Mutation Research, 282 (1992) 231-234
231
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-7992/92/$05.00
MUTLET 0684
Clastogenicity of 2-chlorobenzylidene malonitrile (CS) in V79 Chinese hamster cells M. B a u c h i n g e r a n d E. S c h m i d Institut fiir Strahlenbiologie, GSF-Forschungszentrum fiir Umwelt und Gesundheit mbH, Neuherberg, Germany (Received 8 April 1992) (Accepted 9 April 1992)
Keywords: 2-Chlorobenzylidene malonitrile; V79 cells; Clastogenicity
Summary The clastogenicity of the sensory irritant 2-chlorobenzylidene malonitrile (CS) to V79 Chinese hamster cells was investigated at various exposure conditions. CS efficiently induced chromatid-type aberrations in a dose-dependent manner provided the cells could run through at least one or two S-phases during a 20-h exposure over a 3-h exposure followed by a 20-h recovery period (cell cycle time 8-10 h). The induction of SCEs indicates an S-dependent mechanism. The hydrolysis products o-chlorobenzaldehyde and malonitrile were inactive in these experiments.
2-Chlorobenzylidene malonitrile (CS) is a short-acting sensory irritant which is used as a lacrimatory riot control agent. In previous experiments with a 3-h exposure of V79 Chinese hamster cells we have shown that CS caused c-mitotic spindle damage in a dose-dependent manner (Schmid et al., 1989). When the cells were examined by differential staining of the spindle apparatus and chromosomes, unlike in colcemid-induced c-metaphases, residual spindle or microtubule material was found in the majority of CS-induced c-metaphases (Salassidis et al., 1991). In further experiments with a 20-h exposure of V79 cells to CS we have shown that the induced spindle damage was correlated with the induction
Correspondence: Dr. M. Bauchinger, Institut fiir Strahlenbiologie, GSF-Forschungszentrum fiir Umwelt und Gesundheit mbH, D-8042 Neuherberg, Germany.
of aneuploidy (Schmid and Bauchinger, 1991). Of the hydrolysis products of CS, o-chlorobenzaldehyde (oCB) and malonitrile (MAL), oCB seems to be an important metabolite for the aneuploidogenic effectiveness. In a preliminary communication we have recently reported also on the clastogenic effectiveness of CS (Schmid and Bauchinger, 1992). In the present study detailed results will be presented after various exposure conditions of V79 cells to this compound and metabolites.
Material and methods V79 Chinese hamster cells were cultured in Dulbecco's modified Eagle's minimum essential medium (DMEM, Biochrom, Berlin), supplemented with 10% fetal calf serum, 100 U / m l penicillin and 100/zg/ml streptomycin at 37°C in a humidified atmosphere containing 7% CO 2.
232
For the experiments, slide cultures with 5 × 10 4 cells were set up in Quadriperm dishes (Heraeus, Hanau). After 24 h cells were rinsed with prewarmed phosphate-buffered saline (PBS) solution and exposed to the test agents in serum-free Eagle's medium with Eagle's salts. CS (Klever, Aham), purified by recrystallization (3 x ) from hot ethanol, oCB (purity 98%) and MAL (purity /> 98%) (both purchased from Merck-Schuchardt, H ohenbr unn) w e r e dissolved in dimethyl sulfoxide (DMSO; final concentration in the medium 0.33%) immediately before use. DMSO-treated cultures served as controls. In the first experiment cells were exposed to the test agents for 20 h. In the second experiment cells were exposed to CS for 3 h. Subsequently, cells were washed twice with PBS, fed with complete DMEM without serum and incubated for a further 20 h in the absence of the test compound. For an evaluation of the induction of sister-chromatid exchanges (SCEs), two separate cultures set up for each concentration received 24.2 p.M BrdU. In the third experiment the cells were incubated for 20 h with the supernatant obtained from the 3-h CS exposure of the second experiment. Two hours prior to fixation 0.03 ~ g / m l colcemid (Ciba, Basel) was added to the cultures. The methods for chromosome preparation and for fluorescence plus Giemsa (FPG) staining are described elsewhere (Salassidis and Bauchinger, 1990; Apelt et al., 1981).
