More than half a century has passed since the first FEMA Expert Panel began a program to assess the safety of flavor ingredients for their intended use in food. Under the authority provided by the 1958 Food Additives Amendment to the Food, Drug and Cosmetic Act—Public Law No. 85-929, 72 Stat. 1784 (1958), codified at 21 U.S.C. Sec. 348 (1988)—the program’s primary objective is to evaluate whether or not substances nominated by the flavor industry can be considered “generally recognized as safe” (GRAS) for their intended use as flavor ingredients. In existence since 1960, the FEMA GRAS program has become the longest-running and most widely recognized industry-sponsored GRAS assessment program.
The FEMA GRAS program began with the passage of the Food Additives Amendment, which defined a food additive as: “… any substance … which … may … [become] a component or … [affect] the characteristics of any food … if such substance is not generally recognized, among experts qualified by scientific training and experience to evaluate its safety, as having been adequately shown through scientific procedures … to be safe under the conditions of its intended use.”
This definition removed from consideration as food additives those substances deemed GRAS, therefore explicitly excluding them from mandatory premarket approval by the U.S. Food and Drug Administration (FDA). This allowed the FDA to dedicate resources to food additive issues of greater safety concern.
The GRAS 25 publication includes the results of the Expert Panel’s review of 61 new GRAS flavoring substances (Tables 1, 2, and 3). In addition, based on poundage and use level data provided by the International Chewing Gum Association (ICGA), the Expert Panel determined that new use levels for 560 flavoring substances in the chewing gum category are consistent with their current GRAS status (Table 4).
In this publication, the Panel also critically reviews the results of chronic 2-year bioassays performed at the National Toxicology Program (NTP) for pyridine (FEMA No. 2966). Also included are changes in the GRAS status of two substances recently re-evaluated by the Expert Panel, an update on the GRAS status of common salts of GRAS substances, and an update on the current membership of the Panel.
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ICGA Chewing Gum Survey and FEMA GRAS Re-evaluation
The ICGA (formerly the National Association of Chewing Gum Manufacturers) conducted surveys of the use of flavoring ingredients employed in chewing gum in 19721 and again in 1975. These surveys provided important information on how flavor ingredients were used in chewing gum; for instance, between 1972 and 1975, 252 additional flavoring substances were reported to be used in chewing gum. Although these surveys were part of an ongoing ICGA program to periodically review the use of flavors in chewing gum, this program was superseded by the FDA’s interest in undertaking a comprehensive review of the patterns and volumes of use of food additives and GRAS substances used in food. The chewing gum surveys were thus incorporated into regular flavor poundage surveys performed by the U.S. flavor industry and sponsored by the FDA and the National Academy of Sciences. Although flavor ingredient poundage used in chewing gum was monitored approximately every 10 years as part of the larger flavor poundage surveys, there was no ongoing program to monitor the changes in use levels or the introduction of new flavor ingredients into chewing gum products during this time. Therefore, in 2000, the ICGA decided to perform a comprehensive survey of the addition of flavor ingredients to chewing gum products.
The 2000 ICGA flavor survey2 was designed primarily to determine key statistics relevant to the total use of each flavor ingredient covered by the survey in the chewing gum industry in calendar year 2000, including:
1) Number of manufacturers and number of products in which the substance was used;
2) The average (arithmetic mean) concentration at which the substance was used in chewing gum;
3) The weighted mean concentration at which the substance was used;
4) The median concentration at which the substance was used;
5) The highest concentration at which the substance was used;
6) The total amount of the substance used in 2000 (calendar year), based on the weighted mean concentration of the substance and the volume of chewing gum disappearing into the marketplace.
The final report covered 768 FEMA GRAS flavoring substances for which data related to use in chewing gum were collected for the year 2000. The vast majority of those 768 flavoring substances are also FDA-approved food additives or FDA GRAS substances allowed for use in the U.S. at levels consistent with good manufacturing practice. In the total number reported in the ICGA Survey, numerous flavoring ingredients were determined to exhibit usual and/or maximum levels higher than those reported in the earlier surveys. The Expert Panel had previously recommended (Smith and Ford, 1993) that usual and maximum levels that may potentially result in significantly increased exposure be re-evaluated; therefore, ICGA submitted all substances with higher usual or higher maximum levels for review to ensure their new uses were consistent with current GRAS status.
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While not all uses of flavoring substances in chewing gum were covered by the ICGA 2000 survey, ICGA estimated that more than 95% of the chewing gum products sold at that time in the United States were surveyed. The pattern of use of these substances will obviously vary from time to time as the availability and price of various flavoring substances fluctuates or manufacturers discontinue or modify existing products, or introduce new products.
Good Manufacturing Practice for Chewing Gum Flavoring
Compared to other food uses, a higher level of flavoring substance is required in gum in order to achieve acceptable flavoring properties. Since sufficient flavor must be available to maintain a palatable taste for periods of at least 20 minutes, a far higher concentration of flavoring substance is used in chewing gum compared to use in other food products. Chewing also stimulates an increased flow of saliva, thus requiring a more concentrated flavoring to compensate for dilution. Additionally, a piece of chewing gum weighs far less than an individual serving of most other food products3. Therefore, a proportionately higher concentration of flavoring substance must be used to achieve a given quantity of flavoring in chewing gum than in other food products4.
