Fd Chem. Toxic. Vol. 30, No. 6, pp. 505-515, 1992
0278-6915/92$5.00+ 0.00 Copyright © 1992PergamonPress Ltd
Printed in Great Britain.All rights reserved
GENETIC TOXICOLOGY OF ACRYLIC ACID K. L. MCCARTHY*, W. C THOMAS'[',M. J. AARDEMA~,,J. L. SEYMOUR~,D. L. PUTMAN§,
L. L. YANG~, R. D. CURREN§ and R. VALENClA)I *ToxicologyDepartment, Rohm and Haas Company, Spring House, PA 19477;tToxicology Department, Hoechst Celanese, PO Box 2500, Somerville, NJ 08876; :~Procter & Gamble Company, Miami Valley Laboratories, Cincinnati, OH 45239; §Genetic Toxicology Division, Microbiological Associates, Inc., Rockville, MD 20850 and IIZoologyDepartment, University of Wisconsin, Madison, WI 53706, USA (Accepted 14 January 1992)
Abstract--Acrylic acid was tested for gene mutations in the in vitro CHO/HGPRT assay, for chromosome aberrations in CHO cells in culture, and for potential to induce unscheduled DNA synthesis in rat hepatocytes in culture. In vivo assays performed included the Drosophila sex-linked recessive lethal assay by both the feeding and injection routes, the in vivo cytogenetic assay in rat bone marrow cells after both a 1-day and 5-day oral dosing regimen, and a dominant lethal assay in mice by both an acute and 5-day dosing regimen. All results were negative (non-mutagenic) except for the in vitro chromosome aberration assay. This latter result is consistent with the previously reported possible clastogenic activity suggested by the results of the mouse lymphoma L5178Y TK locus assay in which a predominance of small-colony mutants was observed (Moore et al., Environmental and Molecular Mutagenesis 1988, 11,494i3). The rapid clearance of acrylic acid in animals and the weight of evidenceof genetic toxicity testing, including negative in vivo data in both somatic and germ cells, indicate a lack of genetic toxicity of acrylic acid in vivo.
INTRODUCTION Acrylic acid is a corrosive liquid used in the production of acrylic esters, which are the polymer building blocks of a wide variety of plastics, coatings, paints and fibres. Previously published genetic toxicology evaluations of acrylic acid reported conflicting results in two in vitro systems: acrylic acid tested negative (non-mutagenic) in the Salmonella typhimurium/mammalian activation plate incorporation assay (Lijinsky and Andrews, 1980; Zeiger et al., 1987) and positive (mutagenic) in the in vitro mouse lymphoma L5178Y T K locus assay (Moore et al., 1988). Moore et al. (1988) demonstrated that the mutagenic response in mouse lymphoma cells at the thymidine kinase (TK) locus in the absence of metabolic activation was attributable primarily to small-colony mutants; they also detected a concentration-dependent increase in chromosome aberrations in the same cells. However, the predictive value of a mutagenic response in the mouse lymphoma assay is open to question due to its low specificity, that is, the proportion of negative results for proven non-carcinogens (Ashby and Purchase, 1985; Kier, 1988). More recently, Wiegand et al. (1989) evaluated three different endpoints for acrylic acid in Syrian hamster embryo (SHE) fibroblast cells in culture. Acrylic acid did not induce micronuclei or
Abbreviations: CHO = Chinese hamster ovary; HGPRT
= hypoanthine-guanine phosphoribosyl transferase; NDMA = N-nitrosodimethylamine; SHE = Syrian hamster embryo; TK = thymidine kinase; UDS = unscheduled DNA synthesis.
unscheduled DNA synthesis and no morphological transformation was observed. To investigate further the genetic toxicology of acrylic acid, the potential for gene mutations was evaluated in vitro in the CHO/HGPRT assay and in vivo by a sex-linked recessive lethal assay in Drosophila. The potential for chromosome aberrations was assessed in vitro in Chinese hamster ovary (CHO) cells and in vivo in a rat bone marrow cytogenetic assay and in a dominant lethal assay in mice. In addition, the potential for DNA damage was assessed by the unscheduled DNA synthesis (UDS) assay in primary rat hepatocytes. The results of this battery of assays are reported here. MATERIALS AND METHODS Test materials. Acrylic acid (>99.8% pure) was obtained from Hoechst Celanese Company (Somerville, N J, USA). Other chemicals were obtained from the following sources: ethyl methanesulphonate and 7,12-dimethylbenzanthracene from Eastman Kodak Chemical Co. (Rochester, NY, USA), benzo[a]pyrene and cyclophosphamide from Sigma Chemical Co. (St Louis, MO, USA) N-nitrosodimethylamine (NDMA) from Radian Corp. (Austin, TX, USA) and triethylenemelamine from Polysciences Inc. (Warrington, PA, USA). C l I O / H G P R T mutation assay. The CHO/HGPRT mutation assay was performed according to a protocol developed from published methods (Machanoff et al., 1981; O'Neill et al., 1977) using CHO-KI-BH4 cells obtained from Dr A. Hsie (Oak Ridge National Laboratory, Oak Ridge, TN, USA). Exponentially
505
506
K.L. MCCARTHYet al.
