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Clastogenicity of isoamylene oxide to rat lymphocytes in culture
Mutation Research Letters
ELSEVIER
Mutation Research 347 (1995) 9-12
Clastogenicity of isoamylene oxide to rat lymphocytes in culture B. Bhaskar Gollapudi a, V. Ann Linscombe a, J.W. Wilmer b,, a The Dow Chemical Company, Health and Environmental Sciences, 1803 Building, Midland, M148674, USA b Dow Europe S.A., Health and Environmental Sciences, CH-8810 Horgen, Switzerland
Received 5 January 1995; revised 20 February 1995; accepted 21 February 1995
Abstract
The mutagenic activity of the aliphatic epoxide isoamylene oxide (2-methyl-2,3-epoxybutane) is not readily detectable in the standard Ames test. In this study, the clastogenic potential of isoamylene oxide was evaluated using an in vitro mammalian cell culture system. Approximately 48 h after establishing primary cultures of rat lymphocyte cultures, the cells were treated for 4 h with various concentrations of isoamylene oxide (50, 166.7, 500, 1666.7, and 5000/~g/ml in the initial assay and 500, 1000, 2000, 3000, 4000, and 5000/~g/ml in the confirmatory assay). The cultures were harvested 24 h after termination of the treatment. Based upon the mitotic indices, cultures treated with the three highest concentrations in both the initial and confirmatory assays were evaluated to estimate the chromosomal aberration frequencies. Isoamylene oxide demonstrated a strong clastogenic activity in this assay: up to 29% aberrant cells (without gaps) were observed at the highest concentration analyzed. The presence of an external metabolic activation system ($9) did not seem to influence the magnitude of the response at the dose levels analyzed.
I. Introduction
Aliphatic epoxides belonging to various subclasses were identified as having genotoxic potential in a variety of short-term assays (Ehrenberg and Hussain, 1981; Canter et al., 1986). The mutagenicity of several of these materials was shown to be affected by the extent of substitution of the oxirane ring ( - C - C - ) (Wade et al., 1978). De Meester et al. (1982) reported one such instance in which the substitution of a hydrogen group by a methyl group at the 2-position in 2,3-epoxybutane (derivative 2-methyl-2,3-epoxy* Corresponding author. Tel. (41)-1-728-2996; Fax (41)-1728-2965.
butane, synonym isoamylene oxide) abolished the mutagenic activity of 2,3-epoxybutane in the A m e s test when assayed with a standard plate incorporation method. Isoamylene oxide did not induce a mutagenic response either in the presence or absence of $9 when assayed in a preincubation modification of the A m e s test utilizing TA98, TA100, TA1535, and TA1537 (unpublished data of T h e Dow Chemical Company). However, a report appearing in the literature only as an abstract (Callander, 1987) claimed that the methyl derivative was mutagenic to the base substitution strains TA100 and TA1535 when a vapor phase exposure technique was employed. In the present report, the clastogenic potential of isoamylene oxide was investigated in rat lymphocyte cultures.
0165-7992/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0165-7992(95)00011-9
10
B.B. GoUapudi et al. / Mutation Research 347 (1995) 9-12
2. Materials and methods
Test and positive control materials Isoamylene oxide (CsH100 , CAS No. 5076-197, purity approximately 97%), a clear liquid, was obtained from Degussa AG, Hanau, Germany. The peroxide content of the test material, which was estimated to be 15 to 21 ppm, was considered to be inconsequential for the conduct of the study. Ethyl methanesulfonate (EMS, Sigma, St. Louis, MO) was used as the positive control for the non-activation assay while cyclophosphamide (CP, Sigma) was used for the assay with $9.
Lymphocyte cultures For each experiment, blood samples were collected by cardiac puncture from two male 10-11 week old Sprague-Dawley rats; blood from one rat was used for the assay without $9 and the second one with $9. Whole blood cultures were set up (Sinha et al., 1989) in RPMI 1640 (Gibco, Grand Island, NY) supplemented with 25 mM HEPES, 10% heat-inactivated fetal bovine serum, 0.25/zl/ml fungizone, 100 units/ml penicillin G, 0.1 m g / m l streptomycin sulfate, 2 mM L-glutamine and 20 /zl/ml PHA (HA16, Wellcome, Dartford, UK). Duplicate cultures were set up at each dose level in T25 plastic tissue culture flasks by inoculating approximately 0.5 ml of whole blood/5 ml of culture medium.