All experiments were carried out in duplicate and 2 x 100 metaphases (exceptions are indicated in Table 1) with a chromosome number of 22 _+ 2 were scored for each concentration. 2 x 20 harlequin-stained second-cycle metaphases with 22 chromosomes were scored for SCEs. Cells containing structural chromosome aberrations were classified as S-cells. Since there were no substantial variations between the data of these replicates, the results of the same concentration were pooled. Results and d i s c u s s i o n
The highest concentration of CS which did not impair cell proliferation (Schmid et al., 1989) was 37.5/xM. In the first experiment a 20-h exposure of the cells to CS caused a dose-dependent increase in the number of S-cells and the frequency of chromatid-type aberrations (single breaks, isolocus breaks and exchanges). The frequency of gaps increased in the same manner. Since in aqueous media CS is very rapidly hydrolyzed to oCB and MAL (Cucinell et al., 1971; Leadbeater, 1973), the cells were also incubated with 18.8 and 37.5 tzM of the individual compounds or with an equimolar mixture (18.8 IzM) for 20 h. Table 1 shows that none of these exposures caused a significant clastogenic effect. In the second experiment when the cells were exposed to CS for only 3 h, followed by a recov-
TABLE 1 FREQUENCY OF CHROMOSOMAL CHANGES DETERMINED IN TWO REPLICATES FOR EACH EXPOSURE (20 h) OF V79 CELLS (EXPERIMENT 1) Exposure
0.33% DMSO 9.4/*M CS 18.8/*M CS 37.5/*M CS 18.8 p.M oCB 37.5/*M oCB 18.8 IzM MAL 37.5/*M M A L 18.8/*M oCB + 18.8 tzM MAL
Cells
Gaps
S-cells
Aberrations per cell
scored
per cell
(%)
Chromatid breaks
Isochromatid breaks
Exchanges
300 300 300 250 200 200 200 200
0.037 0.103 0.230 0.420 0.035 0.045 0.035 0.015
1.3 11.0 22.3 24.8 2.5 4.0 0.5 0.5
0.003 0.107 0.273 0.460 0.005 0.020 0 0
0.010 0.037 0.050 0.080 0.020 0.020 0 0.005
0 0.060 0.093 0.100 0 0.005 0.005 0
200
0.025
1.0
0.005
0.005
0
233 TABLE 2 FREQUENCY OF CHROMOSOMAL CHANGES DETERMINED IN TWO REPLICATES FOR EACH EXPOSURE (3 h + 20 h RECOVERY) OF V79 CELLS (EXPERIMENT 2) Exposure
0.33% DMSO 18.8 p.M CS 37.5 IzM CS
Cells scored
Gaps per cell
S-cells (%)
Aberrationsper cell C h r o m a t i d Isochromatid breaks breaks
Exchanges
SCEs per cell _+SEM
200 200 200
0.015 0.055 0.065
0.5 9.0 10.0
0.005 0.070 0.070
0 0.105 0.065
6.50_+0.42 10.10_+0.62 15.23 ± 0.63
ery period of 20 h, the compound was less effective. In the SCE test a dose-dependent increase of SCEs per cell became apparent (Table 2). No clastogenic effect could be observed in cells incubated with the supernatant of the 3-h exposure of the second experiment (Table 3). In our previous study the hydrolysis product of CS, oCB, could be identified as a spindle-damaging and aneuploidogenic compound (Schmid and Bauchinger, 1991). In contrast neither oCB nor M A L proved to be clastogens in the present experiments. The efficient induction of chromosomal damage by CS is unexpected since it can be covalently bound to nuclear proteins but not to D N A (Holmes, 1962; Cucinell et al., 1971; Jones, 1972; von D~iniken et al., 1981). Thus its spindledisturbing action may be a consequence of an interaction of CS itself or of oCB with the preformed spindle microtubules (Salassidis et al., 1991). The observation of Ziegler-Skylakakis et al. (1989) that a 3-h exposure of V79 cells to CS did not cause any detectable increase in repair incorporation is at variance with the present find-
0 0.025 0.005
ings of a high clastogenicity which must be attributed to induced D N A lesions. However, already Ziegler-Skylakakis et al. (1989) suggested that these lesions might be created by mechanisms other than binding of CS or of a metabolite to DNA, e.g., by the formation of reactive oxygen radicals. Since CS reacts very rapidly with water (Patai and Rappaport, 1962) and is hydrolyzed to oCB and MAL, primary D N A lesions must have been induced by this compound or by a yet unidentified metabolite within a relatively short time period. Such an assumption is supported by the results of experiment 3 in which the cells were incubated for 20 h with the supernatant of the 3-h CS exposure of experiment 2. Despite the long-term exposure no significant clastogenic effect could be observed which indicates that no more active compounds were present in the supernatant. Assuming an S-dependent clastogenic mechanism which is supported by the results of the SCE test, D N A lesions should be efficiently converted to structural chromosomal changes, provided that
TABLE 3 FREQUENCY OF CHROMOSOMALCHANGES DETERMINED IN TWO REPLICATESFOR EACH EXPOSURE ~ (20 h) OF V79 CELLS (EXPERIMENT 3) Exposure a
0.33% DMSO 18.8 p.M CS 37.5/~M CS
Cells scored
Gaps per cell
S-cells (%)
Aberrations per cell Chromatid Isochromatid breaks breaks
Exchanges
200 200 200
0.005 0.020 0.010
1.0 0.5 1.5
0.005 0 0
0 0.005 0.015
a Supernatant from corresponding 3-h exposures of experiment 3, given in Table 2.
