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Lack of Mutagenicity of Polycyclic Musk Fragrances in Salmonella typhimurium
Toxicology in Vitro 12 (1998) 389±393
Lack of Mutagenicity of Polycyclic Musk Fragrances in Salmonella typhimurium V. MERSCH-SUNDERMANNO1*, S. KEVEKORDESO2 and C. JENTERO1
1
Institute of Medical Microbiology and Hygiene, Faculty of Clinical Medicine Mannheim, University of Heidelberg, D-68135, Mannheim and 2Medical Institute of General Hygiene and Environmental Health, University of GoÈttingen, Windausweg 2, D-37073 GoÈttingen, Germany (Accepted 7 December 1997)
AbstractÐSynthetic musks are widely used as fragrances and therefore found in various environmental samples. Residues were identi®ed in river and waste water, animal and human tissues and breast milk. In the present study, six arti®cial polycyclic musk fragrancesÐGalaxolide, Tonalide, Celestolide, Phantolide, Cashmeran and TraseolideÐwere tested for mutagenicity using the Salmonella/mammalianmicrosome assay with Salmonella typhimurium strains TA97, TA98, TA100 and TA102 in the presence (+S-9) and absence (ÿS-9) of an exogenous metabolizing system. All compounds tested exhibited no mutagenicity in the Salmonella assay. These results could be rated as one indicator of the biological inactivity of this group of chemicals with respect to genotoxicity. # 1998 Elsevier Science Ltd. All rights reserved Abbreviation: DMSO = dimethyl sulfoxide.
INTRODUCTION
Arti®cial fragrances such as the polycyclic musks Galaxolide, Tonalide, Celestolide, Phantolide, Cashmeran and Traseolide are used in perfumes, lotions and detergents, as food additives, in cigarettes and ®sh baits. In contrast to the synthetic nitro benzenoid musk ambrette, musk ketone and musk xylene (Ippen, 1994a,b; Mersch-Sundermann et al., 1996c) polycyclic musk fragrances are nonnitro benzenoid compounds. Eschke et al. (1994 and 1995a) identi®ed Galaxolide, Tonalide and Celestolide in bream and perch from the Ruhr River, Germany, at average concentrations between 2.5 mg/kg and 4.6 mg/kg fatty tissue. MuÈller et al. (1996) found Galaxolide (12±135 mg/kg) and Celestolide (0.12±3.5 mg/kg) in human fatty tissues. Both Galaxolide and Celestolide, were detected in all 15 samples examined. Eschke et al. (1995b) identi®ed polycyclic musks in the fat fraction of breast milk in average amounts of 335 mg/kg fat for Galaxolide, 270 mg/kg fat for Tonalide, 22 mg/kg fat for Celestoide, 14 mg/ kg fat for Phantolide and 18 mg/kg fat for Traseolide. Cashmeran could not be detected. *Author for correspondence at: Institute of Medical Microbiology and Hygiene, Faculty of Clinical Medicine Mannheim, University of Heidelberg, PO Box 100023, D-68135 Mannheim, Germany.
Rimkus and Wolf (1996) identi®ed similar amounts of residues in human fat tissues and breast milk. Under consideration of the fact that polycyclic musk fragrances are bioaccumulating compounds (Eschke et al., 1995a,b) it is necessary to estimate health risks. Therefore, in the present study the mutagenic potency of six widely used polycyclic musks was examined using the Salmonella/mammalian microsome assay with S. typhimurium strains TA97, TA98, TA100 and TA100 in the presence (+S-9) and absence (ÿS-9) of an exogenous metabolizing system.
MATERIALS AND METHODS
Galaxolide (1,3, 4, 6, 7, 8-hexa -hydro - 4, 6, 6, 7, 8, 8hexamethyl-cyclopenta-(g)-2-benzopyrane; CAS no. 1222-05-5), Tonalide (7-acetyl-1,1,3,4,4,6-hexamethyltetraline; CAS no. 1506-02-1), Celestolide (4-acetyl-1,1-dimethyl-6-tert-butylindane; CAS no. 13171-00-1), Phantolide (6-acetyl-1,1,2,3,3,5-hexamethylindane; CAS no. 15323-35-0), Cashmeran (6,7-dihydro-1,1,2,3,3-pentamethyl-4-(5H) indanone; CAS no. 33704-61-9) and Traseolide (5-acetyl1,1,2,6-tetramethyl-3-isopropylindane; CAS no. 68140-48-7) were from Promochem (Wesel, Germany). The structures of the compounds are given in Fig. 1. 9000-g fractions (S-9) from the livers of Arochlor 1254-treated male Sprague±Dawley rat were
0887-2333/98/$19.00+0.00 # 1998 Elsevier Science Ltd. All rights reserved. Printed in Great Britain PII: S0887-2333(98)00007-1
390
V. Mersch-Sundermann et al.
Fig. 1. Chemical structures, names and CAS numbers of polycyclic musk fragrances tested for mutagenicity in Salmonella typhimurium TA97, TA98, TA100 and TA102.
