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 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 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.

Computer programs for calculation of median effective dose (LD50 or ED50) using the method of moving average interpolation.

Over the past 40 years, toxicologists and pharmacologists have used tables published by Weil for the determination of LD50 (or ED50) values and their associated 95% confidence intervals. With the advances in computer technology, it is now common for investigators to have personal computers in their laboratories. Therefore, two identical programs were developed for determination of the LD50 (or ED50) which may be run on a personal computer. One of these programs was written in BASIC, and the other in FORTRAN. The programs are easy and rapid to use, requiring minimum computer hardware and little, if any, knowledge of programming. They also offer more user flexibility than the previously published tables of Weil, in that there are fewer restrictions on the number of animals and number of dosage levels used in an experiment. The output of the programs may be typed on the screen of a computer monitor, and may be sent to a printer. The two programs calculate the LD50 and 95% confidence intervals for the LD50. These programs should be valuable for many investigators.

Safety of Sanguinaria extract as used in commercial toothpaste and oral rinse products.

This report represents the findings of an Expert Panel on the safety of Sanguinaria extract used in Viadent oral rinse and toothpaste products and represents an independent review of the Sanguinaria extract toxicologic data base. It is based on reviews and discussions of the data base by all members of the Expert Panel on Sanguinaria extract. The Panel concluded that the data base on Sanguinaria extract is substantial and indicates that Sanguinaria extract is safe in its present use in Viadent products based on a large margin of safety between levels of human exposure and levels found to produce minimum effect or to be without adverse effect in animals. The panel further concluded that published literature suggesting an association between human exposure to Sanguinaria extract and potential reproductive, cardiovascular, or ocular toxicity, or carcinogenicity is largely anecdotal, unfounded, and not corroborated by or consistent with the substantial data base that was subjected to peer review.

Dermal oncogenicity studies on two methoxysilanes and two ethoxysilanes in male C3H mice.

The dermal oncogenic potential of beta-(3,4-epoxycyclohexyl)ethyltrimethoxysilane (EEMS), gamma-glycidoxypropyltrimethoxysilane (GPMS), beta-(3,4-epoxycyclohexyl)ethyltriethoxysilane (EEES), and gamma-glycidoxypropyltriethoxysilane (GPES) was assessed by applying 25-microliters aliquots of acetone solutions to the skin of 40 male C3H/HeJ mice. The concentrations applied were 100, 25, 10, and 10% by volume for EEMS, GPMS, EEES, and GPES, respectively. Applications were made thrice weekly until the death of the animals. A negative control group received acetone (solvent) only. No treatment-related skin tumors were observed, nor was there evidence of increased incidence of any internal tumor in the groups that received GPMS, EEES, or GPES. In the group treated with EEMS, four mice were observed with squamous cell carcinomas of the treated skin and two mice had subcutaneous sarcomas outside of the treated area. No skin tumors were observed in the group treated with acetone, but two mice had subcutaneous sarcomas outside of the treated area. The mean survival times were 529, 482, 545, 492, and 502 days for the EEMS, GPMS, EEES, GPES, and acetone control groups, respectively. In no case was the mortality rate significantly different from that of the controls. The results indicate that only EEMS was oncogenic under the conditions of these studies.

Dermal oncogenicity studies on various ethyleneamines in male C3H mice.

The dermal oncogenic potential of diethylenetriamine, high purity and commercial grades (DETA-HP and DE-TA-C), triethylenetetramine (TETA), tetraethylenepentamine (TEPA), and polyamine HPA No. 2 was assessed by applying 25-microliter aliquots of aqueous solutions to the skin of groups of 50 male C3H/HeJ mice. The concentrations applied were 5.0, 5.0, 5.0, 25.0, and 10.0% by volume for DETA-HP, DETA-C, TETA, TEPA, and HPA No. 2, respectively. Applications were made thrice weekly until the death of the animals. A negative control group received deionized water (solvent) only. All animals were individually housed. No treatment-related skin tumors were observed, nor was there evidence of increased incidence of any internal tumor. Twenty TEPA-treated mice had hyperkeratosis and 13 had necrosis of the epidermis, both indicative of skin irritation. Such lesions were absent or occurred very infrequently in the other groups of mice. The mean survival times were 587, 662, 627, 591, 601, and 626 days for the DETA-HP, DETA-C, TETA, TEPA, HPA No. 2, and water control groups, respectively. In no case was the mortality rate significantly different from that of the controls. The results indicate that none of these compounds was oncogenic under the conditions of these studies.