0.005 0 0
234 these lesions persist u n t i l D N A synthesis. Since the c e l l cycle time of o u r V79 cell line is a b o u t 8 - 1 0 h, the cells had a m p l e time to r u n t h r o u g h at least o n e or two S-phases d u r i n g either the 20-h exposure or the 3-h exposure to CS followed by a 20-h recovery period. This may explain why in both e x p e r i m e n t s significant clastogenic effectiveness of CS could be detected.
Acknowledgements W e t h a n k N. G a b e l l i n i a n d S. M e y e r for excellent technical assistance.
References Apelt, F., J. Kolin-Gerresheim and M. Bauchinger (1981) Azathioprines, a clastogen in human somatic cells? Analysis of chromosome damage and SCE in tymphocytes after exposure in vivo and in vitro, Mutation Res., 88, 61-71. Cucinell, S.A., K.C. Swentzel, R. Biskup, N. Snodgrass, S. Lovre, W. Stark, L. Feinsilver and F. Vocci (1971) Biochemical interactions and metabolic fate of riot control agents, Fed. Proc., 30, 86-91. Holmes, H.L. (1962) A theory for the action of benzalmalonitri'les on nerve endings, Part V, Suffield Technical Paper, Ralston, Alberta, 234, 1-22. Jones, G.R.N. (1972) CS and its chemical relatives, Nature (London), 235, 257-261. Leadbeater, L. (1973) The absorption of orthochlorobenzylidene malonitrile (CS) by the respiratory tract, Toxicol. Appl. Pharmacol., 25, 101-110. Patai, S., and Z. Rappaport (1962) Nucleophilic attacks on
carbon-carbon double bonds. Part II. Cleavage or arylmethylenemalononitrileg by water in 95% ethanol, J. Chem. Soc., 383-392. Salassidis, K., and M. Bauchinger (1990) Mitotic spindle damage induced by 3,4-dichloroaniline in V79 Chinese hamster cells examined by differential staining of he spindle apparatus and chromosomes, Mutagenesis, 5, 367-370. Salassidis, K., E. Schmid and M. Bauchinger (1991) Mitotic spindle damage induced by 2-chlorobenzylidene malonitrile (CS) in V79 Chinese hamster cells examined by differential staining of the spindle apparatus and chromosomes, Mutation Res., 262, 263-266. Schmid, E., and M. Bauchinger (1991) Analysis of the aneuploidy inducing capacity of 2-chlorobenzylidine malonitrile (CS) and metabolites in V79 Chinese hamster cells, Mutagenesis, 6, 303-305. Schmid, E., and M. Bauchinger (1992) Cytogenetic effects of the sensory irritant CS and its metabolites, 13th Meeting of the German section of EEMS, Heidelberg, 25.27.9.1991, Mutagenesis, 7, 167 (Abstract). Schmid, E., M. Bauchinger, K. Ziegler-Skylakakis and U. Andrae (1989) 2-Chlorobenzylidene malonitrile (CS) causes spindle disturbances in V79 Chinese hamster cells, Mutation Res., 226, 133-136. Von Diiniken, A., U. Friedrich, W.K. Lutz and C. Schlatter (1981) Tests for mutagenicity in Salmonella and covalent binding to DNA and proteins in the rat of the riot control agent o-chlorobenzylidene malonitrile (CS), Arch. Toxicol., 49, 15-27. Ziegler-Skylakakis, K., K.H. Summer and U. Andrae (1989) Mutagenicity and cytotoxicityof 2-chorobenzylidene malonitrile (CS) and metabolites in V79 Chinese hamster cells, Arch. Toxicol. 63, 314-319.