growing CHO cells were plated in Ham's F-12 medium (supplemented with 5% dialysed foetal bovine serum, 1% L-glutamine and 1% penicillin/ streptomycin) at a density of 5 x 105 cells/25-cm2 flask and incubated at 37°C in a humidified atmosphere of 5% CO 2 in air for 18-24 hr before treatment with the test compound. The test compound was diluted with phosphate buffer before it was added to the culture medium in amounts calculated to achieve the final treatment concentration. The pH of the mixture of treatment solution and medium was monitored and adjusted to pH 7.5 before addition to the cells. Osmolality was measured and dosing solutions of less than 400 mosmol/kg were used. In an initial assay, single cultures were used; in the repeat assay, triplicate cultures were exposed to the test compound with or without a metabolic activation system (Aroclor 1254-induced rat liver S-9) for 5 hr at 37°C. These triplicate cultures were independently subcultured throughout the mutation assay and the reported results are the average of the triplicate values for each treatment level. At the end of the treatment period, the medium was removed, the cells washed and then cultured for an additional 18-24hr. At this point, each treated culture was subcultured with a portion of the cells being used to assess cytotoxicity (cloning efficiency determination from 100 cells/60-mm dish) and the remainder being subcultured at 2-3-day intervals at a density no greater than 106 cells/100-mm dish to allow for the 7-9-day expression period. At the end of the expression period, selection for thioguanine resistant mutants was performed by culturing five plates at a density of 2 x 105 cells/100-mm dish in the presence of 10/~u-thioguanine; concurrently, dishes were plated for determining cloning efficiency at the time of selection. The cytotoxicity effects are expressed as cloning efficiency relative to the vehicle-treated control. The mutant frequency is calculated by dividing the total number of mutant colonies by the number of cells selected (usually 106, five plates at 2 x 105 cells/dish), corrected for the cloning efficiency at the time of selection, and is expressed as thioguanine-resistant mutants per l06 clonable cells. For experimental conditions in which no mutant colonies were observed, mutant frequencies are expressed as less than the frequency obtained with one mutant colony. Drosophila sex-linked recessive lethal assay. The Drosophila sex-linked recessive lethal assay was conducted after exposure by both feeding and injection. In the feeding experiments, 1-day-old males (previously starved for 4 hr) were treated in groups of 15 in shell vials (23 x 90 mm) plugged with rayon-fibre balls. The base of each vial was covered with two discs of glass-fibre filter material (SP141) on which was pipetted approximately 1.0 ml of feeding solution containing the acrylic acid (5% sucrose solution at pH 6). Daily for 3 days, the males were transferred to new treatment vials with freshly prepared feeding
solution. At the end of each 24-hr period, the number of dead flies was noted. Negative control flies were fed by the same procedure on 5% aqueous sucrose plus buffer. Positive control ( N D M A in 5% aqueous sucrose) feedings were for 24 hr only, since 72-hr treatments have been found to induce high post-mating mortality and male sterility, and it is known that 24-hr exposure to N D M A yields a high frequency of mutations. The males were starved for 4 hr and then fed on the third day of test exposures, so that males were of the same age and from the same collection batch as the males treated with acrylic acid. Injection was done using a glass needle attached by polyethylene tubing to a Hamilton 5-#1 syringe, the plunger of which was pushed by a micrometer. Approximately 0.3/~1 test or control solution (0.7% saline at pH 6) was injected into the body of the etherized fly at the base of a wing. The injected males were then held on sucrose-agar medium for 1 day to observe recovery before mating. Treated and control P~ males (of the Canton-S wild-type stock) were mated singly to three virgin females of the balancer stock "Basc", whose X chromosomes carry inversions and are marked with genes for apricot eye (w a) and Bar eye (B). The females were 3-10 days old when mated. Each male was transferred after 3 days to three new virgin females. The fertilized females of brood 1 were kept in the culture vial for 4 more days and then discarded. This transfer process was repeated twice more but the time that the males