Rho1p, a yeast protein at the interface between cell polarization and morphogenesis.

The enzyme that catalyzes the synthesis of the major structural component of the yeast cell wall, beta(1-->3)-D-glucan synthase (also known as 1,3-beta-glucan synthase), requires a guanosine triphosphate (GTP) binding protein for activity. The GTP binding protein was identified as Rho1p. The rho1 mutants were defective in GTP stimulation of glucan synthase, and the defect was corrected by addition of purified or recombinant Rho1p. A protein missing in purified preparations from a rho1 strain was identified as Rho1p. Rho1p also regulates protein kinase C, which controls a mitogen-activated protein kinase cascade. Experiments with a dominant positive PKC1 gene showed that the two effects of Rho1p are independent of each other. The colocalization of Rho1p with actin patches at the site of bud emergence and the role of Rho1p in cell wall synthesis emphasize the importance of Rho1p in polarized growth and morphogenesis.

Environmental risk assessment for the polycyclic musks AHTN and HHCB in the EU. I. Fate and exposure assessment.

For the environmental exposure assessment of the fragrance ingredients 7-acetyl-1,1,3,4,4,6-hexamethyl-1,2,3,4-tetrahydronaphthalene (AHTN) and 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-gamma-2-ben zopyran (HHCB) the following properties were determined: vapour pressure 0.0682 and 0.0727 Pa; water solubility 1.25 and 1.75 mg/l; log K(ow) 5.7 and 5.9; log K(oc) 4.80 and 4.86; bioconcentration factor in fish: 597 and 1584 (fresh weight) for AHTN and HHCB, respectively. Both substances are degraded to more polar metabolites in fish, in soil and during sewage treatment. A review is made of concentrations measured in sludge, in freshwater and marine systems including suspended matter, sediment and fish. The 90th-percentile in more than 200 surface water samples is 0.3 microg/l for AHTN and 0.5 microg/l for HHCB. The 90th-percentile of the concentrations in fish is 0.12 mg/kg fresh weight for both substances (n = 27). These concentrations are lower by a factor of 5-15 than predicted on the basis of the yearly use volumes in Europe, 585 tonnes for AHTN and 1482 tonnes for HHCB. Concentrations in sludge-amended soils and in earthworms are predicted based on concentrations measured in sludge. For AHTN, the predicted values are: PECsoil, 0.029 mg/kg and PECworm, 0.065 mg/kg while for HHCB the corresponding figures are 0.032 and 0.099 mg/kg. These concentrations assume a biodegradation half-life in the soil of 180 days based on preliminary soil biodegradation data.

Environmental risk assessment for the polycyclic musks, AHTN and HHCB. II. Effect assessment and risk characterisation.

Reports of the polycyclic musks AHTN and HHCB in surface water and fish, primarily in Europe, have prompted studies of their environmental effects. These effects then are used, along with the predicted environmental concentrations in a risk assessment according to the approach developed under European Union Regulation 793/93, in line with the Technical Guidance Document for risk assessment of new and existing chemicals. In 72-h studies with algae (Pseudokirchneriella subcapitata), NOECs were 0.374 mg/l (AHTN) and 0.201 mg/l (HHCB). In 21-day reproductive tests with daphnia (Daphnia magna) NOECs were 0.196 (AHTN) and 0.111 mg/l (HHCB). In 21-day growth tests with bluegill sunfish (Lepomis macrochirus), NOECs were 0.067 (AHTN) and 0.068 mg/l (HHCB). And, finally 35-day early life stage tests with fathead minnows (Pimephales promelas) resulted in NOECs of 0.035 (AHTN) and 0.068 mg/l (HHCB). These results lead to Predicted No Effect Concentrations (PNEC) of 3.5 microg/l (AHTN) and 6.8 microg/l (HHCB) for aquatic organisms. For the soil compartment, 8-week studies with earthworms (Eisenia fetida) resulted in NOECs of 105 (AHTN) and 45 mg/kg (HHCB) and 4-week studies with springtails (Folsomia candida) resulted in a NOECs of 45 mg/kg for both substances. These values lead to a PNEC of 0.32 mg/kg dw for both materials. Using mammalian studies, PNECs for fish or worm eating predators of 10 mg/kg fw (AHTN) and 100 mg/kg fw (HHCB) can be derived. For sediment dwelling organisms, PNECs were derived by equilibrium partitioning using the aquatic PNECs. Comparing PNECs with the measured or predicted environmental exposures leads to risk characterisation ratios as follows: aquatic species: AHTN 0.086, HHCB 0.074; sediment organisms: AHTN 0.44, HHCB 0.064; soil organisms: AHTN 0.091, HHCB 0.10; fish eating predators: AHTN 0.012, HHCB 0.001; worm eating predators: AHTN 0.007, HHCB 0.001.

