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Mutagenicity studies on ketone solvents: methyl ethyl ketone, methyl isobutyl ketone, and isophorone
Mutation Research, 206 (1988) 149-161
149
Elsevier MTR 01310
Mutagenicity studies on ketone solvents: methyl ethyl ketone, methyl isobutyl ketone, and isophorone J.L. O ' D o n o g h u e 1, S.R. H a w o r t h 2 R . D . C u r r e n 2, P.E. K i r b y 3 T. L a w l o r 2, E.J. M o r a n 4, R . D . Phillips 5, D . L . P u t n a m 2, A.M~ R o g e r s - B a c k 2, R.S. Slesinski 6 a n d A. T h i l a g a r 3 1 Health and Environment Laboratories, Eastman Kodak Company, Rochester, N Y 14652-3615, 2 Microbiological Associates, Inc., Department of Genetic Toxicology, 5221 River Road, Bethesda, M D 20816, 3 Sitek Research Laboratories, 12111 Parklawn Drive, Rockville, M D 20852, 4 Chemical Manufacturers Association, 2501 M Street, N W , Washington, DC 20037, 5 Exxon Biomedical Sciences Inc., Mettlers Rd., CN2350, East Millstone, NJ 08875-2350, and 6 Bushy Run Research Center, RD 4, Mellow Road, Export, PA 15632 (U.S.A.)
(Received 9 July 1986) (Revision received 18 February 1988) (Accepted 26 February 1988)
Keywords: Ketone solvents; Methyl ethyl ketone; Methyl isobutyl ketone; Isophorone
Summary 3 ketone solvents (methyl ethyl ketone (MEK), methyl isobutyl ketone (MiBK), and isophorone) were tested for potential genotoxicity. The assays of MEK and MiBK included the Salmonella/microsome (Ames) assay, L5178Y/TK ÷/- mouse lymphoma (ML) assay, BALB/3T3 cell transformation (CT) assay, unscheduled DNA synthesis (UDS) assay, and micronucleus (MN) assay. Only the ML, UDS, and MN assays were conducted on samples of isophorone. No genotoxicity was found for MEK or isophorone. The presence of a marginal response only at the highest, cytotoxic concentration tested in the ML assay, the lack of reproducibility in the CT assay, and clearly negative results in the Ames assay, UDS and MN assays, suggest that MiBK is unlikely to be genotoxic in mammalian systems.
Ketone solvents have found wide use in the coatings industry because of their excellent solvation properties. 3 ketone solvents of interest primarily because of production volumes are methyl ethyl ketone (MEK; 343 x 103 tons), methyl isobutyl ketone (MiBK; 95 x 103 tons), and isophorone (10-15 x 103 tons); production volumes are 1979 estimates for the U.S.A. (Chemical Correspondence: Dr. John L. O'Donoghue, Health and Environment Laboratories, B-320, Kodak Park, Eastman Kodak Company, Rochester, NY 14650 (U.S.A.). Tel.: 716588-4741.
Manufacturers Association, 1981). Uses of these materials include coatings, adhesives, and petroleum refining for MEK; coatings, solvent extraction, and metallurgy for MiBK; and coatings, inks, and agricultural formulations for isophorone. Few mutagenicity data are available on these substances. Shirasu et al. (1976) used MEK as a solvent for a group of pesticides tested for mutagenicity utiliz: ing E. coli, Salmonella typhimurium, or H17 Rec ÷ and M45 Rec- strains of Bacillus subtilis without metabolic activation. MEK did not induce mutagenic effects in these test systems. Isophorone did
0165-1218/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
150 not induce mutations when tested using 4 different strains of Salmonella typhimurium with or without exogenous metabolic activation provided by rat or hamster liver $9 preparations (National Toxicology Program, 1982). N o other genetic toxicity data were available at the time these studies were begun; therefore, the present studies were conducted to further elucidate the genotoxic potential of these 3 ketones. Materials and methods
5 assays for genetic activity were conducted for M i B K a n d M E K . T h e assays were the Salmonella/microsome (Ames) assay, L 5 1 7 8 Y / T K ÷/- mouse lymphoma (ML) assay, B A L B / 3 T 3 cell transformation (CT) assay, unscheduled D N A synthesis (UDS) assay, and micronucleus (MN) assay. Since data for the activity of isophorone in the Ames assay already existed (National Toxicology Program, 1982), the assay was not repeated during this study. Due to the volatility of the test substances, all assays were conducted in sealed glass containers to prevent escape of the test material. The M E K (CAS No. 78-93-3) used for these studies was supplied by the Celanese Corporation and was 99.9% pure. MiBK (CAS No. 108-10-1) was supplied by the Exxon Chemical C o m p a n y and was 99.6% pure. The isophorone (CAS No. 78-59-1) sample was supplied by the Exxon Corporation and was > 97% pure. The test materials were blanketed with nitrogen and stored at room temperature.
Salmonella / microsome (Ames) assay Salmonella strains TA98, TA100, TA1535, TA1537, and TA1538 were received from Dr. B. Ames, University of California, Berkeley, CA. The genotypic characteristics of each culture were tested prior to use as recommended by Ames et al. (1975). The experimental procedures were based on the work of Ames et al. (1975) and Yahagi et al. (1977). M E K and MiBK were diluted in dimethyl sulfoxide (DMSO), and based on the results of preliminary cytotoxicity assays, 5 doses of each chemical were tested in at least triplicate plates.
When metabolic activation was required, 500/~1 of S9 mix containing 10% homogenate, 50/~1 of an appropriate tester strain, and 50 #1 of solvent or an appropriate concentration of test article were added to 13 m m x 100 m m glass, Teflon fluoroc a r b o n - l i n e d , s c r e w - c a p p e d culture tubes, preheated to 37 ° C. The $9 fraction was prepared from the livers of male Sprague-Dawley rats given 50 m g / k g of Aroclor 1254. When metabolic activation was not required, 500 /zl of S9 sham mix (0.1 M phosphate buffer) was substituted for the $9 mix. After vortex mixing, the mixture was allowed to incubate in the sealed test tube for 2 h at 37 ° C. 2 ml of selective top agar were then added to each tube, and the tube was mixed and its contents were overlaid onto the surface of 25 ml of Vogel-Bonner bottom agar contained in glass petri dishes. After each dose level was plated and the top agar had solidified, all of the plates of that dose level were sealed into one or more glass chambers and incubated at 37 ° C for 48 h. Plates containing a metabolic activation system were placed in glass chambers separate from plates without the metabolic activation system. Plates which were not counted immediately following the 48-h incubation period were stored at 4 ° C until such time that colony counting could be conducted. The glass containers used for this study were 1 gallon, flint glass jars fitted with a screw cap. Teflon sheeting was placed over the mouth of the jar prior to capping. Handling the plates in this manner ensured that volatilized solvent did not escape during the exposure and expression periods. Combinations of positive control substances and the tester strains were plated along with the test plates (Tables 1 and 2). Positive controls were preincubated for 20 min at 3 7 ° C and were not placed in sealed chambers for the 48-h incubation period. For a test article to have been considered positive, it must have caused at least a doubling in the mean revertants per plate of at least 1 tester strain. This increase in the mean number of revertants per plate must have been accompanied by a dose response to increasing concentrations of the test article. In those cases in which the observed dose-responsive increase in TA1537 or
151 TA1538 mean revertants per plate was less than 3-fold, the response had to have been reproducible.
L5178 Y / TK +/- mouse lymphoma assay This assay was based on the methods of Clive and Spector (1975) and Clive et al. (1979) to measure the forward mutation frequency at the thymidine kinase locus in mouse l y m p h o m a cells. Based on preliminary toxicity tests, the test chemicals (MEK, MiBK, and isophorone) were diluted with DMSO to produce 15 serial eighth log dilutions. This resulted in 16 dose levels decreasing approximately 100-fold from highest to lowest. The highest dose was 100% toxic while the lowest dose was non-toxic. Each test chemical was tested with and without $9 fraction. The $9 fraction was prepared from the livers of male Sprague-Dawley rats injected with 500 m g / k g of a 2 : 1 mixture of Aroclor 1242 and Aroclor 1254. Final cell suspensions were 0.6 x 10 6 cells/ml. Control tubes received D M S O only and positive controls received ethyl methanesulfonate (1.0 or 0.5/~l/ml) or 7,12dimethylbenz[a]anthracene (7.5 or 5.0/~g/ml). All tubes were glass and fitted with Teflon-lined caps to prevent escape of the test articles. 5% CO 2 in air was added to the tubes which were then placed on a roller drum for 4 h at 37 ° C. At the end of the exposure period, the cells were washed and incubated for 2 days to allow for expression of the mutant phenotype. During the expression period, cells were counted daily and adjusted to a cell population density of 0.3 × 106/ml. Following the expression period, cells were cloned on medium containing 0.34% Noble agar. Trifluorothymidine, at a concentration of 3 /~g/ml, was used as the restrictive agent. 3 c o u n t s / p l a t e were made on an automatic colony counter and the median count was recorded. A test article was considered positive if there was a positive dose response and 1 or more of the 3 highest doses exhibited a mutant frequency which was 2-fold greater than the background level. If there was no dose response but any one or more doses exhibited a 2-fold increase in mutant frequency over background, the result was considered equivocal. A negative response was one in which there was no dose response and none of the test cultures exhibited mutant fre-
quencies which were 2-fold greater than background (Clive et al., 1979).
Unscheduled DNA synthesis in rat primary hepatocytes The methods used were adapted from those of Williams (1977, 1979). Primary rat hepatocytes were derived from Sprague-Dawley rats obtained from Charles River Laboratories, Inc., Kingston, NY. Rats were anesthetized with methoxyflurane and their livers perfused with 0.5 m M ethylenebis (oxyethylenenitrilo)-tetraacetic acid (EGTA) followed by a collagenase solution. Dissociated cells were counted and seeded into glass Leighton tubes containing coverslips (2.5 × 105 viable cells/tube). The culture medium was Williams Medium E (WME) buffered with 0.01 M Hepes, supplemented with 10% fetal calf serum, 2 m M L-glutamine, and 50/~g of gentamicin/ml. Cultures were incubated (37°_+ 1°C, 5% CO2 in air) for 2 h, washed, refed with serum-free W M E and used in the assay. 5 or 6 concentrations of each test article were assayed based on preliminary cytotoxicity studies. Survival was assayed by comparing the number of trypan blue-excluding cells after 24-h exposure to the test article with the original number of cells seeded. Doses were chosen to span the range from approximately 50% relative survival (ratio of survival of treated cells to the survival of solvent control cells) to 100% survival. Triplicate cultures were seeded for each chemical. The positive control was 2-acetylaminofluorene (2 and 20 /~g/ml 2-AAF). Solvent controls included D M S O (diluent for M E K and MiBK) and ethanol (diluent for isophorone and 2-AAF). [3H]Thymidine (NEN, 20 C i / m m o l e ) was present at 10 /~Ci/ml in all treated cultures. Tubes treated in parallel for cytotoxicity were not treated with [3H]thymidine. After 18 h of exposure in sealed glass Leighton tubes, the cells were washed, swelled in 1% sodium citrate, and fixed in an ethanol-acetic acid fixative. Dried coverslips were coated with Kodak N T B emulsion, stored at 4 ° C for 8 days, developed in K o d a k D19 developer, fixed, and stained with h e m a t o x y l i n - s o d i u m acetate-eosin stain. Grain counts were made using a Biotran 2 colony counter. Net nuclear grain counts (grain count over the nucleus minus the average grain
152
count from 3 adjacent nuclear sized areas) were calculated for at least 100 randomly selected cells at each dose level. The result for a particular dose level was considered significant if the mean net nuclear count was increased 5 or more counts over the solvent control. A test article was judged positive if it either induced a dose-related response and at least 1 dose produced a significant increase in the average net nuclear grains when compared tO that of the control, or if the test article should show a significant increase in the mean net nuclear grain count in at least 2 successive doses. If a test article showed a significant increase in the net nuclear grain count at 1 dose level without any dose response, the test article was considered to have marginal positive activity. The test article was considered negative if no significant increase in the net nuclear grain counts at any dose level was observed.