obtained from Organon Teknika (Heidelberg, Germany). All media and reagents used for the Salmonella assay were described by Maron and Ames (1983) and Mersch-Sundermann and KraÈmer (1993). All ingredients used for the preparation of the exogenous metabolizing system are described by Maron and Ames (1983). The S. typhimurium His+ strains TA97, TA98, TA100 and TA102 were kindly provided by Bruce N. Ames, Berkeley, CA, USA. Cultures were grown from frozen stocks (ÿ808C) in Oxoid nutrient broth no.2. The polycyclic musks were dissolved in dimethyl sulfoxide (DMSO) to 5 mg/ml. Then the compounds were diluted down to 50 mg/ml. A range of ®ve concentrations between 500 mg and 5.0 mg/plate was analysed. The maximum concentration was chosen considering the limit of solubility of the compounds in the aqueous medium of the assays. The Salmonella mutagenicity test procedure was performed as a plate incorporation assay. As positive controls we used 2,4,7-trinitro-9-¯uorenone (TA97, TA98, ÿS-9), sodium azide (TA100, ÿS-9) methyl methanesulfonate (TA102, ÿS-9) and 2-amino¯uorene (all strains +S-9). To show the lack of mutagenicity of 2-amino¯uorene in the absence of S-9 we used the spot test procedure (Maron and Ames, 1983). All test compounds were examined in the S. typhimurium strains TA97, TA98, TA100 and TA102 in the absence (ÿS-9) and presence (+S-9) of rat liver S-9 mix.
To 100 ml of the compound solution (or DMSO, negative control) 500 ml S-9 mix (corresponding to 20 ml S-9 per plate; assay +S-9) or 500 ml L-medium (assay ÿS-9) and 100 ml bacterial overnight culture (approx. 108 bacteria) were added. Finally, 2 ml top agar (45-508C) supplemented with histidine and biotin (0.05 mM) was added. These mixtures were agitated gently and poured on minimal glucose plates (Vogel-Bonner medium E). The plates were incubated at 378C for 72 hr in the dark. The number of His+ revertants was counted with an image analysing system (Domino colony counter). All chemicals were tested in three independent experiments on three dierent days. Results with a reproducible, dose-related and at least twofold elevation in comparison to the spontaneous revertant count were designated as positives. In the case of positive results the mutagenic potency was determined as the slope of the linear portion of the dose±response curve and expressed as revertants per mg (rev/mg) or revertants per nmol (rev/nmol) of the test chemical.
RESULTS AND DISCUSSION
The chemical structures of the compounds tested are given in Fig. 1. The concentrations tested and the results including the mean values and standard deviations are shown in Table 1.
Table 1. Mutagenicity (His+ revertants/plate) of polycyclic musk fragrances in S. typhimurium strains TA97, TA98, TA100 and TA102 in the presence (+S-9) and absence (ÿS-9) of an exogenous metabolizing system (n = 3) S. typhimurium TA97 +S-9 mg/plate Galaxolide
Tonalide
Celestoide
Phantolide
Cashmeran
Traseolide
0 5 16.6 50 166.6 500 0 5 16.6 50 166.6 500 0 5 16.6 50 166.6 500 0 5 16.6 50 166.6 500 0 5 16.6 50 166.6 500 0 5 16.6 50 166.6 500
m 182.7 184.3 176.3 176.3 169.6 158 182.7 205.7 207.3 200.0 183.7 162.0 182.7 199.7 186.0 154.3 174.0 206.7 182.7 194.0 199.7 184.3 193.3 165.3 182.7 203.0 203.7 182.0 131.1 139.7 182.7 228.3 227.7 194.3 191.3 182.7
S. typhimurium TA98 ÿS-9