Chronic toxicity and oncogenicity studies of ethylene glycol in rats and mice.

These studies were performed to assess the chronic toxicity and oncogenicity of ethylene glycol (EG) in rats and mice. Groups of 130 Fischer 344 rats and 80 CD-1 mice per sex were fed diets yielding approximate dosages of 1.0, 0.2, or 0.04 g/kg/day of EG. Two separate control groups in each study received no EG. Mortality rate was increased in high-dose male rats all of which died by 475 days. The following effects were also observed in high dose male rats: reduced body weight gain, increased water intake, increased blood urea nitrogen and creatinine, reduced erythrocyte count, reduced hematocrit and hemoglobin, increased neutrophil count, increased urine volume, reduced specific gravity and pH. Urinary calcium oxalate crystals and increased kidney weight were seen in all high-dose rats. Uric acid crystals were seen in the urine of high-dose female rats at 18 and 24 months. Histopathologic changes in high-dose male rats included tubular cell hyperplasia, tubular dilation, peritubular nephritis, parathyroid hyperplasia, and generalized soft tissue mineralization. Fatty change of the liver was seen in high- and intermediate-dose female rats. No clinical signs, or gross or microscopic evidence of toxicity was seen in mice at the dosages used. Water intake and clinical pathologic parameters were not measured in the mouse study. In these studies there was no evidence of an oncogenic effect of EG in rodents.

Three-generation reproduction and dominant lethal mutagenesis studies of ethylene glycol in the rat.

To assess the possible effects of ethylene glycol (EG) on reproductive performance and mutagenesis, three-generation reproduction and dominant lethal mutagenesis studies were performed in the Fischer 344 rat. EG was included in the diet at approximate dosages of 1.0, 0.2, and 0.04 g/kg/day during three generations of reproduction. Each generation was bred once. In a dominant lethal mutagenesis study, the F2 males from the reproduction study were bred to three consecutive lots of untreated females at weekly intervals. Concomitantly, another group of untreated F2 males that received a single ip injection of 0.50 mg/kg triethylenemelamine (TEM) were bred similarly to serve as a positive control group. No evidence of reduced fertility or increased fetal death was observed in any of the groups receiving EG. Dominant lethal effects in the TEM group confirmed the susceptibility of the rats to a known mutagen. In conclusion, there were no reproductive or dominant lethal effects associated with the inclusion of as much as 1.0 g/kg/day of EG in the diet.

Influence of housing conditions for mice on the results of a dermal oncogenicity bioassay.

Male C3H/HeJ mice were thrice weekly given 25 microliter applications of 0.25, 0.05, or 0.01% (w/w) benzo(a)pyrene (BaP) in acetone, or acetone alone, to clipped dorsal skin from 12 to 14 weeks of age for the remainder of their life spans. There were two groups of 40 mice for each treatment regimen, one group being housed in conventional stainless-steel wire mesh cages and the other in polycarbonate cages with wood shavings held in an enclosed ventilated cabinet. Under both housing conditions, tumor incidence was directly related and latency inversely related to BaP concentration. The time-adjusted incidence of epidermal neoplasms was significantly greater for the groups housed in polycarbonate cages. Mortality rates were directly related to BaP concentration and were significantly enhanced by the polycarbonate-cage housing conditions for the high and intermediate concentrations. Survival patterns for the two acetone control groups were similar. These findings indicate that differences in housing conditions can influence both the incidence and the latency of local neoplasms produced in response to the chronic application of a carcinogen in dermal oncogenesis bioassays.

Dermal oncogenicity bioassays of monofunctional and multifunctional acrylates and acrylate-based oligomers.

