Mutation Research 416Ž. 1998 129±136

Mutagenicity testing of ž/" -, 1,8-cineole, citral, , ž/y - and terpineol with the Salmonellarmicrosome assay

Maria Regina Gomes-Carneiro a, Israel Felzenszwalb b, Francisco J.R. Paumgartten a,) a LaboratorioÂÂÂÄ de Toxicologia Ambiental, Escola Nacional de Saude Publica, FundacËao Oswaldo Cruz, AÕ. Brasil 4365, Rio de Janeiro, RJ 21045-900, Brazil b Departmento de Biofõsica e Biometria, Instituto de Biologia, UniÕersidade de Estado do Rio de Janeiro, Rio de Janeiro, RJ 20551-030, Brazil Received 3 December 1997; revised 18 May 1998; accepted 20 May 1998

Abstract

The essential oils and their monoterpenoid constituents have been widely used as fragrances in cosmetics, as flavouring food additives, as scenting agents in a variety of household products, as active ingredients in some old drugs, and as intermediates in the synthesis of perfume chemicals. The present study was undertaken to investigate the mutagenic potential of six monoterpenoid compounds: two aldehydesŽ citral and citronellal . , a ketone ŽŽ" .-camphor . , an oxide Ž1,8-cineole, also known as .ŽŽ.. , and two alcohols terpineol and y -menthol . It is part of a more comprehensive toxicological screening of under way at our laboratory. Mutagenicity was evaluated by the Salmonellarmicrosome assay Ž.TA97a, TA98, TA100 and TA102 tester strains , without and with addition of an extrinsic metabolic activation system Ž.lyophilized rat liver S9 fraction induced by Aroclor 1254 . In all cases, the upper limit of the dose interval tested was either the highest non-toxic dose or the lowest dose of the toxic to TA100 strain in the preliminary toxicity test. No mutagenic effect was found with Ž." camphor, citral, citronellal, 1,8-cineole, and Ž.y -menthol. Terpineol caused a slight but dose-related increase in the number of hisq revertants with TA102 tester strain both without and with addition of S9 mixture. The results from this study therefore suggest that, with the exception of terpineol, the monoterpenoid compounds tested are not mutagenic in the Ames test. q 1998 Elsevier Science B.V. All rights reserved.

Keywords: Monoterpene; Essential oil; Genotoxicity; Salmonella typhimurium; Ames test

1. Introduction ous principles obtained from a large variety of plants wx1,2 . The essential oils and their monoterpenoid Monoterpenes are the main chemical constituents constituents are widely used as fragrances in cosmet- of the essential oils which are mixtures of odorifer- ics, as flavouring additives in food and beverages, as scenting agents in a variety of household products ) Corresponding author. Fax: q55-21-5648985; E-mail: Že.g., detergents, soaps, room-air-fresheners, and in- [email protected] sect repellents. , as constituents of some old over-

1383-5718r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII: S1383-5718Ž. 98 00077-1 130 M.R. Gomes-Carneiro et al.rMutation Research 416() 1998 129±136 the-counter drugsŽ. e.g., menthol, cineole, camphor concentration of approximately 1.2=109 bacteria and as intermediates in the manufacture of perfume per milliliter was obtained. chemicalswx 1±3 . The present study was undertaken to evaluate the 2.4. Salmonellarmicrosome reÕerse mutation assay mutagenic potential of Ž." -camphor, citral, citronel- lal, 1,8-cineole, Ž.y -menthol and terpineol. These The Salmonella mutagenicity test was performed monoterpenoid compounds are found in the essential by the plate incorporation methodwx 4 . Briefly, 2 ml oils of edible and medicinal plants and are employed of top-agar was mixed with 100 ml of an overnight in the industry for different purposes. grown culture of S. typhimurium, 100 ml of the test substanceŽ diluted in ethanol analytical grade, Merck, KGaA. , the negative control, or the positive control 2. Material and methods Ž.PC and 500 ml of the phosphate buffer or the S9 mixture. The doses of the monoterpenes ranged from m m 2.1. Chemicals 1 g up to 2500 g per plate. Ethanol served as the negativeŽ. solvent controls, while the positive control substances were: SAŽ.Ž 0.5 mgrplate , NPD 1 Ž." -Camphor, 1,8-cineole Ž also known as euca- mgrplate.Ž , MC 0.5 mgrplate . , 4-NQNO Ž 1 lyptol.Ž. , citral, citronellal, y -menthol and terpineol, mgrplate.Ž , 2AF 10 mgrplate . , 2AA Ž 0.5 or 1 mitomycin CŽ. MC , benzowxa pyrenŽ B-wxa -P. , 4- mgrplate. , and B-wxa -PŽ. 50 mgrplate . SA and MC nitroquinolone-n-oxideŽ. 4-NQNO , nitro-o-pheni- were dissolved in distilled water and dimethyl sul- lene-diamineŽ. NPD were all purchased from Sigma foxidewx DMSO was used as vehicle for the other ChemicalŽ. St. Louis, MO, USA . 2-Aminofluorene positive controls. Plates were incubated at 378C for Ž.2AF and 2-aminoanthracene Ž 2AA . were bought 72 h in the dark and then scored for revertant hisq from Merck KGaA, and sodium azideŽ. SA was from bacteria colonies. Every determination was made in Aldrich Chemical. triplicate and each experiment was repeated at least once in order to check the reproducibility of the () 2.2. Metabolic actiÕation system S9 mixture results.