Dermal absorption and disposition of musk ambrette, musk ketone and musk xylene in rats.

Dermal doses of carbon-14 labelled musk ambrette (MA), musk ketone (MK) or musk xylene (MX) to male Sprague-Dawley CD rats were applied at a nominal dose level of 0.5 mg/kg (11 microg/cm2 of skin) and excess material removed at 6 h. Means of about 40, 31 and 19% of the applied doses of MA, MK and MX, respectively, were absorbed. Most of the absorbed material was excreted within 5 days with only 1-2% of the applied dose remaining in the animal at this time. Tissue concentrations of radiolabel were similar for all three compounds with peak concentrations occurring at 6-8 h. In general, fat and liver contained the highest concentrations at around 0.2 microg nitromusk equivalents/g but concentrations in fat declined fairly rapidly to around 0.005 microg equiv./g at 120 h. Most of the absorbed dose was eliminated in bile mainly in the form of polar conjugated metabolites. Structural characterisation of the major aglycones for MA and MX indicated that they were hydroxylated analogues formed by oxidation of the ring methyl. Repeated daily dosing for 14 days resulted in little bioaccumulation for musk xylene and accumulation of about three-fold for musk ketone.

Evaluation of the oral subchronic toxicity of HHCB (1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-gamma-2 -benzopyran) in the rat.

1,3,4,6,7,8-Hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-gamma-2-ben zopyran (HHCB) is used in a wide variety of fragrances in consumer products. Because of the widespread exposure to this material, a 90-day oral feeding study in accordance with OECD guidelines No. 408, with 4-week recovery periods for selected rats, was conducted. HHCB was added to the diet of rats at levels calculated to result in mean daily doses of 5, 15, 50 or 150 mg HHCB/kg. On completion of the treatment period, three males and three females from the high dose and control groups were maintained for a treatment free period of 4 weeks. No adverse effects were revealed from clinical examination or following extensive histopathological examinations. There were no significant findings at any dose level; a NOAEL of 150 mg/kg per day was concluded. As a supplement to the main study, histopathological examination of the prostate, seminal vesicles, mammary gland and testes of males and ovaries, mammary gland, uterus and vagina of females was undertaken on all animals in all test groups. This examination did not reveal any evidence of hormonal effects.

The systemic exposure to the polycyclic musks, AHTN and HHCB, under conditions of use as fragrance ingredients: evidence of lack of complete absorption from a skin reservoir.

7-Acetyl-1,1,3,4,4,6-hexamethyl-1,2,3,4-tetrahydronaphthalene (AHTN) and 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexa-methylcyclopenta-gamma-2-be nzopyran (HHCB) are two large volume fragrance ingredients widely used in consumer products. As part of the risk evaluation, the systemic exposures to these materials was determined in rats under occlusion and in humans under simulated conditions of exposure. Ring 14C-labeled AHTN or HHCB were applied dermally in alcoholic solutions to rats at doses of 4.5 mg/kg and occluded for 6 h. Urine, feces and air were collected for up to 120 h and analyzed for radioactivity. Pairs of rats were sacrificed periodically for analysis of tissues and organs. The total amount absorbed was approximately 19% for AHTN and 14% for HHCB. In both cases, significant amounts diffused into the skin, most of which was further absorbed but a significant amount of which was lost to surface dressing by reverse diffusion and/or desquamation. Ring 14C-labeled AHTN or HHCB were applied in alcoholic solutions without occlusion to three male volunteers at concentrations approximating that which might be encountered in a typical cologne type product. After a 6-h period, all material was removed from the surface of the skin. Blood, feces and urine were collected over a 5-day period. For both materials, levels in blood and plasma were below limits of detection at all times. Based on excretion, primarily in the urine, the total absorbed dose was approximately 1 and 0.1% for AHTN and HHCB, respectively. However, over the 5-day period, 14.5% of AHTN and 19.5% of HHCB was recovered from the skin in dressings over the site of application indicating that a 'reservoir' had formed in the skin but the material in the reservoir was lost, by desquamation and/or by reverse absorption, and not available systemically. A mean of 24% (AHTN) and 22% (HHCB) was shown to evaporate under the conditions of exposure.