Treatment procedure Isoamylene oxide, EMS and CP were dissolved directly in the treatment medium. The treatment medium was RPMI 1640 with the above mentioned concentrations of HEPES and antibiotics. The in vitro metabolic activation system consisted of $9 liver homogenate prepared from Aroclor 1254 treated male Sprague-Dawley rats and a co-factor mix (O'Neill et al., 1982). The final concentration of $9 during the treatment was 2% (v/v). Approximately 48 h after initiation of the cultures, the culture medium was removed and kept. The cells were treated with the treatment solutions (5 ml aliquots) in the presence a n d / o r absence of $9 for approximately 4 h at 37° C and the exposure was terminated by washing the cells with culture medium (without serum and PHA).
In assay 1, treatments were conducted in .15 ml centrifuge tubes with tightened caps at concentrations of 50, 166.7, 500, 1666.7, and 5000 ~g/ml. The highest concentration tested (i.e., 5000 /xg/ml) was the generally accepted limit dose for the in vitro cytogenetic assays. In assay 2, cells were exposed to the treatment solutions (500, 1000, 2000, 3000, 4000, and 5000/zg/ml) in 5 ml centrifuge tubes. At the end of the treatment, exposures were terminated by washing the cells with the culture medium (without serum and PHA) and the cells were returned to their original culture medium until the time of harvest, i.e., 24 h after the termination of treatment. Colcemid (1 /zg/culture) was added 2.5-3 h prior to harvest. The cells were swollen by hypotonic treatment (0.075 M KCI), fixed with methanol:acetic acid (3:1), dropped onto microscope slides, and stained in Giemsa.
Evaluation of slides Mitotic indices were determined as the number of cells in metaphase among 1000 cells/replicate and expressed as percentages. Chromosomal aberration frequencies were determined from coded slides (Buckton and Evans, 1973; Sinha et al., 1984; Gollapudi et al., 1986). Only those metaphases that contained 42 centromeres were scored with the exception of severely damaged cells (10 or more aberrations/cell), in which case accurate counts of the centromeres were not possible.
Statistical analysis The numbers of cells with cytogenetic abnormalities (excluding gaps) were analyzed by the following method. At each dose, data from the replicates were pooled and analyzed by constructing two-dimensional contingency tables. The total chi-square was partitioned into components of interest. Specifically, statistics were generated to test the two global hypotheses of (1) no difference in average scores (average number of aberrations per cell) among the dose groups and (2) no linear trend of increasing scores with increasing dose. An ordinal metric (0, 1, 2, etc.) was used for the doses in the statistical evaluation. If either statistic was found to be significant at a = 0.01
B.B. Gollapudi et al. / Mutation Research 347 (1995) 9-12
11
Table 1 I n c i d e n c e o f c h r o m o s o m a l a b n o r m a l i t i e s in r a t l y m p h o c y t e c u l t u r e s t r e a t e d w i t h i s o a m y l e n e o x i d e ": a s s a y 1
Mitotic index (%) Chromatid gaps Chromatid breaks Chromatid exchanges Chromosome breaks Chromosome exchanges S e v e r e l y d a m a g e d cells Cells w i t h a b e r r a t i o n s c
Negative control 500.0/~g/ml
1666.7/zg/ml
5000.0/~g/ml
-
+ $9
-
+ $9
-
4.6 1 1 0 0 0 0 1(0.5)
8.0 10 3 0 0 0 0 3(1.5)
6.2 6 1 1 0 0 1 3(1.5)
7.1 19 24 30 6 1 0 27 * (13.5)
$9
6.5 9 2 0 0 0 0 2(1.0)
+ $9
-
$9
5.0 9 3 0 0 0 0 3(1.5)
7.2 8 1 4 0 0 0 2(1.0)
$9
$9
Positive c o n t r o l b + $9
-
$9
+ $9
4.2 6 3 19 1 0 0 11 (5.5)
5.5 15 11 15 2 0 0 25 * (12.5)
3.0 8 8 24 7 0 1 16 * (8.0)
a Pooled data from duplicate cultures. b 1000 / x g / m l E M S w i t h o u t $9 a n d 4 . 2 / x g / m l C P w i t h $ 9 . c Cells w i t h o n l y g a p s a r e n o t i n c l u d e d . N u m b e r s in p a r e n t h e s e s a r e p e r c e n t a g e s . * S i g n i f i c a n t l y ( a _< 0.01) d i f f e r e n t f r o m t h e n e g a t i v e c o n t r o l .
versus a two-sided alternative, pairwise tests (i.e., control vs. treatment) were performed at each dose level and evaluated at a = 0.01 again versus a two-sided alternative.