Lyophilized rat liver S9 fraction induced by Aro- clor 1254 was purchased from MoltoxŽ Molecular 3. Results and discussion Toxicology, Annapolis, USA. . The S9 mixturew 21 x ml was prepared for use as follows: 7.0 ml of Toxicity of monoterpenoid compounds to S. ty- distilled water; 10.5 ml of 100 mM phosphate buffer phimurium was investigated in a preliminary test pH 7.4; 0.84 ml of 100 mM NADP; 0.105 ml of 1 M carried out with TA100 strain without and with q glucose-6-phosphate; 0.420 ml of 1.65 M KCl 0.4 addition of S9 mixtureŽ. Table 1 . In all subsequent M MgCl2 salt solution and 2.1 ml of lyophilized S9 assays, the upper limit of the dose interval tested was fraction reconstituted with distilled water. either the highest non-toxic dose or the lowest toxic dose determined in this preliminary assay. Toxicity 2.3. Bacterial strains was apparent either as a reduction in the number of revertants, and or as an alteration of the auxotrophic TA102, TA100, TA98 and TA97a tester strains of background growthŽ. i.e., background lawn . It is of Salmonella typhimurium were kindly supplied by Dr note that the six monoterpenoid compounds tested B.N. Ames. The bacterial strains were checked for were different regarding their toxicity towards S. their genetic markers immediately before and at the typhimurium tester strains. Citronellal was by far the end of the study. For all assays overnight fresh most toxic monoterpene testedŽ highest non-toxic cultures were prepared from frozen permanentsŽ 200 dose s 200 mgrplate.Ž. . Citral and y -menthol ml of the culture to 20 ml of Oxoid Nutrient Broth Ž.highest non-toxic doses600±700 mgrplate were No. 2. and incubated at 378C with shaking until a less toxic than citronellal, but clearly more toxic than M.R. Gomes-Carneiro et al.rMutation Research 416() 1998 129±136 131 ) ) q 1 30 21 13 14 0 r " " " " " ) S9 q 110 ) 46 20 180 63 742 6± 8 147 " " " " " -Cineole S9 ± 101 ± 102 1,8 y b b ) ) 2104 26 191 69 842 9 13 150 44 " " " " " " S9 ±± ± 76 q 135 ) ) 7152 6184 ) 14 8 40 -Camphor " " " " " S9 " 96 y ) 8146 2145 35 934 591 11 q " " " " 369 S9 r q q 0184 6166 65 464 35 ± ± 132 ±± ± 6±±±± 5± ± ± 172 r 0 " " " " " S9 r ± ± ±±± ± ± ± ±±± ± y q q ± ±±± ± 0±± ± ±±± ± 0±± ) r r 0 0 7±±±± ± 17±± 2669 167 913 ) 10 r r 6 " " " " ) ) " S9 q 77 167 ) ) 12 166 1541 184 591 29 14 120 39 -Menthol Terpineol " " " " " S9 " y ± ± ± ± ±±± ± y Ž. Ž. q 0 r ) 2669 174 924 3±± ± ±±± ± 23±± 75 " " " r 3 ± ± ± ± ±±± ± S9 r 154 q TA100 strain q ab a b ab a b qq 0 03 q 1541 184 591 12 179 r 0 ± 165 r 3 r 29 " " " 