(+)-and (-)CARVONE First draft prepared by Dr J.C. Larsen Institute of Technology, National Food Agency of Denmark 1. EXPLANATION (+)-Carvone and (-)carvone were evaluated for acceptable daily intake for man (ADI) by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 1967, 1979, 1981, 1986 and 1988 (Annex 1, references 15, 50, 56, 62, 73, 83). Toxicological monographs have been issued in 1968 and 1980 (Annex 1, references 15 and 51). At the twenty-third meeting of the committee (Annex 1, reference 50) a temporary ADI of 0-1 mg/kg of body weight was allocated (as sum of the isomers), pending submission of further relevant biochemical and metabolic studies. This temporary ADI was continued by the twenty- fifth meeting (Annex 1, reference 56). At the twenty-seventh meeting (Annex 1, reference 62) the committee was informed that lifetime studies with (+)-carvone in rats and mice were in progress. The committee therefore extended the temporary ADI, pending submission of the results from these studies. The temporary ADI was again extended at the thirtieth and the thirty-third meetings of the committee still awaiting the results of the long term studies. Since the previous evaluation a long term study in mice with (+)-carvone has become available along with other studies. The existing monograph has been extended and is presented in its entirety. 2. BIOLOGICAL DATA 2.1 Biochemical aspects 2.1.1 Absorption, distribution, and excretion Carvone was found to be present in the urine of healthy adult humans. The authors stated that urine profiles were compared over a period of two months. The diets consumed by the subjects were not discussed (Zlatkis & Liebich, 1971; Zlatkis et al., 1973a,b). 2.1.2 Biotransformation When there is unsaturation in the ring, hydroxylation of that ring is the primary metabolic pathway (F.E.M.A., 1976). This has been shown to be true for carvone (Hildebrandt, 1902; Williams, 1959). Carvone was metabolized by the rabbit to 1,5-dimethyl-1,5- hexadien-1,6-dicarboxylic acid and a carbinol in which one ethylene linkage was saturated and the keto group was reduced (Fischer & Bielig, 1940; Opdyke, 1973; Opdyke, 1978). 2.1.3 Effects on enzymes and other biochemical parameters When (-)carvone was administered orally to male albino rats at a dose level of 600 mg/kg bw/day for three days no effects were observed on the levels of either cytochrome P-450 or cytochrome b5 (Moorthy et al., 1989). Carvone (isomer not specified) was administered to three or four male Wistar rats for 14 days at a dietary level of 1%, corresponding to approximately 500 mg/kg bw/day. Increased serum cholesterol and triacylglycerol levels were seen in the dosed rats compared to controls. Decreased food consumption and body weight gain were observed over the 14-day period (Imaizumi et al., 1985). Groups of ten 6-week old male Wistar rats were administered (+)-carvone or (-)-carvone at levels of 80 or 160 mg/kg bw/day for 28 days. Plasma creatinine was determined in all rats on days 0, 13 and 28 using a specific HPLC-method. A dose related decrease in plasma creatinine was observed on day 28 in the rats receiving (+)- carvone. (-)-Carvone had no effect (Lam, 1988). 2.2 Toxicological Studies 2.2.1 Acute toxicity studies LD50 Species Route (mg/kg b.w.) References Mouse s.c. 2675 Wenzel & Ross, 1957 Rat Oral 1640a Jenner et al., 1964 Rat oral 3710b Opdyke, 1978 Guinea-Pig Oral 766c Jenner et al., 1964 Rabbit Dermal 3860 Opdyke, 1978 a Oral LD50 reported on "carvone", identified as p-mentha-6,8-dien-2-one. b Oral LD50 reported on (+)-carvone. c Dermal LD50 reported as 4 ml/kg body weight for (+)-carvone. Single intraperitoneal injections of (+)-carvone to male Swiss- Webster albino mice did not result in any observed hepatotoxic or pulmonary effects. (+)-Carvone was administered in graded doses (not given) up to 800 mg/kg body weight at which level significant convulsive effects occurred (Gordon et al., 1982). 