sd 216.3 213.9 24.7 25.7 28.6 212.3 216.4 26.1 216.8 29.1 213.1 211.1 216.4 211.7 222.7 217.6 211.3 231.5 216.4 215.8 25.9 216.3 222.5 25.2 216.4 28.8 214.7 219.0 224.0 22.4 216.4 225.8 217.8 29.1 212.2 26.9
m
+S-9 sd
125.0 130.7 131.0 138.0 125.0 127.3 125.0 128.3 124.0 127.3 131.0 139.3 125.0 134.3 127.3 123.7 123.0 128.7 125.0 125.0 127.7 118.7 122.0 128.7 125.0 118.3 125.7 120.7 111.7 112.7 125.0 136.0 129.7 132.7 142.7 138.0
216.3 218.2 22.8 219.1 220.1 217.8 216.3 210.1 29.1 213.1 24.6 210.0 216.3 26.1 29.8 212.2 212.0 214.0 216.3 218.2 21.7 211.0 213.6 26.0 216.3 29.2 212.9 220.0 211.3 212.1 216.3 28.0 213.1 24.9 25.1 215.3
m 60.3 63.7 58 55 56.3 55.6 60.3 60.0 68.7 57.3 62.3 53.7 60.3 51.0 51.7 52.3 66.7 58.7 60.3 59.0 47.3 49.0 54.0 50.3 60.3 58.3 60.0 54.7 54.3 57.0 60.3 66.3 58.0 56.0 54.0 46.7
S. typhimurium TA100 +S-9 mg/plate Galaxolide
Tonalide
Celestoide
Phantolide
Cashmeran
Traseolide
0 5 16.6 50 166.6 500 0 5 16.6 50 166.6 500 0 5 16.6 50 166.6 500 0 5 16.6 50 166.6 500 0 5 16.6 50 166.6 500 0 5 16.6 50 166.6 500
m 215.7 237.7 241.3 234.3 243 213 215.7 227.3 238.3 252.3 244.7 202.7 215.7 244.7 239.0 299.7 238.0 266.7 215.7 222.7 247.0 243.3 225.7 199.3 215.7 267.0 248.7 226.7 198.0 129.7 215.7 232.3 238.7 247.0 233.0 196.0
sd
m 185.3 199.3 185.0 176.7 190.7 205.3 185.7 179.0 165.0 187.0 179.7 188.3 185.7 197.7 195.7 188.0 201.0 202.7 185.7 181.0 172.3 192.3 190.7 172.0 185.7 180.3 173.0 183.0 128.7 91.3 185.7 174.3 169.7 178.7 187.7 195.0
25.9 27.9 218.0 22.4 26.6 25.4 25.9 212.8 215.2 23.7 213.7 24.1 25.9 210.8 213.0 28.2 25.4 213.0 25.9 24.6 25.0 28.2 211.0 217.3 25.9 213.9 25.7 214.4 220.3 221.5 25.9 213.1 217.1 214.9 210.4 212.1
m
sd
21.0 22.3 18.3 20.0 23.0 23.3 21.0 16.7 18.7 19.7 24.3 17.7 21.0 22.0 16.7 19.0 25.7 24.3 21.0 21.7 16.3 20.7 18.3 19.0 21.0 20.7 16.0 17.3 22.7 32.3 21.0 17.0 21.7 21.7 20.3 20.0
26.7 22.5 23.7 24.6 26.5 26.0 26.7 25.4 25.4 24.8 24.0 23.1 26.7 26.4 22.6 25.1 25.8 24.9 26.7 23.9 24.8 21.3 24.0 22.9 26.7 23.3 23.6 21.7 24.7 27.0 26.7 22.9 22.9 22.5 23.9 21.4
S. typhimurium TA102 ÿS-9
242.4 236.6 211.6 227.2 227.4 240.4 242.4 223.8 236.6 229.3 226.4 211.6 242.4 264.5 223.5 244.5 258.0 268.7 242.4 244.2 257.7 262.3 239.3 234.9 242.4 253.1 244.6 251.9 242.9 237.8 242.4 255.2 240.2 240.1 250.5 233.8
ÿS-9 sd
+S-9 sd 217.2 24.9 213.4 22.6 214.7 211.3 217.6 225.5 25.0 213.1 212.1 25.0 217.6 22.5 27.1 223.4 215.3 218.5 217.6 215.8 25.9 210.0 23.8 27.8 217.6 212.8 28.1 27.3 224.5 253.4 217.6 225.8 212.7 218.1 212.2 28.0
m 275.0 330.0 321.3 298.3 313.3 269.3 275.0 273.0 276.0 311.0 309.7 269.3 275.0 289.7 265.3 285.3 296.3 302.7 275.0 305.7 285.7 283.3 292.3 282.3 275.0 315.7 330.0 300.7 293.7 285.0 275.0 323.0 324.7 320.0 280.0 251.7
ÿS-9 sd
242.0 251.1 272.0 275.4 294.4 238.1 242.0 243.2 293.1 293.6 283.1 246.3 242.0 283.5 286.4 265.3 269.7 270.8 242.0 275.9 293.7 288.0 284.9 275.8 242.0 286.5 283.3 2100.7 278.2 293.0 242.0 2104.6 2117.3 2106.4 2100.8 236.2
m 174.3 196.7 178.7 171.0 208.3 177.0 174.3 217.0 220.0 200.7 218.7 202.3 174.3 187.7 184.3 203.7 211.0 206.3 174.3 200.3 196.7 200.0 209.3 201.3 174.3 212.3 196.7 204.7 150.7 120.3 174.3 202.7 200.7 215.0 210.7 215.3
sd 223.8 224.4 232.4 22.2 221.0 210.7 223.8 251.1 218.0 219.6 211.5 218.3 223.8 226.7 224.4 223.8 236.6 210.6 223.8 217.5 217.3 231.5 215.2 26.7 223.8 213.0 232.1 210.3 22.5 246.0 223.8 219.1 214.4 228.3 25.9 221.1
+/ ÿ S-9 = in the presence/absence of an exogenous metabolizing system; m=mean value; SD = standard deviation; in bold = bacteriotoxic eects.