Ventricular size and regional cerebral blood flow in schizophrenia: an attempted replication.

We attempted to replicate the finding of Berman et al. (1987) that frontal cortical blood flow correlated inversely with ventricular size in schizophrenia. Computed tomography and high-resolution 99m-Tc-HMPAO single photon emission computed tomography were performed in 25 right-handed chronic schizophrenic men engaged in a word-fluency task. Weak, nonstatistically significant inverse correlations were found between the ventricle-brain ratio (VBR) and frontal cortical blood flow. The VBR was significantly inversely correlated with the ratio of left to right medial frontal blood flow.

Evaluation of the dermal subchronic toxicity of diphenyl ether in the rat.

Diphenyl ether (DPE) was investigated to determine the dermal absorption parameters and subchronic toxicity of this fragrance ingredient. For the absorption, distribution and elimination study, Sprague-Dawley rats received a dermal application of [14C]DPE under a semi-occlusive dressing for 6 h. DPE was diluted in diethyl phthalate (DEP) to administer a total application volume of 2 ml/kg and concentrations of 0.5, 5 and 50% (approximately equal to 10, 100 and 1000 mg DPE/kg body weight). Approximately 17.7% of the administered dose was eliminated in the urine, with small amounts also found in the feces (1.18-3.79%). At 72 h post-dosing, approximately 0.2% of the applied dose was retained in the body with low levels also measured in the liver, kidney and gastrointestinal tract (approximately equal to 0.04, 0.02 and 0.3%, respectively). The 13-week subchronic toxicity study was performed with groups of 12 Sprague-Dawley rats/sex/dose that received semi-occluded daily dermal applications of DPE for 6 h/day. All groups were dosed at a constant 2 ml/kg body weight volume of DPE in the DEP vehicle at concentrations to administer 0, 100, 300 or 1000 mg DPE/kg body weight/day. At the high dose level, there was a slight reduction in body weight gain in males (13%), increase in albumin (5-6%) and phosphate (10-15%) levels in both sexes, a reduction of cholesterol in females (14%), an increase in kidney (17%) and brain (8%) weights in males, and an increase in liver weight (18-19%) in both sexes. No histopathological lesions were seen in any organ examined. At 300 mg/kg body weight/day, the only notable findings were an increase in liver weight (10%) in both sexes and a slight increase in albumin (5%) in females. In addition, skin irritation reactions at the site of application were observed in all DPE dose groups. The systemic no-observed-effect level (NOEL) in this study is 100 mg/kg body weight/day. Owing to mitigating factors, the systemic findings were judged to lack biological significance and the no-observed-adverse-effect level (NOAEL) was determined to be 1000 mg/kg body weight/day.

Safety evaluation of dibenzyl ether.

Dibenzyl ether (FEMA No. 2371, CAS No. 103-50-4) was given in the diet to rats at a rate of 62, 196 or 620 mg/kg/day for 91 consecutive days. Body weights and food consumption were measured weekly; haematological, clinical chemistry and urinalysis values were obtained at wk 6 and 12. Gross and microscopic pathological changes were observed and organ weights recorded. The high-dose females had increased absolute and relative liver weights; this was considered to be related to dose. Other statistically significant events that occurred sporadically within the test groups were unrelated to dose and were considered to be normal adaptive change. No toxicological or pathological effects were noted at any of the dose levels after 91 consecutive days of feeding dibenzyl ether. A no-effect level was achieved at 196 mg/kg/day. In a 60-kg human, this would be equivalent to approximately 11.8 g/day, assuming a direct relationship between dose and body weight across species. Based on the possible average daily intake of 19.2 mg/day, this would confer a safety factor of 600. The safety factor based on the more realistic consumption per capita of 23.6 micrograms/day would be approximately 500,000.