were in broods 2 and 3 was 2 days each. The males were then discarded. Thus, only post-meiotic germ cells were tested. F~ females (heterozygous for the treated X and the balancer X) were mated individually to brothers. An attempt was made to mate 33 females (but no more) from each Pl male per brood, for a total of 99 chromosomes tested for each male. Due to postmating mortality and sterility, the number tested was sometimes less than 99 per male. The F 2 cultures were observed when fully hatched (at 11-15 days after mating) for the presence (nonlethal) or absence (lethal) of wild-type males. Suspected lethal cases were retested by re-mating heterozygous females and observing the F 3. All stocks and matings were maintained on standard yeasted cornmeal-molasses-agar culture medium at room temperature (about 22°C). The recessive lethal data were evaluated by comparing the overall mutation frequency of each treated group with the overall mutation frequency of the concurrent negative control, by applying the Normal test (Margolin et al., 1983). In vitro cytogenetic assay in C H O cells. CHO-KI cells (American Type Culture Collection, repository number CCL61, Rockville, MD, USA) with a stable karyotype were grown in Ham's F-12 medium (supplemented with 10% foetal bovine serum) in a humidified, 5% CO2 atmosphere. For each treatment
Genetic t o x i c o l o g y o f acrylic acid
507
Table I. CHO/HGPRT gene mutation assay of acrylic acid Without metabolic activation Toxicity (% survival)*
Treatment Untreated control Vehicle control (phosphate buffer) Acrylic acid (/~l/ml) 2.8 2.4 1.9 1.5 1.0 0.6 0.3 Positive controls EMS 0.2 #l/ml B[a]P 4.0 #g/ml
Total mutant colonies
With metabolic activation
Mutants/106 clonable cellst
Toxicity (% survival)*
Total mutant colonies
Mutants/106 clonable cellst
110
13
11.5
92
16
13.3
100
1
1.6
100
11
8.2
ND ND 35 59 81 75 94
ND ND 5 6 15 0 10
ND ND 5.0 5.8 16.7 < 1.2 11.7
2 24 35 59 97 ND ND
Too toxic 4 2 0 10 ND ND
Too toxic 4.2 2.2 < 1.3 7.9 ND ND
94 ND
141 ND
126.6 ND
ND 78
ND 244
ND 244.3
ND = not determined EMS = ethyl methanesulphonate B[a]P = benzola]pyrene *Toxicity is expressed as cloning efficiency relative to the vehicle-treated control. Cloning efficiencies at the time of selection ranged from 0.73 to 1.31. tMutant frequency is expressed as thioguanine-resistant mutants/106 clonable cells, which is calculated by total mutant colonies divided by the number of cells selected, corrected for cloning efficiency at the time of selection. All results are the average of triplicate independent cultures carried throughout the assay procedure within the same trial.
group in the cytogenetic assay, approximately 106 cells (harvested in logarithmic growth) were treated in suspension with constant gentle agitation for 4 hr at 37°C either with or without a metabolic activation system (Aroclor 1254-induced rat liver S-9). The highest stock concentration of acrylic acid was adjusted to pH 7 with 1 ~-NaOH and then serially diluted in water to the appropriate dosing solution concentrations. The osmolality of 5000 nl acrylic acid/ml medium was 403 mosmol/kg water. After treatment, cells were collected by centrifugation and plated in T-25 flasks or 60-mm dishes (for a concurrent toxicity assessment, stained 7-8 days later). After 16hr, Colcemid (0.2#g/ml) was added to the T25 cultures and mitotic cells were harvested 2 hr later by mitotic shake-off. Mitotic cells were fixed and slides were prepared and stained with Giemsa for
chromosome analysis. Slides were coded and evaluated blind; 100 metaphase spreads were scored for each treatment group. In vivo cytogenetic assay in rat bone marrow cells.
Acrylic acid was administered to male and female Sprague-Dawley rats according to both acute and repeated dosing regimens. For the acute assay, rats were given a single gavage dose of 1000, 333 or 100 mg acrylic acid/kg body weight, given in water at 3 ml/kg. Body weights were recorded 12 and 24hr after dosing. Five animals per sex in each treatment group were killed for analysis at 6, 12 and 24hr after dosing. For the repeated dosing regimen, rats were provided ad lib. with drinking water containing 5000 or 2000ppm acrylic acid for 5 days; body weights were recorded daily. All animals (both acute and repeated dosing regimens) received colchicine
Table 2. Results of sex-linked recessive lethal assay using adult male Drosophila fed acrylic acid Lethals
No. lethals/no, tested (% lethals) Treatment*
Experiment
Brood 1
Brood 2
Brood 3
Acrylic acid (2%)
330E 331E 334C 338A Sum.
