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An evaluation of genotoxicity tests with musk ketone
Pergamon
Food and Chemical Toxicology 34 (1996) 633-638
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An Evaluation of Genotoxicity Tests with Musk Ketone A. M . A P I * , E. A. P F I T Z E R * a n d R. H. C. S A N t *Research Institute for Fragrance Materials, Inc., 2 University Plaza Drive, Suite 406, Haekensack, NJ 07601, USA and tMicrobiological Associates, Inc., 9900 Blackwell Road, Rockville, MD 20850, USA (Accepted I Februa O' 1996)
Abstract--Musk ketone, a synthetic musk fragrance ingredient that has been found in river water, fish and breast milk, was evaluated for potential genotoxicity in a battery of short-term tests. The mouse lymphoma assay was conducted at musk ketone concentrations ranging from 700 to 4000 pg/ml and 2.0 to 35 pg/ml in the absence and presence of rat liver S-9, respectively. No increased mutant frequencies were noted. An in vitro cytogenetics assay in Chinese hamster ovary cells was conducted at musk ketone concentrations ranging from 4.3 to 34 pg/ml and 1.25 to 10/tg/ml in the absence and presence of rat liver S-9, respectively. On the basis of the non-reproducibility of a statistically significant increase at a single concentration and no increases in other test systems, musk ketone was concluded to be negative for chromosome aberrations. An in vitro unscheduled DNA synthesis (UDS) assay was conducted in primary rat hepatocytes at musk ketone concentrations between 0.5 and 50/~g/ml. No increases in net nuclear grain counts were noted. Musk ketone did not show genotoxic potential based on the negative results in the mouse lymphoma, in vitro cytogenetics and in vitro UDS assays. Copyright (V) 1996 Elsevier Science Ltd.
INTRODUCTION Musk ketone (3,5-dinitro-2,6-dimethyl-4-tert-butylacetophenone; CAS no. 81-14-1) is a synthetic musk fragrance ingredient used both as a fixative and because of its scent. It is one of a number of structurally related nitro musks that are reported to occur in the environment, including river water, fish, and human milk (Eschke et al., 1994; Geyer et al., 1994; Liebl and Ehrenstorfer, 1993; Rimkus et al., 1994; Rimkus and Wolf, 1993 and 1993; Yamagishi et al., 1981 and 19'83). Musk ketone has been reported to be negative in Ames tests conducted on Salmonella typhimurium strains TA97, TA98, TA100, TA1535, TA1537 and TA1538 with and without metabolic activation (Givaudan Corporation, private communication, 1980; Zeiger et al., 1988). Musk ketone was not found to be mutagenic under any of the conditions of these two studies. In this current study further evaluation of the genotoxic potential of musk ketone was conducted in a mouse lymphoma assay, an in vitro cytogenetics assay in Chinese hamster ovary (CHO) cells and an in vitro Abbreviations: CHO=Chinese hamster ovary; C P = cyclophosphamide; DMBA = 7,12-dimethylbenz[a]anthracene; DMSO =: dimethyl sulfoxide; EMS = ethyl methanesulfonate; TEM = triethylenemelamine; TFT = trifluorothymidine; TK = thymidine kinase; UDS = unscheduled DNA synthesis.
unscheduled D N A synthesis (UDS) assay in primary rat hepatocytes. MATERIALS AND METHODS
Musk ketone was supplied by Firmenich Incorporated (Princeton, N J, USA). The material was more than 99% pure and was from lot no. 65659.9305. It was a pale yellowish platelet or crystalline powder, and was stored at 2-6°C and protected from exposure to light. All studies reported here were conducted at Microbiological Associates Inc., Rockville, MD, USA. M o u s e lymphoma assay
Musk ketone was tested for its ability to induce a forward mutation from the T K + / - to the TK-/genotype at the thymidine kinase (TK) locus of L5178 mouse lymphoma cells (clone 3.7.2C). The mouse lymphoma cells were obtained directly from Dr Donald Clive, Burroughs Wellcome Company, Research Triangle Park, NC, USA. The mutation assay was performed according to a protocol described by Clive and Spector (1975). The solvent for musk ketone was acetone and for the positive controls dimethyl sulfoxide (DMSO) (Aldrich). The positive controls were ethyl methanesulfonate (EMS) in the absence of metabolic activation and 7,12-
0278-6915/96/$15.00 + 0.00 Copyright © 1996 Elsevier Science Ltd. All rights reserved. Printed in Great Britain PH S0278-6915(96)00028-2 FCT M/7+-D
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A.M. Api et al.
dimethylbenz[a]anthracene (DMBA) (Eastman Kodak Chemical Company, Rochester, NY, USA) in the presence of metabolic activation. Metabolic activation of the culture was obtained with S-9, namely the supernatant (9000 g) of a 2:1 mixture of Aroclor-1242 and Aroclor-1254-induced liver homogenate, obtained from adult male Sprague-Dawley rats, in sucrose. Preliminary tests were conducted with cells exposed (in the presence and absence of metabolic activation) to the test article, positive control or solvent for 4 hr at 37 + I°C to determine optimal growth rates and cytotoxicity. On the basis of cell population growth relative to the solvent controls from the preliminary tests, the optimal concentration ranges selected for musk ketone were 700-4000 pg/ml in the absence of activation and 2.0-35 #g/ml in the presence of activation. EMS was tested at 0.25 and 0.50 pl/ml and DMBA was tested at 2.5 and 5.0 #g/ ml. Mutant frequencies were expressed as mutants/ 106 surviving cells. They were calculated as ratios between the number of colonies from medium containing trifluorothymidine (TFT) and those without TFT. The mutation frequency for the acetone solvent represents the spontaneous mutations for the culture. Total growth (expressed as a percent of control) was calculated from the growth rate during the period for expression of the mutant phenotype multiplied by the cloning growth rate after incubation in cloning medium without TFT. Dose levels yielding total growth (relative survival) values less than 10% were not evaluated because the changes that occur at such highly toxic concentrations are not considered biologically relevant. Chromosome aberrations in CHO cells using multiple harvest times CHO (CHO-K~ cells, American Type Culture Collection, Rockville, MD, USA) were seeded at approximately 3 x 105 cells/25cm 2 flask (24-hr harvest) or 1 x 105 cells/25 cm 2 flask (48-hr harvest), and were incubated at 37 _+ UC for 16-24 hr. Aroclor 1254-induced rat liver S-9 was used as the metabolic activation system. Musk ketone was dissolved in acetone. Preliminary toxicity tests consisting of determination of cloning efficiency after treatment with musk ketone solution relative to solvent control were performed for the purpose of selecting dose levels. Triethylenemelamine (TEM) (Polysciences, Inc., Warrenton, PA, USA) was used as the positive control in the non-activated study at a final concentration of 0.25/~g/ml in deionized, distilled water. Cyclophosphamide (CP) (Sigma Chemical Company, St Louis, MO, USA) was used as the positive control in the S-9 activated study at a final concentration of 50#g/ml in deionized, distilled water. An untreated control consisting of cells in complete medium or S-9 reaction mixture was also included.