392
V. Mersch-Sundermann et al.
Table 2. Results of positive control compounds used in the Salmonella/mammalian-microsome assay with tester strains of S. typhimurium TA97, TA98, TA100 and TA102 (mean values of top dose levels 2SD)
2,4,7-trinitro-9-¯uorenone TNF (0.2 mg/plate) Sodium azide NAz (1.5 mg/plate) Methyl methanesulfonate MMS (1 ml/plate) 2-Amino¯uorene* 2-AF (10 mg/plate)
TA97 (ÿS-9)
TA98 (ÿS-9)
TA100 (ÿS-9)
TA102 (ÿS-9)
2983 220.4
5441 2 298
NT
NT
NT
NT
1093 2 102
NT
NT
NT
NT
5848 2 304
TA97 (+S-9) 1369 2 173
TA98 (+S-9) 3418 2 358
TA100 (+S-9) 1833 2 289
TA102 (+S-9) 341 2 15
NT = not tested; ÿS-9 = tested in the absence of an exogenous metabolizing system; +S-9 = tested in the presence of an exogenous metabolizing system; * = no mutagenicity in the spot test (ÿS-9).
The results of the positive controls are given in Table 2. The polycyclic musks tested up to the limit of solubility exhibited no mutagenicity in S. typhimurium in the presence (+S-9) and absence (ÿS-9) of an exogenous metabolizing system. Polycyclic musks represent a group of bioaccumulating arti®cial fragrances which were found in river and waste water, food, animals, human tissues and breast milk (Eschke et al., 1995a,b). Their potency to cause adverse eects in biological systems is still unknown. Some of the nitro-containing fragrances such as musk ambrette, musk xylene and musk ketone exhibited biological eects regarding mutagenic and co-genotoxic properties. Emig et al. (1996) demonstrated that musk ambrette possess mutagenic activity in strain TA100 in the absence of an exogenous metabolizing system and that both bacterial and mammalian (S-9) enzymes are responsible for its mutagenicity. Furthermore, MerschSundermann et al. (1996b) showed that musk xylene and musk ketone are strong inducers of xenobiotic toxifying enzymes in rat liver. Kevekordes et al. (1996 and 1997) found no genotoxicity of nitro musks using sister-chromatid exchange and micronucleus induction tests. The role of nitro substitution regarding mutagenic mechanisms in bacterial systems was discussed elsewhere (MerschSundermann et al., 1992 and 1994a). Because of the biological eects of the nitro containing musk compounds in the present study the polycyclic musks were examined using a well known and validated bacterial short-term test (Helma et al., 1996, Mersch-Sundermann et al., 1994b and 1996a). In the present study polycyclic musks did not show mutagenic eects even in high doses when using the Salmonella/mammalian mutagenicity assay. These results could be rated as one indicator of the biological inactivity of these group of compounds with respect to genotoxicity. Further studies are necessary to estimate the genetic risks caused by polycyclic fragrances. AcknowledgementsÐWe thank Bruce N. Ames, Berkeley, CA, USA, for the S. typhimurium strains TA97, TA98, TA100 and TA102. This investigation was supported by
the Research Funds of the Faculty of Clinical Medicine Mannheim of the University of Heidelberg and the ``Stiftung Krebsforschung'', Heidelberg.
REFERENCES
Emig M., Reinhardt A. and Mersch-Sundermann V. (1996) A comparative study of ®ve nitro musk compounds for genotoxicity in the SOS chromotest and Salmonella mutagenicity. Toxicology Letters 85, 151±156. Eschke H. D., Dibowski H. J. and Traud J. (1995a) Untersuchungen zum Vorkommen polycyclischer Moschus-Duftstoe in verschiedenen UmweltkompartimentenÐBefunde in Ober¯aÈchen-, AbwaÈssern und Fischen sowie in Waschmitteln und Kosmetika (2. Mitteilung). Umweltwissenschaften und SchadstoForschung 7, 131±138. Eschke H. D., Dibowski H. J. and Traud J. (1995b) Nachweis und Quanti®zierung von polycyclischen Moschus-Duftstoen mittels Ion-Trap GC/MS/MS in Humanfett und Muttermilch. Deutsche LebensmittelRundschau 12, 375±379. Eschke H. D., Traud J. and Dibowski H. J. (1994) Untersuchungen zum Vorkommen polycyclischer Moschus-Duftstoe in verschiedenen UmweltkompartimentenÐNachweis und Analytik mit GC/MS in Ober¯aÈchen-, AbwaÈssern und Fischen (1. Mitteilung). Umweltwissenschaften und Schadsto-Forschung 6, 183± 189. Helma C., Mersch-Sundermann V., Houk V. S., Glasbrenner U., Klein C., Wenqing L., Kassie F., Schulte-Hermann R. and KnasmuÈller S. (1996) Comparative evaluation of four bacterial assays for the detection of genotoxic eects in the dissolved water phases of aqueous matrices. Environmental Science and Technology 30, 897±907. Ippen H. (1994a) Nitromoschus. 1. Mitteilung. Bundesgesundheitsblatt 6, 255±260. Ippen H. (1994b) Nitromoschus. 2. Mitteilung. Bundesgesundheitsblatt 7, 291±294. Kevekordes S., Grahl K., Zaulig A. and Dunkelberg H. (1996) Genotoxicity testing of nitro musks with the SOS-chromotest and the sister-chromatid exchange test. Environmental Science and Pollution Research 4, 189± 192. Kevekordes S., Zaulig A. and Dunkelberg H. (1997) Genotoxicity of nitro musks in the micronucleus test with human lymphocytes in vitro and the human hepatoma cell line HepG2. Toxicology Letters 91, 13±17. Maron D. M. and Ames B. N. (1983) Revised methods for the Salmonella mutagenicity test. Mutation Research 113, 173±215. Mersch-Sundermann V., Emig M. and Reinhardt A. (1996b) Nitro musks are cogenotoxicants by inducing