Several important components of photocurable coatings were studied for dermal tumorigenic activity by repeated application to the skin of mice. The substances tested were 2-ethylhexyl acrylate (EHA) and methylcarbamoyloxyethyl acrylate (MCEA) (monomers); neopentyl glycol diacrylate (NPGDA), esterdiol-204-diacrylate (EDDA), and pentaerythritol tri(tetra)acrylate (PETA) (cross-linkers); and three acrylated urethane oligomers. For each bioassay, 40 C3H/HeJ male mice were dosed 3 times weekly on the dorsal skin for their lifetime with the highest dose of the test agent that caused no local irritation or reduction in body weight gain. Two negative control groups received acetone (diluent) only. A positive control group received 0.2% methylcholanthrene (MC). NPGDA and EHA had significant tumorigenic activity with tumor yields of eight and six tumor-bearing mice (three and two malignancies), respectively. The MC group had 34 mice with carcinomas and 1 additional mouse with a papilloma. MCEA had no dermal tumorigenic activity but resulted in early mortality. No skin tumors in the treatment area were observed in the other groups. Additional studies will be necessary to elucidate possible relationships between structure and tumorigenic activity for the acrylates.

A subchronic inhalation study on the toxicologic potential of ethylene oxide in B6C3F1 mice.

Four groups of B6C3F1 mice, each containing 30 per sex, were exposed to ethylene oxide (EtO) vapor at target concentrations of 250, 100, 50, 10, or 0 ppm which are equivalent to 450, 180, 90, 18, and 0 mg/m3, respectively. The actual mean chamber concentrations were 236, 104, 48, 10, and 0 ppm, respectively. After an exposure regimen of 6 hr per day, 5 days per week, for 10 weeks (males), or 11 weeks (females), urine and blood samples were taken for clinical pathologic evaluation and selected tissues were weighed and examined by light microscopy. Statistically significant findings that could be indicative of a toxic response were observed in the 250-ppm exposure group. These included minimal changes in certain erythroid parameters, increased liver weight, decreased testicular weight, and decreased spleen weight which was noted also in the 100-ppm group. However, there were no microscopic findings to support or explain any of these apparently treatment-related effects. Results of a neuromuscular screening test indicated that certain reflex responses and locomotor activities were altered in the EtO-exposed animals. A dose-related trend of response in the 250-, 100-, and 50-ppm exposure groups was noted in the evaluation of locomotor function; however, because of the small sample size, it was not possible to determine a threshold concentration for this effect. There were no accompanying histopathologic alterations in muscle and central or peripheral nervous tissue.

A two-year inhalation study of the carcinogenic potential of ethylene oxide in Fischer 344 rats.

Fischer 344 rats were exposed to 100, 33, 10, or 0 ppm of ethylene oxide vapor (EtO) by inhalation for 6 hr per day, 5 days per week, for approximately 2 years. Inhalation of EtO resulted in a significant depression of body weight gain in the 100- and 33-ppm exposure groups and a significant increase in mortality in the 100-ppm group. Through 18 months of exposure to EtO, no statistically significant increases in tumor incidence were observed. After 18 months, the incidence of primary brain tumors was increased for both sexes. Statistical evaluation indicated a treatment-related response, particularly for the male rats, in the 100- and 33-ppm exposure groups. After 24 months of exposure, histologic findings confirmed hematologic evidence that exposure to EtO resulted in an increased prevalence of mononuclear cell leukemia, which is a neoplasm that is common in aged Fischer 344 rats. This increase was dose related and increased for each of the three exposure concentrations. The percentage of female rats with multiple neoplasms was also greater in all three exposure groups than in controls. Furthermore, in both the 100- and 33-ppm exposure groups, the percentage of female rats with at least one malignant neoplasm was increased. An increased frequency of peritoneal mesothelioma was treatment related in the male rats exposed to 100 or 33 ppm of EtO. This study has shown biologically significant adverse effects at all concentrations tested. The incidences of mononuclear cell leukemia, peritoneal mesothelioma, and primary brain tumors in the air-control rats were similar to those reported in the literature. The possible contribution of a sialodacryoadenitis viral outbreak (which occurred during the 15th exposure month) to the EtO exposure-related tumors is unknown, though unlikely.