Micronucleus cytogenetic assay in mice Groups of 5 male and 5 female mice (CD-1, 6 - 8 weeks old, Charles River Laboratories, Inc., Kingston, NY) were given a single dose of the test chemical in corn oil and sacrificed 12, 24 or 48 h later. The test dose was the LD20 of the test article, determined in a preliminary toxicity study. The positive control group was given 0.25 m g / k g of triethylene melamine (TEM) and examined 24 h post dosing. All doses were given intraperitoneally at a rate of 10 ml of test article-corn oil m i x t u r e / k g body weight. At sample collection, the femur was exposed and bone marrow aspirated into a syringe containing fetal calf serum. The cells were washed, centrifuged, resuspended and smears prepared. M a y - G r u e n w a l d - G i e m s a stain was used to stain the bone marrow preparations. 1000 polychromatic erythrocytes were scored on coded slides for the presence of micronuclei. Micronucleated normocytes were also counted. A 1-way analysis of variance and Duncan's multiple range test ( P ~< 0.05) were used to assess the statistical significance of any observed effects. B A L B / 3T3 mouse embryo cell transformation assay B A L B / 3 T 3 clone A31-1 cells obtained from Dr. T. Kakunaga, National Cancer Institute, Be-
thesda, MD, were harvested during exponential growth and suspended in 50-ml glass tubes fitted with Teflon-lined caps (Kakunaga, 1973). Cell density was 2 × 106 cells in 2 ml phosphatebuffered saline with or without Aroclor 1254-induced rat liver $9 fraction. 2 ml of the test article or control substance was added to each tube. N-Methyl-N'-nitro-N-nitrosoguanidine ( M N N G ) was the positive control for the non-activated cultures, and benzo[a]pyrene (BaP) was the positive control for the S9-activated tubes. Doses for the study were chosen based on a preliminary cytotoxicity study. The doses of M E K were 9, 13 and 18 ~tl/ml for non-activated cultures and 6, 8 and 10 # l / m l for the activated cultures. The doses of MiBK were 2.4, 3.6 and 4.8/~l/ml without activation and 1, 2 and 4 # l / m l with activation. In a repeat assay on MiBK, doses were 4, 5, 6 and 7 / d / m l without activation and 2, 3, 4 and 5 / ~ l / m l with activation. A concurrent toxicity study was also conducted for each material. Following a 2-h incubation period in serum-free medium containing the test article, the cells were transferred to complete medium, 15 dishes/treatment. After 4 - 6 weeks incubation, the transformation plates were fixed in 95% methanol, stained with Giemsa stain and scored for type II and type III loci as recommended by Reznikoff et al. (1973). The cytotoxic effects of each treatment condition were expressed relative to the solvent-treated control (relative cloning efficiency). The transformation frequency for each treatment condition was expressed as the number of transformed loci per surviving cell. The confidence level associated with the number of surviving cells employed in the selection process required that the assumption be made that were there more cells utilized for the selection of transformants, the possibility existed that at least I type I I I focus could be obtained. Therefore, for test conditions in which no type III foci were observed, transformation frequencies are expressed as less than the frequency obtained with 1 type III focus. The numbers of type II and type III foci per total dishes scored are also presented. The transforming potential of each treatment condition was compared to that of the solvent control using an application of the Poisson distribution as follows: The proportion ( P ) of all cells not subject to
153 i n d u c e d t r a n s f o r m a t i o n is e q u a l to the n u m b e r of surviving ceils in the solvent c o n t r o l g r o u p d i v i d e d by the total of the surviving cells in the solvent c o n t r o l an d t r e a t m e n t group. Significance is d e t e r m i n e d by: ?/
P(X>~nl/n) =
E(
)P"'(1-P)"-"'
n 1
w he re p is the probability, n is the n u m b e r of i n d u c e d type I I I foci, n 1 is the n u m b e r of s p o n t a ne ous type I I I foci, a n d P is d e f i n e d above. If p were very small ( p ~< 0.05), the hypothesis that the p r o b a b i l i t y of a t r a n s f o r m a t i o n e v e n t was the same for b o t h the treated a n d c o n t r o l groups was rejected an d the i n d u c e d t r a n s f o r m a t i o n f r e q u e n c y was considered significant at the p ~< 0.05 level.
Results and discussion T h e results of the b a t t e r y o f g e n o t o x i c i t y tests for ke t o n i c solvents M E K , M i B K a n d i s o p h o r o n e
are sh o w n in T a b l e s 1 - 9 . T h e assays of M E K an d i s o p h o r o n e were n e g a t i v e an d did n o t d e m o n s t r a t e significant m u t a g e n i c activity. T h e results of the assays for M i B K were n o t as conclusive as those for M E K a n d i s o p h o r o n e . Clearly n e g a t i v e results were o b t a i n e d in the S a l m o n e l l a / m i c r o s o m e assay, the u n s c h e d u l e d D N A synthesis assay, and the m i c r o n u c l e u s assay c o n d u c t e d using M i B K . T h e m o u s e l y m p h o m a assay of M i B K was negative w h e n tested w i t h rat liver $9 m e t a b o l i c a c t i v a t i o n b u t resulted in e q u i v o c a l results w h e n tested at high c o n c e n t r a t i o n s w i t h o u t a c t i v a t i o n ( T a b l e 4). A second m o u s e l y m p h o m a study of M i B K was c o n d u c t e d using replicate cultures (Table 5) an d again the results were eq u i v o cal at the highest dose levels. T h e m u t a n t f r e q u e n c y seen after M i B K e x p o s u r e did n o t follow a dose-related response an d r e p e a t testing w i t h replicate cultures did n o t show a consistent positive effect ( T a b l e 5). T h e greatest r e s p o n s e to M i B K was o b s e r v e d at doses w h i ch resulted in 9 6 - 9 9 % lethality. Clive et al. (1979) h a v e suggested that doses which result in 9 0 - 1 0 0 % lethality are n o t r e l e v a n t in d e t e r m i n -
TABLE 1 SALMONELLA/MICROSOME (AMES) ASSAY RESULTS OF METHYL ETHYL KETONE: REVERTANTS/PLATE Salmonella strain
$9 activation
TA98 TA98
+ --
TA100 TA100
+ -
TA1535 TA1535
DMSO control (50 lal/plate)
MEK concentration (/xl/plate) 0.05
0.1
0.5
2
16
32
1239+104e 1240 + 34 f
29 + 3
37+ 8 21 _+ 6
42+ 8 26 _+ 6 b
27_+ 8b 22 ~- 5 b
31_+ lb 21 q- 5 b
115+ 5 115+12
1201-1-118g 1312+132 e
117+20 98--+17 120+13
97_+61 114-+ 8 b
127_+11 122-+10 b
107--+10b 125+_15 b
2+2 d
+ -
6+ 1 16-+ 3
241-+ 42 h 1040+155 g
11-+ 4 19-+ 6
9-+ 2 17+ 6 b
11-+ 1 15-+ 3 b
7-+ 3 b 17-+ 0 c
1+2 d
20-+ 4
TA1537 TA1537
+ -
6+ 3 4-+ 2
469-+ 24 h 358+153 i
2-+ 2 6-+ 2
5-+ 2 7-+ 4
7-+ 4 9-+ 2 b
5-+ 2 b 6-+ 1 b
1+0 d
7-+ 3
TA1538 TA1538
+ -
20-+ 5 18+ 4
1027-+ 21 e 1835-+ 74 f
15-+ 2 15-+ 3
18-+ 4 13-+ 3
20-+ 1 16-+ 4 b
21-+ 4 b 13-+ 3 b
14+7 a
19+ 5
a b c d e f g h i
34+ 2a 22_+ 9
Positive controls
~ + S.D. revertants/plate. All strains were plated in triplicate except for TA100, which had 4 plates. Background bacterial lawn slightly reduced. Background bacterial lawn moderately reduced. Background bacterial lawn extremely reduced. 2-Aminoanthracene 1.0 lag/plate. 4-Nitro-O-phenylenediamine 10 lag/plate. Sodium azide 5.0 lag/plate. 2-Aminoanthracene 4.0 lag/plate. 9-Aminoacridine 75 lag/plate.
0 d
154 TABLE 2 S A L M O N E L L A / M I C R O S O M E (AMES) ASSAY RESULTS O F M E T H Y L IS O B U TY L K E T O N E : R E V E R T A N T S / P L A T E Salmonella strain
$9 activation
TA98 TA98
+ -
TA100 TA100
+ -
TA1535 TA1535 b TA1535
DMSO control (50/xl/plate)
Positive controls
Mi B K concentration (# 1/plate) 0.04
0.1
0.4
1
4
1106_+ 17 d 1797_+259 e
35_+ 3 20_+ 4
39_+ 9 20-+ 8
47_+ 3 23-+ 6
33_+11 19-t- 7 ~
31+10 c 17+ 1 c
125_+17 129-+24
2798_+580 d 2173-+ 57 f
117_+15 121-+15
148_+16 130-+ 7
141_+20 114+16
108+19 ¢ 155+24 c
116 + 13 c 116+16 c
+ + -
9-+ 3 10-+ 2 26-+ 2
219-+ 20 g 164-+ 9 g 1036-+ 83 f
15-+ 9 12-+ 3 24-+ 8
18+ 6 8+ 2 27_+ 6
23+ 7 9_+ 2 24_+ 1
22+ 4 ¢ 12+ 3 c 26+ 5 ~
10+ 5 c
TA1537 TA1537
+ -
7-+ 2 6-+ 1
226-+ 54 g 1066+135 h
14+ 1 5_+ 1
6-+ 3 4_+ 2
5_+ 3 7_+ 2
9-+ 4 7_+ 3 c
TA1538 Ta1538
+ -
23_+ 2 17-+ 2
757_+ 53 d 2125+127 e
31-+ 3 15-+ 4
25_+ 7 17+ 4
19+ 5 16-+ 6
35_+ 2 ~ 24_+ 6
20_+ 3 16_+ 4
12+ 2 c 23+ 5 ~ 7+ 6+
5~ 1~
29+ 5 ~ 17_+ 4 c
a ~ + S.D. revertants/plate. All strains were plated in triplicate except for TA100, which had 4 plates, TA1538 without $9 activation which had 8 plates and the replicate assay on TA1535 with activation which had 5 plates. b The TA1535 cultures with activation were reassayed to further examine a marginal response in the first assay. c Background bacterial lawn slightly reduced. d 2-Aminoanthracene 1.0 # g / p l a t e . e 4-Nitro-O-phenylenediamine 10 # g / p l a t e . f Sodium azide 5.0 # g / p l a t e . g 2-Aminoanthracene 4.0 # g / p l a t e . h 9-Aminoacridine 75 /~g/plate. TABLE 3 L 5 1 7 8 Y / T K + / - M O U S E L Y M P H O M A ASSAY OF M E T H Y L E T H Y L K E T O N E MEK
Without metabolic activation
conc. (~ l / m l )
Mean mut. colonies/plate
Mutant freq. (//10 4 cells)
With rat liver $9 fraction Total growth (%)
Mean rout. colonies/plate
Mut a nt freq. ( / 1 0 4 cells)
Total growth (%)
12 8.9 6.7 5.0 3.8 2.8 2.1 1.6 1.2 0.89 0.67
52 46 39 35 47 45 41 47 41 54 -
0.6 0.5 0.4 0.4 0.5 0.5 0.4 0.5 0.5 0.6 -
46 65 89 84 96 113 122 112 111 122 -
49 43 62 49 51 56 54 67 59 60
0.6 0.4 0.7 0.5 0.5 0.7 0.7 0.7 0.6 0.7
0 30 116 89 78 100 100 97 112 104 98
Control(DMSO)
52
0.5
100
52
0.7
100
49.3 11.9
0 14
198 193
6.6 3.3
13 52
Ethyl methanesulfonate ( # l / m l ) 1.0 90 0.5 315 7,12-Dimethylbenz[ a ]anthracene (/~g/ml) 7.5 5.0 -
-
At concentrations of 16 and 21 # l / m l , there was no cell survival. These data represents the means of 3 counts from single cultures except for the controls which are the means of 2 cultures.