H 2 0 for the control medium) was interpreted to be inconsequential since rat lymphocytes can withstand far higher osmolalities without any adverse effect on chromosomal integrity (Sinha et al., 1989). The mitotic indices of cultures treated with various concentrations of isoamylene oxide (i.e., 50.0, 166.7, 500.0, 1666.7, 5000.0/zg/ml) in assay 1 indicated no apparent effect of treatment on mitotic activity. Since any induced and dose-related effects on chromosomal aberrations should be evident at the highest concentrations tested in the absence of mitotic delay, it was decided to
3. Results and discussion
The pH of the treatment medium containing 5000 /xg/ml of isoamylene oxide (pH 7.44) was not appreciably different from the culture medium (pH 7.35). The slight increase in osmolality at this dose level (343 m O s m / k g H 2 0 vs. 301 m O s m / k g
Table 2 Incidence o f c h r o m o s o m a l a b n o r m a l i t i e s in rat lymphocyte t r e a t e d with isoamylene oxide a: assay 2 Negative c o n t r o l 3 0 0 0 / ~ g / m l -
$9
Mitotic index (%) 13.6 C h r o m a t i d gaps 2 C h r o m o s o m e gaps 0 C h r o m a t i d breaks 2 C h r o m a t i d exchanges 0 C h r o m o s o m e breaks 2 C h r o m o s o m e exchanges 0 Miscellaneous 0 Severely d a m a g e d cells 0 Cells with a b e r r a t i o n s c 3 (1.5)
+ $9
9.8 1 0 l 0 0 0 0 0 1 (0.5)
-
$9
10.7 14 1 21 11 3 2 1 2 29 * (14.5)
4000/xg/ml
5000 p , g / m l
+ $9
- $9
+ $9
8.1 15 0 17 17 7 1 3 0 26 * (13.0)
6.3 9 0 25 16 14 2 0 1 28 * (14.0)
6.7 20 0 36 25 16 2 0 0 36 * (18.0)
a Pooled d a t a f r o m duplicate cultures. b 1000 / x g / m l EMS without $9 a n d 4 . 2 / x g / m l CP with $ 9 . c Cells with only gaps a r e not included. * Significantly (~ < 0.01) different f r o m the negative control.
- $9
7.5 31 3 32 22 24 5 2 4 47 * (23.5)
Positive control b + $9
- $9
+ $9
3.2 9.8 4.3 19 9 6 1 0 0 54 10 21 43 6 17 16 1 4 5 0 1 2 1 1 5 0 0 57 * (28.5) 15 * (7.5) 20 * (10.0)
12
B.B. Gollapudi et al. / Mutation Research 347 (1995) 9-12
evaluate only slides from the top three concentrations (5000, 1666.7, and 500 /zg/ml) for aberrations. There was a significant increase in the number of ceils with aberrations in cultures treated with 5000 /zg/ml in the absence of $9 (13.5% abnormal cells vs. 1.0% in the negative control, Table 1). Even though the incidence of abnormal ceils in treatments with $9 was not significantly different from the corresponding negative control cultures, there was a noticeable increase in the incidence of exchanges at the 5000 ~g/ml. In a repeat assay conducted to confirm the above positive finding and to examine the existence of a dose response, rat lymphocyte cultures were treated with 500, 1000, 2000, 3000, 4000, or 5000 ~ g / m l of isoamylene oxide. In this assay, unlike the first assay, a depression in the mitotic index was noticed at the two highest concentrations (see Table 2). Since the test material is a volatile liquid (vapor pressure 158 mbar at 20° C, saturated atmosphere 158000 ppm at 20° C), the minimization of head space in assay 2 by treating the cells in a 5 ml tube as opposed to a 15 ml tube in assay 1 might have maintained the targeted concentrations of the test material in the treatment medium. Hence the variability in the mitotic index data is likely related to the differences in the effective concentration of the test material available to the cells during the 4 h treatment period. Cultures treated with the top three concentrations (viz., 5000, 4000, and 3000 ~ g / m l ) were selected to determine the aberration frequencies in assay 2. Significant dose-related increases in aberration rates were observed both with and without $9 activation (Table 2). The difference in the extent of response observed in the presence of $9 in assay 1 vs. assay 2 is most likely due to the minimization of the head space in the second assay. The results of this in vitro study clearly establish the potent clastogenic activity of isoamylene
oxide and further demonstrate that methyl substitution of the oxirane ring in 2,3-epoxybutane does not eliminate its mutagenicity potential as claimed by De Meester et al. (1982). Isoamylene oxide was also found to be positive in the mouse bone marrow micronucleus test and the test for morphological transformation in C 3 H / 1 0 T 1 / 2 cells (personal communication of unpublished data kindly provided by Solvay SA, Brussels, Belgium).