0/211 0/422 0/1601 0/1341 0/3575
0/172 0/433 0/1216 0/1372 0/3193
Controls
330F 331D 334D 338B Sum.
0/409 0/634 0/1539 0/1451 0/4033
NDMA (I 00 ppm)
330G 331F 334E
1/294 5/179 7/182
0/315 0/727 0/1367 2/1423 2/3832 (0.052) 6/242 7/148 12/146
0/158 0/552 1/207 0/1385 1/2302 (0.043) 0/140 2/713 0/459 1/1324 3/2636 (0.114) 3/237 0/145 0/3
338E
0/177
6/191
13/832 (1.56)
31/727 (4.26)
Swm.
Total tests
St
M~,
Total
Percentage lethals
541 1407 3024 4098 9070
0 0 I 0 1
0 0 0 0 0
0 0 1 0 1
0 0 0.033 0 0.011
864 2074 3365 4198 10,501
0 0 0 3 3
0 1(2) 0 0 1(2)
0 2 0 3 5
0 0.096 0 0.071 0.048
773 472 331
5 2 1
10 12 19
1.29 2.54 5.74
17/123
491
1
23
4.68
20/508 (3.93)
2067
9
1(2) 1(3) 1(2) 1(8) 1(2) 1(3) 2(4) 1(5) 1(3) 2(4) 1(5) 1(6) 3(2) 3(3) 4(4) 2(5) 1(6) 1(8)
64
3.09
*Acrylic acid was fed to the Drosophila for 3 days; NDMA (N-nitrosodimethylamine) was fed for only I day. tSingle lethal per family. ::[:Multiple lethals in a family: no. of families (no. of lethals).
508
K. L. MCCARTHY et al. Table 3. Results of sex-linked recessive lethal assay using adult male Drosophila injected with acrylic acid Lethals
No. lethals/no, tested (% lethals) Treatment
Acrylic acid (2%) Controls NDMA (500 ppm)
Experiment
333A 335A Sum... 333B 335B Sum... 333C 335C Sum...
Brood I
Brood 2
Brood 3
Total tests
0/953 0/1188 1/2141 (0.047) 0/1039 0/1265 0/2304 5/318
1/895 0/969 1/1864 (0.054) 0/1021 0/1149 0/2170 17/267
0/620 0/827 0/1447
2468 2984 5452
1 1 2
0 0 0
0/876 0/1278 0/2154 31/300
2936 3692 6628 885
0 0 0 0
2/190 7/508 (1.37)
1/129 18/396 (4.54)
5/189 36/489 (7.36)
508 1393
3 3
0 0 0 1(2) 1(3) 3(5) 3(6) 1(7) 1(8) 1(2) 1(3) 2(2) 2(3) 3(5) 3(6) 1(7) 1(8)
St
M:~
Total
Percentage lethals
1 1 2
0.041 0.034
0 0 0 53
0 0 0 5.98
8 61
1.57 4.37
o.o37§
tSingle lethal per family. :[:Multiple lethals in a family: no. of families (no. of lethals). §Not significantly different from concurrent controls (P = 0.059; Normal test).