Communicated by I.-D. Adler
231
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-7992/92/$05.00
MUTLET 0684
Clastogenicity of 2-chlorobenzylidene malonitrile (CS) in V79 Chinese hamster cells M. B a u c h i n g e r a n d E. S c h m i d Institut fiir Strahlenbiologie, GSF-Forschungszentrum fiir Umwelt und Gesundheit mbH, Neuherberg, Germany (Received 8 April 1992) (Accepted 9 April 1992)
Keywords: 2-Chlorobenzylidene malonitrile; V79 cells; Clastogenicity
Summary The clastogenicity of the sensory irritant 2-chlorobenzylidene malonitrile (CS) to V79 Chinese hamster cells was investigated at various exposure conditions. CS efficiently induced chromatid-type aberrations in a dose-dependent manner provided the cells could run through at least one or two S-phases during a 20-h exposure over a 3-h exposure followed by a 20-h recovery period (cell cycle time 8-10 h). The induction of SCEs indicates an S-dependent mechanism. The hydrolysis products o-chlorobenzaldehyde and malonitrile were inactive in these experiments.
2-Chlorobenzylidene malonitrile (CS) is a short-acting sensory irritant which is used as a lacrimatory riot control agent. In previous experiments with a 3-h exposure of V79 Chinese hamster cells we have shown that CS caused c-mitotic spindle damage in a dose-dependent manner (Schmid et al., 1989). When the cells were examined by differential staining of the spindle apparatus and chromosomes, unlike in colcemid-induced c-metaphases, residual spindle or microtubule material was found in the majority of CS-induced c-metaphases (Salassidis et al., 1991). In further experiments with a 20-h exposure of V79 cells to CS we have shown that the induced spindle damage was correlated with the induction
Correspondence: Dr. M. Bauchinger, Institut fiir Strahlenbiologie, GSF-Forschungszentrum fiir Umwelt und Gesundheit mbH, D-8042 Neuherberg, Germany.
of aneuploidy (Schmid and Bauchinger, 1991). Of the hydrolysis products of CS, o-chlorobenzaldehyde (oCB) and malonitrile (MAL), oCB seems to be an important metabolite for the aneuploidogenic effectiveness. In a preliminary communication we have recently reported also on the clastogenic effectiveness of CS (Schmid and Bauchinger, 1992). In the present study detailed results will be presented after various exposure conditions of V79 cells to this compound and metabolites.
Material and methods V79 Chinese hamster cells were cultured in Dulbecco's modified Eagle's minimum essential medium (DMEM, Biochrom, Berlin), supplemented with 10% fetal calf serum, 100 U / m l penicillin and 100/zg/ml streptomycin at 37°C in a humidified atmosphere containing 7% CO 2.
232
For the experiments, slide cultures with 5 × 10 4 cells were set up in Quadriperm dishes (Heraeus, Hanau). After 24 h cells were rinsed with prewarmed phosphate-buffered saline (PBS) solution and exposed to the test agents in serum-free Eagle's medium with Eagle's salts. CS (Klever, Aham), purified by recrystallization (3 x ) from hot ethanol, oCB (purity 98%) and MAL (purity /> 98%) (both purchased from Merck-Schuchardt, H ohenbr unn) w e r e dissolved in dimethyl sulfoxide (DMSO; final concentration in the medium 0.33%) immediately before use. DMSO-treated cultures served as controls. In the first experiment cells were exposed to the test agents for 20 h. In the second experiment cells were exposed to CS for 3 h. Subsequently, cells were washed twice with PBS, fed with complete DMEM without serum and incubated for a further 20 h in the absence of the test compound. For an evaluation of the induction of sister-chromatid exchanges (SCEs), two separate cultures set up for each concentration received 24.2 p.M BrdU. In the third experiment the cells were incubated for 20 h with the supernatant obtained from the 3-h CS exposure of the second experiment. Two hours prior to fixation 0.03 ~ g / m l colcemid (Ciba, Basel) was added to the cultures. The methods for chromosome preparation and for fluorescence plus Giemsa (FPG) staining are described elsewhere (Salassidis and Bauchinger, 1990; Apelt et al., 1981).