Mutagenicity of polycyclic musks toxifying enzymes in the rat. Mutation Research 356, 237±245. Mersch-Sundermann V., Klopman G. and Rosenkranz H. S. (1992) Structural requirements for the induction of the SOS repair in bacteria by nitrated polycyclic aromatic hydrocarbons and related chemicals. Mutation Research 265, 61±73. Mersch-Sundermann V., Klopman G. and Rosenkranz H. S. (1996c) Chemical structure and genotoxicity: studies of the SOS chromotest. Mutation Research 340, 81± 91. Mersch-Sundermann V. and KraÈmer H. (1993) The in¯uence of culture conditions on the susceptibility of Salmonella typhimurium in the Salmonella mutagenicity test. Zentralblatt Hygiene und Umweltmedizin 193, 471± 480. Mersch-Sundermann V., Reinhardt A. and Emig M. (1996c) Untersuchungen zur MutagenitaÈt,
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GenotoxizitaÈt und KogenotoxizitaÈt umweltrelevanter Nitromoschusver-bindungen. Zentralblatt Hygiene und Umweltmedizin 198, 429±442. Mersch-Sundermann V., Rosenkranz H. S. and Klopmann G. (1994b) The structural basis of the genotoxicity of nitroarenofurans and related compounds. Mutation Research 304, 271±284. Mersch-Sundermann V., Schneider U., Klopmann G. and Rosenkranz H. S. (1994a) SOS induction in Escherichia coli and Salmonella mutagenicity: a comparison using 330 compounds. Mutagenesis 9, 205±224. MuÈller S., Schmid P. and Schlatter C. (1996) Occurrence of nitro and non-nitro benzenoid musk compounds in human adipose tissue. Chemosphere Vol. 33, No. 1. 17± 28. Rimkus G. G. and Wolf M. (1996) Polycyclic musk fragrances in human adipose tissue and human milk. Chemosphere 10, 2033±2043.
Lack of Mutagenicity of Polycyclic Musk Fragrances in Salmonella typhimurium V. MERSCH-SUNDERMANNO1*, S. KEVEKORDESO2 and C. JENTERO1
1
Institute of Medical Microbiology and Hygiene, Faculty of Clinical Medicine Mannheim, University of Heidelberg, D-68135, Mannheim and 2Medical Institute of General Hygiene and Environmental Health, University of GoÈttingen, Windausweg 2, D-37073 GoÈttingen, Germany (Accepted 7 December 1997)
AbstractÐSynthetic musks are widely used as fragrances and therefore found in various environmental samples. Residues were identi®ed in river and waste water, animal and human tissues and breast milk. In the present study, six arti®cial polycyclic musk fragrancesÐGalaxolide, Tonalide, Celestolide, Phantolide, Cashmeran and TraseolideÐwere tested for mutagenicity using the Salmonella/mammalianmicrosome assay with Salmonella typhimurium strains TA97, TA98, TA100 and TA102 in the presence (+S-9) and absence (ÿS-9) of an exogenous metabolizing system. All compounds tested exhibited no mutagenicity in the Salmonella assay. These results could be rated as one indicator of the biological inactivity of this group of chemicals with respect to genotoxicity. # 1998 Elsevier Science Ltd. All rights reserved Abbreviation: DMSO = dimethyl sulfoxide.
INTRODUCTION
Arti®cial fragrances such as the polycyclic musks Galaxolide, Tonalide, Celestolide, Phantolide, Cashmeran and Traseolide are used in perfumes, lotions and detergents, as food additives, in cigarettes and ®sh baits. In contrast to the synthetic nitro benzenoid musk ambrette, musk ketone and musk xylene (Ippen, 1994a,b; Mersch-Sundermann et al., 1996c) polycyclic musk fragrances are nonnitro benzenoid compounds. Eschke et al. (1994 and 1995a) identi®ed Galaxolide, Tonalide and Celestolide in bream and perch from the Ruhr River, Germany, at average concentrations between 2.5 mg/kg and 4.6 mg/kg fatty tissue. MuÈller et al. (1996) found Galaxolide (12±135 mg/kg) and Celestolide (0.12±3.5 mg/kg) in human fatty tissues. Both Galaxolide and Celestolide, were detected in all 15 samples examined. Eschke et al. (1995b) identi®ed polycyclic musks in the fat fraction of breast milk in average amounts of 335 mg/kg fat for Galaxolide, 270 mg/kg fat for Tonalide, 22 mg/kg fat for Celestoide, 14 mg/ kg fat for Phantolide and 18 mg/kg fat for Traseolide. Cashmeran could not be detected. *Author for correspondence at: Institute of Medical Microbiology and Hygiene, Faculty of Clinical Medicine Mannheim, University of Heidelberg, PO Box 100023, D-68135 Mannheim, Germany.