ip at 1 mg/kg approximately 2--4 hr before they were killed. Immediately after killing, bone marrow cells were collected from both femurs, fixed and processed for slide preparation. Slides were prepared and stained by standard procedures, and then coded for blind scoring. The mitotic index was determined for each animal by counting 500 cells. 50 metaphase spreads for each animal were evaluated for chromosome aberrations. Unscheduled DNA synthesis in primary hepatocytes. Primary rat hepatocytes were isolated by in situ collagenase perfusion (Williams, 1977; Williams et al., 1977). After perfusion, the liver was removed and the cells were dissociated, counted, and seeded at 5 x l05 cells/dish in 35-mm dishes that each contained a coverslip and Williams' medium E supplemented with 10% foetal bovine serum, 2 mM-Lglutamine and 50/~ g gentamicin/ml. After incubation at 37°C in a humidified 5% CO2 atmosphere for 1.5-2hr, the coverslip-cuitures were washed with serum-free medium to remove floating cells and then used for the UDS assay. The test substance was diluted in phosphate buffered saline, and the pH was adjusted before the cells were added. The pH at the time of treatment was 7.2-7.4. The osmolality of the dosing solutions was ~
0278-6915/92$5.00+ 0.00 Copyright © 1992PergamonPress Ltd
Printed in Great Britain.All rights reserved
GENETIC TOXICOLOGY OF ACRYLIC ACID K. L. MCCARTHY*, W. C THOMAS'[',M. J. AARDEMA~,,J. L. SEYMOUR~,D. L. PUTMAN§,
L. L. YANG~, R. D. CURREN§ and R. VALENClA)I *ToxicologyDepartment, Rohm and Haas Company, Spring House, PA 19477;tToxicology Department, Hoechst Celanese, PO Box 2500, Somerville, NJ 08876; :~Procter & Gamble Company, Miami Valley Laboratories, Cincinnati, OH 45239; §Genetic Toxicology Division, Microbiological Associates, Inc., Rockville, MD 20850 and IIZoologyDepartment, University of Wisconsin, Madison, WI 53706, USA (Accepted 14 January 1992)
Abstract--Acrylic acid was tested for gene mutations in the in vitro CHO/HGPRT assay, for chromosome aberrations in CHO cells in culture, and for potential to induce unscheduled DNA synthesis in rat hepatocytes in culture. In vivo assays performed included the Drosophila sex-linked recessive lethal assay by both the feeding and injection routes, the in vivo cytogenetic assay in rat bone marrow cells after both a 1-day and 5-day oral dosing regimen, and a dominant lethal assay in mice by both an acute and 5-day dosing regimen. All results were negative (non-mutagenic) except for the in vitro chromosome aberration assay. This latter result is consistent with the previously reported possible clastogenic activity suggested by the results of the mouse lymphoma L5178Y TK locus assay in which a predominance of small-colony mutants was observed (Moore et al., Environmental and Molecular Mutagenesis 1988, 11,494i3). The rapid clearance of acrylic acid in animals and the weight of evidenceof genetic toxicity testing, including negative in vivo data in both somatic and germ cells, indicate a lack of genetic toxicity of acrylic acid in vivo.
INTRODUCTION Acrylic acid is a corrosive liquid used in the production of acrylic esters, which are the polymer building blocks of a wide variety of plastics, coatings, paints and fibres. Previously published genetic toxicology evaluations of acrylic acid reported conflicting results in two in vitro systems: acrylic acid tested negative (non-mutagenic) in the Salmonella typhimurium/mammalian activation plate incorporation assay (Lijinsky and Andrews, 1980; Zeiger et al., 1987) and positive (mutagenic) in the in vitro mouse lymphoma L5178Y T K locus assay (Moore et al., 1988). Moore et al. (1988) demonstrated that the mutagenic response in mouse lymphoma cells at the thymidine kinase (TK) locus in the absence of metabolic activation was attributable primarily to small-colony mutants; they also detected a concentration-dependent increase in chromosome aberrations in the same cells. However, the predictive value of a mutagenic response in the mouse lymphoma assay is open to question due to its low specificity, that is, the proportion of negative results for proven non-carcinogens (Ashby and Purchase, 1985; Kier, 1988). More recently, Wiegand et al. (1989) evaluated three different endpoints for acrylic acid in Syrian hamster embryo (SHE) fibroblast cells in culture. Acrylic acid did not induce micronuclei or
Abbreviations: CHO = Chinese hamster ovary; HGPRT
= hypoanthine-guanine phosphoribosyl transferase; NDMA = N-nitrosodimethylamine; SHE = Syrian hamster embryo; TK = thymidine kinase; UDS = unscheduled DNA synthesis.
unscheduled DNA synthesis and no morphological transformation was observed. To investigate further the genetic toxicology of acrylic acid, the potential for gene mutations was evaluated in vitro in the CHO/HGPRT assay and in vivo by a sex-linked recessive lethal assay in Drosophila. The potential for chromosome aberrations was assessed in vitro in Chinese hamster ovary (CHO) cells and in vivo in a rat bone marrow cytogenetic assay and in a dominant lethal assay in mice. In addition, the potential for DNA damage was assessed by the unscheduled DNA synthesis (UDS) assay in primary rat hepatocytes. The results of this battery of assays are reported here. MATERIALS AND METHODS Test materials. Acrylic acid (>99.8% pure) was obtained from Hoechst Celanese Company (Somerville, N J, USA). Other chemicals were obtained from the following sources: ethyl methanesulphonate and 7,12-dimethylbenzanthracene from Eastman Kodak Chemical Co. (Rochester, NY, USA), benzo[a]pyrene and cyclophosphamide from Sigma Chemical Co. (St Louis, MO, USA) N-nitrosodimethylamine (NDMA) from Radian Corp. (Austin, TX, USA) and triethylenemelamine from Polysciences Inc. (Warrington, PA, USA). C l I O / H G P R T mutation assay. The CHO/HGPRT mutation assay was performed according to a protocol developed from published methods (Machanoff et al., 1981; O'Neill et al., 1977) using CHO-KI-BH4 cells obtained from Dr A. Hsie (Oak Ridge National Laboratory, Oak Ridge, TN, USA). Exponentially
505
506
K.L. MCCARTHYet al.