Dermal oncogenicity bioassays of acrylic acid, ethyl acrylate, and butyl acrylate.

Male C3H/HeJ mice (40 per group) were treated with 25-microliter applications of undiluted ethyl acrylate, 1% acrylic acid, or 1% butyl acrylate on the dorsal skin 3 times weekly for their lifetime. A negative control group received acetone (diluent) only, and a positive control group received 0.1% 3-methylcholanthrene (MC). No epidermal tumors were observed in the animals that received any of the three test substances. In the positive control group, 39 animals had skin tumors, including 33 with confirmed squamous-cell carcinomas. Nonneoplastic skin changes such as dermatitis, dermal fibrosis, epidermal necrosis, and hyperkeratosis were observed in several mice that received ethyl acrylate. No statistically significant effects on survival were seen. Therefore, there was no evidence for local carcinogenic activity of acrylic acid, ethyl acrylate, or butyl acrylate under the conditions of these studies.

Dermal oncogenicity bioassays of di-sec-butoxyacetophenone and diethoxyacetophenone.

Groups of 40 male C3H/HeJ mice were treated 3 times per week for their lifetime on the skin of the back with undiluted di-sec-butoxyacetophenone (DBAP), or with diethoxyacetophenone (DEAP), undiluted and as a 50% dilution in acetone. Approximate doses per application were 22.4 mg for DBAP, and 25.0 or 11.3 mg for DEAP. Two negative control groups received acetone only. Both DBAP and DEAP (undiluted) had weak tumorigenic activity. In the DBAP group, one mouse developed a squamous-cell papilloma, whereas one mouse in the high-dose DEAP group had a squamous-cell carcinoma, both tumors appearing in the treatment area. No skin tumors were observed in the other groups. Both DEAP-treated groups also had substantial incidences of hyperkeratosis, epidermal hyperplasia, and dermatitis. No significant reduction in survival was observed in the test groups. The occurrence of the skin tumors in the DBAP- and DEAP-treated groups is considered to be treatment-related and suggestive of oncogenic potential, because of the extremely low historical control incidence of skin tumors in similar studies.

Acute and subchronic toxicity of ethylenediamine in laboratory animals.

As part of a comprehensive toxicology program on ethylenediamine (EDA), acute, short-term repeated and subchronic toxicity studies were conducted. Ethylenediamine dihydrochloride (EDA X 2HCl) was used in these studies. EDA X 2HCl was slightly to moderately toxic to laboratory rats, mice or rabbits in the following acute tests: Peroral intubation, percutaneous administration, primary skin irritation and eye injury. Following dietary inclusion of EDA X 2HCl for 7 days at up to 2.70 g/kg/day to Fischer 344 rats or B6C3F1 mice, body weight gain and some organ weights of the animals were depressed in both sexes at the highest dose level. When Fischer 344 rats were fed EDA X 2HCl at 0, 0.05, 0.25, 1.00 g/kg/day for 3 months, marked decreases in body weight gain were observed in both sexes at the highest dose level. Other dose-related effects observed in either or both sexes primarily at the highest dose level, and for some at the intermediate dose level, included organ weight changes and alterations in some clinical chemistry, hematology and urinalysis parameters. No deaths occurred during the exposure period, nor were there any significant gross lesions in any of the animals. Histologic findings indicate a dose-related increase in hepatocellular pleomorphism and mild hepatocellular degeneration.

Economical LD50 and slope determinations.

Many regulatory standards and guidelines required LD50 determinations and dose-response curve slopes, using at least 5 animals per sex per dosage level and at least 3 levels. In contrast, the current trend is to use as few animals as necessary for toxicological studies. The moving average method for calculation of the LD50 has been available since 1947 but, as it does not involve curve fitting, has not been used to determine the slope. An accurate adaptation of this is presented so that both the LD50 and slope can be determined using very few animals and using levels with 0 or 100% kills; not usable with probit determinations.