155 TABLE 4 L5178Y/TK + / - MOUSE LYMPHOMA ASSAY OF METHYL ISOBUTYL K E T O N E MiBK
Without metabolic activation
conc. (/.t 1/ml)
Mean mutant colonies/plate
4.2 3.2 2.4 1.8 1.3 1.0 0.75 0.56 0.42 0.32
136 82 62 82 61 76 74 70 45 49
Control (DMSO)
42
Ethyl methanesulfonate (/~l/ml) 1.0 184 0.5 _
MiBK
With rat liver $9 fraction
Total growth (%)
conc. (/.t l / m l )
Mean mutant colonies/plate
1.9 * 0.8 * 0.6 0.8 * 0.5 0.6 0.6 0.'l 0.2 0.5
3 31 52 58 86 90 95 81 157 87
4.2 3.2 2.4 1.8 1.3 1.0 0.75 0.56 0.42 0.32
69 70 69 69 71 66 78 62 66 68
0.6 0.6 0.6 0.6 a 0.6 0.7 0.5 0.6 0.6
23 33 59 84 _ 92 78 95 95 85
0.4
100
Control (DMSO)
59
0.5
100
15.2 a
6 -
7,12-Dimethylbenz[a]anthracene (/~g/ml) 7.5 213 9.0 5.0 168 6.6
9 18
Mutant freq. ( / 1 0 4 cells)
Mutant freq. ( / 1 0 4 cells)
Total growth (%)
a Culture lost. * Significant increase in mutant frequency; 2-fold control level. MiBK concentration of 5.6 and 7.5/xl/ml resulted in no net cell growth in activated and non-activated cultures. These data represent the means of 3 counts from single cultures except for the controls which are the means of 2 cultures.
TABLE 5 REPEAT L5178Y/TK + / - MOUSE LYMPHOMA ASSAY OF METHYL ISOBUTYL K E T O N E MiBK
Without metabolic activation
conc. (/~ 1/ml)
Mean mutant colonies/plate
Mutant freq. (/104 cells)
3.7 3.7 2.9 2.9 2.1 2.1 1.4 1.4 0.6 0.6
70 43 56 52 46 47 43 42 41 43
28.0 3.4 1.3 1.6 1.9 1.2 1.1 1.2 1.0 0.8
Control (DMSO)
43
0.8
100
4.4 8.3
4 24
Ethyl methanesulfonate (~ 1/ml) 1.0 40 0.5 174
* *
Total growth (%)
* *
1 4 42 32 31 51 60 46 68 80
MiBK
With rat liver S9 fraction
conc. (/L1/ml)
Mean mutant colonies/plate
3.4 3.4 3.0 3.0 2.5 2.5 1.9 1.9 1.4 1.4
64 61 40 57 43 49 48 48 52 52
Control (DMSO)
48
Mutant freq. ( / 1 0 4 cells)
Total growth (%)
0.8 1.0 1.4 1.1 1.0 1.2 1.1
35 37 28 34 51 47 62
1.3
63 _
0.9
100
7,12-Dimethylbenz[ a ]anthracene ( ~ g / m l ) 7.5 163 15.5 5.0 120 4.2
10 43
a
--
a
a Culture lost. * Significant increase in mutant frequency; 2-fold control level. MiBK concentrations of 4.5 and 5.2 ~tl/ml in non-activated cultures and 4 # l / m l in activated cultures resulted in no net cell growth. These data represent the means of 3 counts from single cultures except for the controls which are the means of 2 cultures.
156 TABLE 6 L5178Y/TK +/- MOUSE LYMPHOMA ASSAY OF ISOPHORONE Isophorone conc. (/~l/ml)
Without metabolic activation Mean mutant Mutant colonies/plate freq. (/10 4 cells)
Total growth (%)
Isophorone conc. (t~1/ml)
With rat liver $9 fraction Mean mutant colonies/plate
Mutant freq. (/104 cells)
Total growth (%)
1.3 1.0 0.75 0.56 0.42 0.32 0.24 0.18 0.13
54 73 54 55 60 54 54 58 42
1.1 1.1 0.7 0.7 0.7 0.8 0.7 0.8 0.6
12 44 73 77 94 84 90 111 110
0.89 0.67 0.50 0.38 0.28 0.21 0.16 0.12 0.089
64 47 66 56 49 49 51 56 60
1.0 0.6 1.0 0.8 0.8 0.6 0.7 0.8 0.9
9 28 25 42 49 81 75 66 75
Control (DMSO)
39 -
0.6 -
100 -
0.067 Control (DMSO)
50 51
0.7 0.7
86 100
24.0 8.8
2 31
7,12-Dimethylbenz[a]anthracene (/~g/ml) 7.5 200 5.2 5.0 178 3.9
24 45
Ethyl methanesulfonate (#l/ml) 1.0 120 0.5 292
In non-activated cultures, isophorone concentrations of 1.8 and 2.4/~l/ml produced 100% lethality. In activated cultures, isophorone concentrations of 1.2 and 1.6/~l/ml produced 100% lethality. These data represent the means of 3 counts from single cultures except for the controls which are means of 2 cultures.
ing significant mutagenicity. If doses resulting in greater t h a n 90% lethality are n o t considered, then the few r e m a i n i n g increases were n o t c o n c e n t r a tion d e p e n d e n t a n d the results w o u l d b e considered negative. Of further i m p o r t a n c e in assessing these results, is the v a r i a t i o n a m o n g the D M S O c o n t r o l cultures for the 8 m o u s e l y m p h o m a assays; the range o f m u t a n t frequencies was 0 . 4 - 0 . 9 / 1 0 4 survivors, a m o r e t h a n 2-fold difference. Thus, n o r m a l v a r i a t i o n in the c o n t r o l m u t a n t frequency m a y alter the i n t e r p r e t a t i o n a n d significance of the m u t a t i o n response. T h e m u t a n t frequencies in the c o n t r o l cultures were w i t h i n n o r m a l historical ranges for the l a b o r a t o r y . A b sence of M i B K - r e l a t e d increases o u t s i d e the negative c o n t r o l range p r o v i d e s a d d i t i o n a l evidence for a negative c o n c l u s i o n for m u t a g e n i c p o t e n t i a l . T h e response o f B A L B / 3 T 3 cells to M i B K was also ambiguous. I n the first assay, an M i B K conc e n t r a t i o n of 4 . 8 / ~ l / m l resulted in 3 t y p e I I I foci in 15 dishes ( T a b l e 9). This n u m b e r o f t y p e I I I foci, c o u p l e d with a r e d u c e d cloning efficiency, resulted in a positive statistical analysis in the n o n - a c t i v a t e d system. W i t h m e t a b o l i c activation,
there was n o t r a n s f o r m i n g activity. O n a r e p e a t assay, M i B K at a c o n c e n t r a t i o n of 5 / ~ l / m l resulted in 2 t y p e I I I foci for 15 p l a t e s with 100% cell survival. T h e resulting t r a n s f o r m a t i o n f r e q u e n c y was n o t statistically i n c r e a s e d over the negative control. Thus, the results of the first B A L B / 3 T 3 a s s a y of M i B K , in the a b s e n c e of m e t a b o l i c activity, c o u l d n o t b e c o n f i r m e d . O t h e r d a t a a p p e a r to s u p p o r t the conclusions d r a w n f r o m these studies; that M E K , M i B K , a n d i s o p h o r o n e have relatively low p o t e n t i a l s for genotoxicity. Z i m m e r m a n n et al. (1985) i n c l u d e d M E K in a series o f solvents s t u d i e d for g e n o t o x i c activity in Saccharomyces cerevisiae strain D61.M. I n this testing p a r a d i g m , M E K was f o u n d to ind u c e a n e u p l o i d y , b u t the r e s p o n s e was s t r o n g o n l y if relatively high c o n c e n t r a t i o n s (3.54%) of M E K were used, M E K was p r e s e n t in the cultures continuously, a n d cultures were held in ice u n d e r c o n d i t i o n s which w o u l d i n h i b i t r e a s s e m b l y of m i t o t i c s p i n d l e tubulin. T h e r e was n o evidence t h a t M E K resulted in p o i n t m u t a t i o n s o r m i t o t i c r e c o m b i n a t i o n s , suggesting that the o b s e r v e d effects were n o t d u e to an i n t e r a c t i o n b e t w e e n M E K
157 TABLE 7 RESULTS OF THE U N S C H E D U L E D D N A SYNTHESIS ASSAYS Treatment (Itl/ml)
RS a
Avg. net grains/nucleus ( X ± S.D.) b
Methyl ethyl ketone 5.0 2.5 1.0 0.5 0.1 DMSO 20.0 It g / m l 2-AAF 2.0 It g / m l 2-AAF
12.7 37.3 71.1 74.7 87.3
1.6+2.3 0.4+1.3 -0.2+1.1 -0.1+1.5 0.5 + 1.6
100.0
-0.2+1.2
25.2 43.6
25.0 ± 5.0 21.3 _+4.9
Methyl isobutyl ketone 100 10 1.0 0.10 0.010
0 1.7 28.5 85.7 88.4
DMSO
100.0
0.6 _+0.2
61.6 65.4
58.6 ± 0.9 49.2 _+0.8
20.0 It g / m l 2-AAF 2.0 It g / m l 2-AAF
Too toxic to count Too toxic to count 1.9 ± 0.4 0.9±0.2 1.2 _+0.2
Isophorone 0.40 0.20 0.10 0.05 0.01 0.005
0 38.4 42.5 60.0 75.9 100.0
Too toxic to count 1.4 _+1.9 0.7 _+1.4 0.1 ± 1.2 0.2± 1.6 - 0.3 _+1.2
EtOH
100.0
- 0.1 _+1.0
35.9 45.0
25.5 ± 7.3 19.4±4.6
20.0 I t g / m l 2-AAF 2.0 I t g / m l 2-AAF
a Relative survival = avg. viable cells/tube cells plated/tube = avg. viable cells/solvent control tube × 100. cells plated/solvent control tube b 100 nuclei were counted at each dose level.
and genetic material, but rather were due to effects on tubulin assembly resulting in chromosomal malsegregation. Under these conditions, the significance of chromosomal malsegregation in Saccharomyces cerevisiae for assessing genotoxicity in man or other species in unclear. M E K has not been observed to interfere with tubulin polymerization in mammalian cells or tissues which should be sensitive indicators of such
an effect. Agutter and Mack (1979) studied the effects of M E K (1 mM) on the ability of sheep brain tubulin to polymerize and depolymerize in vitro and found no effect due to MEK. It might also be postulated that if M E K were to alter tubulin polymerization or depolymerization in mammalian cells, that growth and differentiation of mitotically active cells should be altered. Selkoe et al. (1978) studied the effects of M E K and other ketones on the cytotoxicity and growth of the murine neuroblastoma cell line. At M E K concentrations of up to 0.5%, cells proliferated well and differentiated normally. Cavender et al. (1983) examined the subchronic toxicity of M E K after exposures of Fischer 344 rats to airborne concentrations of up to 5000 ppm for 90 days and found only minor effects on body weight, liver weight, and liver enzyme levels. Significantly, no effects were observed in the testes, bone marrow, or gastrointestinal tract which are mitotically active tissues that might be expected to be adversely affected by an inhibitor of tubulin polymerization. The metabolism of M E K and M i B K (DiVincenzo et al., 1976) proceeds by pathways which would be unlikely to result in genotoxic metabolites. Both materials undergo oxidation to their respective hydroxy ketones, 3-hydroxy-2-butanone and 4-hydroxy-4-methyl-2-pentanone and to a lesser extent undergo reduction to their respective alcohols, 2-butanol and 4-methyl-2-pentanol. M E K is also metabolized to 2,3-butanediol. The parent compounds and their metabolites are rapidly cleared from the serum within 16 h (DiVincenzo et al., 1976). Subchronic toxicity associated with M E K and MiBK exposure is slight. Cavender et al. (1983) found no specific organ toxicity, and only mild adaptive change, in Fischer 344 rats after inhalation exposure to 5000 ppm M E K for 90 days. Similarly, organ effects after MiBK exposure were mild. Exposure of Fischer 344 rats and B6C3F t mice to atmospheres of 1000 ppm MiBK for 14 weeks resulted in increased liver weights in both species and the presence of hyalin droplets in the kidneys of male rats (Phillips et al., 1987). The National Toxicology Program (NTP, 1986) completed a series of studies on the genotoxicity, chronic toxicity, and carcinogen±city of isophorone. Genotoxicity was examined in a series of
158 TABLE 8 M I C R O N U C L E A T E D P O L Y C H R O M A T I C E R Y T H R O C Y T E S IN BONE M A R R O W CELLS F R O M RATS T R E A T E D W I T H K E T O N I C SOLVENTS Treatment
Sex
Time (h)
N u m b e r of mice
Proportion of PCE
Micronucleated polychromatic erythrocytes Number/PCEs scored
Range per animal
N u m b e r / 1 0 0 0 PCEs (mean +- S.D.)