References Buckton, K.E. and H.J. Evans (1973) Methods for the Analysis of Human Chromosome Aberrations, World Health Organization, Geneva. Callander, R.D. (1987) 2-Methyl-2,3-epoxybutane is mutagenic to Salmonella, Mutation Res., 181, 313. Canter, D.A., E. Zeiger, S. Haworth, T. Lawlor, K. Mortelmans and W. Speck (1986) Comparative mutagenicity of aliphatic epoxides in Salmonella, Mutation Res., 172, 105138. De Meester, C., M. Mercier and F. Poncelet (1982) Nonmutagenicity of 2-methyl-2,3-epoxybutane and factors influencing the mutagenicity of 2,3-epoxybutane, J. Appl. Toxicol., 2, 284-288. Ehrenberg, L. and S. Hussain (1981) Genetic toxicity of some important epoxides, Mutation Res., 86, 1-113. Gollapudi, B.B., D.J. Sutcliffe and A.K. Sinha (1986) Assessment of cytogenetic response to folic acid deprivation in rat lymphocytes, In Vitro, 22, 681-684. O'Neill, J.P., R. Machanoff, R. San Sebastian and A.W. Hsie (1982) Cytotoxicity and mutagenicity of dimethylnitrosamine in mammalian cells ( C H O / H G P R T system): enhancement by calcium phosphate, Environ. Mutagen., 4, 7-18. Sinha, A.K., V.A. Linscombe, B.B. Gollapudi, M.L. McClintock, R.E. Flake and K.M. Bodner (1984) The incidence of spontaneous cytogenetic aberrations in lymphocytes cultured from normal humans for 48 and 72 h, Can. J. Genet. Cytol., 26, 528-531. Sinha, A.K., B.B. GoUapudi, V.A. Linscombe and M.L. McClintock (1989) Utilization of rat lymphocytes for the in vitro chromosomal aberration assay, Mutagenesis, 4, 147153. Wade, D.R., S.C. Airy and J.E. Sinsheimer (1978) Mutagenicity of aliphatic epoxides, Mutation Res., 58, 217-223. Communicated by I.-D. Adler
ELSEVIER
Mutation Research 347 (1995) 9-12
Clastogenicity of isoamylene oxide to rat lymphocytes in culture B. Bhaskar Gollapudi a, V. Ann Linscombe a, J.W. Wilmer b,, a The Dow Chemical Company, Health and Environmental Sciences, 1803 Building, Midland, M148674, USA b Dow Europe S.A., Health and Environmental Sciences, CH-8810 Horgen, Switzerland
Received 5 January 1995; revised 20 February 1995; accepted 21 February 1995
Abstract
The mutagenic activity of the aliphatic epoxide isoamylene oxide (2-methyl-2,3-epoxybutane) is not readily detectable in the standard Ames test. In this study, the clastogenic potential of isoamylene oxide was evaluated using an in vitro mammalian cell culture system. Approximately 48 h after establishing primary cultures of rat lymphocyte cultures, the cells were treated for 4 h with various concentrations of isoamylene oxide (50, 166.7, 500, 1666.7, and 5000/~g/ml in the initial assay and 500, 1000, 2000, 3000, 4000, and 5000/~g/ml in the confirmatory assay). The cultures were harvested 24 h after termination of the treatment. Based upon the mitotic indices, cultures treated with the three highest concentrations in both the initial and confirmatory assays were evaluated to estimate the chromosomal aberration frequencies. Isoamylene oxide demonstrated a strong clastogenic activity in this assay: up to 29% aberrant cells (without gaps) were observed at the highest concentration analyzed. The presence of an external metabolic activation system ($9) did not seem to influence the magnitude of the response at the dose levels analyzed.