Safety evaluation of benzophenone.

Benzophenone (FEMA No. 2134; CAS No. 119-61-9) was administered in the diet to rats at target dose levels of 20 mg/kg body weight/day for 90 days and 100 or 500 mg/kg/day for 28 days. Body weights and food consumption were measured weekly; haematology, clinical chemistry and urinalysis values were obtained at 4 wk and at the end of the study. Gross and microscopic pathological examinations were conducted and organ weights were recorded. Treatment-related changes occurred in erythrocyte count, haemoglobin, haematocrit, bilirubin, total protein and albumin at the mid- and high-dose levels, although all changes did not occur in both groups in both sexes. There were indications of increased absolute and relative liver and kidney weights in the mid- and high-dose groups, but this was not statistically consistent for absolute kidney weights. Histopathology of the liver in the mid- and high-dose groups showed hepatocellular enlargement with an associated clumping of cytoplasmic basophilic material around the central vein. A no-effect level was demonstrated at 20 mg/kg/day for 90 days of administration. This would be equivalent to an intake of 1200 mg/day for a 60-kg human. On the basis of the calculated Possible Average Daily Intake of 0.33 mg/day, a safety factor of greater than 3600 is demonstrated. The safety factor based on the more realistic per capita consumption of 0.32 microgram/day would be approximately 3.7 million.

An evaluation of musk xylene in a battery of genotoxicity tests.

Musk xylene (CAS no. 81-15-2), a synthetic musk fragrance ingredient, was evaluated in a battery of short-term genotoxicity tests that included the mouse lymphoma assay, an in vitro cytogenetics assay in Chinese hamster ovary (CHO) cells, the in vitro unscheduled DNA synthesis (UDS) assay in primary rat hepatocytes and an in vivo UDS assay. Musk xylene gave uniformly negative results in these genotoxicity tests. These observation, combined with previously reported negative Ames tests, suggest a non-genotoxic mechanism for the induction of mouse liver tumours by musk xylene.

Correlation of structural class with no-observed-effect levels: a proposal for establishing a threshold of concern.

The relationship between chemical structure and toxicity was explored through the compilation of a large reference database consisting of over 600 chemical substances tested for a variety of endpoints resulting in over 2900 no-observed-effect levels (NOELs). Each substance in the database was classified into one of three structural classes using a decision tree approach. The resulting cumulative distributions of NOELs for each of the structural classes differed significantly from one another, supporting the contention that chemical structure defines toxicity. The database was used to derive a threshold of acceptable human exposure for each of the structural classes that could be applied in the absence of specific toxicity data on a substance within one of the three structural classes. The human exposure thresholds provide guidance on the degree of testing and evaluation required for substances that lack toxicity data.

Allergic contact sensitization potential of hydroxycitronellal in humans.

Hydroxycitronellal, an important ingredient in fragrances, was studied for its sensitizing potential in human skin. Fifteen human maximization tests were conducted with hydroxycitronellal obtained from four different sources at induction concentrations from 5 to 12%. No reactions were induced at 5% in two separate panels while 10% sensitized 2/25 panelists in one test but none in a second. Induction at 12% produced sensitization in 8 of 11 tests. Impurities do not appear to be a sensitizing factor. There is some evidence that the l-stereoisomer is a less potent sensitizer than the d-stereoisomer. In an initial modified human repeat-insult patch-test two positive reactions to challenge were observed among 197 panelists, one at a concentration of 5% and the other at 7.5%. When 100 of the non-reacting panelists were re-exposed in the same way, allergic sensitization reactions appeared during the induction period with concentrations as low as 2.5%. When 28 sensitized panelists were exposed to 1% concentrations in a simulated use test, there were three reactors. A no-effect level for sensitization has not been determined although the lowest concentrations tested were in the product usage range.

90-day dermal toxicity study and neurotoxicity evaluation of nitromusks in the albino rat.