Methyl ethyl ketone 1.96 m l / k g
M F M F M F
12 12 24 24 48 48
5 5 5 5 5 5
0.47 0.47 0.47 0.47 0.46 0.46
8/5000 8/5000 9/5000 14/5000 8/5000 6/5000
000100-
3 4 4 4 4 3
1.6+_ 1.6_+ 1.8+2.8_+ 1.6_+ 1.2_+
1.1 1.8 1.6 1.3 1.5 1.1
Cornoil 10 m l / k g
M F M F M F
12 12 24 24 48 48
5 5 5 5 5 5
0.47 0.48 0.47 0.48 0.47 0.47
4/5000 10/5000 5/5000 5/5000 8/5000 13/5000
010001-
2 3 2 2 4 4
0.8_+ 2.0+1.0_+ 1.0_+ 1.6+ 2.6+
0.8 1.0 1.0 0.7 1.7 1.1
TEM 0.25 m g / k g
M F
24 24
5 5
0.47 0.46
173/5000 124/5 000
24-50 11-43
34.6_+ 9.8 * 24.8 _+12.2 *
Methyl isobutyl ketone 0.73 m l / k g
M F M F M F
12 12 24 24 48 48
5 5 5 5 5 5
0.47 0.48 0.46 0.47 0.46 0.47
7 / 5 000 5/4000 4/5000 7 / 5 000 10/5000 10/5000
000100-
4 2 2 2 4 4
1.4 +1.3+ 0.8+_ 1.4 _+ 2.0+2.0-+
1.7 1.0 0.8 0.6 1.9 1.6
Corn oil 10 m l / k g
M F M F M F
12 12 24 24 48 48
5 5 5 5 5 5
0.47 0.48 0.47 0.48 0.47 0.47
4/5000 10/5000 5 / 5 000 5/5000 8 / 5 000 13/5000
010001-
2 3 2 2 4 4
0.8+2.0+1.0_+ 1.0+ 1.6 +2.6_+
0.8 1.0 1.0 0.7 1.7 1.1
TEM 0.25 m g / k g
M F
24 24
5 5
0.47 0.47
173/5000 124/5 000
0.54 m l / k g
M F M F M F
12 12 24 24 48 48
5 5 5 5 5 5
0.50 0.51 0.50 0.50 0.51 0.52
7/5000 6/5000 2/5000 4/5000 7/5000 6/5000
100011-
2 3 1 2 2 3
1.4_+ 1.2_+ 0.4_+ 0.8_+ 1.4_+ 1.2_+
0.5 1.3 0.5 1.1 0.5 1.3
Corn oil
M F M F M F
12 12 24 24 48 48
5 5 5 5 5 5
0.51 0.51 0.50 0.51 0.50 0.51
3/5000 6/5000 4/5000 4/5000 4/5000 6/5000
000000-
1 3 2 2 1 2
0.6_+ 1.2+ 0.8_+ 0.8+0.8+1.2+
0.5 1.3 1.1 0.8 0.4 1.1
TEM 0.25 m g / k g
M F
24 24
5 5
0.50 0.52
188/5000 177/5000
24-50 11-43
34.6_+ 9.8 * 24.8 + 12.2 *
Isophorone
31-43 30-40
37.6_+ 4.3 * 35.4+- 3.7 *
Dose levels were equal to the LD20 for each ketone and are reported as ml test article/kg body weight when administered in a total volume of 10 ml test article-vehicle m i x t u r e / k g body weight. * P ~< 0.01, Student's t test.
159 TABLE9 TRANSFORMATION POTENTIAL OF KETONIC SOLVENTS USING BALB/3T3 CLONE A31-1 CELLS Treatment (/.tl/ml)
Total foci/total dishes Type II Type III
Transformation frequency ( x l 0 -4)
Relative cloning efficiency (%)
18 13 9
0/15 2/15 1/14
2/15 0/15 1/14
0.58 < 0.19 0.17
51 80 93
PBS control
1/15
0/15
< 0.15
100
0.5 ~ g / m l M N N G
7/15
19/15
Methylethylketone
12.67 *
22
Methyl ethyl ketone with metabolic activation 10 8 6 PBS control
1/15 0/14 0/15 2/15
0/15 0/14 0/15 0/15
< < < <
0.20 0.19 0.16 0.14
67 76 86 100
12.5 ~g/ml BaP
8/15
12/15
3.08 *
53
4.8 3.6 2.4
0/15 0/15 1/15
3/15 3/15 2/15
0.87 * 0.56 0.34
51 80 87
PBS control
1/15
0/15
0.5 ~ g / m l M N N G
7/15
19/15
Methyl ~obu~l ketone
< 0.15 12.67 *
100 22
Methyl isobutyl ketone (repeat assay) 7 6 5 4
1/15 2/15 0/15 0/14
0/15 0/15 2/15 0/14
To~c < 0.95 0.33 < 0.19
<1 17 100 93
PBS control
0/14
0/14
< 0.17
100
0.5 ~ g / m l M N N G
5/15
11/15
5.64 *
32
Methyl~obu~lkewne with metaboficactivatwn 4 2 1
1/14 0/13 1/14
0/14 1/13 1/14
< 0.22 0.21 0.17
66 76 84
PBS control
2/15
0/15
< 0/14
100
12.5 ~ g / m l B a P
8/15
12/15
3.08 *
53
Methyl isobutyl ketone with metabolic activation (repeat assay) 5 4 3 2
0/15 1/15 1/15 0/15
1/15 1/15 4/15 1/15
0.18 0.16 0.59 0.14
75 82 88 96
PBS control
0/14
0/14
< 0.14
100
12.5 ~ g / m l B a P
0/15
5/15
1.23 *
53
* P < 0.01, modified Poisson distribution. Relative cloning efficiency is reported as the number of colonies per treatment condition relative to 100% survival in the solvent control. number of type III loci Transformation frequency 10 000 cells x cloning efficiency × dishes scored"
160 studies including Ames assays with and without $9 from Aroclor 1254-induced rat or hamster livers, M L assays without metabolic activation, assays for sister-chromatid exchange in Chinese hamster ovary ( C H O ) cells with and without $9 from Aroclor 1254-induced rat livers, and in assays to detect induction of c h r o m o s o m a l aberrations in C H O cells with and without rat liver $9. Although negative in most of the assays, weak mutagenic activity was present in the M L assay and sister-chromatid exchanges were induced in C H O cells in the absence of $9. The reasons for the differing results when the N T P M L assay is c o m p a r e d to the present study are unclear. Both assays were conducted at similar isophorone concentrations, and other factors in the protocols including the carrier solvent were similar, but the results were not reproducible between the laboratories. 3-year exposure of Fischer 3 4 4 / N rats and B6C3F 1 mice to 500 m g / k g of isophorone in corn oil resulted in decreased survival of male rats after 96 weeks of exposure and slight nephrotoxicity in male rats (National Toxicology Program, 1986). There was no evidence of carcinogenicity in female animals of either species. The responses in male animals were classified as ' s o m e evidence of carcinogenicity' for male rats and 'equivocal evidence of carcinogenicity' for male mice. The male rat responses included increased renal tumors in rats given 250 or 500 m g / k g isophorone and a low incidence of preputial gland tumors in males given 500 m g / k g . The responses in male mice included liver tumors and mesenchymal tumors in the integumentary system in mice given 500 m g / k g and malignant l y m p h o m a s in mice given 250 m g / k g , although the increased incidence of l y m p h o m a s was marginal. Overall, the data suggest that exposure to M E K , MiBK, and isophorone are unlikely to result in significant toxicity. The data for M E K do not indicate any genotoxic activity. The presence of a marginal response only at the highest cytotoxic concentration tested in the mouse l y m p h o m a assay and the lack of reproducibility in the B A L B 3T3 transformation assay, and clearly negative results in the Ames, UDS, and M N assays suggest that M i B K is unlikely to be genotoxic in m a m malian systems. The weight-of-the-evidence on
isophorone genotoxicity would also suggest that it is unlikely to be a genotoxic hazard for mammals. The only positive indicator of genotoxicity was the induction of sister-chromatid exchanges in C H O cells. The results of the mouse l y m p h o m a assays without metabolic activation while negative in the present study were positive in the N T P study and thus were not reproducible between laboratories. All other assays of isophorone for genotoxic activity were negative. These included the Ames, mouse l y m p h o m a with metabolic activation, CT, MN, sister-chromatid exchange in C H O cells with metabolic activation, and c h r o m o s o m a l aberration in C H O cells assays.
References Agutter, P.S., and A.P. Mack (1979) The effects of afl-dicarbonyl compounds on the polymerization of sheep brain tubulin in vitro, Biochem. Soc. Trans., 7, 691-693. Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/ mammalian-microsome mutagenicity test, Mutation Res., 31, 347-364. Cavender, F.L., H.W. Casey, H. Salem, J.A. Swenburg and E.J. Gralla (1983) A 90-day vapor inhalation toxicity study of methyl ethyl ketone, Fundam. AppL Toxicol., 3, 264-270. Chemical Manufacturers Association Ketones Program Panel Report to the U.S. Environmental Protection Agency, October 21, 1981. Clive, D., and J.F.S. Spector (1975) Laboratory procedure for assessing specific locus mutations at the TK locus in cultured L5178Y mouse lymphoma cells, Mutation Res., 31, 17-29. Clive, D., K.O. Johnson, J.F.S. Spector, A.G. Batson and M.M.M. Brown (1979) Validation and characterization of the L5178Y/TK +/- mouse lymphoma mutagen assay system, Mutation Res., 59, 61-108. DiVincenzo, G.D., C.J. Kaplan and J. Dedinas (1976) Characterization of the metabolites of methyl n-butyl ketone, methyl isobutyl ketone, and methyl ethyl ketone in guinea pig serum and their clearance, Toxicol. Appl. Pharmacol., 36,511-522. Kakunaga, T. (1973) A quantitative system for assay of malignant transformation by chemical carcinogens using a clone derived from BALB/3T3, Int. J. Cancer, 12, 463-473. National Toxicology Program (1982) Mutagenicity results on isophorone, Dept. of Health and Human Services, Washington, DC, p. 1-6. National Toxicology Program (1986) Toxicology and carcinogenesis studies of isophorone in F344/N rats and B6C3Fl mice, NIH Publ. No. 86-2547, Washington, DC. Phillips, R.D., E.J. Moran, D.E. Dodd, E.H. Fowler, C.D. Kary and J.L. O'Donoghue (1987) A 14-week vapor inhalation toxicity study of methyl isobutyl ketone, Fundam. Appl. Toxicol., 9, 380-388.
161 berger (1973) Quantitative and qualitative studies of chemical transformation of cloned C3H mouse embryo cells sensitive to post-confluence inhibition of cell division, Cancer Res., 33, 3239-3249. Selkoe, D.J., L. Luckenbill-Edds and M.L. Shelanski (1978) Effects of neurotoxic industrial solvents on cultured neuroblastoma cells: methyl n-butyl ketone, n-hexane and derivatives, J. Neuropathol. Exp. Neurol., 36, 768-789. Shirasu, Y., M. Moriya, K. Kato, A. Furyhashi and T. Kada (1976) Mutagenicity screening of pesticides in the microbial system, Mutation Res., 40, 19-30. Williams, G.M. (1977) Carcinogen-induced DNA repair in primary rat liver cell cultures, a possible screen for chemical-carcinogens, Cancer Lett., 1, 231-237.
Williams, G.M. (1979) The detection of chemical mutagens/ carcinogens by DNA repair and mutagenesis in liver cultures, in: F.J. de Serres and A. Hollaender (Eds.), Chemical Mutagens, Plenum, New York, pp. 71-79. Yahagi, T., M. Nagao, Y. Seino, T. Matsushima, T. Sugimura and M. Okada (1977) Mutagenicities of N-nitrosamines on Salmonella, Mutation Res., 48, 121-130. Zimmermann, F.K., V.W. Mayer, I. Scheel and M.A. Resnick (1985) Acetone, methyl ethyl ketone, ethyl acetate, acetonitrile and other polar aprotic solvents are strong inducers of aneuploidy in Saccharomyces cereoisiae, Mutation Res., 149, 339-351.