2.2.2 Short-term studies 2.2.2.1 Mouse Groups of five B6CBF1 mice (7-8 weeks old) of each sex were administered 0, 150, 328, 723, 1,590 or 3,500 mg/kg body weight (+)- carvone in corn oil by gavage, 5 days per week, total 12 doses over 16 days. Animals were housed five per cage. Water and feed were given ad libitum. The mice were observed twice per day and were weighed before the study and on days 1, 7 and 14 and at the end of the study. A necropsy was performed on all animals. All mice receiving 1,590 or 3,500 mg/kg body weight died on day 2 or 3. At necropsy the mean body weights of the treated animals were similar to controls. Ataxia, impaired grasping reflex, ocular discharge, corneal opacity, body tremors, prostration, gasping, clonic or tonic convulsions, or impaired righting reflex were observed at 1,590 and 3,500 mg/kg body weight; and lacrimation, piloerection, hypoactivity, bradypnea, or ptosis were observed at 723, 1,590 and 3,500 mg/kg body weight. Relative liver weights were increased in all groups of dosed male mice and relative thymus weights were decreased in all groups of dosed female mice. Relative weights of brain, heart, right kidney, lungs and bronchi were not changed. Histological examinations were carried out on all animals in the control group and the highest dosed group. However, the lungs and livers of all dosed mice were examined. No compound-related changes were seen (details on pathology not given) (NTP, 1989). Groups of 10 female and 30 male B6CBF1 mice (5-7 weeks old) were given 0, 93, 375 or 1,500 mg/kg body weight (+)-carvone in corn oil by gavage, 5 days per week, for 13 weeks. The 20 extra males per group were included to be used for other studies, and except the highest dosed group they are not relevant for this evaluation. Additional groups of 10 mice of each sex were administered 187 or 750 mg/kg body weight on the same schedule. Animals were housed five per cage and feed and water were available ad libitum. The mice were observed twice per day. The animals were weighed at the beginning of the study and then once per week, and at the end of the study. A necropsy was performed on all animals. All male and 8/10 female mice that received 1,500 mg/kg body weight and 1/10 males at 93 mg/kg body weight died during the first week of the study. An additional female mouse died during week 12 of the study. Final mean body weights of dosed and control animals were similar. Hypoactivity, ataxia, and hypersensitivity to touch were seen at 1,500 mg/kg body weight. The female that received 1,500 mg/kg body weight and survived to the end of the study had body tremors, hypersensitivity to touch, and impaired hind limb function during the study. The relative liver weights for male and female mice that received 750 mg/kg body weight were significantly greater than of vehicle controls. Relative weights of brain, thymus, lungs/bronchi, heart, right kidney, and right testis (males) were not altered. Histological examinations were carried out on vehicle controls, mice receiving 750 mg/kg body weight, and all animals that died or were killed before the end of the study. No compound-related changes were seen (details on pathology not given) (NTP, 1989). 2.2.2.2 Rat Groups of five male and five female Osborne-Mendel rats were maintained on diets containing carvone (p-mentha-6,8-dien-2-one) (isomer not specified) at a level of 1000 ppm (approximately equivalent to 50 mg/kg bw/day) for 27-28 weeks, and at a level of 2500 ppm (approximately equivalent to 125 mg/kg bw/day) for one year. No adverse effects were observed in these rats as judged by growth, appearance, food intake, haematology, final body weights, or gross and microscopic examination of the major organs. Rats maintained on a diet containing carvone at a level of 10,000 ppm (approximately equivalent to 500 mg/kg bw/day) for 16 weeks showed growth depression and testicular atrophy (Hagan et al., 1967). Total doses of 2450-2950 mg carvone/kg body weight (isomer not specified) through seven daily subcutaneous injections were not able to increase liver regeneration in five to 15 partially hepatectomized Charles River rats (Gershbein, 1977). Groups of five F344/N rats (6-7 weeks old) of each sex were administered 0, 150, 328, 723, 1,590 or 3,500 mg/kg body weight (+)- carvone in corn oil by gavage, 5 days per week, for 12 doses over 16 days. The animals were housed five per cage and water and feed were given ad libitum. The rats were observed twice per day and were weighed on days 1, 7 and 14 and at the end of the study. A necropsy was performed on all animals. All rats that received 1,590 or 3,500 mg/kg body weight died during the first week of the study. The male rats that received 723 mg/kg body weight had 9% lower mean necropsy bodyweight than controls. Relative liver