I. Introduction
Aliphatic epoxides belonging to various subclasses were identified as having genotoxic potential in a variety of short-term assays (Ehrenberg and Hussain, 1981; Canter et al., 1986). The mutagenicity of several of these materials was shown to be affected by the extent of substitution of the oxirane ring ( - C - C - ) (Wade et al., 1978). De Meester et al. (1982) reported one such instance in which the substitution of a hydrogen group by a methyl group at the 2-position in 2,3-epoxybutane (derivative 2-methyl-2,3-epoxy* Corresponding author. Tel. (41)-1-728-2996; Fax (41)-1728-2965.
butane, synonym isoamylene oxide) abolished the mutagenic activity of 2,3-epoxybutane in the A m e s test when assayed with a standard plate incorporation method. Isoamylene oxide did not induce a mutagenic response either in the presence or absence of $9 when assayed in a preincubation modification of the A m e s test utilizing TA98, TA100, TA1535, and TA1537 (unpublished data of T h e Dow Chemical Company). However, a report appearing in the literature only as an abstract (Callander, 1987) claimed that the methyl derivative was mutagenic to the base substitution strains TA100 and TA1535 when a vapor phase exposure technique was employed. In the present report, the clastogenic potential of isoamylene oxide was investigated in rat lymphocyte cultures.
0165-7992/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0165-7992(95)00011-9
10
B.B. GoUapudi et al. / Mutation Research 347 (1995) 9-12
2. Materials and methods
Test and positive control materials Isoamylene oxide (CsH100 , CAS No. 5076-197, purity approximately 97%), a clear liquid, was obtained from Degussa AG, Hanau, Germany. The peroxide content of the test material, which was estimated to be 15 to 21 ppm, was considered to be inconsequential for the conduct of the study. Ethyl methanesulfonate (EMS, Sigma, St. Louis, MO) was used as the positive control for the non-activation assay while cyclophosphamide (CP, Sigma) was used for the assay with $9.
Lymphocyte cultures For each experiment, blood samples were collected by cardiac puncture from two male 10-11 week old Sprague-Dawley rats; blood from one rat was used for the assay without $9 and the second one with $9. Whole blood cultures were set up (Sinha et al., 1989) in RPMI 1640 (Gibco, Grand Island, NY) supplemented with 25 mM HEPES, 10% heat-inactivated fetal bovine serum, 0.25/zl/ml fungizone, 100 units/ml penicillin G, 0.1 m g / m l streptomycin sulfate, 2 mM L-glutamine and 20 /zl/ml PHA (HA16, Wellcome, Dartford, UK). Duplicate cultures were set up at each dose level in T25 plastic tissue culture flasks by inoculating approximately 0.5 ml of whole blood/5 ml of culture medium.
Treatment procedure Isoamylene oxide, EMS and CP were dissolved directly in the treatment medium. The treatment medium was RPMI 1640 with the above mentioned concentrations of HEPES and antibiotics. The in vitro metabolic activation system consisted of $9 liver homogenate prepared from Aroclor 1254 treated male Sprague-Dawley rats and a co-factor mix (O'Neill et al., 1982). The final concentration of $9 during the treatment was 2% (v/v). Approximately 48 h after initiation of the cultures, the culture medium was removed and kept. The cells were treated with the treatment solutions (5 ml aliquots) in the presence a n d / o r absence of $9 for approximately 4 h at 37° C and the exposure was terminated by washing the cells with culture medium (without serum and PHA).