On the basis of the results of the preliminary toxicity tests, test concentrations were selected so that the high dose gave approximately 50% reduction in cloning efficiency relative to that of the solvent control. Dose levels of 4.3, 8.5, 17 and 34 #g/ml were selected for the non-activated portion of the assay. Dose levels of 1.25, 2.5, 5 and 10/~g/ml were selected for the activated portion of the assay. A confirmatory assay was conducted for the activated portion of the assay to verify effects seen at the high dose in the 24-hr harvest. The confirmatory assay was conducted at dose levels of 8, 10, 12 and 14 #g/ml for the 24-hr harvest only. In the non-activated study, the cells were continuously exposed to treatment in the incubator at 37 + I°C prior to harvest at approximately 24 or 48 hr. In the S-9 activated study, the cells were exposed to treatment in the incubator for 4 hr at 37 _+ I°C. After the exposure period, the treatment medium was removed, the cells washed with calcium and magnesium-free phosphate buffered saline, re-fed with complete medium and returned to the incubator until harvest at approximately 24 or 48 hr. 2 hr prior to the scheduled cell harvest, Colcemid was added to duplicate flasks for each treatment condition (activated and non-activated studies) at a final concentration of 0.1 pg/ml and the flasks incubated for an additional 2 hr. The metaphase cells were then harvested for both the non-activated and S-9 activated studies by trypsinization. Cells were collected approximately 24 and 48 hr after initiation of treatment. Metaphase cells with 20 + 2 centromeres were examined under oil immersion without prior knowledge of treatment groups. Whenever possible, a minimum of 200 metaphase spreads (100/duplicate flask) were examined and scored for chromatid-type and chromosome-type aberrations. Chromatid and isochromatid gaps were recorded but not included in the analysis. Polyploidy was evaluated as the percentage of polyploid cells/100 metaphase cells counted. The mitotic index was recorded as the percentage of cells in mitosis/500 cells counted. In vitro UDS in rat primary hepatocytes Primary rat hepatocytes derived from the livers of normal adult male Fischer 344 rats (Harlan Sprague Dawley, Inc., Frederick, MD, USA) were used in this study. The procedure used for obtaining rat hepatocyte cultures was essentially that of Williams (1976). A preliminary cytotoxicity test was performed to establish an appropriate dose range for musk ketone. Acetone, which was used to dissolve the musk ketone, was used as the solvent control. The positive control, DMBA, was dissolved in DMSO, used at 3.0 and 10/~g/ml, but scored at a single concentration of 3/~g/ml. DMSO was used as the solvent control for the positive control. Three replicate dishes/dose level were seeded with 5 x l0 s rat hepatocytes/dish and were treated with
Genotoxicity tests with musk ketone 0.05-150 #g/ml of test article. Acetone, which was used to dissolve the test article, was used as the solvent control for the test article. Media controls consisted of cultures treated only with serum-free Williams' medium E. Each test article and control dish received 3H-thy:aaidine at a final concentration of 10 /~Ci/ml. In parallel with the test dishes, two cultures/dilution were treated with the test article and control compounds for a parallel toxicity test. The cells were treated for 18-20 hr. The parallel toxicity dishes were harvested by removal of a portion of the medium for lactate dehydrogenase determinations to obtain the relative survivals and relative toxicities as compared with those of the solvent control. The slides were scored without prior knowledge of treatment. Nuclear grains were counted in 50 cells in random areas on each of three coverslips/treatment where possible. The net nuclear counts were determined by counting three nucleus-sized areas adjacent to each nucleus and subtracting the average cytoplasmic count from the nuclear count. Nuclei in replicative DNA synthesis (completely blackened with grains) and nuclei exhibiting toxic effects of treatment (such as dark or uneven staining, disrupted membranes, or irregtdar shape) were not counted. RESULTS
Mouse lymphoma assay
Table 1 shows a dose-related increase in cytotoxicity with musk ketone (as measured by total growth as a percent of control) in the presence of S-9 metabolic activation. ;Some cytotoxicity was observed in the absence of S-9 metabolic activation, although
Table 1. L5178Y TK-~-/- mouse lymphoma cells treated with musk ketone
Treatment No S-9 activation Musk ketone (,ug/rrl) 35 30 25 20 10 Acetone DMBA (l~g/ml) 5 2.5 No S-9 activation Musk ketone (/tg/ml) 4000 3000 2000 1000 700 Acetone EMS (/~g/ml) 0.5 0.25
Total growth (%)"
Mutant frequency ( x l&) b
24 40 73 86 102 100
38 33 26 33 22 33
6 59
491 22 I
77 55 63 61 53 100
22 24 24 27 19 22
32 67
808 395
"Percent total growth (relative survival) reflects toxicity of test articles relative to that of the solvent control. bMutant frequency is the ratio of mutant colonies (resistant to TFT) to viable ceU colonies/106 surviving cells.
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the profile was not definitively dose related. None of the cultures treated with musk ketone and cloned in the presence or absence of S-9 metabolic activation exhibited a mutant frequency that was at least twice the mean mutant frequency of that of the solvent controls. The acetone solvent control and the positive controls gave expected results to validate the assay. Chromosome aberrations in CHO cells
The activity of musk ketone in the induction of chromosome aberrations in CHO cells when treated in the presence or absence of metabolic activation is presented in Table 2 (five of the 10 concentrations studied are shown as representative of the range of results). The positive and negative controls fulfilled the requirements for a valid test. Cytotoxicity based on percent relative cloning efficiency was satisfactory to provide adequate metaphase cells for examination of chromosomal aberrations. Musk ketone at the dose level of 10.0~tg/ml showed a statistically significant increase (12%) in structural aberrations, but not numerical aberrations, only in the presence of S-9 metabolic activation at the 24-hr harvest. In an attempt to confirm this observation a confirmatory assay was run at dose levels ranging from 8.0 to 14.0/lg/ml; the results are shown in Table 3. There was no significant increase in structural or numerical aberrations in the confirmatory assay. On the basis of the overall findings and the non-reproducibility at a single dose level, it was concluded that musk ketone is negative in the mammalian cytogenetic assay using CHO cells. In vitro UDS assay
The results from the UDS assay are summarized in Table 4. After an examination of the fixed and stained cells treated with 150 #g/ml musk ketone, it was decided that UDS could not be evaluated at this dose level because of excessive toxicity. The next five lower dose levels were evaluated despite some dose-related toxicity. The positive control, DMBA, at 3.0 ~g/ml, induced a significant increase in the average net nuclear count of silver grains. All criteria for a valid test were met. Musk ketone did not cause a significant increase in UDS as measured by the mean number of net nuclear grain counts (i.e. an increase of at least five counts over the solvent control) at any dose level. Therefore, musk ketone was considered to be negative in this study. DISCUSSION
Because of the reports of musk ketone in the environment and the potential for bioaccumulation, it was considered necessary to assess the potential for genotoxicity as part of the overall safety evaluation. On the basis of the findings of these studies, musk ketone was concluded to be negative in a mouse lymphoma assay, an in vitro cytogenetics assay in CHO cells and an in vitro UDS assay in primary rat
A . M . Api et al.