149
Elsevier MTR 01310
Mutagenicity studies on ketone solvents: methyl ethyl ketone, methyl isobutyl ketone, and isophorone J.L. O ' D o n o g h u e 1, S.R. H a w o r t h 2 R . D . C u r r e n 2, P.E. K i r b y 3 T. L a w l o r 2, E.J. M o r a n 4, R . D . Phillips 5, D . L . P u t n a m 2, A.M~ R o g e r s - B a c k 2, R.S. Slesinski 6 a n d A. T h i l a g a r 3 1 Health and Environment Laboratories, Eastman Kodak Company, Rochester, N Y 14652-3615, 2 Microbiological Associates, Inc., Department of Genetic Toxicology, 5221 River Road, Bethesda, M D 20816, 3 Sitek Research Laboratories, 12111 Parklawn Drive, Rockville, M D 20852, 4 Chemical Manufacturers Association, 2501 M Street, N W , Washington, DC 20037, 5 Exxon Biomedical Sciences Inc., Mettlers Rd., CN2350, East Millstone, NJ 08875-2350, and 6 Bushy Run Research Center, RD 4, Mellow Road, Export, PA 15632 (U.S.A.)
(Received 9 July 1986) (Revision received 18 February 1988) (Accepted 26 February 1988)
Keywords: Ketone solvents; Methyl ethyl ketone; Methyl isobutyl ketone; Isophorone
Summary 3 ketone solvents (methyl ethyl ketone (MEK), methyl isobutyl ketone (MiBK), and isophorone) were tested for potential genotoxicity. The assays of MEK and MiBK included the Salmonella/microsome (Ames) assay, L5178Y/TK ÷/- mouse lymphoma (ML) assay, BALB/3T3 cell transformation (CT) assay, unscheduled DNA synthesis (UDS) assay, and micronucleus (MN) assay. Only the ML, UDS, and MN assays were conducted on samples of isophorone. No genotoxicity was found for MEK or isophorone. The presence of a marginal response only at the highest, cytotoxic concentration tested in the ML assay, the lack of reproducibility in the CT assay, and clearly negative results in the Ames assay, UDS and MN assays, suggest that MiBK is unlikely to be genotoxic in mammalian systems.
Ketone solvents have found wide use in the coatings industry because of their excellent solvation properties. 3 ketone solvents of interest primarily because of production volumes are methyl ethyl ketone (MEK; 343 x 103 tons), methyl isobutyl ketone (MiBK; 95 x 103 tons), and isophorone (10-15 x 103 tons); production volumes are 1979 estimates for the U.S.A. (Chemical Correspondence: Dr. John L. O'Donoghue, Health and Environment Laboratories, B-320, Kodak Park, Eastman Kodak Company, Rochester, NY 14650 (U.S.A.). Tel.: 716588-4741.
Manufacturers Association, 1981). Uses of these materials include coatings, adhesives, and petroleum refining for MEK; coatings, solvent extraction, and metallurgy for MiBK; and coatings, inks, and agricultural formulations for isophorone. Few mutagenicity data are available on these substances. Shirasu et al. (1976) used MEK as a solvent for a group of pesticides tested for mutagenicity utiliz: ing E. coli, Salmonella typhimurium, or H17 Rec ÷ and M45 Rec- strains of Bacillus subtilis without metabolic activation. MEK did not induce mutagenic effects in these test systems. Isophorone did
0165-1218/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
150 not induce mutations when tested using 4 different strains of Salmonella typhimurium with or without exogenous metabolic activation provided by rat or hamster liver $9 preparations (National Toxicology Program, 1982). N o other genetic toxicity data were available at the time these studies were begun; therefore, the present studies were conducted to further elucidate the genotoxic potential of these 3 ketones. Materials and methods
5 assays for genetic activity were conducted for M i B K a n d M E K . T h e assays were the Salmonella/microsome (Ames) assay, L 5 1 7 8 Y / T K ÷/- mouse lymphoma (ML) assay, B A L B / 3 T 3 cell transformation (CT) assay, unscheduled D N A synthesis (UDS) assay, and micronucleus (MN) assay. Since data for the activity of isophorone in the Ames assay already existed (National Toxicology Program, 1982), the assay was not repeated during this study. Due to the volatility of the test substances, all assays were conducted in sealed glass containers to prevent escape of the test material. The M E K (CAS No. 78-93-3) used for these studies was supplied by the Celanese Corporation and was 99.9% pure. MiBK (CAS No. 108-10-1) was supplied by the Exxon Chemical C o m p a n y and was 99.6% pure. The isophorone (CAS No. 78-59-1) sample was supplied by the Exxon Corporation and was > 97% pure. The test materials were blanketed with nitrogen and stored at room temperature.
Salmonella / microsome (Ames) assay Salmonella strains TA98, TA100, TA1535, TA1537, and TA1538 were received from Dr. B. Ames, University of California, Berkeley, CA. The genotypic characteristics of each culture were tested prior to use as recommended by Ames et al. (1975). The experimental procedures were based on the work of Ames et al. (1975) and Yahagi et al. (1977). M E K and MiBK were diluted in dimethyl sulfoxide (DMSO), and based on the results of preliminary cytotoxicity assays, 5 doses of each chemical were tested in at least triplicate plates.
When metabolic activation was required, 500/~1 of S9 mix containing 10% homogenate, 50/~1 of an appropriate tester strain, and 50 #1 of solvent or an appropriate concentration of test article were added to 13 m m x 100 m m glass, Teflon fluoroc a r b o n - l i n e d , s c r e w - c a p p e d culture tubes, preheated to 37 ° C. The $9 fraction was prepared from the livers of male Sprague-Dawley rats given 50 m g / k g of Aroclor 1254. When metabolic activation was not required, 500 /zl of S9 sham mix (0.1 M phosphate buffer) was substituted for the $9 mix. After vortex mixing, the mixture was allowed to incubate in the sealed test tube for 2 h at 37 ° C. 2 ml of selective top agar were then added to each tube, and the tube was mixed and its contents were overlaid onto the surface of 25 ml of Vogel-Bonner bottom agar contained in glass petri dishes. After each dose level was plated and the top agar had solidified, all of the plates of that dose level were sealed into one or more glass chambers and incubated at 37 ° C for 48 h. Plates containing a metabolic activation system were placed in glass chambers separate from plates without the metabolic activation system. Plates which were not counted immediately following the 48-h incubation period were stored at 4 ° C until such time that colony counting could be conducted. The glass containers used for this study were 1 gallon, flint glass jars fitted with a screw cap. Teflon sheeting was placed over the mouth of the jar prior to capping. Handling the plates in this manner ensured that volatilized solvent did not escape during the exposure and expression periods. Combinations of positive control substances and the tester strains were plated along with the test plates (Tables 1 and 2). Positive controls were preincubated for 20 min at 3 7 ° C and were not placed in sealed chambers for the 48-h incubation period. For a test article to have been considered positive, it must have caused at least a doubling in the mean revertants per plate of at least 1 tester strain. This increase in the mean number of revertants per plate must have been accompanied by a dose response to increasing concentrations of the test article. In those cases in which the observed dose-responsive increase in TA1537 or
151 TA1538 mean revertants per plate was less than 3-fold, the response had to have been reproducible.
L5178 Y / TK +/- mouse lymphoma assay This assay was based on the methods of Clive and Spector (1975) and Clive et al. (1979) to measure the forward mutation frequency at the thymidine kinase locus in mouse l y m p h o m a cells. Based on preliminary toxicity tests, the test chemicals (MEK, MiBK, and isophorone) were diluted with DMSO to produce 15 serial eighth log dilutions. This resulted in 16 dose levels decreasing approximately 100-fold from highest to lowest. The highest dose was 100% toxic while the lowest dose was non-toxic. Each test chemical was tested with and without $9 fraction. The $9 fraction was prepared from the livers of male Sprague-Dawley rats injected with 500 m g / k g of a 2 : 1 mixture of Aroclor 1242 and Aroclor 1254. Final cell suspensions were 0.6 x 10 6 cells/ml. Control tubes received D M S O only and positive controls received ethyl methanesulfonate (1.0 or 0.5/~l/ml) or 7,12dimethylbenz[a]anthracene (7.5 or 5.0/~g/ml). All tubes were glass and fitted with Teflon-lined caps to prevent escape of the test articles. 5% CO 2 in air was added to the tubes which were then placed on a roller drum for 4 h at 37 ° C. At the end of the exposure period, the cells were washed and incubated for 2 days to allow for expression of the mutant phenotype. During the expression period, cells were counted daily and adjusted to a cell population density of 0.3 × 106/ml. Following the expression period, cells were cloned on medium containing 0.34% Noble agar. Trifluorothymidine, at a concentration of 3 /~g/ml, was used as the restrictive agent. 3 c o u n t s / p l a t e were made on an automatic colony counter and the median count was recorded. A test article was considered positive if there was a positive dose response and 1 or more of the 3 highest doses exhibited a mutant frequency which was 2-fold greater than the background level. If there was no dose response but any one or more doses exhibited a 2-fold increase in mutant frequency over background, the result was considered equivocal. A negative response was one in which there was no dose response and none of the test cultures exhibited mutant fre-
quencies which were 2-fold greater than background (Clive et al., 1979).
Unscheduled DNA synthesis in rat primary hepatocytes The methods used were adapted from those of Williams (1977, 1979). Primary rat hepatocytes were derived from Sprague-Dawley rats obtained from Charles River Laboratories, Inc., Kingston, NY. Rats were anesthetized with methoxyflurane and their livers perfused with 0.5 m M ethylenebis (oxyethylenenitrilo)-tetraacetic acid (EGTA) followed by a collagenase solution. Dissociated cells were counted and seeded into glass Leighton tubes containing coverslips (2.5 × 105 viable cells/tube). The culture medium was Williams Medium E (WME) buffered with 0.01 M Hepes, supplemented with 10% fetal calf serum, 2 m M L-glutamine, and 50/~g of gentamicin/ml. Cultures were incubated (37°_+ 1°C, 5% CO2 in air) for 2 h, washed, refed with serum-free W M E and used in the assay. 5 or 6 concentrations of each test article were assayed based on preliminary cytotoxicity studies. Survival was assayed by comparing the number of trypan blue-excluding cells after 24-h exposure to the test article with the original number of cells seeded. Doses were chosen to span the range from approximately 50% relative survival (ratio of survival of treated cells to the survival of solvent control cells) to 100% survival. Triplicate cultures were seeded for each chemical. The positive control was 2-acetylaminofluorene (2 and 20 /~g/ml 2-AAF). Solvent controls included D M S O (diluent for M E K and MiBK) and ethanol (diluent for isophorone and 2-AAF). [3H]Thymidine (NEN, 20 C i / m m o l e ) was present at 10 /~Ci/ml in all treated cultures. Tubes treated in parallel for cytotoxicity were not treated with [3H]thymidine. After 18 h of exposure in sealed glass Leighton tubes, the cells were washed, swelled in 1% sodium citrate, and fixed in an ethanol-acetic acid fixative. Dried coverslips were coated with Kodak N T B emulsion, stored at 4 ° C for 8 days, developed in K o d a k D19 developer, fixed, and stained with h e m a t o x y l i n - s o d i u m acetate-eosin stain. Grain counts were made using a Biotran 2 colony counter. Net nuclear grain counts (grain count over the nucleus minus the average grain
152
count from 3 adjacent nuclear sized areas) were calculated for at least 100 randomly selected cells at each dose level. The result for a particular dose level was considered significant if the mean net nuclear count was increased 5 or more counts over the solvent control. A test article was judged positive if it either induced a dose-related response and at least 1 dose produced a significant increase in the average net nuclear grains when compared tO that of the control, or if the test article should show a significant increase in the mean net nuclear grain count in at least 2 successive doses. If a test article showed a significant increase in the net nuclear grain count at 1 dose level without any dose response, the test article was considered to have marginal positive activity. The test article was considered negative if no significant increase in the net nuclear grain counts at any dose level was observed.
