/kg/day. In the wheat plant CCC or its metabolites appear not to be bound to nucleic acidsc. CCC seems to be excreted rather quickly in rats, a relatively large fraction of the remaining compound being lett in the gonads l. Chronic toxicity experiments in rats were negative, but in their testes the growth of abnormally big cells was demonstrated3. In male as well as in female locusts meiosis was almost completely inhibited after feeding CCC at a total dosage of 5 mg/g body weight, or after injection at dosages of 0.2 or 2 mg/g body weight; in the cotton stainer (Dysdercus cardinal&) CCC caused a decrease in fertility2y3. Based on these data and on the suspicion that CCC, since it is a quaternary ammonium compound, could have alkylating properties, it was considered worthwhile to test the compound for mutagenicity. Mutagenicity testing was carried out with Drosophila melanogaster (P. G. N. KRAMERS AND A. G. A. C. KNAAP) and with two species of bacteria : Klebsiella pneunzoniae and Citrobacter freundii (C. E. VOOGD). Drosophila tests
Two types of genetic damage were measured in male germ cells, namely (1) sexlinked recessive lethal mutations and (2) dominant lethal mutations. CCC was administered by injection (approx. 0.2 ,ul) into the abdomen of 4-day-old males, in concentrations of I and IO mg/ml in saline, to which 0.5% DMSO was added. In the sex-linked recessive lethal experiments the flies were of the following genetic constitution: R(l)2 y B/yfY(JJ) and Inscy; bw; st pp($?$?). Atter treatment the males were individually mated every two days to three virgins, in order to sample different germ cell stages. Assuming no delay in spermatogenesis following treatment the first three broods were considered to represent post-meiotic stages and the last three broods meiotic and pre-meiotic ones. The frequencies of sex-linked recessive lethal mutations were scored in the F,, according to standard procedures. The results of these tests are given in Table I. Since no significant differences existed between the broods, the data of the postmeiotic broods (A-C) as well as those pertaining to the meiotic and premeiotic broods (D-F) respectively, were pooled. It is evident, that in these experiments the injection of CCC did not increase the mutation rate over the control frequency. Actually, with the numbers of germ cells tested, a z.5-fold increase of the mutation frequency in the CCC series over the control rate would have been detected as a significant increase at the 5% level’,lo. Abbreviations:CCC, (z-chloroethyl)trimethylammonium
dimethyl sulphoxide.
chloride (chlormequat
chloride)
; DMSO,
SHORT COMMUNICATIONS
66
TABLE I FREQUENcIEs OF SEX-LINKED RECESSIVE L.ETH.4L.S AFTERINJECTIoNOFccc Cont. (mg/ml)
Broods A-C Days o-6
Broods D-F Days 7-12
Number of yO lethals chrom. Control (2 expts.) I (I expt.) IO (2 expts.)
3374 2479 5396
0.24 0.08 0.07
95% conf.& limits
Number of yO lethals chrom.
0.1-0.5 0.01-0.3
1579 1149 4364
0.02-0.2
a Confidence limits are calculated according to STEVENS
0.38 0.0 0.16
95% conf.” limits 0.1-0.8 0.0-0.3 0.06-0.3
(ref.IO)
TABLE II AMOUNTS Cont.
(mglml)
OFCCC
FOUND
Time between injection and fixation
IN THE FLIES AFTERINJECTION
Number of flies
Total amount”
ofCCCinWed
(h) 0
0
50
0
IO
0
IO0
10
3
50
75
150
Cd
OfCCC
Total amountb
Percentage of the injected quantity determined
8
_
z
59
determined (Mls)
70
& Based on an average injected volume of
0.2 ~1 per fly. b With this method amounts below IO ,ug cannot be measured accurately.