Another key factor that determines flavor concentration is the retention of flavoring substance within the chewing gum base. The gum base retains a portion of the flavoring throughout the chewing period. The rate and amount of release of a flavoring agent from a specific gum base during chewing, referred to as “chewout,” is also determined in part by the structure and physicochemical properties of the flavoring substance. Since saliva is aqueous, the water solubility of a substance may be related to the amount of chew-out, provided that the flavoring substance does not undergo chemical changes that affect these properties. ICGA developed a program to analyze chew-out data for 44 representative flavor ingredients that are used in chewing gum, and provided that information to the Panel. The Panel incorporated these data where relevant to refine intake estimates for the applicable flavor ingredients used in chewing gums that were re-evaluated for FEMA GRAS status.
Determination of Intake of Flavor Ingredients from Chewing Gum
In order to make meaningful comparison of the use and intake of flavoring substances from chewing gum with the use and intake of these substances in other foods, several additional factors were considered by the Expert Panel. A simple comparison of the concentrations (in ppm) of flavoring substances in chewing gum with the concentrations (in ppm) in other foods is misleading. More relevant information is obtained by comparing the weight of the substance consumed on a per serving basis or a per day basis. For chewing gum, a “serving” is a piece of gum chewed for about 20 minutes. If the stick is chewed for a lesser time, it is equivalent to part of a serving because a smaller portion of the flavoring substance is released during a shorter period.
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As one example, consider a 2 g (2,000 mg) stick of gum that contains 0.8% (8,000 ppm) peppermint oil, which itself contains 2% menthofuran. The 2 g stick of gum would contain 16 mg of peppermint oil and 0.32 mg of menthofuran. Upon chewing for 20 minutes, only 10%, or 0.032 mg of the menthofuran, is chewed out. If the person chewing weighs 60 kg, the intake of menthofuran would be less than 0.0005 mg/kg body weight (0.5 μg/kg body weight). By comparison, if a 20 g portion of soft candy contained 100 ppm peppermint oil that contain 2% menthofuran, the intake of peppermint oil would be 2 mg, and the intake of menthofuran would be 0.04 mg/person or for a 60 kg body weight, 0.0006 mg/kg body weight (0.6 μg/kg body weight). Although both intakes are relatively small, intake of menthofuran (0.5 μg/kg body weight) from chewing a 2 g stick of gum containing 8,000 ppm peppermint oil for 20 minutes is roughly equivalent to the intake of menthofuran from consuming a 20 g portion of soft candy containing 100 ppm of the same peppermint oil.
FEMA GRAS Re-evaluation
The Panel re-evaluated all relevant metabolic and toxicity data for these flavor ingredients within the context of their increased use levels in chewing gum. The intake of flavoring substance from chewing gum—given its light weight per serving, flavor retention rates, but seemingly high added concentrations—may be roughly equivalent to the intake of these same substances at lower concentration in foods that are consumed over a shorter time period and have a much greater portion size. Therefore, in cases where the relevant data were available, the intake related to use of these flavor ingredients in chewing gum was evaluated in the context of the effects of portion size, level of chew-out, and other factors affecting the intake of the flavor ingredient. Based on their review of the intake data, and the relevant metabolic and toxicity data for these substances, the FEMA Expert Panel determined that the new or higher use levels in chewing gum for the 560 flavoring ingredients listed in Table 4 are consistent with their current GRAS status.
Safety Assessment of Pyridine (FEMA No. 2966)
At ppm levels, the aroma of pyridine is described as warm, burnt, and smoky (Arctander, 1969). Based upon a reported annual volume of pyridine of 54 kg in the United States (Gavin et al., 2008), the daily per capita intake (“eaters only”)5 of pyridine from use as a flavor ingredient is calculated to be 7 μg/person/day. Pyridine has been isolated in the volatile components from cooked beef (sukiyaki) in Japan (Shibamoto et al., 1981); fried chicken in the U.S. (Tang et al., 1983); fried bacon (Ho et al., 1983); Beaufort cheese (Dumont and Adda, 1978); black tea aroma (Vitzthum et al., 1975); and coffee aroma (Aeschbacher et al., 1989). An 8 oz cup of fresh brewed coffee may contain up to 5 μg of pyridine. Concentrations in cocoa (0.5 ppm), coffee (37–49 ppm), and shrimp (4.1–9.9 ppm) represent principal food sources of pyridine (Nijssen et al., 2010). Based on quantitative natural occurrence data, the intake of pyridine from consumption of all traditional foods exceeds its intake as an added flavor ingredient by a factor of at least 100 (Stofberg and Kirschman, 1985; Stofberg and Grundschober, 1987).
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In animals, pyridine is metabolized primarily by two biotransformation routes: oxidation at the nitrogen atom to give pyridine N-oxide and methylation of the nitrogen atom to yield the quaternary ammonium ion, N-methylpyridinium. Both polar metabolites are excreted in the urine (D’Souza et al., 1980; Damani et al., 1982). To a lesser extent, ring oxidation may occurto give 2- and 4-pyridone and 3-hydroxypyridine. In vitro experiments with human and rat microsomal and cytosolic liver fractions, and human microsomal andcytosolic kidney and lung fractions, show the formation of these three ring-oxidized metabolites (Wilke et al., 1989). In vivo studies in rats demonstrate that CYP2E1 is induced by pyridine and is responsible for formation of pyridine N-oxide (Kim et al., 1988; Kaul and Novak, 1987). Other pyridineinduced isoforms include rat renal CYP1A1 and CYP1A2 (Kim et al., 1995), rat hepatic CYP2B1/2B2 (Kim et al., 1993), and rabbit liver CYP2B1/2B2 (Kim et al., 1991).