Micronucleus cytogenetic assay in mice Groups of 5 male and 5 female mice (CD-1, 6 - 8 weeks old, Charles River Laboratories, Inc., Kingston, NY) were given a single dose of the test chemical in corn oil and sacrificed 12, 24 or 48 h later. The test dose was the LD20 of the test article, determined in a preliminary toxicity study. The positive control group was given 0.25 m g / k g of triethylene melamine (TEM) and examined 24 h post dosing. All doses were given intraperitoneally at a rate of 10 ml of test article-corn oil m i x t u r e / k g body weight. At sample collection, the femur was exposed and bone marrow aspirated into a syringe containing fetal calf serum. The cells were washed, centrifuged, resuspended and smears prepared. M a y - G r u e n w a l d - G i e m s a stain was used to stain the bone marrow preparations. 1000 polychromatic erythrocytes were scored on coded slides for the presence of micronuclei. Micronucleated normocytes were also counted. A 1-way analysis of variance and Duncan's multiple range test ( P ~< 0.05) were used to assess the statistical significance of any observed effects. B A L B / 3T3 mouse embryo cell transformation assay B A L B / 3 T 3 clone A31-1 cells obtained from Dr. T. Kakunaga, National Cancer Institute, Be-
thesda, MD, were harvested during exponential growth and suspended in 50-ml glass tubes fitted with Teflon-lined caps (Kakunaga, 1973). Cell density was 2 × 106 cells in 2 ml phosphatebuffered saline with or without Aroclor 1254-induced rat liver $9 fraction. 2 ml of the test article or control substance was added to each tube. N-Methyl-N'-nitro-N-nitrosoguanidine ( M N N G ) was the positive control for the non-activated cultures, and benzo[a]pyrene (BaP) was the positive control for the S9-activated tubes. Doses for the study were chosen based on a preliminary cytotoxicity study. The doses of M E K were 9, 13 and 18 ~tl/ml for non-activated cultures and 6, 8 and 10 # l / m l for the activated cultures. The doses of MiBK were 2.4, 3.6 and 4.8/~l/ml without activation and 1, 2 and 4 # l / m l with activation. In a repeat assay on MiBK, doses were 4, 5, 6 and 7 / d / m l without activation and 2, 3, 4 and 5 / ~ l / m l with activation. A concurrent toxicity study was also conducted for each material. Following a 2-h incubation period in serum-free medium containing the test article, the cells were transferred to complete medium, 15 dishes/treatment. After 4 - 6 weeks incubation, the transformation plates were fixed in 95% methanol, stained with Giemsa stain and scored for type II and type III loci as recommended by Reznikoff et al. (1973). The cytotoxic effects of each treatment condition were expressed relative to the solvent-treated control (relative cloning efficiency). The transformation frequency for each treatment condition was expressed as the number of transformed loci per surviving cell. The confidence level associated with the number of surviving cells employed in the selection process required that the assumption be made that were there more cells utilized for the selection of transformants, the possibility existed that at least I type I I I focus could be obtained. Therefore, for test conditions in which no type III foci were observed, transformation frequencies are expressed as less than the frequency obtained with 1 type III focus. The numbers of type II and type III foci per total dishes scored are also presented. The transforming potential of each treatment condition was compared to that of the solvent control using an application of the Poisson distribution as follows: The proportion ( P ) of all cells not subject to
153 i n d u c e d t r a n s f o r m a t i o n is e q u a l to the n u m b e r of surviving ceils in the solvent c o n t r o l g r o u p d i v i d e d by the total of the surviving cells in the solvent c o n t r o l an d t r e a t m e n t group. Significance is d e t e r m i n e d by: ?/
P(X>~nl/n) =
E(
)P"'(1-P)"-"'
n 1
w he re p is the probability, n is the n u m b e r of i n d u c e d type I I I foci, n 1 is the n u m b e r of s p o n t a ne ous type I I I foci, a n d P is d e f i n e d above. If p were very small ( p ~< 0.05), the hypothesis that the p r o b a b i l i t y of a t r a n s f o r m a t i o n e v e n t was the same for b o t h the treated a n d c o n t r o l groups was rejected an d the i n d u c e d t r a n s f o r m a t i o n f r e q u e n c y was considered significant at the p ~< 0.05 level.
Results and discussion T h e results of the b a t t e r y o f g e n o t o x i c i t y tests for ke t o n i c solvents M E K , M i B K a n d i s o p h o r o n e
are sh o w n in T a b l e s 1 - 9 . T h e assays of M E K an d i s o p h o r o n e were n e g a t i v e an d did n o t d e m o n s t r a t e significant m u t a g e n i c activity. T h e results of the assays for M i B K were n o t as conclusive as those for M E K a n d i s o p h o r o n e . Clearly n e g a t i v e results were o b t a i n e d in the S a l m o n e l l a / m i c r o s o m e assay, the u n s c h e d u l e d D N A synthesis assay, and the m i c r o n u c l e u s assay c o n d u c t e d using M i B K . T h e m o u s e l y m p h o m a assay of M i B K was negative w h e n tested w i t h rat liver $9 m e t a b o l i c a c t i v a t i o n b u t resulted in e q u i v o c a l results w h e n tested at high c o n c e n t r a t i o n s w i t h o u t a c t i v a t i o n ( T a b l e 4). A second m o u s e l y m p h o m a study of M i B K was c o n d u c t e d using replicate cultures (Table 5) an d again the results were eq u i v o cal at the highest dose levels. T h e m u t a n t f r e q u e n c y seen after M i B K e x p o s u r e did n o t follow a dose-related response an d r e p e a t testing w i t h replicate cultures did n o t show a consistent positive effect ( T a b l e 5). T h e greatest r e s p o n s e to M i B K was o b s e r v e d at doses w h i ch resulted in 9 6 - 9 9 % lethality. Clive et al. (1979) h a v e suggested that doses which result in 9 0 - 1 0 0 % lethality are n o t r e l e v a n t in d e t e r m i n -
TABLE 1 SALMONELLA/MICROSOME (AMES) ASSAY RESULTS OF METHYL ETHYL KETONE: REVERTANTS/PLATE Salmonella strain
$9 activation
TA98 TA98
+ --
TA100 TA100
+ -
TA1535 TA1535
DMSO control (50 lal/plate)
MEK concentration (/xl/plate) 0.05
0.1
0.5
2
16
32
1239+104e 1240 + 34 f
29 + 3
37+ 8 21 _+ 6
42+ 8 26 _+ 6 b
27_+ 8b 22 ~- 5 b
31_+ lb 21 q- 5 b
115+ 5 115+12
1201-1-118g 1312+132 e
117+20 98--+17 120+13
97_+61 114-+ 8 b
127_+11 122-+10 b
107--+10b 125+_15 b
2+2 d
+ -
6+ 1 16-+ 3
241-+ 42 h 1040+155 g
11-+ 4 19-+ 6
9-+ 2 17+ 6 b
11-+ 1 15-+ 3 b
7-+ 3 b 17-+ 0 c
1+2 d
20-+ 4
TA1537 TA1537
+ -
6+ 3 4-+ 2
469-+ 24 h 358+153 i
2-+ 2 6-+ 2
5-+ 2 7-+ 4
7-+ 4 9-+ 2 b
5-+ 2 b 6-+ 1 b
1+0 d
7-+ 3
TA1538 TA1538
+ -
20-+ 5 18+ 4
1027-+ 21 e 1835-+ 74 f
15-+ 2 15-+ 3
18-+ 4 13-+ 3
20-+ 1 16-+ 4 b
21-+ 4 b 13-+ 3 b
14+7 a
19+ 5
a b c d e f g h i
34+ 2a 22_+ 9
Positive controls
~ + S.D. revertants/plate. All strains were plated in triplicate except for TA100, which had 4 plates. Background bacterial lawn slightly reduced. Background bacterial lawn moderately reduced. Background bacterial lawn extremely reduced. 2-Aminoanthracene 1.0 lag/plate. 4-Nitro-O-phenylenediamine 10 lag/plate. Sodium azide 5.0 lag/plate. 2-Aminoanthracene 4.0 lag/plate. 9-Aminoacridine 75 lag/plate.
0 d
154 TABLE 2 S A L M O N E L L A / M I C R O S O M E (AMES) ASSAY RESULTS O F M E T H Y L IS O B U TY L K E T O N E : R E V E R T A N T S / P L A T E Salmonella strain
$9 activation
TA98 TA98
+ -
TA100 TA100
+ -
TA1535 TA1535 b TA1535
DMSO control (50/xl/plate)
Positive controls
Mi B K concentration (# 1/plate) 0.04
0.1
0.4
1
4
1106_+ 17 d 1797_+259 e
35_+ 3 20_+ 4
39_+ 9 20-+ 8
47_+ 3 23-+ 6
33_+11 19-t- 7 ~
31+10 c 17+ 1 c
125_+17 129-+24
2798_+580 d 2173-+ 57 f
117_+15 121-+15
148_+16 130-+ 7
141_+20 114+16
108+19 ¢ 155+24 c
116 + 13 c 116+16 c
+ + -
9-+ 3 10-+ 2 26-+ 2
219-+ 20 g 164-+ 9 g 1036-+ 83 f
15-+ 9 12-+ 3 24-+ 8
18+ 6 8+ 2 27_+ 6
23+ 7 9_+ 2 24_+ 1
22+ 4 ¢ 12+ 3 c 26+ 5 ~
10+ 5 c
TA1537 TA1537
+ -
7-+ 2 6-+ 1
226-+ 54 g 1066+135 h
14+ 1 5_+ 1
6-+ 3 4_+ 2
5_+ 3 7_+ 2
9-+ 4 7_+ 3 c
TA1538 Ta1538
+ -
23_+ 2 17-+ 2
757_+ 53 d 2125+127 e
31-+ 3 15-+ 4
25_+ 7 17+ 4
19+ 5 16-+ 6
35_+ 2 ~ 24_+ 6
20_+ 3 16_+ 4
12+ 2 c 23+ 5 ~ 7+ 6+
5~ 1~
29+ 5 ~ 17_+ 4 c
a ~ + S.D. revertants/plate. All strains were plated in triplicate except for TA100, which had 4 plates, TA1538 without $9 activation which had 8 plates and the replicate assay on TA1535 with activation which had 5 plates. b The TA1535 cultures with activation were reassayed to further examine a marginal response in the first assay. c Background bacterial lawn slightly reduced. d 2-Aminoanthracene 1.0 # g / p l a t e . e 4-Nitro-O-phenylenediamine 10 # g / p l a t e . f Sodium azide 5.0 # g / p l a t e . g 2-Aminoanthracene 4.0 # g / p l a t e . h 9-Aminoacridine 75 /~g/plate. TABLE 3 L 5 1 7 8 Y / T K + / - M O U S E L Y M P H O M A ASSAY OF M E T H Y L E T H Y L K E T O N E MEK
Without metabolic activation
conc. (~ l / m l )
Mean mut. colonies/plate
Mutant freq. (//10 4 cells)
With rat liver $9 fraction Total growth (%)
Mean rout. colonies/plate
Mut a nt freq. ( / 1 0 4 cells)
Total growth (%)
12 8.9 6.7 5.0 3.8 2.8 2.1 1.6 1.2 0.89 0.67
52 46 39 35 47 45 41 47 41 54 -
0.6 0.5 0.4 0.4 0.5 0.5 0.4 0.5 0.5 0.6 -
46 65 89 84 96 113 122 112 111 122 -
49 43 62 49 51 56 54 67 59 60
0.6 0.4 0.7 0.5 0.5 0.7 0.7 0.7 0.6 0.7
0 30 116 89 78 100 100 97 112 104 98
Control(DMSO)
52
0.5
100
52
0.7
100
49.3 11.9
0 14
198 193
6.6 3.3
13 52
Ethyl methanesulfonate ( # l / m l ) 1.0 90 0.5 315 7,12-Dimethylbenz[ a ]anthracene (/~g/ml) 7.5 5.0 -
-
At concentrations of 16 and 21 # l / m l , there was no cell survival. These data represents the means of 3 counts from single cultures except for the controls which are the means of 2 cultures.