All experiments were carried out in duplicate and 2 x 100 metaphases (exceptions are indicated in Table 1) with a chromosome number of 22 _+ 2 were scored for each concentration. 2 x 20 harlequin-stained second-cycle metaphases with 22 chromosomes were scored for SCEs. Cells containing structural chromosome aberrations were classified as S-cells. Since there were no substantial variations between the data of these replicates, the results of the same concentration were pooled. Results and d i s c u s s i o n
The highest concentration of CS which did not impair cell proliferation (Schmid et al., 1989) was 37.5/xM. In the first experiment a 20-h exposure of the cells to CS caused a dose-dependent increase in the number of S-cells and the frequency of chromatid-type aberrations (single breaks, isolocus breaks and exchanges). The frequency of gaps increased in the same manner. Since in aqueous media CS is very rapidly hydrolyzed to oCB and MAL (Cucinell et al., 1971; Leadbeater, 1973), the cells were also incubated with 18.8 and 37.5 tzM of the individual compounds or with an equimolar mixture (18.8 IzM) for 20 h. Table 1 shows that none of these exposures caused a significant clastogenic effect. In the second experiment when the cells were exposed to CS for only 3 h, followed by a recov-
TABLE 1 FREQUENCY OF CHROMOSOMAL CHANGES DETERMINED IN TWO REPLICATES FOR EACH EXPOSURE (20 h) OF V79 CELLS (EXPERIMENT 1) Exposure
0.33% DMSO 9.4/*M CS 18.8/*M CS 37.5/*M CS 18.8 p.M oCB 37.5/*M oCB 18.8 IzM MAL 37.5/*M M A L 18.8/*M oCB + 18.8 tzM MAL
Cells
Gaps
S-cells
Aberrations per cell
scored
per cell
(%)
Chromatid breaks
Isochromatid breaks
Exchanges
300 300 300 250 200 200 200 200
0.037 0.103 0.230 0.420 0.035 0.045 0.035 0.015
1.3 11.0 22.3 24.8 2.5 4.0 0.5 0.5
0.003 0.107 0.273 0.460 0.005 0.020 0 0
0.010 0.037 0.050 0.080 0.020 0.020 0 0.005
0 0.060 0.093 0.100 0 0.005 0.005 0
200
0.025
1.0
0.005
0.005
0
233 TABLE 2 FREQUENCY OF CHROMOSOMAL CHANGES DETERMINED IN TWO REPLICATES FOR EACH EXPOSURE (3 h + 20 h RECOVERY) OF V79 CELLS (EXPERIMENT 2) Exposure
0.33% DMSO 18.8 p.M CS 37.5 IzM CS
Cells scored
Gaps per cell
S-cells (%)
Aberrationsper cell C h r o m a t i d Isochromatid breaks breaks
Exchanges
SCEs per cell _+SEM
200 200 200
0.015 0.055 0.065
0.5 9.0 10.0
0.005 0.070 0.070
0 0.105 0.065
6.50_+0.42 10.10_+0.62 15.23 ± 0.63
ery period of 20 h, the compound was less effective. In the SCE test a dose-dependent increase of SCEs per cell became apparent (Table 2). No clastogenic effect could be observed in cells incubated with the supernatant of the 3-h exposure of the second experiment (Table 3). In our previous study the hydrolysis product of CS, oCB, could be identified as a spindle-damaging and aneuploidogenic compound (Schmid and Bauchinger, 1991). In contrast neither oCB nor M A L proved to be clastogens in the present experiments. The efficient induction of chromosomal damage by CS is unexpected since it can be covalently bound to nuclear proteins but not to D N A (Holmes, 1962; Cucinell et al., 1971; Jones, 1972; von D~iniken et al., 1981). Thus its spindledisturbing action may be a consequence of an interaction of CS itself or of oCB with the preformed spindle microtubules (Salassidis et al., 1991). The observation of Ziegler-Skylakakis et al. (1989) that a 3-h exposure of V79 cells to CS did not cause any detectable increase in repair incorporation is at variance with the present find-
0 0.025 0.005
ings of a high clastogenicity which must be attributed to induced D N A lesions. However, already Ziegler-Skylakakis et al. (1989) suggested that these lesions might be created by mechanisms other than binding of CS or of a metabolite to DNA, e.g., by the formation of reactive oxygen radicals. Since CS reacts very rapidly with water (Patai and Rappaport, 1962) and is hydrolyzed to oCB and MAL, primary D N A lesions must have been induced by this compound or by a yet unidentified metabolite within a relatively short time period. Such an assumption is supported by the results of experiment 3 in which the cells were incubated for 20 h with the supernatant of the 3-h CS exposure of experiment 2. Despite the long-term exposure no significant clastogenic effect could be observed which indicates that no more active compounds were present in the supernatant. Assuming an S-dependent clastogenic mechanism which is supported by the results of the SCE test, D N A lesions should be efficiently converted to structural chromosomal changes, provided that
TABLE 3 FREQUENCY OF CHROMOSOMALCHANGES DETERMINED IN TWO REPLICATESFOR EACH EXPOSURE ~ (20 h) OF V79 CELLS (EXPERIMENT 3) Exposure a
0.33% DMSO 18.8 p.M CS 37.5/~M CS
Cells scored
Gaps per cell
S-cells (%)
Aberrations per cell Chromatid Isochromatid breaks breaks
Exchanges
200 200 200
0.005 0.020 0.010
1.0 0.5 1.5
0.005 0 0
0 0.005 0.015
a Supernatant from corresponding 3-h exposures of experiment 3, given in Table 2.