In two separate 90-day studies, groups of 10 male and 10 female F344/N or 10 male Wistar rats were provided drinking water containing pyridine at concentrations of 0, 50, 100, 250, 500, or 1,000 ppm (equivalent to average daily doses of 5, 10, 25, 55, or 90 mg pyridine/kg body weight) (NTP, 2000). For the F344/N rats, in addition to increased mortality (2 females at 1,000 ppm), dehydration and decreased body weights were reported at the two highest concentrations. On day 5, erythrocytosis was reported in males at concentrations of 100 ppm and greater. This observation is consistent with dehydration, which can cause relative erythrocytosis due to decreased blood volume and hemoconcentration (Jain, 1986). On day 20, the erythrocytosis was replaced by a developing normocytic, normochromic, non-responsive anemia, demonstrated by decreased hematocrit values, hemoglobin concentrations, and erythrocyte counts relative to controls in males and females exposed to concentrations of 250 ppm or greater. At study termination, evidence of anemia persisted in the 500 and 1,000 ppm males and all exposed groups of females. At the two highest concentrations, there was evidence of hepatocellular injury and/or altered hepatic function (increased serum alanine aminotransferase and sorbitol dehydrogenase activities and bile acid concentrations). Liver weights of males and females exposed to 250 ppm or greater were significantly greater than controls. The incidences of hepatic centrilobular degeneration, hypertrophy, chronic inflammation, and pigmentation were generally increased in 500 and 1,000 ppm males and females relative to controls. In the kidney, the incidences of granular casts and hyaline degeneration (hyaline droplets) were significantly increased in 1,000 ppm males and slightly increased in 500 ppm males; these lesions suggested alpha-2u-globulin-type nephropathy. Additionally, there were increased incidences and/or severities of protein casts, chronic inflammation, mineralization, and regeneration primarily in 500 and 1,000 ppm males. Based on the appearance of erythrocytosis in males at 100 ppm, the NOAEL (no observed adverse effect level) was concluded to be 50 ppm or approximately 5 mg/kg body weight per day.
Responses (lower mean body weights, dehydration, and hepatic injury) in male Wistar rats provided pyridine in drinking water were similar to those in F344/N rats and occurred in a similar concentration range, and the NOAEL in Wistar rats was at a concentration of 100 ppm, or approximately 10 mg/kg body weight per day. Groups of 10 male and 10 female B6C3F1 mice were exposed to the same concentrations of pyridine as used in the 90-day rat study. Based on increased liver weights relative to controls in males exposed to 100 ppm or greater, the NOAEL was concluded to be 50 ppm or approximately 5 mg/kg body weight per day.
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In a 2-year bioassay performed by the National Toxicology Program (NTP, 2000), groups of 50 B6C3F1 mice of both sexes were exposed to pyridine in drinking water at concentrations calculated to provide an average daily intake of 0, 35, 65, or 110 mg/kg body weight for males and 15, 25, or 70 mg/kg body weight for females. Based on the results of the 2-year drinking water study in B6C3F1 mice, the NTP reached the following conclusion: “There was clear evidence of carcinogenic activity of pyridine in male and female B6C3F1 mice based on increased incidences of malignant hepatocellular neoplasms.”
Hepatocellular adenomas occurred at an increased incidence in males (29/50 in controls, 40/50 (p = 0.003), 34/49, and 39/50 (p = 0.011) in low-, mid-, and high-dose groups, respectively. The incidence of hepatocellular carcinomas in males was 15/50 in controls, 35/50 in the low-, 41/49 in the mid-, and 40/50 in the high-dose group, respectively (p < 0.001, pairwise comparisons for all treated groups). The incidence of hepatoblastomas in males was 2/50, 18/50, 22/49, and 15/50 (p < 0.001, pairwise comparisons for all treated groups) in control, low-, mid-, and high-dose groups, respectively. In female mice, the incidence of hepatocellular carcinomas was increased in a dose-related manner: 13/49 in controls, 23/50, 33/50 (p = 0.014), and 41/50 (p < 0.001) in the low- mid-, and high-dose groups, respectively. The incidence of hepatoblastomas was also dose-dependent and significantly increased: 1/49 in controls, 2/50, 9/50 (p = 0.007), and 16/50 (p < 0.001) in the low-, mid-, and high-dose groups, respectively (NTP, 2000).
The statistical evidence of increased incidence of liver neoplasms must be evaluated in the context of strain and species-specific effects recently elucidated in the B6C3F1 mouse (Turosov, 2002). Shortly after publication of the 2-year bioassay with pyridine, the NTP reported an unexpected 5-fold increase in the incidence of spontaneous hepatoblastomas in control B6C3F1 mice during the period 1994–2002. This was accompanied by an increase in the incidence of chemically induced hepatoblastomas in B6C3F1 mice in 2-year NTP studies compared to the previous 7 years. There was a positive association between an increased incidence of mice with hepatoblastoma and an increased incidence of mice with hepatocellular tumors. Although a variety of chemicals caused an increased incidence of mice with hepatoblastoma, there was no apparent association between a specific chemical structure and their capacity to induce hepatoblastomas. Hepatoblastoma is a component of the spectrum of hepatocellular tumors that are identified separately in addition to hepatocellular adenoma and carcinoma in a study. Chemicals that induce hepatoblastomas and hepatocellular carcinomas are not necessarily more potent than chemicals that induce hepatocellular carcinomas alone. Because hepatoblastomas frequently arise within hepatocellular adenomas and hepatocellular carcinomas, the study authors (Turosov et al., 2002) concluded that it is reasonable to combine the incidence of mice with hepatoblastomas with the incidence of mice with hepatocellular adenomas and hepatocellular carcinomas in the overall evaluation for hazard classification.