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Table 2. Cytogenetic analysis (CHO cells) with musk ketone Cells with aberrations (%) Treatment
Mitotic index
S-9 activation--24 hr harvest Untreated Acetone Musk ketone (/~g/ml) 1.25 2.5 5.0 10.0 CP (/~g/ml) 50.0 S-9 activationS8 hr harvest Untreated Acetone Musk ketone (pg/ml) 1.25 2.5 5.0 10.0 CP (itg/ml) 50.0 No S-9 activation--24 hr harvest Untreated Acetone Musk ketone (l~g/ml) 4.3 8.5 17.0 34.0 TEM (,ug/ml) 0.25 No S-9 activationS8 hr harvest Untreated Acetone Musk ketone (,ug/ml) 4.3 8.5 17.0 34.0 TEM (,ug/ml) 0.25
Aberrations/ cell" (mean ± SD)
Numerical
Structural
8.7 9.2
0.040 ± 0.196 0.030 + 0.171
0.5 0.5
4.0 3.0
7.8 8.8 8.1 5.7
0.040 + 0.015 + 0.040 + 0.140 +
0.221 0.122 0.281 0.414
2.0 1.0 1.5 2.0
3.5 1.5 2.5 12.0"
8.8
0.950 ± 1.448
2.0
48.0"
10.4 9.5
0.010 ± 0.100 0.040 + 0.242
0.5 1.0
1.0 3.0
8.9 9.8 10.7 9.4
0.015 ± 0.025 ± 0.045 + 0.050 ±
0.158 0.211 0.252 0.260
1.5 0.5 2.0 1.5
1.0 1.5 3.5 4.0
8.1
0.130 ± 0.494
2.5
7.5"
5.7 6.0
0.005 +_ 0.071 0.015 ± 0.158
0.0 1.0
0.5 1.0
5.4 5.6 4.8 3.0
0.005 ± 0.005 ± 0.020 ± 0.005 +
0.071 0.071 0.140 0.071
0.5 1.5 2.0 1.5
0.5 0.5 2.0 0.5
4.6
0.865 ± 1.247
0.0
39.5*
5.1 6.0
0.015 _+0.158 0.005 ± 0.071
0.5 1.0
1.0 0.5
5.2 6.6 2.9 3.3
0.025 ± 0.020 ± 0.025 ± 0.030 ±
0.211 0.172 0.186 0.264
0.5 1.5 1.5 2.0
1.5 1.5 2.0 1.5
4.8
2.165 + 2.439
2.5
60.5*
"Severely damaged cells were counted as 10 aberrations. "P ~<0.025, one-sided Fisher's exact test. h e p a t o c y t e s . T h e s e findings, c o m b i n e d w i t h the negative results in the A m e s tests ( G i v a u d a n C o r p o r a t i o n , p r i v a t e c o m m u n i c a t i o n , 1980: Zeiger et al., 1988), leads to the c o n c l u s i o n t h a t m u s k k e t o n e h a s n o significant potential to act as a g e n o t o x i c carcinogen. It h a s been e s t i m a t e d t h a t if a c o n s u m e r used all p r o d u c t types c o n t a i n i n g m u s k k e t o n e at m a x i m u m c o n c e n t r a t i o n s a n d w i t h extensive freq u e n c y o f use, the m a x i m u m e x p o s u r e w o u l d be 0.25 m g / k g / d a y ( R I F M , u n p u b l i s h e d data, 1994). G i v e n the low d e r m a l a b s o r p t i o n , less t h a n 0 . 5 % after 6 h r b y h u m a n s u n d e r s i m u l a t e d use c o n d i t i o n s
(D. R. H a w k i n s , D. K i r k p a t r i c k , P. W. Scott, C. W. F i n n , B. C o n w a y a n d M. E. F o r r e s t , u n p u b l i s h e d data, 1984), the expected systemic e x p o s u r e to m u s k k e t o n e as it is u s e d in f r a g r a n c e s is e s t i m a t e d to be less t h a n 0,0013 m g / k g . A 90-day s u b c h r o n i c d e r m a l toxicity s t u d y in rats at d o s e levels o f 7.5, 24, 75 a n d 240 m g / k g / d a y ( F o r d et al., 1990) resulted in a n o - o b s e r v e d - e f f e c t level o f 75 m g / k g / d a y . T h e only significant effect d u e to m u s k k e t o n e w a s a n increase in liver w e i g h t at the highest d o s e o f 240 m g / k g / d a y , a l t h o u g h this increase w a s n o t associated with a n y p a t h o l o g i c a l findings. T h e results o f this in vivo toxicity s t u d y are p a r t i c u l a r l y relevant in view o f the
Table 3. Confirmatory assay: cytogenetic analysis (CHO cells) with musk ketone (S-9 activation) (24-hr harvest) Cells with aberrations (%) Treatment Untreated Acetone Musk ketone (l~g/ml) 8.0 10.0 12.0 14.0 CP (/~g/ml) 25.0
Mitotic index
Aberrations/ cell~ (mean + SD)
Numerical
Structural
8.5 6.2
0.035 + 0.232 0.020 ± 0.172
2.5 2.5
2.5 1.5
6.3 6.9 5.5 8.6
0.040 _+0.221 0.030 ± 0.171 0.055 + 0.269 0.025 + 0.157
6.0 6.0 5.5 8.0
3.5 3.0 4.5 2.5
7.3
0.180 __ 0.819
4.5
11.5"
~Severely damaged cells were counted as 10 aberrations. "P ~<0.025, one-sided Fisher's exact test.