In assay 1, treatments were conducted in .15 ml centrifuge tubes with tightened caps at concentrations of 50, 166.7, 500, 1666.7, and 5000 ~g/ml. The highest concentration tested (i.e., 5000 /xg/ml) was the generally accepted limit dose for the in vitro cytogenetic assays. In assay 2, cells were exposed to the treatment solutions (500, 1000, 2000, 3000, 4000, and 5000/zg/ml) in 5 ml centrifuge tubes. At the end of the treatment, exposures were terminated by washing the cells with the culture medium (without serum and PHA) and the cells were returned to their original culture medium until the time of harvest, i.e., 24 h after the termination of treatment. Colcemid (1 /zg/culture) was added 2.5-3 h prior to harvest. The cells were swollen by hypotonic treatment (0.075 M KCI), fixed with methanol:acetic acid (3:1), dropped onto microscope slides, and stained in Giemsa.
Evaluation of slides Mitotic indices were determined as the number of cells in metaphase among 1000 cells/replicate and expressed as percentages. Chromosomal aberration frequencies were determined from coded slides (Buckton and Evans, 1973; Sinha et al., 1984; Gollapudi et al., 1986). Only those metaphases that contained 42 centromeres were scored with the exception of severely damaged cells (10 or more aberrations/cell), in which case accurate counts of the centromeres were not possible.
Statistical analysis The numbers of cells with cytogenetic abnormalities (excluding gaps) were analyzed by the following method. At each dose, data from the replicates were pooled and analyzed by constructing two-dimensional contingency tables. The total chi-square was partitioned into components of interest. Specifically, statistics were generated to test the two global hypotheses of (1) no difference in average scores (average number of aberrations per cell) among the dose groups and (2) no linear trend of increasing scores with increasing dose. An ordinal metric (0, 1, 2, etc.) was used for the doses in the statistical evaluation. If either statistic was found to be significant at a = 0.01
B.B. Gollapudi et al. / Mutation Research 347 (1995) 9-12
11
Table 1 I n c i d e n c e o f c h r o m o s o m a l a b n o r m a l i t i e s in r a t l y m p h o c y t e c u l t u r e s t r e a t e d w i t h i s o a m y l e n e o x i d e ": a s s a y 1
Mitotic index (%) Chromatid gaps Chromatid breaks Chromatid exchanges Chromosome breaks Chromosome exchanges S e v e r e l y d a m a g e d cells Cells w i t h a b e r r a t i o n s c
Negative control 500.0/~g/ml
1666.7/zg/ml
5000.0/~g/ml
-
+ $9
-
+ $9
-
4.6 1 1 0 0 0 0 1(0.5)
8.0 10 3 0 0 0 0 3(1.5)
6.2 6 1 1 0 0 1 3(1.5)
7.1 19 24 30 6 1 0 27 * (13.5)
$9
6.5 9 2 0 0 0 0 2(1.0)
+ $9
-
$9
5.0 9 3 0 0 0 0 3(1.5)
7.2 8 1 4 0 0 0 2(1.0)
$9
$9
Positive c o n t r o l b + $9
-
$9
+ $9
4.2 6 3 19 1 0 0 11 (5.5)
5.5 15 11 15 2 0 0 25 * (12.5)
3.0 8 8 24 7 0 1 16 * (8.0)
a Pooled data from duplicate cultures. b 1000 / x g / m l E M S w i t h o u t $9 a n d 4 . 2 / x g / m l C P w i t h $ 9 . c Cells w i t h o n l y g a p s a r e n o t i n c l u d e d . N u m b e r s in p a r e n t h e s e s a r e p e r c e n t a g e s . * S i g n i f i c a n t l y ( a _< 0.01) d i f f e r e n t f r o m t h e n e g a t i v e c o n t r o l .
versus a two-sided alternative, pairwise tests (i.e., control vs. treatment) were performed at each dose level and evaluated at a = 0.01 again versus a two-sided alternative.