0 S9 r r r ± ± 159 y SD of three plates. S. typhimurium " ) 12 1 21 52 1458 174 924 6 0 ± ± 134 " " " " " S9 r Ž. ±± ± 93 q 12 28 150 2483 173 886 23 102 qq 12 148 ) 5 " " " " ) S9 " " y Mutant counts of individual plates. plate Toxicity assays carried out concomitantly shared the same solvent- and positive controls. Toxicity apparent as an alteration of the background lawn. . r . g q ±Dosenottested. a,b m ) r 200 ± ± 139 Table 1 Toxicity of monoterpenoid compounds to DoseŽ. 3000 Citral2000 ±900 ±800 ±700 Citronellal 500 ± ±400 7 300 ± 155 0178 94 ±PC ± 143 ±Ž. ±Ž Ž. 728 ±Ž ± ±Data are shown as mutant counts mean ± ± 82 ± ± ± ± ± 101 68 0 2500 ± ± ± ± ± ± ± 180 2750 ± ± ± ± ± ± ± ± 94 600 165 1500 ± ± ± ± ± ± 126 12501000 ± ± ± ± ± ± ± ± ± ± ± ± 145 164 132 M.R. Gomes-Carneiro et al.rMutation Research 416() 1998 129±136 Ž plate ; -P 50 r w a g wx m S9, B- rq S9, NPD 1 microsome assay TA100, ry r plate ; TA102 r g m Salmonella plate ; TA98 r g m S9, MC 0.5 ry S9, 2AA 1 rq plate ; TA102 r g plate ; TA100 m r g m Ž . Ž. Ž. S9, 2AF 10 S9, SA 0.5 rq ry plate ; TA97a r g m Ž.Ž. Ž. S9, 4-NQNO 1 ry x l ethanol PA; PCÐPositive Control: TA100 m plate ; TA97a r g m SD of three plates of one out of two representative experiment. " Ž. Ž. Ž. Ž. S9; 2AA 0.5 . rq plate . Toxicity apparent as an alteration of the background lawn. r g ± Dose not tested. ) m Ž. Ž. Values are the means TA98 Dose OÐNegative Control: 100 Table 2 TA98, TA97a and TA102 tester strains Mutagenicity testing of the monoterpenoid citral 3,7-dimethyl-2,6-octadienal and citronellal 3,7-dimethyl-6-octenal in the M.R. Gomes-Carneiro et al.rMutation Research 416() 1998 129±136 133 Ž plate ; -P 50 r a g wx microsome m r S9, B- rq S9, NPD 1 Salmonella ry plate ; TA102 r g m plate ; TA98 r g m -menth-1-en-ol in the p S9, MC 0.5 ry S9, 2AA 1 rq plate ; TA102 r g plate ; TA100 m r g m Ž . Ž. Ž. S9, 2AF 10 S9, SA 0.5 rq ry plate ; TA97a r g m -menthol 5-methyl-2- 1-methyl-ethyl cyclohexanol and terpineol y Ž. Ž Ž . . Ž . Ž. S9, 4-NQNO 1 ry l ethanol PA; PCÐPositive Control: TA100 m plate ; TA97a r g m SD of three plates of one out of two representative experiment. " Ž. Ž. Ž. Ž. S9; 2AA 0.5 . mutant counts of individual plates. wx rq plate . . Toxicity apparent as an alteration of the background lawn. r q g ± Dose not tested. ) r m Ž. Ž. Ž TA98 assay TA100, TA98, TA97a and TA102 tester strains Dose OÐNegative Control: 100 Values are the means Table 3 Mutagenicity testing of the monoterpenoid alcohols 134 M.R. Gomes-Carneiro et al.rMutation Research 416() 1998 129±136