growing CHO cells were plated in Ham's F-12 medium (supplemented with 5% dialysed foetal bovine serum, 1% L-glutamine and 1% penicillin/ streptomycin) at a density of 5 x 105 cells/25-cm2 flask and incubated at 37°C in a humidified atmosphere of 5% CO 2 in air for 18-24 hr before treatment with the test compound. The test compound was diluted with phosphate buffer before it was added to the culture medium in amounts calculated to achieve the final treatment concentration. The pH of the mixture of treatment solution and medium was monitored and adjusted to pH 7.5 before addition to the cells. Osmolality was measured and dosing solutions of less than 400 mosmol/kg were used. In an initial assay, single cultures were used; in the repeat assay, triplicate cultures were exposed to the test compound with or without a metabolic activation system (Aroclor 1254-induced rat liver S-9) for 5 hr at 37°C. These triplicate cultures were independently subcultured throughout the mutation assay and the reported results are the average of the triplicate values for each treatment level. At the end of the treatment period, the medium was removed, the cells washed and then cultured for an additional 18-24hr. At this point, each treated culture was subcultured with a portion of the cells being used to assess cytotoxicity (cloning efficiency determination from 100 cells/60-mm dish) and the remainder being subcultured at 2-3-day intervals at a density no greater than 106 cells/100-mm dish to allow for the 7-9-day expression period. At the end of the expression period, selection for thioguanine resistant mutants was performed by culturing five plates at a density of 2 x 105 cells/100-mm dish in the presence of 10/~u-thioguanine; concurrently, dishes were plated for determining cloning efficiency at the time of selection. The cytotoxicity effects are expressed as cloning efficiency relative to the vehicle-treated control. The mutant frequency is calculated by dividing the total number of mutant colonies by the number of cells selected (usually 106, five plates at 2 x 105 cells/dish), corrected for the cloning efficiency at the time of selection, and is expressed as thioguanine-resistant mutants per l06 clonable cells. For experimental conditions in which no mutant colonies were observed, mutant frequencies are expressed as less than the frequency obtained with one mutant colony. Drosophila sex-linked recessive lethal assay. The Drosophila sex-linked recessive lethal assay was conducted after exposure by both feeding and injection. In the feeding experiments, 1-day-old males (previously starved for 4 hr) were treated in groups of 15 in shell vials (23 x 90 mm) plugged with rayon-fibre balls. The base of each vial was covered with two discs of glass-fibre filter material (SP141) on which was pipetted approximately 1.0 ml of feeding solution containing the acrylic acid (5% sucrose solution at pH 6). Daily for 3 days, the males were transferred to new treatment vials with freshly prepared feeding
solution. At the end of each 24-hr period, the number of dead flies was noted. Negative control flies were fed by the same procedure on 5% aqueous sucrose plus buffer. Positive control ( N D M A in 5% aqueous sucrose) feedings were for 24 hr only, since 72-hr treatments have been found to induce high post-mating mortality and male sterility, and it is known that 24-hr exposure to N D M A yields a high frequency of mutations. The males were starved for 4 hr and then fed on the third day of test exposures, so that males were of the same age and from the same collection batch as the males treated with acrylic acid. Injection was done using a glass needle attached by polyethylene tubing to a Hamilton 5-#1 syringe, the plunger of which was pushed by a micrometer. Approximately 0.3/~1 test or control solution (0.7% saline at pH 6) was injected into the body of the etherized fly at the base of a wing. The injected males were then held on sucrose-agar medium for 1 day to observe recovery before mating. Treated and control P~ males (of the Canton-S wild-type stock) were mated singly to three virgin females of the balancer stock "Basc", whose X chromosomes carry inversions and are marked with genes for apricot eye (w a) and Bar eye (B). The females were 3-10 days old when mated. Each male was transferred after 3 days to three new virgin females. The fertilized females of brood 1 were kept in the culture vial for 4 more days and then discarded. This transfer process was repeated twice more but the time that the males