weights for male rats in the group receiving 723 mg/kg body weight and the female mice in the groups receiving 328 or 723 mg/kg body weigh were significantly higher than those of the vehicle controls. Relative kidney weights for rats of either sex that received 328 or 723 mg/kg body weight were significantly higher than those for the vehicle controls. The relative weights of the thymus of male rats at 723 mg/kg body weight and of brain in female rats at 723 mg/kg body weight were decreased. No histopathology was reported (NTP, 1989). Groups of 10 female and 30 male F 344/N rats (4-5 weeks old) were administered 0, 93, 375, or 1,500 mg/kg body weight (+)-carvone in corn oil by gavage, 5 days per week, for 13 weeks. The 20 extra males per group were included to be used for other studies, and are, except those in the high dose group, of no relevance for this study. Additional groups of 10 rats of each sex were included at dose levels of 187 or 750 mg/kg body weight on the same schedule. Animals were housed five per cage and allowed feed and water ad libitum. The rats were observed twice per day and were weighed at the beginning of the study, once per week thereafter, and at the end of the study. A necropsy was performed on all animals. All rats that received 1,500 mg/kg body weight died during the first weeks and 9/10 males and 10/10 females that received 70 mg/kg body weight died before the termination of the study (weeks 7-12). Final mean body weights of male rats that received 187 or 375 mg/kg body weight were lower (7% or 11%, respectively) than those of the controls. The relative liver weights for dosed males and females that received 93, 187 or 375 mg/kg body weight were increased. The relative kidney weights of males administered 187 or 375 mg/kg, and of females that received 375 mg/kg body weight, were also increased. The relative weight of the right testis and lungs/bronchi of males were increased at dose levels of 187 or 375 mg/kg body weight. In females receiving 375 mg/kg body weight the relative brain weights were decreased. Histopathology was performed on vehicle controls, rats receiving 375 mg/kg body weight and all animals dying or killed before the end of the study. Testicular degeneration was seen in the male rats at 750 mg/kg body weight. This lesion was characterized by moderate to marked loss of germinal epithelium in the seminiferous tubules and an absence of sperm in the epididymal ducts. In some rats, syncytial giant cells of spermatids were present in seminiferous tubules. Atrophy of the thymic cortex was present in rats of each sex which received 750 mg/kg body weight. Hyaline droplets and granular casts commonly associated with the accumulation of alpha2u-globulin were not observed in the kidney tissue of dosed rats. No further information was given on pathology. A planned 2 year chronic toxicity/carcinogenicity study in rats using doses of 0, 187 or 375 mg/kg body weight (+)-carvone in corn oil by gavage, 5 days per week, had to be terminated after 60 weeks due to low survival of dosed animals (males: vehicle control, 48/50; low dose, 41/50; high dose, 16/50; females: 50/50; 48/50; 38/50). No further details are given (NTP, 1989). 2.2.3 Long-term/carcinogenicity studies 2.2.3.1 Mouse Groups of 50 male and 50 female B6C3F1 (C57BL/6N, female x C3H/HeN MTV male) mice (7 week old) were administered 0, 375, or 750 mg/kg body weight (+)-carvone in corn oil by gavage, 5 days per week for 103 weeks. Animals were housed five per cage. Feed and water were available ad libitum. All animals were observed twice per day for clinical signs. No clinical signs were observed. Body weights were recorded once per week for the first 13 weeks of the study, and then at least once per month thereafter. The mean body weights of dosed and control male mice were similar throughout most of the study; the mean body weights of the dosed female mice were within 7% of those of the vehicle controls throughout most of the study. The survival of both the low dose (29/50 after week 101) and the high dose (38/50 after week 92) groups of female mice was significantly greater than that of the controls (14/50). This was probably caused by a very high death rate in the control females. The low survival of female vehicle control mice was related to a high incidence (52%) of ovarian abscesses similar to those seen in studies associated