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The overall rates of tumor incidence for combined hepatocellular adenomas, carcinomas, and blastomas for male B6C3F1 mice exposed to pyridine was 38/50 (76%) in controls, 47/50 (94%) in the low-, 46/49 (94%) in the mid-, and 47/50 (94%) in the high-dose groups. The combined tumor incidence for female B6C3F1 mice was 41/49 (84%) in controls, 42/50 (84%) in the low-, 45/50 (90%) in the mid-, and 44/50 (88%) in the high-dose groups. Therefore, there are no statistically significant differences in tumor incidence between test and control groups of either sex of mice. The increase in spontaneous background incidence of liver neoplasms in male B6C3F1 mice in the 2-year NTP bioassay is now well recognized. This phenomenon has been reported in 2-year studies for other flavor ingredients (Smith et al., 2009; Adams et al., 2005). It is also generally well accepted that male and female B6C3F1 mouse liver tumors that arise in 2-year bioassays with various agents (e.g., chloroform; see Meek et al., 2003) can be the result of dose-related chronic toxicity and resulting regenerative cellular proliferation. In the absence of this chronic toxicity at exposure levels in humans, the occurrence of these tumors does not provide evidence that pyridine represents a significant risk for humans (Cohen et al., 2004).
There is substantial evidence that the appearance of male B6C3F1 mouse liver tumors is not relevant to a human risk assessment. First, there was no statistical evidence of an increased incidence of total hepatic tumors in male or female rats related to administration of pyridine. Second, all dose groups of male and female B6C3F1 mice suffered chronic hepatic toxicity prior to the development of liver adenomas, carcinomas, or blastomas, as evidenced by the results of the 90-day and 2-year studies. Hepatocellular tumors also occurred late in the life span of both male and female mice. From a biological perspective, the increase in the incidence of tumors in B6C3F1 mice reflects the impact of high-dose liver damage to an organ already prone to spontaneous development of liver neoplasms (Smith et al., 2009; Haseman et al., 1986; Haseman, 1990).
Therefore, it can be concluded that the carcinogenic potential in this sensitive strain and sex of laboratory mouse is a secondary biological response to dose-dependent hepatotoxicity, and is not relevant to humans who consume pyridine at low nontoxic levels (<0.0005 mg/kg body weight per day) from intended use as a flavoring ingredient or as a constituent of food. The 90-day NOAEL of either 5 or 10 mg/kg body weight per day in Fisher F344/N or male Wistar rats, respectively, (NTP, 2000) is approximately 1,000 times the daily per capita intake (“eaters only”) intake of 0.007 mg/kg body weight per day from use of pyridine as a flavor ingredient. Also, these levels of intake are at least four orders of magnitude lower than those used in the NTP bioassay that resulted in hepatic toxicity and neoplasms.
In the rat 2-year bioassay, groups of 50 F344/N rats of both sexes were exposed to pyridine in the drinking water at concentrations calculated to provide an average daily intake of 0, 7, 14, or 33 mg/kg body weight per day. In a second strain of rats, groups of male Wistar rats were maintained on drinking water containing pyridine at concentrations calculated to providean average daily intake of 0, 8, 17, or 36 mg/kg body weight per day.
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Consistent with the 13-week studies, the target organs included the liver and kidney in male F344/N rats, the liver and testes in Wistar rats, and the liver in female F344/N rats. Although, in general, these liver lesions were more severe in Wistar rats than in F344/N rats, there was no evidence of an increase in the incidence of hepatocellular neoplasms in either sex or strain of treated rats compared to controls. The only evidence of neoplasms occurred in the kidney where there was an increased incidence of renal tubule adenomas in male F344/N rats [2/50 (4%) in the controls, 3/48 (4%) in the low-, 6/50 (12%) in the mid-, and 10/50 (20%) in the high-dose groups] in the 2-year bioassay based on evaluation of single and multiple sections combined. Based on these results, the NTP concluded: “Under the conditions of these 2-year drinking water studies, there was some evidence of carcinogenic activity of pyridine in male F344/N rats based on increased incidences of renal tubule neoplasms.”
Although the NTP noted the presence of hyaline droplets, granular casts, and immunohistochemical evidence of alpha 2u-globulin nephropathy, the droplets and casts were clearly less severe than for other alpha 2u-globulin nephropathy inducers and there were no linear foci of mineralization within the renal medulla in this study, a change normally characteristic of alpha 2u-globulin-associated nephropathy. Based primarily on these observations, the NTP suggested that the renal neoplastic response in the male F344/N rat kidney was not attributable to alpha 2u-globulin nephropathy.