Genotoxicity tests with musk ketone
637
Table 4. In vitro UDS Assay with musk ketone Relative Net nuclear Cells with five or survival" grain counts more net Treatment (%)" (mean + SD) nuclear grains (%) Musk ketone (/~g/ml) 150 47 Toxic -50 52 -0.9 __. 1.9 0 15 55 0.2 +_ 1.9 1 5.0 89 -0.6 _+2.1 0 1.5 94 -0.6 + 1.9 0 0.5 89 -0.6 + 2.3 1 ]:)MBA (,ug/ml) 3.0 89 38.7 + 12.7 97 DMSO~ (/~l/ml) 10.0 100 -0.8 _+2.3 I Acetone¢ (/~l/ml) 10.0 100 -0.5 _+2.4 1 WME 104 -0.7 _+2.0 1 WME = Williams' medium E control "Relativesurvival was based on comparison of lactate dehydrogenaserelease from hepatocyte cultures as a measure of cytotoxicity. bSolvent control for DMBA. ~Solvent control for musk ketone.
m a r k e d increase in cytotoxicity observed with the L5178 m o u s e l y m p h o m a cells with metabolic activation. A n analogue c o m p o u n d , namely m u s k xylene, t h a t c o n t a i n s a third nitro g r o u p r a t h e r t h a n the ketone g r o u p o f m u s k ketone has been reported to cause a n increase in t u m o u r s in mice ( M a e k a w a et al., 1990). Following a d m i n i s t r z t i o n in the diet o f B6C3F~ mice at levels o f 0.075 or I).15% for 18 m o n t h s , increased incidences o f hepatocellular a d e n o m a s a n d carcin o m a s in male mice a n d o f a d e n o m a s in female mice were observed. N o genotoxic potential was observed with m u s k xylene in a battery of tests (Api et al., 1995). F u r t h e r m o r e , it has been s h o w n t h a t m u s k xylene is b o t h a n inhibitor a n d a n inducer o f C Y P 2 B enzymes (as well as a weak inducer o f C Y P 1 A enzymes) when administered orally to mice at the dose k n o w n to cause liver t u m o u r s ( L e h m a n M c K e e m a n et al., 1995). Despite being a n inhibitor o f C Y P 2 B enzymes, rausk xylene caused general liver changes, including a significant increase in liver weight, hepatocellulzLr h y p e r t r o p h y a n d induction o f reduced nicotinamide adenine dinucleotide phosp h a t e / N A D P H c y t o c h r o m e c reductase. These results lead Lo the consideration that m u s k xylene m a y cause liver t u m o u r s by a non-genotoxic m e c h a n i s m similar to t h a t observed with p h e n o b a r b i tal, a n d t h a t the effect is of little relevance to humans. In addition, it has been reported (N. J. T h a t c h e r a n d J. Caldwell, 10th International S y m p o s i u m o n Microsomes a n d D r u g Oxidations, T o r o n t o , 1821 July 1994) t h a t the induction o f C Y P 1 A enzymes at dietary doses of 0.1)45 a n d 0.15% o f m u s k xylene was reversible within 14 days a n d t h a t there was n o i n d u c t i o n observed aL doses o f 0.015%. Moreover, it is significant t h a t b o t h m u s k xylene a n d m u s k k e t o n e gave negative results with strain TA100 in the A m e s assay. The n i t r o a r o m a t i c c o m p o u n d 2,6-dinitrotoluene was hepatocarcinogenic in rats a n d positive in strain TA100, but n o t in nitroreductase-
deficient TA100 (Rickert et al., 1984). It was established t h a t bacterial nitroreductase in T A 100 (or in intestinal microflora) was necessary to metabolize the nitro g r o u p o n the ring to a n a m i n o group, a n d t h a t this was not accomplished by m a m m a l i a n nitroreductase from S-9 mix (or in rat hepatocytes). Thus, the negative effects in TA100 support the consideration that musk xylene carcinogenicity was not related to metabolism by nitroreductase. These considerations o f the analogue, musk xylene, a n d the negative observations for m u s k ketone o f the current study s u p p o r t the conclusion that m u s k ketone has n o significant potential for genotoxic or carcinogenic liability u n d e r current conditions of exposure to humans. REFERENCES
Api A. M., Ford R. A. and San R. H. C. (1995) An evaluation of musk xylene in a battery of genotoxicity tests. Food and Chemical Toxicology 33, 1039-1045. Clive D. and Spector J. F. S. (1975) Laboratory procedure for assessing specific locus mutations at the TK locus in cultured L5178Y mouse lymphoma cells. Mutation Research 31, 7-29. Eschke H., Traud J. and Dibowski H. (1994) Artificial nitromusk substances in surface water, wastewater and fish of the Ruhr Basin--analysis and results. Vom Wasser 83, 1-18. Ford R. A., Api A. M. and Newberne P. M. (1990) 90-Day dermal toxicity study and neurotoxicity evaluation of nitromusks in the albino rat. Food and Chemical Toxicology 28, 55-61. Geyer H. J., Rimkus G., Wolf M., Attar A., Steinberg C. and Kettrup A. (1994) Synthetische nitromoschus-duftstoffe und bromocyclen. Umweltwissenschaften und Schadstoff-Forschung: Zeitschrift fuer Umweltchemie Oekotoxikologie 6, 9-17. Lehman-McKeeman L. D., Caudill D., Young J. A. and Dierckman T. A. (1995) Musk xylene induces and inhibits mouse hepatic cytochrome P-450 2B enzymes. Biochemical and Biophysical Research Communications 206, 975-980. Liebl B. and Ehrenstorfer S. (1993) Nitro musks in human milk. Chemosphere 27, 2253-2260.
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A. M. Api et al.
Maekawa A., Matsushima Y., Onodera H., Shibutani M., Ogasawara H., Kodama Y. and Kuroka Y. (1990) Long-term toxicity/carcinogenicity of musk xylol in B6C3F~ mice. Food and Chemical Toxicology 28, 581-586. Rickert D. E., Butterworth B. E. and Popp J. A. (1984) Dinitrotoluene: acute toxicity, oncogenicity, genotoxicity and metabolism. CRC Critical Reviews in Toxicology 13, 217-234. Rimkus G., Rimkus B. and Wolf M. (1994) Nitro musks in human adipose tissue and breast milk. Chemosphere 28, 421-432. Rimkus G. and Wolf M. (1993) Contaminants in fish from aquaculture. Part 2: musk xylene and musk ketone contaminants in fish. Deutsche Lebensmittel-Rundschau 89, 171-175. Rimkus G. G. and Wolf M. (1995) Nitro musk fragrances in biota from freshwater and marine environment. Chemosphere 30, 641-651.
Williams G. M. (1976) Carcinogen-induced DNA repair in primary rat liver cell cultures, a possible screen for chemical-carcinogens. Cancer Letters 1, 231-235. Yamagishi T., Miyazaki T., Horii S. and Kaneko S. (1981) Identification of musk xylene and musk ketone in freshwater fish collected from the Tama River, Tokyo. Bulletin of Environmental Contamination and Toxicology 26, 656-662. Yamagishi T., Miyazaki T., Horii S. and Akiyama K. (1983) Synthetic musk residues in biota and water from Tama River and Tokyo Bay (Japan). Archives of Environmental Contamination and Toxicology 12, 83-89. Zeiger E., Anderson B., Haworth S., Lawlor T. and Mortelmans K. (1988) Salmonella mutagenicity tests: IV. Results from the testing of 300 chemicals. Environmental and Molecular Mutagenesis 11, Suppl. 12, 1-158.