H 2 0 for the control medium) was interpreted to be inconsequential since rat lymphocytes can withstand far higher osmolalities without any adverse effect on chromosomal integrity (Sinha et al., 1989). The mitotic indices of cultures treated with various concentrations of isoamylene oxide (i.e., 50.0, 166.7, 500.0, 1666.7, 5000.0/zg/ml) in assay 1 indicated no apparent effect of treatment on mitotic activity. Since any induced and dose-related effects on chromosomal aberrations should be evident at the highest concentrations tested in the absence of mitotic delay, it was decided to
3. Results and discussion
The pH of the treatment medium containing 5000 /xg/ml of isoamylene oxide (pH 7.44) was not appreciably different from the culture medium (pH 7.35). The slight increase in osmolality at this dose level (343 m O s m / k g H 2 0 vs. 301 m O s m / k g
Table 2 Incidence o f c h r o m o s o m a l a b n o r m a l i t i e s in rat lymphocyte t r e a t e d with isoamylene oxide a: assay 2 Negative c o n t r o l 3 0 0 0 / ~ g / m l -
$9
Mitotic index (%) 13.6 C h r o m a t i d gaps 2 C h r o m o s o m e gaps 0 C h r o m a t i d breaks 2 C h r o m a t i d exchanges 0 C h r o m o s o m e breaks 2 C h r o m o s o m e exchanges 0 Miscellaneous 0 Severely d a m a g e d cells 0 Cells with a b e r r a t i o n s c 3 (1.5)
+ $9
9.8 1 0 l 0 0 0 0 0 1 (0.5)
-
$9
10.7 14 1 21 11 3 2 1 2 29 * (14.5)
4000/xg/ml
5000 p , g / m l
+ $9
- $9
+ $9
8.1 15 0 17 17 7 1 3 0 26 * (13.0)
6.3 9 0 25 16 14 2 0 1 28 * (14.0)
6.7 20 0 36 25 16 2 0 0 36 * (18.0)
a Pooled d a t a f r o m duplicate cultures. b 1000 / x g / m l EMS without $9 a n d 4 . 2 / x g / m l CP with $ 9 . c Cells with only gaps a r e not included. * Significantly (~ < 0.01) different f r o m the negative control.
- $9
7.5 31 3 32 22 24 5 2 4 47 * (23.5)
Positive control b + $9
- $9
+ $9
3.2 9.8 4.3 19 9 6 1 0 0 54 10 21 43 6 17 16 1 4 5 0 1 2 1 1 5 0 0 57 * (28.5) 15 * (7.5) 20 * (10.0)
12
B.B. Gollapudi et al. / Mutation Research 347 (1995) 9-12
evaluate only slides from the top three concentrations (5000, 1666.7, and 500 /zg/ml) for aberrations. There was a significant increase in the number of ceils with aberrations in cultures treated with 5000 /zg/ml in the absence of $9 (13.5% abnormal cells vs. 1.0% in the negative control, Table 1). Even though the incidence of abnormal ceils in treatments with $9 was not significantly different from the corresponding negative control cultures, there was a noticeable increase in the incidence of exchanges at the 5000 ~g/ml. In a repeat assay conducted to confirm the above positive finding and to examine the existence of a dose response, rat lymphocyte cultures were treated with 500, 1000, 2000, 3000, 4000, or 5000 ~ g / m l of isoamylene oxide. In this assay, unlike the first assay, a depression in the mitotic index was noticed at the two highest concentrations (see Table 2). Since the test material is a volatile liquid (vapor pressure 158 mbar at 20° C, saturated atmosphere 158000 ppm at 20° C), the minimization of head space in assay 2 by treating the cells in a 5 ml tube as opposed to a 15 ml tube in assay 1 might have maintained the targeted concentrations of the test material in the treatment medium. Hence the variability in the mitotic index data is likely related to the differences in the effective concentration of the test material available to the cells during the 4 h treatment period. Cultures treated with the top three concentrations (viz., 5000, 4000, and 3000 ~ g / m l ) were selected to determine the aberration frequencies in assay 2. Significant dose-related increases in aberration rates were observed both with and without $9 activation (Table 2). The difference in the extent of response observed in the presence of $9 in assay 1 vs. assay 2 is most likely due to the minimization of the head space in the second assay. The results of this in vitro study clearly establish the potent clastogenic activity of isoamylene
oxide and further demonstrate that methyl substitution of the oxirane ring in 2,3-epoxybutane does not eliminate its mutagenicity potential as claimed by De Meester et al. (1982). Isoamylene oxide was also found to be positive in the mouse bone marrow micronucleus test and the test for morphological transformation in C 3 H / 1 0 T 1 / 2 cells (personal communication of unpublished data kindly provided by Solvay SA, Brussels, Belgium).
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