Ž." -camphor, 1,8-cineole and terpineol Ž highest of metabolic activation, also gave negative results in non-toxic doses2000±2500 mgrplate. . In all cases, three of the four strains used. In contrast to the TA102 proved to be the most susceptible tester absence of mutagenicity towards TA97a, TA98 and strain. TA100 strains, terpineol produced a dose-related in- Table 2 shows the results of the Salmonella muta- crease in the number of TA102 revertants. The maxi- genicity assay with the aldehydes citralŽ a mixture of mum effects were a 2.0-fold increase in the number isomers neral and geranial. and citronellal. Citral and TA102 revertants at doses as high as 750 mgrplate citronellal did not induce any increase in the number in the absence of S9 mixture, and a 2.2-fold increase of revertant hisq colonies over negative control at doses as high as 1250 mgrplate in the presence of values obtained for strains TA97a, TA98, TA100 S9 mixture. The negative as well as the positive and TA102, either in the presence or in the absence results presented in Table 3 were reproduced by a of extrinsic metabolic activationŽ. S9 mix . The nega- confirmatory experiment. As shown in Table 4, ter- tive results presented in Table 2 were confirmed by a pineol also induced a dose related increase in the second experimentŽ. results not shown . number of TA102 revertants in this second experi- Citral was regarded as a possible carcinogenr ment. mutagen due to its a,b-unsaturated func- Menthol, a constituent of the peppermint oil, had tion. However, a recent prediction for citral carcino- been previously evaluated in the Salmonellarmicro- genicity made on the basis of computer testsŽ COM- some assay with the tester strains TA92, TA1535, PACT: Computer Optimised Molecular Parametric TA100, TA1537, TA94 and TA98 and no mutagenic Analysis for Chemical Toxicity. was negativewx 5 . effect was foundwx 6,9 . The present study findings The absence of citral-induced mutagenicity was pre- adding TA97a and TA102 strains to the testing viously reported by Ishidate et al.wx 6 with the battery, confirmed that menthol is not mutagenic in Salmonellarmicrosome assayŽ strains TA92; the Ames test. TA1535, TA100, TA1537, TA94 and TA98. without No previous study of the mutagenicity of terpi- and with S9 mixture. Citral was also tested for its neol was found in the literature. The present study potential to increase the frequencies of sister chro- showed that terpineol was weakly mutagenic with matid exchanges and chromosome aberrations in V79 TA102 strain and that exogenous metabolic activa- cellsŽ. without and with addition of S9 mix and tion is not required for this effect. This positive negative results were foundwx 7 . Kamasaki et al. wx 8 evaluated the mutagenic potential of citronellal with the Salmonella assayŽ. TA 98 and TA100 . Negative Table 4 results were obtained by these authors with the two Mutagenicity of terpineol to TA102 tester strain in the confirma- tory experiment S. typhimurium strains but they also reported that r y q citronellal induced an increase in the frequency of Dose Ž.mg plate S9 S9 chromosome aberrations in Chinese hamster B241 2000 ± 572"28) " cellswx 8 . Our results also including the TA97a and 1500 ± 1562 123 1250 ± 1526"219 TA102 tester strains therefore confirm literature data 1000 1050"38 1114"562 suggesting that the monoterpenoid aldehydes citral 750 1088"32 1057"74 and citronellal are not mutagenic compounds. 500 786"58 824"6 The results of the mutagenicity assay with the 250 742"24 ± Ž.y 100 634"26 ± monoterpenoid alcohols -menthol and terpineol " Ž.y 50 600 32 ± are shown in Table 3. -Menthol was tested in 25 630"54 ± doses up to 600±700 mgrplateŽ TA97a, TA98 and 0 613"24 790"120 TA100.Ž. and 200±400 mgrplate TA102 , and no PC 5820"311 1627"260 increase in the number of revertant colonies was Ž.m observed without and with addition of S9 mixture. Dose OÐnegative control 100 l ethanol PA ; PCÐpositive controls, yS9Ž.