Food and Chemical Toxicology 34 (1996) 633-638
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An Evaluation of Genotoxicity Tests with Musk Ketone A. M . A P I * , E. A. P F I T Z E R * a n d R. H. C. S A N t *Research Institute for Fragrance Materials, Inc., 2 University Plaza Drive, Suite 406, Haekensack, NJ 07601, USA and tMicrobiological Associates, Inc., 9900 Blackwell Road, Rockville, MD 20850, USA (Accepted I Februa O' 1996)
Abstract--Musk ketone, a synthetic musk fragrance ingredient that has been found in river water, fish and breast milk, was evaluated for potential genotoxicity in a battery of short-term tests. The mouse lymphoma assay was conducted at musk ketone concentrations ranging from 700 to 4000 pg/ml and 2.0 to 35 pg/ml in the absence and presence of rat liver S-9, respectively. No increased mutant frequencies were noted. An in vitro cytogenetics assay in Chinese hamster ovary cells was conducted at musk ketone concentrations ranging from 4.3 to 34 pg/ml and 1.25 to 10/tg/ml in the absence and presence of rat liver S-9, respectively. On the basis of the non-reproducibility of a statistically significant increase at a single concentration and no increases in other test systems, musk ketone was concluded to be negative for chromosome aberrations. An in vitro unscheduled DNA synthesis (UDS) assay was conducted in primary rat hepatocytes at musk ketone concentrations between 0.5 and 50/~g/ml. No increases in net nuclear grain counts were noted. Musk ketone did not show genotoxic potential based on the negative results in the mouse lymphoma, in vitro cytogenetics and in vitro UDS assays. Copyright (V) 1996 Elsevier Science Ltd.
INTRODUCTION Musk ketone (3,5-dinitro-2,6-dimethyl-4-tert-butylacetophenone; CAS no. 81-14-1) is a synthetic musk fragrance ingredient used both as a fixative and because of its scent. It is one of a number of structurally related nitro musks that are reported to occur in the environment, including river water, fish, and human milk (Eschke et al., 1994; Geyer et al., 1994; Liebl and Ehrenstorfer, 1993; Rimkus et al., 1994; Rimkus and Wolf, 1993 and 1993; Yamagishi et al., 1981 and 19'83). Musk ketone has been reported to be negative in Ames tests conducted on Salmonella typhimurium strains TA97, TA98, TA100, TA1535, TA1537 and TA1538 with and without metabolic activation (Givaudan Corporation, private communication, 1980; Zeiger et al., 1988). Musk ketone was not found to be mutagenic under any of the conditions of these two studies. In this current study further evaluation of the genotoxic potential of musk ketone was conducted in a mouse lymphoma assay, an in vitro cytogenetics assay in Chinese hamster ovary (CHO) cells and an in vitro Abbreviations: CHO=Chinese hamster ovary; C P = cyclophosphamide; DMBA = 7,12-dimethylbenz[a]anthracene; DMSO =: dimethyl sulfoxide; EMS = ethyl methanesulfonate; TEM = triethylenemelamine; TFT = trifluorothymidine; TK = thymidine kinase; UDS = unscheduled DNA synthesis.
unscheduled D N A synthesis (UDS) assay in primary rat hepatocytes. MATERIALS AND METHODS
Musk ketone was supplied by Firmenich Incorporated (Princeton, N J, USA). The material was more than 99% pure and was from lot no. 65659.9305. It was a pale yellowish platelet or crystalline powder, and was stored at 2-6°C and protected from exposure to light. All studies reported here were conducted at Microbiological Associates Inc., Rockville, MD, USA. M o u s e lymphoma assay
Musk ketone was tested for its ability to induce a forward mutation from the T K + / - to the TK-/genotype at the thymidine kinase (TK) locus of L5178 mouse lymphoma cells (clone 3.7.2C). The mouse lymphoma cells were obtained directly from Dr Donald Clive, Burroughs Wellcome Company, Research Triangle Park, NC, USA. The mutation assay was performed according to a protocol described by Clive and Spector (1975). The solvent for musk ketone was acetone and for the positive controls dimethyl sulfoxide (DMSO) (Aldrich). The positive controls were ethyl methanesulfonate (EMS) in the absence of metabolic activation and 7,12-
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dimethylbenz[a]anthracene (DMBA) (Eastman Kodak Chemical Company, Rochester, NY, USA) in the presence of metabolic activation. Metabolic activation of the culture was obtained with S-9, namely the supernatant (9000 g) of a 2:1 mixture of Aroclor-1242 and Aroclor-1254-induced liver homogenate, obtained from adult male Sprague-Dawley rats, in sucrose. Preliminary tests were conducted with cells exposed (in the presence and absence of metabolic activation) to the test article, positive control or solvent for 4 hr at 37 + I°C to determine optimal growth rates and cytotoxicity. On the basis of cell population growth relative to the solvent controls from the preliminary tests, the optimal concentration ranges selected for musk ketone were 700-4000 pg/ml in the absence of activation and 2.0-35 #g/ml in the presence of activation. EMS was tested at 0.25 and 0.50 pl/ml and DMBA was tested at 2.5 and 5.0 #g/ ml. Mutant frequencies were expressed as mutants/ 106 surviving cells. They were calculated as ratios between the number of colonies from medium containing trifluorothymidine (TFT) and those without TFT. The mutation frequency for the acetone solvent represents the spontaneous mutations for the culture. Total growth (expressed as a percent of control) was calculated from the growth rate during the period for expression of the mutant phenotype multiplied by the cloning growth rate after incubation in cloning medium without TFT. Dose levels yielding total growth (relative survival) values less than 10% were not evaluated because the changes that occur at such highly toxic concentrations are not considered biologically relevant. Chromosome aberrations in CHO cells using multiple harvest times CHO (CHO-K~ cells, American Type Culture Collection, Rockville, MD, USA) were seeded at approximately 3 x 105 cells/25cm 2 flask (24-hr harvest) or 1 x 105 cells/25 cm 2 flask (48-hr harvest), and were incubated at 37 _+ UC for 16-24 hr. Aroclor 1254-induced rat liver S-9 was used as the metabolic activation system. Musk ketone was dissolved in acetone. Preliminary toxicity tests consisting of determination of cloning efficiency after treatment with musk ketone solution relative to solvent control were performed for the purpose of selecting dose levels. Triethylenemelamine (TEM) (Polysciences, Inc., Warrenton, PA, USA) was used as the positive control in the non-activated study at a final concentration of 0.25/~g/ml in deionized, distilled water. Cyclophosphamide (CP) (Sigma Chemical Company, St Louis, MO, USA) was used as the positive control in the S-9 activated study at a final concentration of 50#g/ml in deionized, distilled water. An untreated control consisting of cells in complete medium or S-9 reaction mixture was also included.