Rimkus and Wolf (1996) identi®ed similar amounts of residues in human fat tissues and breast milk. Under consideration of the fact that polycyclic musk fragrances are bioaccumulating compounds (Eschke et al., 1995a,b) it is necessary to estimate health risks. Therefore, in the present study the mutagenic potency of six widely used polycyclic musks was examined using the Salmonella/mammalian microsome assay with S. typhimurium strains TA97, TA98, TA100 and TA100 in the presence (+S-9) and absence (ÿS-9) of an exogenous metabolizing system.
MATERIALS AND METHODS
Galaxolide (1,3, 4, 6, 7, 8-hexa -hydro - 4, 6, 6, 7, 8, 8hexamethyl-cyclopenta-(g)-2-benzopyrane; CAS no. 1222-05-5), Tonalide (7-acetyl-1,1,3,4,4,6-hexamethyltetraline; CAS no. 1506-02-1), Celestolide (4-acetyl-1,1-dimethyl-6-tert-butylindane; CAS no. 13171-00-1), Phantolide (6-acetyl-1,1,2,3,3,5-hexamethylindane; CAS no. 15323-35-0), Cashmeran (6,7-dihydro-1,1,2,3,3-pentamethyl-4-(5H) indanone; CAS no. 33704-61-9) and Traseolide (5-acetyl1,1,2,6-tetramethyl-3-isopropylindane; CAS no. 68140-48-7) were from Promochem (Wesel, Germany). The structures of the compounds are given in Fig. 1. 9000-g fractions (S-9) from the livers of Arochlor 1254-treated male Sprague±Dawley rat were
0887-2333/98/$19.00+0.00 # 1998 Elsevier Science Ltd. All rights reserved. Printed in Great Britain PII: S0887-2333(98)00007-1
390
V. Mersch-Sundermann et al.
Fig. 1. Chemical structures, names and CAS numbers of polycyclic musk fragrances tested for mutagenicity in Salmonella typhimurium TA97, TA98, TA100 and TA102.
obtained from Organon Teknika (Heidelberg, Germany). All media and reagents used for the Salmonella assay were described by Maron and Ames (1983) and Mersch-Sundermann and KraÈmer (1993). All ingredients used for the preparation of the exogenous metabolizing system are described by Maron and Ames (1983). The S. typhimurium His+ strains TA97, TA98, TA100 and TA102 were kindly provided by Bruce N. Ames, Berkeley, CA, USA. Cultures were grown from frozen stocks (ÿ808C) in Oxoid nutrient broth no.2. The polycyclic musks were dissolved in dimethyl sulfoxide (DMSO) to 5 mg/ml. Then the compounds were diluted down to 50 mg/ml. A range of ®ve concentrations between 500 mg and 5.0 mg/plate was analysed. The maximum concentration was chosen considering the limit of solubility of the compounds in the aqueous medium of the assays. The Salmonella mutagenicity test procedure was performed as a plate incorporation assay. As positive controls we used 2,4,7-trinitro-9-¯uorenone (TA97, TA98, ÿS-9), sodium azide (TA100, ÿS-9) methyl methanesulfonate (TA102, ÿS-9) and 2-amino¯uorene (all strains +S-9). To show the lack of mutagenicity of 2-amino¯uorene in the absence of S-9 we used the spot test procedure (Maron and Ames, 1983). All test compounds were examined in the S. typhimurium strains TA97, TA98, TA100 and TA102 in the absence (ÿS-9) and presence (+S-9) of rat liver S-9 mix.
To 100 ml of the compound solution (or DMSO, negative control) 500 ml S-9 mix (corresponding to 20 ml S-9 per plate; assay +S-9) or 500 ml L-medium (assay ÿS-9) and 100 ml bacterial overnight culture (approx. 108 bacteria) were added. Finally, 2 ml top agar (45-508C) supplemented with histidine and biotin (0.05 mM) was added. These mixtures were agitated gently and poured on minimal glucose plates (Vogel-Bonner medium E). The plates were incubated at 378C for 72 hr in the dark. The number of His+ revertants was counted with an image analysing system (Domino colony counter). All chemicals were tested in three independent experiments on three dierent days. Results with a reproducible, dose-related and at least twofold elevation in comparison to the spontaneous revertant count were designated as positives. In the case of positive results the mutagenic potency was determined as the slope of the linear portion of the dose±response curve and expressed as revertants per mg (rev/mg) or revertants per nmol (rev/nmol) of the test chemical.
RESULTS AND DISCUSSION
The chemical structures of the compounds tested are given in Fig. 1. The concentrations tested and the results including the mean values and standard deviations are shown in Table 1.