Musk ketone, musk xylene, musk tibetene and moskene, synthetic musks used in fragrances, were applied dermally to rats in daily doses of 240 (musk ketone and musk xylene only), 75, 24 or 7.5 mg/kg body weight for 90 days. The chemically related musk ambrette, a known neurotoxin in rats, was used as a positive control. While musk ambrette was clearly neurotoxic and caused testicular atrophy, as had been previously reported, the other compounds tested caused neither effect. The only effects of application of these materials were some organ weight changes at the higher doses, but these were not associated with histopathological changes in any of the tissues. The no-effect levels were: musk ketone, 75 mg/kg for males and females; musk xylene, 75 mg/kg for males and 24 mg/kg for females; moskene, 24 mg/kg for males and 75 mg/kg (highest dose administered) for females; and musk tibetene, 75 mg/kg (highest dose) for males and females.

Comparative metabolism and kinetics of coumarin in mice and rats.

Coumarin, a well recognized rat hepatotoxicant, also causes acute, selective necrosis of terminal bronchiolar Clara cells in the mouse lung. Further, chronic oral gavage administration of coumarin at 200 mg/kg, a dose that causes Clara cell death, resulted in a statistically significant increased incidence of alveolar/bronchiolar adenomas and carcinomas in B6C3F1 mice. In contrast, mouse lung tumors were not observed at the 100 and 50 mg/kg dose levels in the oral gavage study, or in CD-1 mice following chronic intake of coumarin at levels equivalent to 276 mg/kg in diet. The current studies were designed to determine the impact of oral gavage vs dietary administration on the pharmacokinetics and metabolism of coumarin in CD-1 and B6C3F1 mice and F344 rats. Following the administration of 200 mg/kg 14C-coumarin via oral gavage, lung C(max) values (total 14C-associated radioactivity) were five- and 37-fold greater than those resulting from a 50 mg/kg oral gavage dose or 1000 ppm in diet, respectively. Coumarin (200 mg/kg) pharmacokinetics and metabolism was also examined in F344 rats following oral gavage dosing. Total 14C-coumarin associated radioactivity in plasma was 3.5-fold lower than in the mouse, and the plasma half-life in rats was five-times longer than in mice. Using non-radiolabeled compound (200 mg/kg), coumarin and products of the coumarin 3,4-epoxidation pathway were quantitated in plasma and urine after oral gavage administration to mice and rats. 7-Hydroxycoumarin (7-HC) was quantitated in mouse plasma and urine. o-Hydroxyphenylacetic acid (o-HPAA) reached a concentration of 37 microg/ml in plasma, and accounted for 41% of the dose in the urine, whereas the C(max) for 7-hydroxycoumarin was 3 microg/ml, and represented 7% of the administered dose. In the rat, the plasma C(max) for o-HPAA was 6 microg/ml, and accounted for 12% of the dose. The coumarin C(max) in rat plasma was comparable to that in mouse. Coumarin 3,4-epoxide (CE) and its rearrangement product o-hydroxyphenylacetaldehyde (o-HPA) and o-hydroxyphenylethanol (o-HPE), were not detected at any time point in plasma or urine. This analysis of coumarin and CE pharmacokinetics in rodents suggests that the differential tumor response in the mouse oral gavage and dietary bioassays is a function of the route of exposure, whereas species differences in lung toxicity between mice and rats result from heightened local bioactivation in the mouse lung.

The in vivo dermal absorption and metabolism of [4-14C] coumarin by rats and by human volunteers under simulated conditions of use in fragrances.

The disposition and metabolic fate of [4-14C]coumarin in a 70% aqueous ethanol solution was studied in male Lister Hooded rats after occluded dermal application and in three male volunteers after an exposure designed to simulate that which may be encountered when using an alcohol-based perfumed product. In both cases, the 6-h exposure was 0.02 mg/cm(2) (rats 0.023 mg/kg and humans 0.77 mg/kg). In both, coumarin was quickly absorbed, distributed and excreted in urine and feces, although fecal excretion of coumarin in humans was only 1% of the applied dose as opposed to 21% in rats. Total absorption was 72% of the applied dose with rats and 60% with humans. Peak plasma radioactivity in both was at 1 h. The mean plasma half-life of coumarin and metabolites was approximately 1.7 h for humans and 5 h for rats. In humans, coumarin was primarily metabolized to and excreted in urine as 7-hydroxycoumarin glucuronide and 7-hydroxycoumarin sulfate. Small amounts of unconjugated 7-hydroxycoumarin and o-hydroxyphenylacetic acid (o-HPAA) were also excreted. In rats, about twenty metabolites were present, but only o-HPAA was identified. These studies show the rat is a very poor model for humans and toxicity in the rat cannot be extrapolated to humans.