On the basis of the results of the preliminary toxicity tests, test concentrations were selected so that the high dose gave approximately 50% reduction in cloning efficiency relative to that of the solvent control. Dose levels of 4.3, 8.5, 17 and 34 #g/ml were selected for the non-activated portion of the assay. Dose levels of 1.25, 2.5, 5 and 10/~g/ml were selected for the activated portion of the assay. A confirmatory assay was conducted for the activated portion of the assay to verify effects seen at the high dose in the 24-hr harvest. The confirmatory assay was conducted at dose levels of 8, 10, 12 and 14 #g/ml for the 24-hr harvest only. In the non-activated study, the cells were continuously exposed to treatment in the incubator at 37 + I°C prior to harvest at approximately 24 or 48 hr. In the S-9 activated study, the cells were exposed to treatment in the incubator for 4 hr at 37 _+ I°C. After the exposure period, the treatment medium was removed, the cells washed with calcium and magnesium-free phosphate buffered saline, re-fed with complete medium and returned to the incubator until harvest at approximately 24 or 48 hr. 2 hr prior to the scheduled cell harvest, Colcemid was added to duplicate flasks for each treatment condition (activated and non-activated studies) at a final concentration of 0.1 pg/ml and the flasks incubated for an additional 2 hr. The metaphase cells were then harvested for both the non-activated and S-9 activated studies by trypsinization. Cells were collected approximately 24 and 48 hr after initiation of treatment. Metaphase cells with 20 + 2 centromeres were examined under oil immersion without prior knowledge of treatment groups. Whenever possible, a minimum of 200 metaphase spreads (100/duplicate flask) were examined and scored for chromatid-type and chromosome-type aberrations. Chromatid and isochromatid gaps were recorded but not included in the analysis. Polyploidy was evaluated as the percentage of polyploid cells/100 metaphase cells counted. The mitotic index was recorded as the percentage of cells in mitosis/500 cells counted. In vitro UDS in rat primary hepatocytes Primary rat hepatocytes derived from the livers of normal adult male Fischer 344 rats (Harlan Sprague Dawley, Inc., Frederick, MD, USA) were used in this study. The procedure used for obtaining rat hepatocyte cultures was essentially that of Williams (1976). A preliminary cytotoxicity test was performed to establish an appropriate dose range for musk ketone. Acetone, which was used to dissolve the musk ketone, was used as the solvent control. The positive control, DMBA, was dissolved in DMSO, used at 3.0 and 10/~g/ml, but scored at a single concentration of 3/~g/ml. DMSO was used as the solvent control for the positive control. Three replicate dishes/dose level were seeded with 5 x l0 s rat hepatocytes/dish and were treated with
Genotoxicity tests with musk ketone 0.05-150 #g/ml of test article. Acetone, which was used to dissolve the test article, was used as the solvent control for the test article. Media controls consisted of cultures treated only with serum-free Williams' medium E. Each test article and control dish received 3H-thy:aaidine at a final concentration of 10 /~Ci/ml. In parallel with the test dishes, two cultures/dilution were treated with the test article and control compounds for a parallel toxicity test. The cells were treated for 18-20 hr. The parallel toxicity dishes were harvested by removal of a portion of the medium for lactate dehydrogenase determinations to obtain the relative survivals and relative toxicities as compared with those of the solvent control. The slides were scored without prior knowledge of treatment. Nuclear grains were counted in 50 cells in random areas on each of three coverslips/treatment where possible. The net nuclear counts were determined by counting three nucleus-sized areas adjacent to each nucleus and subtracting the average cytoplasmic count from the nuclear count. Nuclei in replicative DNA synthesis (completely blackened with grains) and nuclei exhibiting toxic effects of treatment (such as dark or uneven staining, disrupted membranes, or irregtdar shape) were not counted. RESULTS
Mouse lymphoma assay
Table 1 shows a dose-related increase in cytotoxicity with musk ketone (as measured by total growth as a percent of control) in the presence of S-9 metabolic activation. ;Some cytotoxicity was observed in the absence of S-9 metabolic activation, although
Table 1. L5178Y TK-~-/- mouse lymphoma cells treated with musk ketone
Treatment No S-9 activation Musk ketone (,ug/rrl) 35 30 25 20 10 Acetone DMBA (l~g/ml) 5 2.5 No S-9 activation Musk ketone (/tg/ml) 4000 3000 2000 1000 700 Acetone EMS (/~g/ml) 0.5 0.25
Total growth (%)"
Mutant frequency ( x l&) b
24 40 73 86 102 100
38 33 26 33 22 33
6 59
491 22 I
77 55 63 61 53 100
22 24 24 27 19 22
32 67
808 395
"Percent total growth (relative survival) reflects toxicity of test articles relative to that of the solvent control. bMutant frequency is the ratio of mutant colonies (resistant to TFT) to viable ceU colonies/106 surviving cells.
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the profile was not definitively dose related. None of the cultures treated with musk ketone and cloned in the presence or absence of S-9 metabolic activation exhibited a mutant frequency that was at least twice the mean mutant frequency of that of the solvent controls. The acetone solvent control and the positive controls gave expected results to validate the assay. Chromosome aberrations in CHO cells
The activity of musk ketone in the induction of chromosome aberrations in CHO cells when treated in the presence or absence of metabolic activation is presented in Table 2 (five of the 10 concentrations studied are shown as representative of the range of results). The positive and negative controls fulfilled the requirements for a valid test. Cytotoxicity based on percent relative cloning efficiency was satisfactory to provide adequate metaphase cells for examination of chromosomal aberrations. Musk ketone at the dose level of 10.0~tg/ml showed a statistically significant increase (12%) in structural aberrations, but not numerical aberrations, only in the presence of S-9 metabolic activation at the 24-hr harvest. In an attempt to confirm this observation a confirmatory assay was run at dose levels ranging from 8.0 to 14.0/lg/ml; the results are shown in Table 3. There was no significant increase in structural or numerical aberrations in the confirmatory assay. On the basis of the overall findings and the non-reproducibility at a single dose level, it was concluded that musk ketone is negative in the mammalian cytogenetic assay using CHO cells. In vitro UDS assay
The results from the UDS assay are summarized in Table 4. After an examination of the fixed and stained cells treated with 150 #g/ml musk ketone, it was decided that UDS could not be evaluated at this dose level because of excessive toxicity. The next five lower dose levels were evaluated despite some dose-related toxicity. The positive control, DMBA, at 3.0 ~g/ml, induced a significant increase in the average net nuclear count of silver grains. All criteria for a valid test were met. Musk ketone did not cause a significant increase in UDS as measured by the mean number of net nuclear grain counts (i.e. an increase of at least five counts over the solvent control) at any dose level. Therefore, musk ketone was considered to be negative in this study. DISCUSSION
Because of the reports of musk ketone in the environment and the potential for bioaccumulation, it was considered necessary to assess the potential for genotoxicity as part of the overall safety evaluation. On the basis of the findings of these studies, musk ketone was concluded to be negative in a mouse lymphoma assay, an in vitro cytogenetics assay in CHO cells and an in vitro UDS assay in primary rat
A . M . Api et al.