Although the incidence and severity of renal tubular neoplasms associated with the formation of hyaline droplets is evident in treated males, the lack of any evidence of renal tubular adenomas and carcinomas in female F344 rats and male Wistar rats and the lack of renal changes in mice clearly establishes that the carcinogenic response is sex-, strain-, and species-specific. One carcinoma was recorded in male F344/N rats, and that result is not considered to be treatment related in that it occurred only in the low-dose group. Renal tubule hyperplasia was slightly increased in male F344/N rats [9/50 (18%) in the controls, 7/48 (15%) in the low-, 11/50 (22%) in the mid-, and 15/50 (30%) in the high-dose groups]. The hyperplastic and the benign tumorigenic response (adenomas only) in male F344/N rats are weak compared to that for other alpha 2u-globulin inducers. Although the proliferative response in the male F344/N rats is slight and there is a lack of known mineralization characteristic of alpha 2u-globulin nephropathy, the immunohistochemical evidence from the 13-week study at higher concentrations cannot be dismissed. Pyridine is nongenotoxic based on mainly negative results in both in vitro and in vivo assays (Pai et al., 1978; NTP, 2000; MacGregor et al., 2000; Harper et al., 1984; Kawachi et al., 1980; Warren et al., 1981; Riebe et al., 1982; Florin et al., 1980; Haworth et al., 1983; Abe and Sasaki, 1977; Ishidate and Odashima, 1977), so the mode of action for induction of the renal tumors is non-genotoxic and dependent on dose-related increase in proliferation. The specific mode of action for these pyridine-induced renal tumors remains to be established.
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The other carcinogenic effect reported by NTP was an increased incidence of mononuclear cell-leukemia in female F344/N rats. They noted that incidences of mononuclear cell leukemia in female rats were slightly but significantly increased in the 200 and 400 ppm groups compared to controls [12/50 (24%) in controls, 16/50 (32%) in the low-, 22/50 (44%) in the mid-, and 23/50 (46%) in the high-dose groups], and that the incidence in the 400 ppm group exceeded the historical control range. The mean historical incidence in 2-year drinking water studies with untreated control groups is 30.9% (± 10.0%) with a range of 16–44%. In males, there was no statistically significant difference in the incidence of mononuclear cell leukemia, but the rates were all higher than in females (0 ppm, 29/50; 100 ppm, 32/50; 200 ppm, 26/50; 400 ppm, 27/50). There was no increased incidence of leukemia in male Wistar rats: this strain exhibits a low spontaneous incidence of this type of tumor. In all animals with this neoplasm, neoplastic cells were found in the spleen and usually also in the liver. Infiltrations in the lung, bone marrow, lymph nodes, adrenal gland, and kidney were also common. Incidences of mononuclear cell leukemia in male rats were similar to those in controls and in the same range as for females at the two higher dose levels (0 ppm, 29/50; 100 ppm, 32/50; 200 ppm, 26/50; 400 ppm, 27/50). Based on the marginal increase in leukemia in females at 200 and 400 ppm, the NTP concluded that: “There was equivocal evidence of carcinogenic activity of pyridine in female F344/N rats based on increased incidences of mononuclear cell leukemia.”
More than 20 years ago, members of NTP reported (Rao et al., 1990) that the incidence of mononuclear cell leukemia in female F344/N rats in drinking water studies had been steadily increasing. From a low of 2.1% in 1971, control rates had increased to over 30% in females. The trend is more pronounced in males, where rates had climbed from 7.9% to as high as 52% (Haseman and Rao, 1992). More recently, as a result of a workshop that focused on whether the choice of animal models used by the NTP should be changed (King-Herbert and Thayer, 2006), the NTP has now concluded that the Harlan Sprague-Dawley rat will replace the F344/N strain as the animal model in future bioassays (King-Herbert et al., 2010). In the NTP study on pyridine using F344/N rats, while statistically significant increases in the incidence of mononuclear cell neoplasms in female rats were reported, the biological significance of these tumors and their relevance to humans remains uncertain.
Pyridine was reaffirmed as GRAS (GRASr) in 2010 based upon its efficient detoxification in humans; its low level of flavor use; the lack of genotoxic and mutagenic potential; the safety factor calculated from results of subchronic studies (NTP, 2000) indicating a margin of safety of at least 1,000; the conclusion that the statistically significant findings in the NTP mouse bioassay, of an increased incidence of hepatocellular neoplasms in male and female B6C3F1 mice were secondary to pronounced hepatotoxicity at high dose levels; the conclusion that the increased incidence of renal neoplasms in male F344/N rats occurs via a dose-dependent non-genotoxic mode of action; and the conclusion that the increased incidence of mononuclear cell leukemia in female F344/N rats is likely species- and sex-specific, the biological significance of which remains uncertain. Based on these conclusions, the use of pyridine as a flavor ingredient is not considered to produce any significant risk to human health. This evaluation is supported by the occurrence of pyridine as a natural component of traditional foods resulting in concentrations in the diet producing a “natural intake” that is at least 100 times higher than that which occurs when pyridine is employed as a flavor ingredient.
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ReGRAS of 2-Methyl-5-vinylpyrazine and 2-Hexyl-4-acetoxytetrahydrofuran
The substance 2-methyl-5-vinylpyrazine (formerly FEMA No. 3211) was removed from the GRAS list in 1984 (Oser et al., 1984). At that time, the substance was reevaluated by the panel and found to require additional data including toxicological testing for continuation of GRAS status. Additional metabolism data supporting side chain oxidation of the substance and toxicity data from a 90-day study on the structurally related substance 2-vinylpyridine were presented to the Panel in October 2009. Based upon an evaluation of these data, the Panel concluded that 2-methyl-5-vinylpyrazine is GRAS under conditions of intended use as a flavor ingredient. The Panel restored the GRAS status of 2-methyl-5-vinylpyrazine with its original FEMA GRAS number.