155 TABLE 4 L5178Y/TK + / - MOUSE LYMPHOMA ASSAY OF METHYL ISOBUTYL K E T O N E MiBK
Without metabolic activation
conc. (/.t 1/ml)
Mean mutant colonies/plate
4.2 3.2 2.4 1.8 1.3 1.0 0.75 0.56 0.42 0.32
136 82 62 82 61 76 74 70 45 49
Control (DMSO)
42
Ethyl methanesulfonate (/~l/ml) 1.0 184 0.5 _
MiBK
With rat liver $9 fraction
Total growth (%)
conc. (/.t l / m l )
Mean mutant colonies/plate
1.9 * 0.8 * 0.6 0.8 * 0.5 0.6 0.6 0.'l 0.2 0.5
3 31 52 58 86 90 95 81 157 87
4.2 3.2 2.4 1.8 1.3 1.0 0.75 0.56 0.42 0.32
69 70 69 69 71 66 78 62 66 68
0.6 0.6 0.6 0.6 a 0.6 0.7 0.5 0.6 0.6
23 33 59 84 _ 92 78 95 95 85
0.4
100
Control (DMSO)
59
0.5
100
15.2 a
6 -
7,12-Dimethylbenz[a]anthracene (/~g/ml) 7.5 213 9.0 5.0 168 6.6
9 18
Mutant freq. ( / 1 0 4 cells)
Mutant freq. ( / 1 0 4 cells)
Total growth (%)
a Culture lost. * Significant increase in mutant frequency; 2-fold control level. MiBK concentration of 5.6 and 7.5/xl/ml resulted in no net cell growth in activated and non-activated cultures. These data represent the means of 3 counts from single cultures except for the controls which are the means of 2 cultures.
TABLE 5 REPEAT L5178Y/TK + / - MOUSE LYMPHOMA ASSAY OF METHYL ISOBUTYL K E T O N E MiBK
Without metabolic activation
conc. (/~ 1/ml)
Mean mutant colonies/plate
Mutant freq. (/104 cells)
3.7 3.7 2.9 2.9 2.1 2.1 1.4 1.4 0.6 0.6
70 43 56 52 46 47 43 42 41 43
28.0 3.4 1.3 1.6 1.9 1.2 1.1 1.2 1.0 0.8
Control (DMSO)
43
0.8
100
4.4 8.3
4 24
Ethyl methanesulfonate (~ 1/ml) 1.0 40 0.5 174
* *
Total growth (%)
* *
1 4 42 32 31 51 60 46 68 80
MiBK
With rat liver S9 fraction
conc. (/L1/ml)
Mean mutant colonies/plate
3.4 3.4 3.0 3.0 2.5 2.5 1.9 1.9 1.4 1.4
64 61 40 57 43 49 48 48 52 52
Control (DMSO)
48
Mutant freq. ( / 1 0 4 cells)
Total growth (%)
0.8 1.0 1.4 1.1 1.0 1.2 1.1
35 37 28 34 51 47 62
1.3
63 _
0.9
100
7,12-Dimethylbenz[ a ]anthracene ( ~ g / m l ) 7.5 163 15.5 5.0 120 4.2
10 43
a
--
a
a Culture lost. * Significant increase in mutant frequency; 2-fold control level. MiBK concentrations of 4.5 and 5.2 ~tl/ml in non-activated cultures and 4 # l / m l in activated cultures resulted in no net cell growth. These data represent the means of 3 counts from single cultures except for the controls which are the means of 2 cultures.
156 TABLE 6 L5178Y/TK +/- MOUSE LYMPHOMA ASSAY OF ISOPHORONE Isophorone conc. (/~l/ml)
Without metabolic activation Mean mutant Mutant colonies/plate freq. (/10 4 cells)
Total growth (%)
Isophorone conc. (t~1/ml)
With rat liver $9 fraction Mean mutant colonies/plate
Mutant freq. (/104 cells)
Total growth (%)
1.3 1.0 0.75 0.56 0.42 0.32 0.24 0.18 0.13
54 73 54 55 60 54 54 58 42
1.1 1.1 0.7 0.7 0.7 0.8 0.7 0.8 0.6
12 44 73 77 94 84 90 111 110
0.89 0.67 0.50 0.38 0.28 0.21 0.16 0.12 0.089
64 47 66 56 49 49 51 56 60
1.0 0.6 1.0 0.8 0.8 0.6 0.7 0.8 0.9
9 28 25 42 49 81 75 66 75
Control (DMSO)
39 -
0.6 -
100 -
0.067 Control (DMSO)
50 51
0.7 0.7
86 100
24.0 8.8
2 31
7,12-Dimethylbenz[a]anthracene (/~g/ml) 7.5 200 5.2 5.0 178 3.9
24 45
Ethyl methanesulfonate (#l/ml) 1.0 120 0.5 292
In non-activated cultures, isophorone concentrations of 1.8 and 2.4/~l/ml produced 100% lethality. In activated cultures, isophorone concentrations of 1.2 and 1.6/~l/ml produced 100% lethality. These data represent the means of 3 counts from single cultures except for the controls which are means of 2 cultures.
ing significant mutagenicity. If doses resulting in greater t h a n 90% lethality are n o t considered, then the few r e m a i n i n g increases were n o t c o n c e n t r a tion d e p e n d e n t a n d the results w o u l d b e considered negative. Of further i m p o r t a n c e in assessing these results, is the v a r i a t i o n a m o n g the D M S O c o n t r o l cultures for the 8 m o u s e l y m p h o m a assays; the range o f m u t a n t frequencies was 0 . 4 - 0 . 9 / 1 0 4 survivors, a m o r e t h a n 2-fold difference. Thus, n o r m a l v a r i a t i o n in the c o n t r o l m u t a n t frequency m a y alter the i n t e r p r e t a t i o n a n d significance of the m u t a t i o n response. T h e m u t a n t frequencies in the c o n t r o l cultures were w i t h i n n o r m a l historical ranges for the l a b o r a t o r y . A b sence of M i B K - r e l a t e d increases o u t s i d e the negative c o n t r o l range p r o v i d e s a d d i t i o n a l evidence for a negative c o n c l u s i o n for m u t a g e n i c p o t e n t i a l . T h e response o f B A L B / 3 T 3 cells to M i B K was also ambiguous. I n the first assay, an M i B K conc e n t r a t i o n of 4 . 8 / ~ l / m l resulted in 3 t y p e I I I foci in 15 dishes ( T a b l e 9). This n u m b e r o f t y p e I I I foci, c o u p l e d with a r e d u c e d cloning efficiency, resulted in a positive statistical analysis in the n o n - a c t i v a t e d system. W i t h m e t a b o l i c activation,
there was n o t r a n s f o r m i n g activity. O n a r e p e a t assay, M i B K at a c o n c e n t r a t i o n of 5 / ~ l / m l resulted in 2 t y p e I I I foci for 15 p l a t e s with 100% cell survival. T h e resulting t r a n s f o r m a t i o n f r e q u e n c y was n o t statistically i n c r e a s e d over the negative control. Thus, the results of the first B A L B / 3 T 3 a s s a y of M i B K , in the a b s e n c e of m e t a b o l i c activity, c o u l d n o t b e c o n f i r m e d . O t h e r d a t a a p p e a r to s u p p o r t the conclusions d r a w n f r o m these studies; that M E K , M i B K , a n d i s o p h o r o n e have relatively low p o t e n t i a l s for genotoxicity. Z i m m e r m a n n et al. (1985) i n c l u d e d M E K in a series o f solvents s t u d i e d for g e n o t o x i c activity in Saccharomyces cerevisiae strain D61.M. I n this testing p a r a d i g m , M E K was f o u n d to ind u c e a n e u p l o i d y , b u t the r e s p o n s e was s t r o n g o n l y if relatively high c o n c e n t r a t i o n s (3.54%) of M E K were used, M E K was p r e s e n t in the cultures continuously, a n d cultures were held in ice u n d e r c o n d i t i o n s which w o u l d i n h i b i t r e a s s e m b l y of m i t o t i c s p i n d l e tubulin. T h e r e was n o evidence t h a t M E K resulted in p o i n t m u t a t i o n s o r m i t o t i c r e c o m b i n a t i o n s , suggesting that the o b s e r v e d effects were n o t d u e to an i n t e r a c t i o n b e t w e e n M E K
157 TABLE 7 RESULTS OF THE U N S C H E D U L E D D N A SYNTHESIS ASSAYS Treatment (Itl/ml)
RS a
Avg. net grains/nucleus ( X ± S.D.) b
Methyl ethyl ketone 5.0 2.5 1.0 0.5 0.1 DMSO 20.0 It g / m l 2-AAF 2.0 It g / m l 2-AAF
12.7 37.3 71.1 74.7 87.3
1.6+2.3 0.4+1.3 -0.2+1.1 -0.1+1.5 0.5 + 1.6
100.0
-0.2+1.2
25.2 43.6
25.0 ± 5.0 21.3 _+4.9
Methyl isobutyl ketone 100 10 1.0 0.10 0.010
0 1.7 28.5 85.7 88.4
DMSO
100.0
0.6 _+0.2
61.6 65.4
58.6 ± 0.9 49.2 _+0.8
20.0 It g / m l 2-AAF 2.0 It g / m l 2-AAF
Too toxic to count Too toxic to count 1.9 ± 0.4 0.9±0.2 1.2 _+0.2
Isophorone 0.40 0.20 0.10 0.05 0.01 0.005
0 38.4 42.5 60.0 75.9 100.0
Too toxic to count 1.4 _+1.9 0.7 _+1.4 0.1 ± 1.2 0.2± 1.6 - 0.3 _+1.2
EtOH
100.0
- 0.1 _+1.0
35.9 45.0
25.5 ± 7.3 19.4±4.6
20.0 I t g / m l 2-AAF 2.0 I t g / m l 2-AAF
a Relative survival = avg. viable cells/tube cells plated/tube = avg. viable cells/solvent control tube × 100. cells plated/solvent control tube b 100 nuclei were counted at each dose level.
and genetic material, but rather were due to effects on tubulin assembly resulting in chromosomal malsegregation. Under these conditions, the significance of chromosomal malsegregation in Saccharomyces cerevisiae for assessing genotoxicity in man or other species in unclear. M E K has not been observed to interfere with tubulin polymerization in mammalian cells or tissues which should be sensitive indicators of such
an effect. Agutter and Mack (1979) studied the effects of M E K (1 mM) on the ability of sheep brain tubulin to polymerize and depolymerize in vitro and found no effect due to MEK. It might also be postulated that if M E K were to alter tubulin polymerization or depolymerization in mammalian cells, that growth and differentiation of mitotically active cells should be altered. Selkoe et al. (1978) studied the effects of M E K and other ketones on the cytotoxicity and growth of the murine neuroblastoma cell line. At M E K concentrations of up to 0.5%, cells proliferated well and differentiated normally. Cavender et al. (1983) examined the subchronic toxicity of M E K after exposures of Fischer 344 rats to airborne concentrations of up to 5000 ppm for 90 days and found only minor effects on body weight, liver weight, and liver enzyme levels. Significantly, no effects were observed in the testes, bone marrow, or gastrointestinal tract which are mitotically active tissues that might be expected to be adversely affected by an inhibitor of tubulin polymerization. The metabolism of M E K and M i B K (DiVincenzo et al., 1976) proceeds by pathways which would be unlikely to result in genotoxic metabolites. Both materials undergo oxidation to their respective hydroxy ketones, 3-hydroxy-2-butanone and 4-hydroxy-4-methyl-2-pentanone and to a lesser extent undergo reduction to their respective alcohols, 2-butanol and 4-methyl-2-pentanol. M E K is also metabolized to 2,3-butanediol. The parent compounds and their metabolites are rapidly cleared from the serum within 16 h (DiVincenzo et al., 1976). Subchronic toxicity associated with M E K and MiBK exposure is slight. Cavender et al. (1983) found no specific organ toxicity, and only mild adaptive change, in Fischer 344 rats after inhalation exposure to 5000 ppm M E K for 90 days. Similarly, organ effects after MiBK exposure were mild. Exposure of Fischer 344 rats and B6C3F t mice to atmospheres of 1000 ppm MiBK for 14 weeks resulted in increased liver weights in both species and the presence of hyalin droplets in the kidneys of male rats (Phillips et al., 1987). The National Toxicology Program (NTP, 1986) completed a series of studies on the genotoxicity, chronic toxicity, and carcinogen±city of isophorone. Genotoxicity was examined in a series of
158 TABLE 8 M I C R O N U C L E A T E D P O L Y C H R O M A T I C E R Y T H R O C Y T E S IN BONE M A R R O W CELLS F R O M RATS T R E A T E D W I T H K E T O N I C SOLVENTS Treatment
Sex
Time (h)
N u m b e r of mice
Proportion of PCE
Micronucleated polychromatic erythrocytes Number/PCEs scored
Range per animal
N u m b e r / 1 0 0 0 PCEs (mean +- S.D.)