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Table 2. Cytogenetic analysis (CHO cells) with musk ketone Cells with aberrations (%) Treatment
Mitotic index
S-9 activation--24 hr harvest Untreated Acetone Musk ketone (/~g/ml) 1.25 2.5 5.0 10.0 CP (/~g/ml) 50.0 S-9 activationS8 hr harvest Untreated Acetone Musk ketone (pg/ml) 1.25 2.5 5.0 10.0 CP (itg/ml) 50.0 No S-9 activation--24 hr harvest Untreated Acetone Musk ketone (l~g/ml) 4.3 8.5 17.0 34.0 TEM (,ug/ml) 0.25 No S-9 activationS8 hr harvest Untreated Acetone Musk ketone (,ug/ml) 4.3 8.5 17.0 34.0 TEM (,ug/ml) 0.25
Aberrations/ cell" (mean ± SD)
Numerical
Structural
8.7 9.2
0.040 ± 0.196 0.030 + 0.171
0.5 0.5
4.0 3.0
7.8 8.8 8.1 5.7
0.040 + 0.015 + 0.040 + 0.140 +
0.221 0.122 0.281 0.414
2.0 1.0 1.5 2.0
3.5 1.5 2.5 12.0"
8.8
0.950 ± 1.448
2.0
48.0"
10.4 9.5
0.010 ± 0.100 0.040 + 0.242
0.5 1.0
1.0 3.0
8.9 9.8 10.7 9.4
0.015 ± 0.025 ± 0.045 + 0.050 ±
0.158 0.211 0.252 0.260
1.5 0.5 2.0 1.5
1.0 1.5 3.5 4.0
8.1
0.130 ± 0.494
2.5
7.5"
5.7 6.0
0.005 +_ 0.071 0.015 ± 0.158
0.0 1.0
0.5 1.0
5.4 5.6 4.8 3.0
0.005 ± 0.005 ± 0.020 ± 0.005 +
0.071 0.071 0.140 0.071
0.5 1.5 2.0 1.5
0.5 0.5 2.0 0.5
4.6
0.865 ± 1.247
0.0
39.5*
5.1 6.0
0.015 _+0.158 0.005 ± 0.071
0.5 1.0
1.0 0.5
5.2 6.6 2.9 3.3
0.025 ± 0.020 ± 0.025 ± 0.030 ±
0.211 0.172 0.186 0.264
0.5 1.5 1.5 2.0
1.5 1.5 2.0 1.5
4.8
2.165 + 2.439
2.5
60.5*
"Severely damaged cells were counted as 10 aberrations. "P ~<0.025, one-sided Fisher's exact test. h e p a t o c y t e s . T h e s e findings, c o m b i n e d w i t h the negative results in the A m e s tests ( G i v a u d a n C o r p o r a t i o n , p r i v a t e c o m m u n i c a t i o n , 1980: Zeiger et al., 1988), leads to the c o n c l u s i o n t h a t m u s k k e t o n e h a s n o significant potential to act as a g e n o t o x i c carcinogen. It h a s been e s t i m a t e d t h a t if a c o n s u m e r used all p r o d u c t types c o n t a i n i n g m u s k k e t o n e at m a x i m u m c o n c e n t r a t i o n s a n d w i t h extensive freq u e n c y o f use, the m a x i m u m e x p o s u r e w o u l d be 0.25 m g / k g / d a y ( R I F M , u n p u b l i s h e d data, 1994). G i v e n the low d e r m a l a b s o r p t i o n , less t h a n 0 . 5 % after 6 h r b y h u m a n s u n d e r s i m u l a t e d use c o n d i t i o n s
(D. R. H a w k i n s , D. K i r k p a t r i c k , P. W. Scott, C. W. F i n n , B. C o n w a y a n d M. E. F o r r e s t , u n p u b l i s h e d data, 1984), the expected systemic e x p o s u r e to m u s k k e t o n e as it is u s e d in f r a g r a n c e s is e s t i m a t e d to be less t h a n 0,0013 m g / k g . A 90-day s u b c h r o n i c d e r m a l toxicity s t u d y in rats at d o s e levels o f 7.5, 24, 75 a n d 240 m g / k g / d a y ( F o r d et al., 1990) resulted in a n o - o b s e r v e d - e f f e c t level o f 75 m g / k g / d a y . T h e only significant effect d u e to m u s k k e t o n e w a s a n increase in liver w e i g h t at the highest d o s e o f 240 m g / k g / d a y , a l t h o u g h this increase w a s n o t associated with a n y p a t h o l o g i c a l findings. T h e results o f this in vivo toxicity s t u d y are p a r t i c u l a r l y relevant in view o f the
Table 3. Confirmatory assay: cytogenetic analysis (CHO cells) with musk ketone (S-9 activation) (24-hr harvest) Cells with aberrations (%) Treatment Untreated Acetone Musk ketone (l~g/ml) 8.0 10.0 12.0 14.0 CP (/~g/ml) 25.0
Mitotic index
Aberrations/ cell~ (mean + SD)
Numerical
Structural
8.5 6.2
0.035 + 0.232 0.020 ± 0.172
2.5 2.5
2.5 1.5
6.3 6.9 5.5 8.6
0.040 _+0.221 0.030 ± 0.171 0.055 + 0.269 0.025 + 0.157
6.0 6.0 5.5 8.0
3.5 3.0 4.5 2.5
7.3
0.180 __ 0.819
4.5
11.5"
~Severely damaged cells were counted as 10 aberrations. "P ~<0.025, one-sided Fisher's exact test.