The substance 2-hexyl-4-acetoxytetrahydrofuran (formerly FEMA No. 2566) was removed from the GRAS list in 1970 due to questions concerning certain isomeric components. In May 2010, clarification of chemical identity and purity was submitted to the Panel together with data supporting the hydrolysis of the ester. The Panel concluded that its FEMA GRAS status should be restored with its original FEMA GRAS number.
Panel Statement on Salt Forms of FEMA GRAS Substances
The FEMA Expert Panel concludes that the neutral, hydrated, and salt forms of GRAS substances that are physiologically equivalent are assigned the same GRAS number. Such salt forms include the chlorides, sulfates, carbonates, bicarbonates, and phosphates of quaternary ammonium and sulfonium salts. The same GRAS number would also be applied to the sodium, potassium, calcium, ferrous, ferric, ammonium, and quaternary ammonium salts derived from FEMA GRAS amines, carboxylates, sulfonates, sulfamates, or sulfates, provided the level of intake of the respective mineral in the salt is sufficiently low compared to dietary intake of these minerals.
NOTES
1 For a description of previous surveys conducted by the NACGM, see “Introduction to NACGM Flavor Survey Final Compilation of Data,” Aug. 1, 1972, Exhibit 20 to “A Comprehensive Survey of Industry on the Use of Food Chemicals Generally Recognized as Safe (GRAS),” Subcommittee on Review of the GRAS list—Phase II, Committee on Food Protection, Food and Nutrition Board, Division of Biology and Agriculture, National Research Council (National Academy of Sciences, September, 1972).
2 Full details related to the methodology and results of the survey, as well as data related to retention/release of flavor ingredients from chewing gum matrices, are expected to be reported elsewhere.
3 A stick of ordinary slab chewing gum weighs about 2 g, and a piece of bubblegum weighs about 5 g. The flavor ordinarily represents 0.5%–1.5% of the product, but in some instances ranges up to 3.0% or even higher.
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4 It should be noted in passing that the very same factors which require a much higher concentration of flavoring in chewing gum than in other foods also substantially reduce the potential toxicity of the use of these higher levels.
5 Intake (μg/person/day) calculated as follows: (((annual volume, kg) x (1 x 109 μg/kg))/(population x survey correction factor x 365 days)), where population (10%, “eaters only”) = 28 x 106 for the U.S.; where correction factor = 0.8 represents the assumption that only 80% of the flavor volume was reported in the 2005 survey (Gavin et al., 2008). Intake (μg/kg body weight per day) calculated as follows: [(μg/person/day)/body weight], where body weight = 60 kg. Slight variations may occur from rounding off.
FEMA GRAS Lists published in Food Technology, in chronological order
Hall, R.L. 1960. Recent progress in the consideration of flavoring ingredients under the Food Additives Amendment. Food Technol. 14: 488-495.
Hall, L. and Oser, B.L. 1961. Recent progress in the consideration of flavoring ingredients under the Food Additives Amendment. II. Food Technol. 15(12): 20, 22-26.
Hall, R.L. and Oser, B.L. 1965. Recent progress in the consideration of flavoring ingredients under the Food Additives Amendment. 3. GRAS substances. Food Technol. 19(2, Part 2): 151-197.
Hall, R.L. and Oser, B.L. 1970. Recent progress in the consideration of flavoring ingredients under the Food Additives Amendment. 4. GRAS substances. Food Technol. 24(5): 25-34.
Oser, B.L. and Hall, R.L. 1972. Recent progress in the consideration of flavoring ingredients under the Food Additives Amendment. 5. GRAS substances. Food Technol. 26(5): 35-42.
Oser, B.L. and Ford, R.A. 1973a. Recent progress in the consideration of flavoring ingredients under the Food Additives Amendment. 6. GRAS substances. Food Technol. 27(1): 64-67.
Oser, B.L. and Ford, R.A. 1973b. Recent progress in the consideration of flavoring ingredients under the Food Additives Amendment. 7. GRAS substances. Food Technol. 27(11): 56-57.
Oser, B.L. and Ford, R.A. 1974. Recent progress in the consideration of flavoring ingredients under the Food Additives Amendment. 8. GRAS substances. Food Technol. 28(9): 76-80.
Oser, B.L. and Ford, R.A. 1975. Recent progress in the consideration of flavoring ingredients under the Food Additives Amendment. 9. GRAS substances. Food Technol. 29(8): 70-72.
Oser, B.L. and Ford, R.A. 1977. Recent progress in the consideration of flavoring ingredients under the Food Additives Amendment. 10. GRAS substances. Food Technol. 31(1): 65-74.
Oser, B.L. and Ford, R.A. 1978. Recent progress in the consideration of flavoring ingredients under the Food Additives Amendment. 11. GRAS substances. Food Technol. 32(2): 60-70.
Oser, B.L. and Ford, R.A. 1979. Recent progress in the consideration of flavoring ingredients under the Food Additives Amendment. 12. GRAS substances. Food Technol. 33(7): 65-73.
Oser, B.L., Ford, R.A., and Bernard, B.K. 1984. Recent progress in the consideration of flavoring ingredients under the Food Additives Amendment. 13. GRAS substances. Food Technol. 38(10): 66-89.
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Oser, B.L., Weil, C.L., Woods, L.A., and Bernard, B.K. 1985. Recent progress in the consideration of flavoring ingredients under the Food Additives Amendment. 14. GRAS substances. Food Technol. 39(11): 108-117.