Influence of dose and sex on the disposition and hepatic effects of cinnamyl anthranilate in the B6C3F1 mouse.

Cinnamyl anthranilate is a synthetic food flavouring and fragrance agent, formerly used at very low levels. There is currently some concern over the potential risk to man from its use, since it has been found to cause liver tumours in mice, following the administration of very large doses. This paper reports studies of its disposition and hepatic effects in B6C3F1 mice in relation to dose. Following a single oral dose of 500 mg cinnamyl anthranilate/kg body weight to B6C3F1 mice peak plasma levels of unchanged compound were reached in 30 min, and were higher in males than in females. Unchanged cinnamyl anthranilate in the urine accounted for 0.3-0.4% of the dose. Anthranilic acid (c. 17%) and hippuric acid (35%; the major metabolite of cinnamyl alcohol) were present in the urine, and recoveries of both were higher in females. Groups of male and female B6C3F1 mice were given 0, 10, 100, 1000, 5000, 15,000 and 30,000 ppm cinnamyl anthranilate in the diet. After 4 days, the diet was removed and urine collected for 24 hr. This contained cinnamyl anthranilate (more in males) hippuric and anthranilic acids (more in females) in concentrations that increased with dose. Other animals were given these diets for 19 days and then killed. Relative liver weight and hepatic microsomal cytochrome P-450 increased with increasing dose above 1000 ppm cinnamyl anthranilate, more markedly in males than in females although the maximum response (roughly twofold) was very similar in the two sexes. SDS-PAGE examination of the microsomes revealed the induction of a cytochrome P-450 isozyme of 53.1 kDa, but the aniline hydroxylase and p-nitroanisole O-demethylase activities of the 9000 g supernatant of liver were not induced. The data are discussed in terms of their significance for the human safety evaluation of cinnamyl anthranilate. It is important to note that liver hypertrophy, microsomal enzyme induction and the excretion of unchanged cinnamyl anthranilate all have the same dose-threshold for their appearance. This suggests that the hepatic effects of cinnamyl anthranilate may be mediated by unhydrolysed cinnamyl anthranilate, which is present only at very high doses due to the saturation of its hydrolysis.

The FEMA GRAS assessment of furfural used as a flavour ingredient. Flavor and Extract Manufacturers' Association.

The Expert Panel of the Flavor and Extract Manufacturers' Association (FEMA) has assessed the safety of furfural for its continued use as a flavour ingredient. The safety assessment takes into account the current scientific information on exposure, metabolism, pharmacokinetics, toxicology, carcinogenicity and genotoxicity. Furfural was reaffirmed as GRAS (GRASr) as a flavour ingredient under conditions of intended use based on: (1) its mode of metabolic detoxication in humans; (2) its low level of flavour use compared with higher intake levels as a naturally occurring component of food; (3) the safety factor calculated from results of subchronic and chronic studies, (4) the lack of reactivity with DNA; and (5) the conclusion that the only statistically significant finding in the 2-year NTP bioassays, an increased incidence of hepatocellular adenomas and carcinomas in the high-dose group of male mice, was secondary to pronounced hepatotoxicity. Taken together, these data do not indicate any risk to human health under conditions of use as a flavour ingredient. This evidence of safety is supported by the occurrence of furfural as a natural component of traditional foods, at concentrations in the diet resulting in a 'natural intake' that is at least 100 times higher than the intake of furfural from use as a flavour ingredient.

The FEMA GRAS assessment of trans-anethole used as a flavouring substance. Flavour and Extract Manufacturer's Association.