Genotoxicity tests with musk ketone
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Table 4. In vitro UDS Assay with musk ketone Relative Net nuclear Cells with five or survival" grain counts more net Treatment (%)" (mean + SD) nuclear grains (%) Musk ketone (/~g/ml) 150 47 Toxic -50 52 -0.9 __. 1.9 0 15 55 0.2 +_ 1.9 1 5.0 89 -0.6 _+2.1 0 1.5 94 -0.6 + 1.9 0 0.5 89 -0.6 + 2.3 1 ]:)MBA (,ug/ml) 3.0 89 38.7 + 12.7 97 DMSO~ (/~l/ml) 10.0 100 -0.8 _+2.3 I Acetone¢ (/~l/ml) 10.0 100 -0.5 _+2.4 1 WME 104 -0.7 _+2.0 1 WME = Williams' medium E control "Relativesurvival was based on comparison of lactate dehydrogenaserelease from hepatocyte cultures as a measure of cytotoxicity. bSolvent control for DMBA. ~Solvent control for musk ketone.
m a r k e d increase in cytotoxicity observed with the L5178 m o u s e l y m p h o m a cells with metabolic activation. A n analogue c o m p o u n d , namely m u s k xylene, t h a t c o n t a i n s a third nitro g r o u p r a t h e r t h a n the ketone g r o u p o f m u s k ketone has been reported to cause a n increase in t u m o u r s in mice ( M a e k a w a et al., 1990). Following a d m i n i s t r z t i o n in the diet o f B6C3F~ mice at levels o f 0.075 or I).15% for 18 m o n t h s , increased incidences o f hepatocellular a d e n o m a s a n d carcin o m a s in male mice a n d o f a d e n o m a s in female mice were observed. N o genotoxic potential was observed with m u s k xylene in a battery of tests (Api et al., 1995). F u r t h e r m o r e , it has been s h o w n t h a t m u s k xylene is b o t h a n inhibitor a n d a n inducer o f C Y P 2 B enzymes (as well as a weak inducer o f C Y P 1 A enzymes) when administered orally to mice at the dose k n o w n to cause liver t u m o u r s ( L e h m a n M c K e e m a n et al., 1995). Despite being a n inhibitor o f C Y P 2 B enzymes, rausk xylene caused general liver changes, including a significant increase in liver weight, hepatocellulzLr h y p e r t r o p h y a n d induction o f reduced nicotinamide adenine dinucleotide phosp h a t e / N A D P H c y t o c h r o m e c reductase. These results lead Lo the consideration that m u s k xylene m a y cause liver t u m o u r s by a non-genotoxic m e c h a n i s m similar to t h a t observed with p h e n o b a r b i tal, a n d t h a t the effect is of little relevance to humans. In addition, it has been reported (N. J. T h a t c h e r a n d J. Caldwell, 10th International S y m p o s i u m o n Microsomes a n d D r u g Oxidations, T o r o n t o , 1821 July 1994) t h a t the induction o f C Y P 1 A enzymes at dietary doses of 0.1)45 a n d 0.15% o f m u s k xylene was reversible within 14 days a n d t h a t there was n o i n d u c t i o n observed aL doses o f 0.015%. Moreover, it is significant t h a t b o t h m u s k xylene a n d m u s k k e t o n e gave negative results with strain TA100 in the A m e s assay. The n i t r o a r o m a t i c c o m p o u n d 2,6-dinitrotoluene was hepatocarcinogenic in rats a n d positive in strain TA100, but n o t in nitroreductase-
deficient TA100 (Rickert et al., 1984). It was established t h a t bacterial nitroreductase in T A 100 (or in intestinal microflora) was necessary to metabolize the nitro g r o u p o n the ring to a n a m i n o group, a n d t h a t this was not accomplished by m a m m a l i a n nitroreductase from S-9 mix (or in rat hepatocytes). Thus, the negative effects in TA100 support the consideration that musk xylene carcinogenicity was not related to metabolism by nitroreductase. These considerations o f the analogue, musk xylene, a n d the negative observations for m u s k ketone o f the current study s u p p o r t the conclusion that m u s k ketone has n o significant potential for genotoxic or carcinogenic liability u n d e r current conditions of exposure to humans. REFERENCES
Api A. M., Ford R. A. and San R. H. C. (1995) An evaluation of musk xylene in a battery of genotoxicity tests. Food and Chemical Toxicology 33, 1039-1045. Clive D. and Spector J. F. S. (1975) Laboratory procedure for assessing specific locus mutations at the TK locus in cultured L5178Y mouse lymphoma cells. Mutation Research 31, 7-29. Eschke H., Traud J. and Dibowski H. (1994) Artificial nitromusk substances in surface water, wastewater and fish of the Ruhr Basin--analysis and results. Vom Wasser 83, 1-18. Ford R. A., Api A. M. and Newberne P. M. (1990) 90-Day dermal toxicity study and neurotoxicity evaluation of nitromusks in the albino rat. Food and Chemical Toxicology 28, 55-61. Geyer H. J., Rimkus G., Wolf M., Attar A., Steinberg C. and Kettrup A. (1994) Synthetische nitromoschus-duftstoffe und bromocyclen. Umweltwissenschaften und Schadstoff-Forschung: Zeitschrift fuer Umweltchemie Oekotoxikologie 6, 9-17. Lehman-McKeeman L. D., Caudill D., Young J. A. and Dierckman T. A. (1995) Musk xylene induces and inhibits mouse hepatic cytochrome P-450 2B enzymes. Biochemical and Biophysical Research Communications 206, 975-980. Liebl B. and Ehrenstorfer S. (1993) Nitro musks in human milk. Chemosphere 27, 2253-2260.
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Maekawa A., Matsushima Y., Onodera H., Shibutani M., Ogasawara H., Kodama Y. and Kuroka Y. (1990) Long-term toxicity/carcinogenicity of musk xylol in B6C3F~ mice. Food and Chemical Toxicology 28, 581-586. Rickert D. E., Butterworth B. E. and Popp J. A. (1984) Dinitrotoluene: acute toxicity, oncogenicity, genotoxicity and metabolism. CRC Critical Reviews in Toxicology 13, 217-234. Rimkus G., Rimkus B. and Wolf M. (1994) Nitro musks in human adipose tissue and breast milk. Chemosphere 28, 421-432. Rimkus G. and Wolf M. (1993) Contaminants in fish from aquaculture. Part 2: musk xylene and musk ketone contaminants in fish. Deutsche Lebensmittel-Rundschau 89, 171-175. Rimkus G. G. and Wolf M. (1995) Nitro musk fragrances in biota from freshwater and marine environment. Chemosphere 30, 641-651.
Williams G. M. (1976) Carcinogen-induced DNA repair in primary rat liver cell cultures, a possible screen for chemical-carcinogens. Cancer Letters 1, 231-235. Yamagishi T., Miyazaki T., Horii S. and Kaneko S. (1981) Identification of musk xylene and musk ketone in freshwater fish collected from the Tama River, Tokyo. Bulletin of Environmental Contamination and Toxicology 26, 656-662. Yamagishi T., Miyazaki T., Horii S. and Akiyama K. (1983) Synthetic musk residues in biota and water from Tama River and Tokyo Bay (Japan). Archives of Environmental Contamination and Toxicology 12, 83-89. Zeiger E., Anderson B., Haworth S., Lawlor T. and Mortelmans K. (1988) Salmonella mutagenicity tests: IV. Results from the testing of 300 chemicals. Environmental and Molecular Mutagenesis 11, Suppl. 12, 1-158.