were in broods 2 and 3 was 2 days each. The males were then discarded. Thus, only post-meiotic germ cells were tested. F~ females (heterozygous for the treated X and the balancer X) were mated individually to brothers. An attempt was made to mate 33 females (but no more) from each Pl male per brood, for a total of 99 chromosomes tested for each male. Due to postmating mortality and sterility, the number tested was sometimes less than 99 per male. The F 2 cultures were observed when fully hatched (at 11-15 days after mating) for the presence (nonlethal) or absence (lethal) of wild-type males. Suspected lethal cases were retested by re-mating heterozygous females and observing the F 3. All stocks and matings were maintained on standard yeasted cornmeal-molasses-agar culture medium at room temperature (about 22°C). The recessive lethal data were evaluated by comparing the overall mutation frequency of each treated group with the overall mutation frequency of the concurrent negative control, by applying the Normal test (Margolin et al., 1983). In vitro cytogenetic assay in C H O cells. CHO-KI cells (American Type Culture Collection, repository number CCL61, Rockville, MD, USA) with a stable karyotype were grown in Ham's F-12 medium (supplemented with 10% foetal bovine serum) in a humidified, 5% CO2 atmosphere. For each treatment
Genetic t o x i c o l o g y o f acrylic acid
507
Table I. CHO/HGPRT gene mutation assay of acrylic acid Without metabolic activation Toxicity (% survival)*
Treatment Untreated control Vehicle control (phosphate buffer) Acrylic acid (/~l/ml) 2.8 2.4 1.9 1.5 1.0 0.6 0.3 Positive controls EMS 0.2 #l/ml B[a]P 4.0 #g/ml
Total mutant colonies
With metabolic activation
Mutants/106 clonable cellst
Toxicity (% survival)*
Total mutant colonies
Mutants/106 clonable cellst
110
13
11.5
92
16
13.3
100
1
1.6
100
11
8.2
ND ND 35 59 81 75 94
ND ND 5 6 15 0 10
ND ND 5.0 5.8 16.7 < 1.2 11.7
2 24 35 59 97 ND ND
Too toxic 4 2 0 10 ND ND
Too toxic 4.2 2.2 < 1.3 7.9 ND ND
94 ND
141 ND
126.6 ND
ND 78
ND 244
ND 244.3
ND = not determined EMS = ethyl methanesulphonate B[a]P = benzola]pyrene *Toxicity is expressed as cloning efficiency relative to the vehicle-treated control. Cloning efficiencies at the time of selection ranged from 0.73 to 1.31. tMutant frequency is expressed as thioguanine-resistant mutants/106 clonable cells, which is calculated by total mutant colonies divided by the number of cells selected, corrected for cloning efficiency at the time of selection. All results are the average of triplicate independent cultures carried throughout the assay procedure within the same trial.
group in the cytogenetic assay, approximately 106 cells (harvested in logarithmic growth) were treated in suspension with constant gentle agitation for 4 hr at 37°C either with or without a metabolic activation system (Aroclor 1254-induced rat liver S-9). The highest stock concentration of acrylic acid was adjusted to pH 7 with 1 ~-NaOH and then serially diluted in water to the appropriate dosing solution concentrations. The osmolality of 5000 nl acrylic acid/ml medium was 403 mosmol/kg water. After treatment, cells were collected by centrifugation and plated in T-25 flasks or 60-mm dishes (for a concurrent toxicity assessment, stained 7-8 days later). After 16hr, Colcemid (0.2#g/ml) was added to the T25 cultures and mitotic cells were harvested 2 hr later by mitotic shake-off. Mitotic cells were fixed and slides were prepared and stained with Giemsa for
chromosome analysis. Slides were coded and evaluated blind; 100 metaphase spreads were scored for each treatment group. In vivo cytogenetic assay in rat bone marrow cells.
Acrylic acid was administered to male and female Sprague-Dawley rats according to both acute and repeated dosing regimens. For the acute assay, rats were given a single gavage dose of 1000, 333 or 100 mg acrylic acid/kg body weight, given in water at 3 ml/kg. Body weights were recorded 12 and 24hr after dosing. Five animals per sex in each treatment group were killed for analysis at 6, 12 and 24hr after dosing. For the repeated dosing regimen, rats were provided ad lib. with drinking water containing 5000 or 2000ppm acrylic acid for 5 days; body weights were recorded daily. All animals (both acute and repeated dosing regimens) received colchicine
Table 2. Results of sex-linked recessive lethal assay using adult male Drosophila fed acrylic acid Lethals
No. lethals/no, tested (% lethals) Treatment*
Experiment
Brood 1
Brood 2
Brood 3
Acrylic acid (2%)
330E 331E 334C 338A Sum.