with Klebsiella sp. infections. Whether the lower incidence of ovarian abscesses in dosed female mice was related to (+)-carvone administration is uncertain. No differences in survival were observed between any groups of male mice. A necropsy was performed on all animals, including those found dead, except for tissues that were autolyzed. All organs and tissues were examined for grossly visible lesions and subjected to histopathology. Several lesions occurred in the nasal cavities of male and female mice with dose-related increased incidences and/or severity. Atrophy of the olfactory epithelium and hyperplasia of the underlying Bowman's glands occurred together with high incidence in either sex in both dosed groups. The effect on the nasal mucosa was probably due to a local effect of (+)-carvone caused by reflux of the gavage material when the gavage needle was withdrawn, since inflammatory exudate and evidence of the corn oil vehicle was found in over 50% of all dosed and control animals. No increases in tumour incidences were seen in mice administered (+)-carvone. The incidence of female control mice with primary neoplasms was 9/50 and the total number of primary neoplasms was 9. These numbers are low compared to 15/50 and 18 in the low dose female mice and 14/50 and 18 in the high dose mice and may be related to the early deaths of female control mice. The incidences of male mice with primary neoplasms (control, 27/50; low dose, 15/50; high dose, 16/50) and the total numbers of primary neoplasms (control, 38; low dose, 18; high dose, 20) were significantly lower in dosed groups than in vehicle controls. The lowered incidence of tumours in dosed male mice was primarily due to a lower incidence of subcutaneous fibromas, sarcomas, fibrosarcomas, or neurofibrosarcomas (NTP, 1989). 2.2.4 Reproduction studies No information available. 2.2.5 Special studies on in vitro toxicity The toxicity of carvone (isomer not specified) to Ascites sarcoma BP8 cells (originating from inoculated C3H mice) cultured in suspension was examined. Carvone was incubated with the cells at 37°C for 48 hours at levels of 0.1 or 1.0 uM. The percent culture growth inhibition at these levels was 13 and 100%, respectively (Pilotti et al., 1975). The effect of carvone (isomer not specified) on cell metabolism was investigated by measuring the inhibition of noradrenaline- induced oxidative metabolism in isolated hamster brown fat cells. At a level of 1 mM carvone, there was a 42% inhibition of noradrenaline-induced oxidative metabolism (Petterson et al., 1980). Carvone (isomer not specified) was reported to be ciliotoxic at a concentration of 5 mM in chicken tracheal organ cultures, with complete cessation of ciliary activity occurring six minutes after the addition of carvone to the culture (Petterson et al., 1982). Carvone, (isomer not specified), at a concentration of 25 mM, was incubated for 30 minutes at 37 °C with labelled human lung fibroblasts. The degree of membrane damage, as measured by the release of an intracellular nucleotide marker, was significantly increased, with 95% of the nucleotides being released in 30 minutes (Thelestam et al., 1980). When carvone (isomer not specified) was incubated with microsomes from phenobarbital pretreated rats a 25% conversion of cytochrome P-450 to cytochrome P-420 was observed. This conversion was not observed when rats were dosed (dose not specified) with carvone in vivo (Madyastha et al., 1985). The eggs of the insect Earias vittella were arranged in groups of 20 and exposed in vitro to various terpenoids. Appropriate controls were conducted and each experiment was replicated five times. Carvone (isomer not specified) did not inhibit embryonic development at any of the concentrations (not given) tested (Metha, 1979). 