Ž MC: 0.5 mgrplate ; qS9 B-wxa -P: 50 mgrplate. . Terpineol, tested in doses up to 1500±2000 Ž.) Toxicity apparent as an alteration of the background lawn. mgrplate, in the absence as well as in the presence Values are revertant countsŽ. mean"SD of three plates . M.R. Gomes-Carneiro et al.rMutation Research 416() 1998 129±136 135 Ž plate ; -P 50 r a g wx wx m S9, B- rq -menthane oxide in p S9, NPD 1 ry -epoxy- 1,8 plate ; TA102 Ž. r g m plate ; TA98 r g -cineole m 1,8 S9, MC 0.5 ry S9, 2AA 1 wx rq . plate ; TA102 r g plate ; TA100 m r g m wx Ž . Ž. Ž. S9, 2AF 10 S9, SA 0.5 rq ry plate ; TA97a r g m -camphor 1,7,7-trimethyl-bicyclo 2.2.1 -heptan-2-one ketone and " Ž. Ž Ž. S9, 4-NQNO 1 ry l ethanol PA; PCÐPositive Control: TA100 wx m plate ; TA97a r g m SD of three plates of one out of two representative experiment. " Ž. Ž. Ž. Ž. microsome assay TA100, TA98, TA97a and TA102 tester strains r S9; 2AA 0.5 . Mutant counts of individual plates. rq plate . . Toxicity apparent as an alteration of the background lawn. Salmonella r q g ± Dose not tested. ) r m Ž. Ž. Ž TA98 Dose OÐNegative Control: 100 Mutagenicity testing of the monoterpenoid compounds Values are the means Table 5 the 136 M.R. Gomes-Carneiro et al.rMutation Research 416() 1998 129±136 result obtained only with the TA102 tester strain FIOCRUZ. IF and FJRP are recipients of CNPq indicated that terpineol induced a base-pair substitu- fellowships. tion affecting a A±T base pair. We have added TA102 to the tester strain battery routinely used at our laboratory because it has been demonstrated that References it detects a variety of oxidants and cross-linking agents as mutagens which are not detectable by other wx1 O. Sticher, Plant mono-, di- and sesquiterpenoids with phar- Salmonella strainswx 10 . macological or therapeutical activity, in: H. Wagner, P. WolffŽ. Eds. , New Natural Products and Plant Drugs with Table 5 presents the results of mutagenicity test- Pharmacological, Biological or Therapeutic Activity, ing of monoterpenoid compounds with two different Springer-Verlag, Berlin, 1977, pp. 137±176. functional groups, i.e., Ž." -camphor, a ketone, and wx2 R.E. Erikson, The industrial importance of monoterpenes and 1,8-cineole, an oxide. Ž." -Camphor and 1,8-cineole essential oils, Lloydia 39Ž. 1976 8±20. wx were tested in doses up to 1500±2500 mgrplate and 3 A.Y. Leung, Encyclopedia of Common Natural Ingredients Used in Food, Drugs and Cosmetics, Wiley, New York, no mutagenic effect was observed with the four 1980. bacterial tester strains, in the absence as well as in wx4 D.M. Maron, B.N. Ames, Revised methods for Salmonella the presence of extrinsic metabolic activation. In mutagenicity test, Mutat. Res. 113Ž. 1983 173±215. both cases, negative results were also obtained by an wx5 D.F.V. Lewis, C. Ionnides, D.V. Parke, COMPACT and independent confirmatory experiment. molecular structure in toxicity assessment: a prospective evaluation of 30 chemicals currently being tested for carcino- In spite of its large scale use, studies on the genicity by the NCIrNTP, Environ. Health Perspect. 104 mutagenic potential of Ž." -camphor are scarce. Ž.1996 1011±1016, Suppl. 5. Kanematsu and Shibatawx 11 reported that endodontic wx6 M. Ishidate, T. Sofuni, K. Yoshikawa, M. Hayashi, T. Nohmi, products containing camphor presented a positive M. Sawada, A. Matsuoka, Primary mutagenicity screening of result in the `rec-assay' with B. subtilis.Onthe food additives currently used in Japan, Food Chem. Toxicol. wx 22Ž. 1981 623±636. other hand, Goel et al. 12 demonstrated that cam- wx7 H.P.S. Zamith, M.N.P. Vidal, G. Speit, F.J.R. Paumgartten, phor had a radiomodifying effect, i.e., camphor an- Evaluation of the Genotoxicity of Citral in Mammalian Cells tagonized the radiation-induced increase in SCE fre- in Vitro, Proceedings of the 22nd Annual Meeting of the quency in mice bone marrow cells. European Environmental Mutagen Society, Berlin, Germany, As far as the authors are aware, no study on the August 31st±September 4th, 1992Ž. Abstract . wx8 A. Kamasaki, H. Takahashi, N. Tsumura, J. Niwa, T. Fujita, mutagenicity of 1,8-cineole has been published in S. Urasawa, Genotoxicity of flavoring agents, Mutat. Res. the literature so far. 105Ž. 1982 387±392. In conclusion, results from the present study thus wx9 P.H. Andersen, N.J. Jensen, Mutagenic investigation of pep- suggest that citral, citronellal, Ž." -camphor, Ž.y - permint oil in the Salmonellarmammalian-microsome test, menthol and 1,8-cineole are not mutagenic in the Mutat. Res. 138Ž. 1984 17±20. wx10 D.E. Levin, M.C. Hollstein, M.F. Christman, E.A. Schwiers, Ames test and that terpineol is weakly mutagenic to B.N. Ames, A new Salmonella tester strainŽ. TA102 with TA102 tester strain. A±T base pairs at the site of mutation detects oxidative mutagens, Proc. Natl. Acad. Sci. USA 79Ž. 1982 7445±7449. wx11 N. Kanematsu, K.I. Shibata, Investigation of DNA reactivity Acknowledgements of endodontic agents by rec-assay, Gifu-Shika-Gakkai-Zasshi 17Ž. 1990 592±597. wx12 H.C. Goel, S. Singh, S.P. Singh, Radiomodifying influence This work was supported by grants from CNPq of camphor on sister-chromatid exchange induction in mouse Ž.Brazilian National Research Council and PAPES- bone marrow, Mutat. Res. 224Ž. 1989 157±1609.