Burdock, G.A., Wagner, B.M., Smith, R.L., Munro, I.C., and Newberne, P.M. 1990. Recent progress in the consideration of flavoring ingredients under the Food Additives Amendment. 15. GRAS substances. Food Technol. 44(2): 78, 80, 82, 84, 86.
Smith, R.L. and Ford, R.A. 1993. Recent progress in the consideration of flavoring ingredients under the Food Additives Amendment. 16. GRAS substances. Food Technol. 47(6): 104-117.
Smith, R.L., Newberne, P., Adams, T.B., Ford, R.A., Hallagan, J.B., and the FEMA Expert Panel. 1996a. GRAS flavoring substances 17. Food Technol. 50(10): 72-78, 80-81.
Smith, R.L., Newberne, P., Adams, T.B., Ford, R.A., Hallagan, J.B., and the FEMA Expert Panel. 1996b. Correction to GRAS flavoring substances 17. Food Technol. 51(2): 32.
Newberne, P., Smith, R.L., Doull, J., Goodman, J.I., Munro, I.C., Portoghese, P.S., Wagner, B.M., Weil, C.S., Woods, L.A., Adams, T.B., Hallagan, J.B., and Ford, R.A. 1998. GRAS flavoring substances 18. Food Technol. 52(9): 65-66, 68, 70, 72, 74, 76, 79-92.
Newberne, P., Smith, R.L., Doull, J., Goodman, J.I., Munro, I.C., Portoghese, P.S., Wagner, B.M., Weil, C.S., Woods, L.A., Adams, T.B., Hallagan, J.B., and Ford, R.A. 1999. Correction to GRAS flavoring substances 18. Food Technol. 53(3): 104.
Newberne, P., Smith, R.L., Doull, J., Feron, V.J., Goodman, J.I., Munro, I.C., Portoghese, P.S., Waddell, W.J., Wagner, B.M., Weil, C.S., Adams, T.B., and Hallagan, J.B. 2000. GRAS flavoring substances 19. Food Technol. 54(6): 66, 68-69, 70, 72-74, 76-84.
Smith, R.L., Doull, J., Feron, V.J., Goodman, J.I., Munro, I.C., Newberne, P.M., Portoghese, P.S., Waddell, W.J., Wagner, B.M., Adams, T.B., and McGowen, M.M. 2001. GRAS flavoring substances 20. Food Technol. 55(12): 34-36, 38, 40, 42, 44-55.
Smith, R.L., Cohen, S.M., Doull, J., Feron, V.J., Goodman, J.I., Marnett, I.J., Portoghese, P.S., Waddell, W.J., Wagner, B.M., and Adams, T.B. 2003. GRAS flavoring substances 21. Food Technol. 57(5): 46-48, 50, 52-54, 56-59.
Smith, R.L., Cohen, S.M., Doull, J., Feron, V.J., Goodman, J.I., Marnett, I.J., Portoghese, P.S., Waddell, W.J., Wagner, B.M., and Adams, T.B. 2005. GRAS flavoring substances 22. Food Technol. 59(8): 24-28, 31-32, 34, 36-62.
Waddell, W.J., Cohen, S.M., Feron, V.J., Goodman, J.I., Marnett, L.J., Portoghese, P.S., Rietjens, I.M.C.M., Smith, R.L., Adams, T.B., Gavin, C. Lucas, McGowen, M.M., and Williams, M.C. 2007. GRAS flavoring substances 23. Food Technol. 61(8): 22-24, 26-28, 30-49.
Smith, R.L., Waddell, W.J., Cohen, S.M., Feron, V.J., Marnett, L.J., Portoghese, P.S., Rietjens, I.M.C.M., Adams, T.B., Lucas Gavin, C., McGowen, M.M., Taylor, S.V., and Williams, M.C. 2009. GRAS flavoring substances 24. Food Technol. 63(6): 46-48, 51-52, 55-56, 58, 60, 62, 64-66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98-105.
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Expert Panel Member Changes
In May 2010, Phillip Portoghese of the University of Minnesota retired from the Panel. Portoghese spent more than a quarter-century in service to the Expert Panel and the flavor industry. His experience in biochemistry, medicinal chemistry, and metabolism provided the Panel with expertise that contributed significantly to the long-standing success of the Panel.
In October 2010, William Waddell of the University of Louisville retired from the Panel after more than a decade of service. With extensive experience in pharmacology, toxicology, medicine, and mechanisms of carcinogenicity, Waddell made numerous key contributions to the work of the Panel. Portoghese and Waddell are now recognized as Emeritus Members of the Panel.
In September 2010, Stephen Hecht, Professor of Cancer Prevention at the University of Minnesota, joined the Panel. His work focuses on the role of carcinogen metabolism and DNA binding in mechanisms of carcinogenesis.
In September 2010, Shoji Fukushima, Director at the Japan Bioassay Research Center, joined the Panel. Fukushima has published numerous articles with a research focus on the mechanisms of carcinogenesis.