2.2.6 Special studies on carcinogenicity Groups of female A/He mice were given intraperitoneal injections of (+)- or (-)-carvone three times weekly for eight weeks. The mice were then observed for a further 16 weeks until the end of the study. The total doses given of each compound were 1200 or 6000 mg/kg body weight. No increases in tumour incidences in the lung, liver, kidney, spleen, thymus, intestine, salivary or endocrine glands were observed. Approximately 20 and 40% of the (+)- and (-)-carvone treated animals, respectively, did not survive until the end of the study regardless of dose. Approximately 20% of the control animals died. The vehicle, tricaprylin, used to administer carvone was found to be impure and resulted in a weight loss of three to four grams in the control animals during the first week of the study (Stoner et al., 1973). A group of approximately 15 female A/J mice was administered 0.2 mmol (approximately 30 mg) (+)-carvone by gavage one hour before oral administration of 20 mg/N-nitrosodiethylamine (NDEA)/kg body weight. This dosing schedule was repeated once a week for eight weeks, and the experiment terminated 26 weeks after the first dose of NDEA. Forestomach tumour formation was significantly inhibited in (+)-carvone treated mice, with more than a 63% reduction in the mean number of papillomas per mouse compared to the controls. A significant reduction (34%) in the number of pulmonary adenomas per mouse was also observed in the (+)-carvone treated mice (Wattenberg et al., 1989). 2.2.7 Special studies on genotoxicity (+)-Carvone at concentrations up to 333 µg/plate was negative for induction of gene mutations in Salmonella typhimurium strains TA 100, TA 1535, TA 1537 and TA 98 when tested in a preincubation assay in the presence or absence of Aroclor 1254 induced male Sprague Dawley rat or Syrian hamster liver S9 (Mortelmans et al., 1986; NTP, 1989). (+)-Carvone induced slight increases in sister chromatid exchanges and chromosomal aberrations in Chinese hamster ovary (CHO) cells with and without addition of Aroclor 1254 induced male Sprague Dawley rat liver S9. Results were statistically positive but in two of three sister chromatid exchange trials there were no correlation of dose with response. This phenomenon of no dose/response relations also occurred in the second trial of the chromosomal aberration test conducted without S9. In the chromosomal aberration test without S9 a chemical-induced delay in cell cycle was observed. This was not observed in the test for sister chromatid exchanges (NTP, 1989). 2.3 Observations in humans No irritation was observed in humans exposed to concentrations of 1% (-)-carvone in petrolatum in a 48-hour closed patch test (Opdyke, 1973). A maximization test was carried out on 25 volunteers. (-)-Carvone at a concentration of 1% in a petrolatum produced no sensitization reactions (Opdyke, 1973). In one of 80 patients suffering from the condition of orofacial granulomatosis, characterized by swelling of the lips and lower half of the face, an association with intolerance to carvone (isomer not specified) was indicated from patch testing (Patton, et al., 1985). Three out of 33 patients tested for contact allergy to toothpaste flavors, experienced a positive allergic response in a patch-test using 5% carvone (isomer not specified) in petrolatum (Anderson, 1978). 3. COMMENTS At its present meeting, the Committee considered that optical enantiomers should not per se be regarded as toxicologically identical compounds, and therefore (+)-carvone and (-)-carvone should be evaluated separately. Although both compounds were placed on the agenda, sufficient data for toxicological evaluation had been submitted only for (+)-carvone. In a long-term toxicity/carcinogenicity study in mice in which (+)-carvone was administered by gavage, no evidence of tumorigenicity was observed. Additional short-term studies in mice and rats, in which (+)-carvone was administered by gavage, and in vitro mutagenicity tests, were also considered . Rats appear to be more susceptible to (+)-carvone than mice. The no-observed- effect level for (+)-carvone in rats was 93 mg per kg of body weight based on the study in which the substance was administered by gavage for 3 months. The Committee noted that, in an earlier 1-year feeding study in rats in which a no-effect-level of 125 mg per kg of body weight per day was observed, it was not specified which isomer had been used. The Committee noted that only limited data were available on the metabolism and pharmacokinetics of (+)-carvone. 4. EVALUATION An ADI of 0-1 mg per kg of body weight per day was established for (+)-carvone based on a no-observed-effect level of 93 mg per kg of body weight per day in rats. The temporary ADI for (-)-carvone was not extended, because insufficient data were available for toxicological evaluation. 5. 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See Also: Toxicological Abbreviations