Table 1. Mutagenicity (His+ revertants/plate) of polycyclic musk fragrances in S. typhimurium strains TA97, TA98, TA100 and TA102 in the presence (+S-9) and absence (ÿS-9) of an exogenous metabolizing system (n = 3) S. typhimurium TA97 +S-9 mg/plate Galaxolide
Tonalide
Celestoide
Phantolide
Cashmeran
Traseolide
0 5 16.6 50 166.6 500 0 5 16.6 50 166.6 500 0 5 16.6 50 166.6 500 0 5 16.6 50 166.6 500 0 5 16.6 50 166.6 500 0 5 16.6 50 166.6 500
m 182.7 184.3 176.3 176.3 169.6 158 182.7 205.7 207.3 200.0 183.7 162.0 182.7 199.7 186.0 154.3 174.0 206.7 182.7 194.0 199.7 184.3 193.3 165.3 182.7 203.0 203.7 182.0 131.1 139.7 182.7 228.3 227.7 194.3 191.3 182.7
S. typhimurium TA98 ÿS-9
sd 216.3 213.9 24.7 25.7 28.6 212.3 216.4 26.1 216.8 29.1 213.1 211.1 216.4 211.7 222.7 217.6 211.3 231.5 216.4 215.8 25.9 216.3 222.5 25.2 216.4 28.8 214.7 219.0 224.0 22.4 216.4 225.8 217.8 29.1 212.2 26.9
m
+S-9 sd
125.0 130.7 131.0 138.0 125.0 127.3 125.0 128.3 124.0 127.3 131.0 139.3 125.0 134.3 127.3 123.7 123.0 128.7 125.0 125.0 127.7 118.7 122.0 128.7 125.0 118.3 125.7 120.7 111.7 112.7 125.0 136.0 129.7 132.7 142.7 138.0
216.3 218.2 22.8 219.1 220.1 217.8 216.3 210.1 29.1 213.1 24.6 210.0 216.3 26.1 29.8 212.2 212.0 214.0 216.3 218.2 21.7 211.0 213.6 26.0 216.3 29.2 212.9 220.0 211.3 212.1 216.3 28.0 213.1 24.9 25.1 215.3
m 60.3 63.7 58 55 56.3 55.6 60.3 60.0 68.7 57.3 62.3 53.7 60.3 51.0 51.7 52.3 66.7 58.7 60.3 59.0 47.3 49.0 54.0 50.3 60.3 58.3 60.0 54.7 54.3 57.0 60.3 66.3 58.0 56.0 54.0 46.7
S. typhimurium TA100 +S-9 mg/plate Galaxolide
Tonalide
Celestoide
Phantolide
Cashmeran
Traseolide
0 5 16.6 50 166.6 500 0 5 16.6 50 166.6 500 0 5 16.6 50 166.6 500 0 5 16.6 50 166.6 500 0 5 16.6 50 166.6 500 0 5 16.6 50 166.6 500
m 215.7 237.7 241.3 234.3 243 213 215.7 227.3 238.3 252.3 244.7 202.7 215.7 244.7 239.0 299.7 238.0 266.7 215.7 222.7 247.0 243.3 225.7 199.3 215.7 267.0 248.7 226.7 198.0 129.7 215.7 232.3 238.7 247.0 233.0 196.0
sd
m 185.3 199.3 185.0 176.7 190.7 205.3 185.7 179.0 165.0 187.0 179.7 188.3 185.7 197.7 195.7 188.0 201.0 202.7 185.7 181.0 172.3 192.3 190.7 172.0 185.7 180.3 173.0 183.0 128.7 91.3 185.7 174.3 169.7 178.7 187.7 195.0
25.9 27.9 218.0 22.4 26.6 25.4 25.9 212.8 215.2 23.7 213.7 24.1 25.9 210.8 213.0 28.2 25.4 213.0 25.9 24.6 25.0 28.2 211.0 217.3 25.9 213.9 25.7 214.4 220.3 221.5 25.9 213.1 217.1 214.9 210.4 212.1
m
sd
21.0 22.3 18.3 20.0 23.0 23.3 21.0 16.7 18.7 19.7 24.3 17.7 21.0 22.0 16.7 19.0 25.7 24.3 21.0 21.7 16.3 20.7 18.3 19.0 21.0 20.7 16.0 17.3 22.7 32.3 21.0 17.0 21.7 21.7 20.3 20.0
26.7 22.5 23.7 24.6 26.5 26.0 26.7 25.4 25.4 24.8 24.0 23.1 26.7 26.4 22.6 25.1 25.8 24.9 26.7 23.9 24.8 21.3 24.0 22.9 26.7 23.3 23.6 21.7 24.7 27.0 26.7 22.9 22.9 22.5 23.9 21.4
S. typhimurium TA102 ÿS-9
242.4 236.6 211.6 227.2 227.4 240.4 242.4 223.8 236.6 229.3 226.4 211.6 242.4 264.5 223.5 244.5 258.0 268.7 242.4 244.2 257.7 262.3 239.3 234.9 242.4 253.1 244.6 251.9 242.9 237.8 242.4 255.2 240.2 240.1 250.5 233.8
ÿS-9 sd
+S-9 sd 217.2 24.9 213.4 22.6 214.7 211.3 217.6 225.5 25.0 213.1 212.1 25.0 217.6 22.5 27.1 223.4 215.3 218.5 217.6 215.8 25.9 210.0 23.8 27.8 217.6 212.8 28.1 27.3 224.5 253.4 217.6 225.8 212.7 218.1 212.2 28.0
m 275.0 330.0 321.3 298.3 313.3 269.3 275.0 273.0 276.0 311.0 309.7 269.3 275.0 289.7 265.3 285.3 296.3 302.7 275.0 305.7 285.7 283.3 292.3 282.3 275.0 315.7 330.0 300.7 293.7 285.0 275.0 323.0 324.7 320.0 280.0 251.7
ÿS-9 sd
242.0 251.1 272.0 275.4 294.4 238.1 242.0 243.2 293.1 293.6 283.1 246.3 242.0 283.5 286.4 265.3 269.7 270.8 242.0 275.9 293.7 288.0 284.9 275.8 242.0 286.5 283.3 2100.7 278.2 293.0 242.0 2104.6 2117.3 2106.4 2100.8 236.2