Methyl ethyl ketone 1.96 m l / k g
M F M F M F
12 12 24 24 48 48
5 5 5 5 5 5
0.47 0.47 0.47 0.47 0.46 0.46
8/5000 8/5000 9/5000 14/5000 8/5000 6/5000
000100-
3 4 4 4 4 3
1.6+_ 1.6_+ 1.8+2.8_+ 1.6_+ 1.2_+
1.1 1.8 1.6 1.3 1.5 1.1
Cornoil 10 m l / k g
M F M F M F
12 12 24 24 48 48
5 5 5 5 5 5
0.47 0.48 0.47 0.48 0.47 0.47
4/5000 10/5000 5/5000 5/5000 8/5000 13/5000
010001-
2 3 2 2 4 4
0.8_+ 2.0+1.0_+ 1.0_+ 1.6+ 2.6+
0.8 1.0 1.0 0.7 1.7 1.1
TEM 0.25 m g / k g
M F
24 24
5 5
0.47 0.46
173/5000 124/5 000
24-50 11-43
34.6_+ 9.8 * 24.8 _+12.2 *
Methyl isobutyl ketone 0.73 m l / k g
M F M F M F
12 12 24 24 48 48
5 5 5 5 5 5
0.47 0.48 0.46 0.47 0.46 0.47
7 / 5 000 5/4000 4/5000 7 / 5 000 10/5000 10/5000
000100-
4 2 2 2 4 4
1.4 +1.3+ 0.8+_ 1.4 _+ 2.0+2.0-+
1.7 1.0 0.8 0.6 1.9 1.6
Corn oil 10 m l / k g
M F M F M F
12 12 24 24 48 48
5 5 5 5 5 5
0.47 0.48 0.47 0.48 0.47 0.47
4/5000 10/5000 5 / 5 000 5/5000 8 / 5 000 13/5000
010001-
2 3 2 2 4 4
0.8+2.0+1.0_+ 1.0+ 1.6 +2.6_+
0.8 1.0 1.0 0.7 1.7 1.1
TEM 0.25 m g / k g
M F
24 24
5 5
0.47 0.47
173/5000 124/5 000
0.54 m l / k g
M F M F M F
12 12 24 24 48 48
5 5 5 5 5 5
0.50 0.51 0.50 0.50 0.51 0.52
7/5000 6/5000 2/5000 4/5000 7/5000 6/5000
100011-
2 3 1 2 2 3
1.4_+ 1.2_+ 0.4_+ 0.8_+ 1.4_+ 1.2_+
0.5 1.3 0.5 1.1 0.5 1.3
Corn oil
M F M F M F
12 12 24 24 48 48
5 5 5 5 5 5
0.51 0.51 0.50 0.51 0.50 0.51
3/5000 6/5000 4/5000 4/5000 4/5000 6/5000
000000-
1 3 2 2 1 2
0.6_+ 1.2+ 0.8_+ 0.8+0.8+1.2+
0.5 1.3 1.1 0.8 0.4 1.1
TEM 0.25 m g / k g
M F
24 24
5 5
0.50 0.52
188/5000 177/5000
24-50 11-43
34.6_+ 9.8 * 24.8 + 12.2 *
Isophorone
31-43 30-40
37.6_+ 4.3 * 35.4+- 3.7 *
Dose levels were equal to the LD20 for each ketone and are reported as ml test article/kg body weight when administered in a total volume of 10 ml test article-vehicle m i x t u r e / k g body weight. * P ~< 0.01, Student's t test.
159 TABLE9 TRANSFORMATION POTENTIAL OF KETONIC SOLVENTS USING BALB/3T3 CLONE A31-1 CELLS Treatment (/.tl/ml)
Total foci/total dishes Type II Type III
Transformation frequency ( x l 0 -4)
Relative cloning efficiency (%)
18 13 9
0/15 2/15 1/14
2/15 0/15 1/14
0.58 < 0.19 0.17
51 80 93
PBS control
1/15
0/15
< 0.15
100
0.5 ~ g / m l M N N G
7/15
19/15
Methylethylketone
12.67 *
22
Methyl ethyl ketone with metabolic activation 10 8 6 PBS control
1/15 0/14 0/15 2/15
0/15 0/14 0/15 0/15
< < < <
0.20 0.19 0.16 0.14
67 76 86 100
12.5 ~g/ml BaP
8/15
12/15
3.08 *
53
4.8 3.6 2.4
0/15 0/15 1/15
3/15 3/15 2/15
0.87 * 0.56 0.34
51 80 87
PBS control
1/15
0/15
0.5 ~ g / m l M N N G
7/15
19/15
Methyl ~obu~l ketone
< 0.15 12.67 *
100 22
Methyl isobutyl ketone (repeat assay) 7 6 5 4
1/15 2/15 0/15 0/14
0/15 0/15 2/15 0/14
To~c < 0.95 0.33 < 0.19
<1 17 100 93
PBS control
0/14
0/14
< 0.17
100
0.5 ~ g / m l M N N G
5/15
11/15
5.64 *
32
Methyl~obu~lkewne with metaboficactivatwn 4 2 1
1/14 0/13 1/14
0/14 1/13 1/14
< 0.22 0.21 0.17
66 76 84
PBS control
2/15
0/15
< 0/14
100
12.5 ~ g / m l B a P
8/15
12/15
3.08 *
53
Methyl isobutyl ketone with metabolic activation (repeat assay) 5 4 3 2
0/15 1/15 1/15 0/15
1/15 1/15 4/15 1/15
0.18 0.16 0.59 0.14
75 82 88 96
PBS control
0/14
0/14
< 0.14
100
12.5 ~ g / m l B a P
0/15
5/15
1.23 *
53
* P < 0.01, modified Poisson distribution. Relative cloning efficiency is reported as the number of colonies per treatment condition relative to 100% survival in the solvent control. number of type III loci Transformation frequency 10 000 cells x cloning efficiency × dishes scored"
160 studies including Ames assays with and without $9 from Aroclor 1254-induced rat or hamster livers, M L assays without metabolic activation, assays for sister-chromatid exchange in Chinese hamster ovary ( C H O ) cells with and without $9 from Aroclor 1254-induced rat livers, and in assays to detect induction of c h r o m o s o m a l aberrations in C H O cells with and without rat liver $9. Although negative in most of the assays, weak mutagenic activity was present in the M L assay and sister-chromatid exchanges were induced in C H O cells in the absence of $9. The reasons for the differing results when the N T P M L assay is c o m p a r e d to the present study are unclear. Both assays were conducted at similar isophorone concentrations, and other factors in the protocols including the carrier solvent were similar, but the results were not reproducible between the laboratories. 3-year exposure of Fischer 3 4 4 / N rats and B6C3F 1 mice to 500 m g / k g of isophorone in corn oil resulted in decreased survival of male rats after 96 weeks of exposure and slight nephrotoxicity in male rats (National Toxicology Program, 1986). There was no evidence of carcinogenicity in female animals of either species. The responses in male animals were classified as ' s o m e evidence of carcinogenicity' for male rats and 'equivocal evidence of carcinogenicity' for male mice. The male rat responses included increased renal tumors in rats given 250 or 500 m g / k g isophorone and a low incidence of preputial gland tumors in males given 500 m g / k g . The responses in male mice included liver tumors and mesenchymal tumors in the integumentary system in mice given 500 m g / k g and malignant l y m p h o m a s in mice given 250 m g / k g , although the increased incidence of l y m p h o m a s was marginal. Overall, the data suggest that exposure to M E K , MiBK, and isophorone are unlikely to result in significant toxicity. The data for M E K do not indicate any genotoxic activity. The presence of a marginal response only at the highest cytotoxic concentration tested in the mouse l y m p h o m a assay and the lack of reproducibility in the B A L B 3T3 transformation assay, and clearly negative results in the Ames, UDS, and M N assays suggest that M i B K is unlikely to be genotoxic in m a m malian systems. The weight-of-the-evidence on
isophorone genotoxicity would also suggest that it is unlikely to be a genotoxic hazard for mammals. The only positive indicator of genotoxicity was the induction of sister-chromatid exchanges in C H O cells. The results of the mouse l y m p h o m a assays without metabolic activation while negative in the present study were positive in the N T P study and thus were not reproducible between laboratories. All other assays of isophorone for genotoxic activity were negative. These included the Ames, mouse l y m p h o m a with metabolic activation, CT, MN, sister-chromatid exchange in C H O cells with metabolic activation, and c h r o m o s o m a l aberration in C H O cells assays.
References Agutter, P.S., and A.P. Mack (1979) The effects of afl-dicarbonyl compounds on the polymerization of sheep brain tubulin in vitro, Biochem. Soc. Trans., 7, 691-693. Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/ mammalian-microsome mutagenicity test, Mutation Res., 31, 347-364. Cavender, F.L., H.W. Casey, H. Salem, J.A. Swenburg and E.J. Gralla (1983) A 90-day vapor inhalation toxicity study of methyl ethyl ketone, Fundam. AppL Toxicol., 3, 264-270. Chemical Manufacturers Association Ketones Program Panel Report to the U.S. Environmental Protection Agency, October 21, 1981. Clive, D., and J.F.S. Spector (1975) Laboratory procedure for assessing specific locus mutations at the TK locus in cultured L5178Y mouse lymphoma cells, Mutation Res., 31, 17-29. Clive, D., K.O. Johnson, J.F.S. Spector, A.G. Batson and M.M.M. Brown (1979) Validation and characterization of the L5178Y/TK +/- mouse lymphoma mutagen assay system, Mutation Res., 59, 61-108. DiVincenzo, G.D., C.J. Kaplan and J. Dedinas (1976) Characterization of the metabolites of methyl n-butyl ketone, methyl isobutyl ketone, and methyl ethyl ketone in guinea pig serum and their clearance, Toxicol. Appl. Pharmacol., 36,511-522. Kakunaga, T. (1973) A quantitative system for assay of malignant transformation by chemical carcinogens using a clone derived from BALB/3T3, Int. J. Cancer, 12, 463-473. National Toxicology Program (1982) Mutagenicity results on isophorone, Dept. of Health and Human Services, Washington, DC, p. 1-6. National Toxicology Program (1986) Toxicology and carcinogenesis studies of isophorone in F344/N rats and B6C3Fl mice, NIH Publ. No. 86-2547, Washington, DC. Phillips, R.D., E.J. Moran, D.E. Dodd, E.H. Fowler, C.D. Kary and J.L. O'Donoghue (1987) A 14-week vapor inhalation toxicity study of methyl isobutyl ketone, Fundam. Appl. Toxicol., 9, 380-388.
161 berger (1973) Quantitative and qualitative studies of chemical transformation of cloned C3H mouse embryo cells sensitive to post-confluence inhibition of cell division, Cancer Res., 33, 3239-3249. Selkoe, D.J., L. Luckenbill-Edds and M.L. Shelanski (1978) Effects of neurotoxic industrial solvents on cultured neuroblastoma cells: methyl n-butyl ketone, n-hexane and derivatives, J. Neuropathol. Exp. Neurol., 36, 768-789. Shirasu, Y., M. Moriya, K. Kato, A. Furyhashi and T. Kada (1976) Mutagenicity screening of pesticides in the microbial system, Mutation Res., 40, 19-30. Williams, G.M. (1977) Carcinogen-induced DNA repair in primary rat liver cell cultures, a possible screen for chemical-carcinogens, Cancer Lett., 1, 231-237.
Williams, G.M. (1979) The detection of chemical mutagens/ carcinogens by DNA repair and mutagenesis in liver cultures, in: F.J. de Serres and A. Hollaender (Eds.), Chemical Mutagens, Plenum, New York, pp. 71-79. Yahagi, T., M. Nagao, Y. Seino, T. Matsushima, T. Sugimura and M. Okada (1977) Mutagenicities of N-nitrosamines on Salmonella, Mutation Res., 48, 121-130. Zimmermann, F.K., V.W. Mayer, I. Scheel and M.A. Resnick (1985) Acetone, methyl ethyl ketone, ethyl acetate, acetonitrile and other polar aprotic solvents are strong inducers of aneuploidy in Saccharomyces cereoisiae, Mutation Res., 149, 339-351.