0.005 0 0
234 these lesions persist u n t i l D N A synthesis. Since the c e l l cycle time of o u r V79 cell line is a b o u t 8 - 1 0 h, the cells had a m p l e time to r u n t h r o u g h at least o n e or two S-phases d u r i n g either the 20-h exposure or the 3-h exposure to CS followed by a 20-h recovery period. This may explain why in both e x p e r i m e n t s significant clastogenic effectiveness of CS could be detected.
Acknowledgements W e t h a n k N. G a b e l l i n i a n d S. M e y e r for excellent technical assistance.
References Apelt, F., J. Kolin-Gerresheim and M. Bauchinger (1981) Azathioprines, a clastogen in human somatic cells? Analysis of chromosome damage and SCE in tymphocytes after exposure in vivo and in vitro, Mutation Res., 88, 61-71. Cucinell, S.A., K.C. Swentzel, R. Biskup, N. Snodgrass, S. Lovre, W. Stark, L. Feinsilver and F. Vocci (1971) Biochemical interactions and metabolic fate of riot control agents, Fed. Proc., 30, 86-91. Holmes, H.L. (1962) A theory for the action of benzalmalonitri'les on nerve endings, Part V, Suffield Technical Paper, Ralston, Alberta, 234, 1-22. Jones, G.R.N. (1972) CS and its chemical relatives, Nature (London), 235, 257-261. Leadbeater, L. (1973) The absorption of orthochlorobenzylidene malonitrile (CS) by the respiratory tract, Toxicol. Appl. Pharmacol., 25, 101-110. Patai, S., and Z. Rappaport (1962) Nucleophilic attacks on
carbon-carbon double bonds. Part II. Cleavage or arylmethylenemalononitrileg by water in 95% ethanol, J. Chem. Soc., 383-392. Salassidis, K., and M. Bauchinger (1990) Mitotic spindle damage induced by 3,4-dichloroaniline in V79 Chinese hamster cells examined by differential staining of he spindle apparatus and chromosomes, Mutagenesis, 5, 367-370. Salassidis, K., E. Schmid and M. Bauchinger (1991) Mitotic spindle damage induced by 2-chlorobenzylidene malonitrile (CS) in V79 Chinese hamster cells examined by differential staining of the spindle apparatus and chromosomes, Mutation Res., 262, 263-266. Schmid, E., and M. Bauchinger (1991) Analysis of the aneuploidy inducing capacity of 2-chlorobenzylidine malonitrile (CS) and metabolites in V79 Chinese hamster cells, Mutagenesis, 6, 303-305. Schmid, E., and M. Bauchinger (1992) Cytogenetic effects of the sensory irritant CS and its metabolites, 13th Meeting of the German section of EEMS, Heidelberg, 25.27.9.1991, Mutagenesis, 7, 167 (Abstract). Schmid, E., M. Bauchinger, K. Ziegler-Skylakakis and U. Andrae (1989) 2-Chlorobenzylidene malonitrile (CS) causes spindle disturbances in V79 Chinese hamster cells, Mutation Res., 226, 133-136. Von Diiniken, A., U. Friedrich, W.K. Lutz and C. Schlatter (1981) Tests for mutagenicity in Salmonella and covalent binding to DNA and proteins in the rat of the riot control agent o-chlorobenzylidene malonitrile (CS), Arch. Toxicol., 49, 15-27. Ziegler-Skylakakis, K., K.H. Summer and U. Andrae (1989) Mutagenicity and cytotoxicityof 2-chorobenzylidene malonitrile (CS) and metabolites in V79 Chinese hamster cells, Arch. Toxicol. 63, 314-319.
Communicated by I.-D. Adler