This publication is the fourth in a series of safety evaluations performed by the Expert Panel of the Flavour and Extract Manufacturers' Association (FEMA). In 1993, the Panel initiated a comprehensive program to re-evaluate the safety of more than 1700 GRAS flavouring substances under conditions of intended use. In this review, scientific data relevant to the safety evaluation of trans-anethole (i.e. 4-methoxypropenylbenzene) as a flavouring substance is critically evaluated by the FEMA Expert Panel. The evaluation uses a mechanism-based approach in which production of the hepatotoxic metabolite anethole epoxide (AE) is used to interpret the pathological changes observed in different species and sexes of laboratory rodents in chronic and subchronic dietary studies. Female Sprague Dawley rats metabolize more trans-anethole to AE than mice or humans and, therefore, are the most conservative model for evaluating the potential for AE-induced hepatotoxicity in humans exposed to trans-anethole from use as a flavouring substance. At low levels of exposure, trans-anethole is efficiently detoxicated in rodents and humans primarily by O-demethylation and omega-oxidation, respectively, while epoxidation is only a minor pathway. At high dose levels in rats, particularly females, a metabolic shift occurs resulting in increased epoxidation and formation of AE. Lower activity of the "fast" acting detoxication enzyme epoxide hydrolase in the female is associated with more pronounced hepatotoxicity compared to that in the male. The continuous intake of high dose levels of trans-anethole (i.e. cumulative exposure) has been shown in dietary studies to induce a continuum of cytotoxicity, cell necrosis and cell proliferation. In chronic dietary studies in rats, hepatotoxicity was observed when the estimated daily hepatic production of AE exceeded 30 mg AE/kg body weight. In female rats, chronic hepatotoxicity and a low incidence of liver tumours were reported at a dietary intake of 550 mg trans-anethole/kg body weight/day. Under these conditions, daily hepatic production of AE exceeded 120 mg/kg body weight. Additionally, neither trans-anethole nor AE show any evidence of genotoxicity. Therefore, the weight of evidence supports the conclusion that hepatocarcinogenic effects in the female rat occur via a non-genotoxic mechanism and are secondary to hepatotoxicity caused by continuous exposure to high hepatocellular concentrations of AE. trans-Anethole was reaffirmed as GRAS (GRASr) based on (1) its low level of flavour intake (54 microg/kg body weight/day); (2) its metabolic detoxication pathway in humans at levels of exposure from use as a flavouring substance; (3) the lack of mutagenic or genotoxic potential; (4) the NOAEL of 120 mg trans-anethole/kg body weight/day in the female rat reported in a 2 + -year study which produces a level of AE (i.e. 22 mg AE/kg body weight/day) at least 10,000 times the level (0.002 mg AE/kg body weight day) produced from the intake of trans-anethole from use as a flavouring substance; and (5) the conclusion that a slight increase in the incidence of hepatocellular tumours in the high dose group (550 mg trans-anethole/kg body weight/day) of female rats was the only significant neoplastic finding in a 2+ -year dietary study. This finding is concluded to be secondary to hepatotoxicity induced by high hepatocellular concentrations of AE generated under conditions of the study. Because trans-anethole undergoes efficient metabolic detoxication in humans at low levels of exposure, the neoplastic effects in rats associated with dose-dependent hepatotoxicity are not indicative of any significant risk to human health from the use of trans-anethole as a flavouring substance.

The FEMA GRAS assessment of alicyclic substances used as flavour ingredients.

For over 35 years, an independent panel of expert scientists has served as the primary body for evaluating the safety of flavour ingredients. This group, the Expert Panel of the Flavor and Extract Manufacturers' Association (FEMA), has achieved international recognition from the flavour industry, government regulatory bodies including the Food and Drug Administration, and the toxicology community for its unique contributions. To date, the Expert Panel has evaluated the safety of more than 1700 flavour ingredients and determined the vast majority to be "generally recognized as safe" (GRAS). Elements that are fundamental to the safety evaluation of flavour ingredients include exposure, structural analogy, metabolism, pharmacokinetics and toxicology. Flavour ingredients are evaluated individually taking into account the available scientific information on the group of structurally related substances. The elements of the GRAS assessment program as they have been applied by the Expert Panel to the group of 119 alicyclic substances used as flavour ingredients, and the relevant scientific data which provide the basis for the GRAS status of these substances, are described herein.