0/211 0/422 0/1601 0/1341 0/3575
0/172 0/433 0/1216 0/1372 0/3193
Controls
330F 331D 334D 338B Sum.
0/409 0/634 0/1539 0/1451 0/4033
NDMA (I 00 ppm)
330G 331F 334E
1/294 5/179 7/182
0/315 0/727 0/1367 2/1423 2/3832 (0.052) 6/242 7/148 12/146
0/158 0/552 1/207 0/1385 1/2302 (0.043) 0/140 2/713 0/459 1/1324 3/2636 (0.114) 3/237 0/145 0/3
338E
0/177
6/191
13/832 (1.56)
31/727 (4.26)
Swm.
Total tests
St
M~,
Total
Percentage lethals
541 1407 3024 4098 9070
0 0 I 0 1
0 0 0 0 0
0 0 1 0 1
0 0 0.033 0 0.011
864 2074 3365 4198 10,501
0 0 0 3 3
0 1(2) 0 0 1(2)
0 2 0 3 5
0 0.096 0 0.071 0.048
773 472 331
5 2 1
10 12 19
1.29 2.54 5.74
17/123
491
1
23
4.68
20/508 (3.93)
2067
9
1(2) 1(3) 1(2) 1(8) 1(2) 1(3) 2(4) 1(5) 1(3) 2(4) 1(5) 1(6) 3(2) 3(3) 4(4) 2(5) 1(6) 1(8)
64
3.09
*Acrylic acid was fed to the Drosophila for 3 days; NDMA (N-nitrosodimethylamine) was fed for only I day. tSingle lethal per family. ::[:Multiple lethals in a family: no. of families (no. of lethals).
508
K. L. MCCARTHY et al. Table 3. Results of sex-linked recessive lethal assay using adult male Drosophila injected with acrylic acid Lethals
No. lethals/no, tested (% lethals) Treatment
Acrylic acid (2%) Controls NDMA (500 ppm)
Experiment
333A 335A Sum... 333B 335B Sum... 333C 335C Sum...
Brood I
Brood 2
Brood 3
Total tests
0/953 0/1188 1/2141 (0.047) 0/1039 0/1265 0/2304 5/318
1/895 0/969 1/1864 (0.054) 0/1021 0/1149 0/2170 17/267
0/620 0/827 0/1447
2468 2984 5452
1 1 2
0 0 0
0/876 0/1278 0/2154 31/300
2936 3692 6628 885
0 0 0 0
2/190 7/508 (1.37)
1/129 18/396 (4.54)
5/189 36/489 (7.36)
508 1393
3 3
0 0 0 1(2) 1(3) 3(5) 3(6) 1(7) 1(8) 1(2) 1(3) 2(2) 2(3) 3(5) 3(6) 1(7) 1(8)
St
M:~
Total
Percentage lethals
1 1 2
0.041 0.034
0 0 0 53
0 0 0 5.98
8 61
1.57 4.37
o.o37§
tSingle lethal per family. :[:Multiple lethals in a family: no. of families (no. of lethals). §Not significantly different from concurrent controls (P = 0.059; Normal test).
ip at 1 mg/kg approximately 2--4 hr before they were killed. Immediately after killing, bone marrow cells were collected from both femurs, fixed and processed for slide preparation. Slides were prepared and stained by standard procedures, and then coded for blind scoring. The mitotic index was determined for each animal by counting 500 cells. 50 metaphase spreads for each animal were evaluated for chromosome aberrations. Unscheduled DNA synthesis in primary hepatocytes. Primary rat hepatocytes were isolated by in situ collagenase perfusion (Williams, 1977; Williams et al., 1977). After perfusion, the liver was removed and the cells were dissociated, counted, and seeded at 5 x l05 cells/dish in 35-mm dishes that each contained a coverslip and Williams' medium E supplemented with 10% foetal bovine serum, 2 mM-Lglutamine and 50/~ g gentamicin/ml. After incubation at 37°C in a humidified 5% CO2 atmosphere for 1.5-2hr, the coverslip-cuitures were washed with serum-free medium to remove floating cells and then used for the UDS assay. The test substance was diluted in phosphate buffered saline, and the pH was adjusted before the cells were added. The pH at the time of treatment was 7.2-7.4. The osmolality of the dosing solutions was ~