m 174.3 196.7 178.7 171.0 208.3 177.0 174.3 217.0 220.0 200.7 218.7 202.3 174.3 187.7 184.3 203.7 211.0 206.3 174.3 200.3 196.7 200.0 209.3 201.3 174.3 212.3 196.7 204.7 150.7 120.3 174.3 202.7 200.7 215.0 210.7 215.3
sd 223.8 224.4 232.4 22.2 221.0 210.7 223.8 251.1 218.0 219.6 211.5 218.3 223.8 226.7 224.4 223.8 236.6 210.6 223.8 217.5 217.3 231.5 215.2 26.7 223.8 213.0 232.1 210.3 22.5 246.0 223.8 219.1 214.4 228.3 25.9 221.1
+/ ÿ S-9 = in the presence/absence of an exogenous metabolizing system; m=mean value; SD = standard deviation; in bold = bacteriotoxic eects.
392
V. Mersch-Sundermann et al.
Table 2. Results of positive control compounds used in the Salmonella/mammalian-microsome assay with tester strains of S. typhimurium TA97, TA98, TA100 and TA102 (mean values of top dose levels 2SD)
2,4,7-trinitro-9-¯uorenone TNF (0.2 mg/plate) Sodium azide NAz (1.5 mg/plate) Methyl methanesulfonate MMS (1 ml/plate) 2-Amino¯uorene* 2-AF (10 mg/plate)
TA97 (ÿS-9)
TA98 (ÿS-9)
TA100 (ÿS-9)
TA102 (ÿS-9)
2983 220.4
5441 2 298
NT
NT
NT
NT
1093 2 102
NT
NT
NT
NT
5848 2 304
TA97 (+S-9) 1369 2 173
TA98 (+S-9) 3418 2 358
TA100 (+S-9) 1833 2 289
TA102 (+S-9) 341 2 15
NT = not tested; ÿS-9 = tested in the absence of an exogenous metabolizing system; +S-9 = tested in the presence of an exogenous metabolizing system; * = no mutagenicity in the spot test (ÿS-9).
The results of the positive controls are given in Table 2. The polycyclic musks tested up to the limit of solubility exhibited no mutagenicity in S. typhimurium in the presence (+S-9) and absence (ÿS-9) of an exogenous metabolizing system. Polycyclic musks represent a group of bioaccumulating arti®cial fragrances which were found in river and waste water, food, animals, human tissues and breast milk (Eschke et al., 1995a,b). Their potency to cause adverse eects in biological systems is still unknown. Some of the nitro-containing fragrances such as musk ambrette, musk xylene and musk ketone exhibited biological eects regarding mutagenic and co-genotoxic properties. Emig et al. (1996) demonstrated that musk ambrette possess mutagenic activity in strain TA100 in the absence of an exogenous metabolizing system and that both bacterial and mammalian (S-9) enzymes are responsible for its mutagenicity. Furthermore, MerschSundermann et al. (1996b) showed that musk xylene and musk ketone are strong inducers of xenobiotic toxifying enzymes in rat liver. Kevekordes et al. (1996 and 1997) found no genotoxicity of nitro musks using sister-chromatid exchange and micronucleus induction tests. The role of nitro substitution regarding mutagenic mechanisms in bacterial systems was discussed elsewhere (MerschSundermann et al., 1992 and 1994a). Because of the biological eects of the nitro containing musk compounds in the present study the polycyclic musks were examined using a well known and validated bacterial short-term test (Helma et al., 1996, Mersch-Sundermann et al., 1994b and 1996a). In the present study polycyclic musks did not show mutagenic eects even in high doses when using the Salmonella/mammalian mutagenicity assay. These results could be rated as one indicator of the biological inactivity of these group of compounds with respect to genotoxicity. Further studies are necessary to estimate the genetic risks caused by polycyclic fragrances. AcknowledgementsÐWe thank Bruce N. Ames, Berkeley, CA, USA, for the S. typhimurium strains TA97, TA98, TA100 and TA102. This investigation was supported by
the Research Funds of the Faculty of Clinical Medicine Mannheim of the University of Heidelberg and the ``Stiftung Krebsforschung'', Heidelberg.
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