The ability of CCC to induce dominant lethal mutations was measured according to the method described by SANKARANARAYANAN~,using wild type flies (Oregon-K). In two experiments a total of 5233 eggs were counted in the CCC series and 2630 in the control. The proportion of unhatched eggs in the CCC series was equal to that in the control series (11.8%, confidence limits of the control group: 10.6-13.1%). This result provides no evidence for the induction of dominant lethal mutations by CCC. In order to investigate whether this lack of mutagenicity in Drosophila was due to CCC being rapidly metabolized, a quantitative determination of the compound was carried out in the flies at two different times after the treatment. These analyses were performed by Dr. DEKHUIJZEN (Wageningen), according to the method described in ref. 4. The results given in Table II show that most of the injected compound was still present 3 h after injection; this finding excludes the possibility that the lack of mutagenic effect ot CCC was due to a rapid metabolic breakdown of the compound. The dosages used in our experiments are in the same range--at a mg/g body weight basis-as those inhibiting meiosis in grasshoppers, as shown by CARLISLE et a1.293. If CCC had a similar effect in Drosophila, this should have caused severe sterility in broods E and F, which, however, was not found. Bacterial tests To test for the mutagenic
potentials OI CCC in bacteria a fluctuation test was carried out as described by VOOGD et al. Il. As test organisms we used Klebsiella pneumoniae, requiring uracil and proline for growth, and Citrobncter freundii 425. This test was performed by dividing nutrient broth containing the substance under test and seeded with IOO bacteria/ml into rag portions of equal volume in culture tubes. After incubation for 20 h at 37”, the total number of streptomycin-
SHORTCOMMUNICATIONS
67
resistant and -dependent bacteria was determined in IOO portions by the pour-plate technique using nutrient agar supplemented with streptomycin (IOO pg/ml) and 3 days of incubation at 37“. The number of bacteria present in the 5 remaining tubes was determined by using nutrient agar, without streptomycin. From the number of portions without streptomycin-resistant or -dependent mutants the mutation rate was calculated by the Poisson distribution. As it is essential to the test to obtain a sufficient number of portions without mutants, the volumes per tube (portion) were 2.5 ml using Klebsiella pneumoniae and 0.5 m.l using Citrobacter freundii. The compound was applied in concentrations up to 5 mg/ml in the culture medium. Table III shows the results of the experiments. As in the Drosophila tests, there is no difference in mutation rate between the treated and the control series. The absence of a mutagenic effect of acridine orange in this test suggests the system’s inability to detect frameshift mutagens. Therefore, the negative results obtained with CCC are likely to apply only to base-pair substitutions. TABLE III MUTAGENICACTION OF CCC ON Klebsiella pneumoniae AND ON Citrobacter freundii
(mslml)
Portions without mutants
Kl. pneumoniae
5 2 O&
C. freundii
56 49 50
5 0”
::
Test organism
Average a 0.1676
Cone.
Number of
Mutation frequency
bacteria/ml
1.18.109 1.27.10~ 1.03~10~ 1.97’109 2.89.10~
0.137.10-9 O.I55.10_9 0.187. IO-~ 0.231.10-9 o.200~Io-9
spontaneous mutation frequency currently obtained * 0.0304. IO-~; b 0.2616 + 0.0760. IO-~.
in control series with these strains:
Conclusion It is concluded that the present data provide no evidence for a mutagenic activity of CCC in Drosophila melanogaster, or in the bacteria Klebsiella pneumoniae and Citrobacter freundii. This work was supported by the Netherlands Ministry of Public Health and Environmental Hygiene. We thank Dr. H. M. DEKHUIJZENfor his suggestion to test CCC for mutagenicity. We are indebted to Prof. F. H. SOBELSfor his encouraging advice and to Dr. K. SANKARANARAYANAN for reading the manuscript. We want to acknowledge the technical assistance of RIA Los, NETTY PEX and WILMA SEGERS. Department
of Radiation Genetics and Chemical
Mutagenesis, State University of Leiden, Wassenaarseweg 62, Leiden and
PIETERG. N. KRAMERS ADA G. A. C. KNAAP “C. E. VOOGD
* National Institute of Public Health, Bilthoven (The Netherlands) I BLINN, R., Biochemical behaviour of z-chloroethyl-trimethylammonium chloride in wheat and in rats, J. Agr. Food Chem., 15 (1967) 984-988. 2 CARLISLE, D. B., AND P. E. ELLIS, z-Chloroethyl-trimethylammonium chloride inhibits gametogenesis in locusts, Nature, 220 (1968) 189.
SHORT
68
COMMUNICATIONS
3 CARLISLE, D. B., P. E. ELLIS, AND D. J. OSBORNE, Effects of plant growth regulators on locusts and cotton stainer bugs, J. Sci. Food Agr., 20 (1969) 391-393. and persistence of chlormequat 4 DEKHUIJZEN, H. M., AND K. B. A. BODLAENDER, Distribution in potato plants, Pesticide Sci., 4 (1973) 619-627. and degradation of chlormequat in 5 DEKHUIJZEN, H. M., AND C. R. VONK, The distribution wheat plants, Pesticide Biochem. Physiol., 4 (1974) in press. of chlorocholine in plants, Z. Pflanzennachr., 114 6 JUNG, J,, AND M.E. EL-FOULY, Degradation (1966) 128-134. the statistical significance of 7 KASTENBAUM, M. A., AND K. 0. BOWMAN, Tables for determining mutation frequencies, Mutation Res., g (1970) 527-549. of chlorocholinechloride residues in 8 MOONEY. R. P.. AND N. R. PASARELA, Determination wheat grain, straw, green wheat foliage,. J. Agr. Food Chem., 15 (1967) 989-995. K., The effect of nitrogen and oxygen treatments on the frequencies of 9 SANKARANARAYANAN, X-ray-induced dominant lethals and on the physiology of the sperm in Drosophila nzelano-
gaster, Mutation Res., 4 (1967) 641-661. 10 STEVENS, W. L., Accuracy of mutation rates, J. Genet., 43 (1942) 301-307. II VOOGD, C. E., J. J. J. A. A. JACOBS, AND J. J. VAN DER STEL, On the mutagenic chlorvos, Mutation Res., 16 (1972) 413-416.
Received March zgth, 1974 Revision received August zy-d, 1974
action
of di-