Robert L. Smith, Chairman of the FEMA Expert Panel, is Professor, Emeritus, and Senior Research Fellow in Molecular Toxicology, Imperial College London. William J. Waddell, Past Chairman of the FEMA Expert Panel, is Professor and Chair, Emeritus, Dept. of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, Ky. Other members of the FEMA Expert Panel are Samuel M. Cohen, Havlik-Wall Professor, Dept. of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Neb.; Shoji Fukushima, Japan Bioassay Research Center, Kanagawa, Japan; Nigel J. Gooderham, Professor in Molecular Toxicology, Dept. of Surgery and Cancer, Imperial College London; Stephen S. Hecht, Professor and Wallin Chair in Cancer Prevention, Dept. of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, Minn.; Lawrence J. Marnett, Dept. of Biochemistry, Vanderbilt School of Medicine, Vanderbilt University, Nashville, Tenn.; Philip S. Portoghese, Professor, College of Pharmacy, University of Minnesota, Minneapolis, Minn.; and Ivonne M.C.M. Rietjens, Professor and Chair, Dept. of Toxicology, Wageningen University, Wageningen, The Netherlands.
Timothy B. Adams is the Scientific Secretary for the FEMA Expert Panel and Scientific Director of FEMA. Christie Lucas Gavin, Margaret McGowen, and Sean Taylor, Assistant Scientific Director of FEMA, are associated with the Flavor and Extract Manufacturers Association, 1620 I Street, NW, Suite 925, Washington, DC 20006. Send reprint requests to author Adams ([email protected]).
Abe, S. and Sasaki, M. 1977. Chromosome aberrations and sister chromatid exchanges in Chinese hamster cells exposed to various chemicals. J. Natl. Cancer Inst. 58(6): 1635-1641.
Adams, T.B., Cohen, S.M., Doull, J., Feron, V.J., Goodman, J.I., Marnett, L.J., Munro, I.C., Portoghese, P.S., Smith, R.L., Waddell, W.J., and Wagner, B.M. 2005. The FEMA GRAS assessment of benzyl derivatives used as flavor ingredients. Food Chem. Toxicol. 43: 1207-1240.
Aeschbacher, H.U., Wolleb, U., Loliger, J., Spadone, J.C., and Liardon, R. 1989. Contribution of coffee aroma constituents to the mutagenicity of coffee. Food Chem. Toxicol. 27(4): 227-232.
Arctander, S. 1969.Perfume and Flavor Chemicals (Aroma Chemicals). Steffen Arctander, Montclair, N.J. Volume II: 2776.
Cohen, S.M., Klaunig, J., Meek, M.E., Hill, R.N., Pastoor, T., Lehman-McKeeman, L., Bucher, J., Longfellow, D.G., Seed, J., and Dellarco, V. 2004. Evaluating the human relevance of chemically induced animal tumors. Toxicol. Sci. 78: 181–186.
Damani, L.A., Crooks, P.A., Shaker, M.S., Caldwell, J., D’Souza, J., and Smith, R.L. 1982. Species differences in the metabolic C- and N-oxidation, and N-methylation of [14C] pyridine in vivo. Xenobiotica 12(8): 527-534.
D’Souza, J., Caldwell, J., and Smith, R.L. 1980. Species variations in the N-methylation and quaternization of [14C]pyridine. Xenobiotica 10(2): 151-157.
Dumont, J.-P. and Adda, J. 1978. Occurrence of sesquiterpenes in mountain cheese volatiles. J. Agric. Food Chem. 26: 364–367.
Florin, I., Rutberg, L., Curvall, M., and Enzell, C.R. 1980. Screening of tobacco smoke constituents for mutagenicity using the Ames’ test. Toxicology 158: 219-232.
Gavin, C.L, Williams, M.C., and Hallagan, J.B. 2008. Flavor and Extract Manufacturers Association of the United States 2005 Poundage and Technical Effects Update Survey, Flavor and Extract Manufacturers Association of the United States, Washington, D.C.
Harper, B.L., Sadagopa Ramanujam, V.M., Gad-El-Karim, M.M., and Legator, M.S. 1984. The influence of simple aromatics on benzene clastogenicity. Mutat. Res. 128: 105-114.
Haseman, J.K., Tharrington, E.C., Huff, J.E., and McConnell, E.E. 1986. Comparison of site-specific and overall tumor incidence analyses for 81 recent national toxicology program carcinogenicity studies. Regul. Toxicol. Pharm. 6: 155-170.
Haseman, J.K. 1990. Letter to the Editor. Fund. Appl. Toxicol. 15: 207-211.
Haseman, J.K., and Rao, G.N. 1992. Effects of corn oil, time-related changes, and inter-laboratory variability on tumor occurrence in control Fischer 344 (F344/N) rats. Toxicol. Pathol. 20(1): 52-60.
Haworth, S., Lawlor, T., Mortelmans, K., Speck, W., and Zeiger, E. 1983. Salmonella mutagenicity test results for 250 chemicals. Environ. Mutagen. 5(Suppl. 1): 3-142.
Ho, C.-T., Lee, K.N., and Jin, Q.Z. 1983. Isolation and identification of volatile flavor compounds in fried bacon. J. Agric. Fd. Chem. 31(2): 336–342.
Ishidate, M., Jr. and Odashima, S. 1977. Chromosome tests with 134 compounds on Chinese hamster cells in vitro—A screening for chemical carcinogens. Mutat. Res. 48: 337-354.
Jain, N.C. 1986. Clinical and laboratory evaluation of anemias and polycythemias. In Schalm’s Veterinary Hematology, pp. 563-576. Lea & Febiger, Philadelphia.
Kawachi, T., Komatsu, T., Kada, T., Ishidate, M., Sasaki, M., Sugiyama, T., and Tazima, Y. 1980. Results of recent studies on the relevance of various short-term screening tests in Japan. In The Predictive Value of Short-Term Screening Tests in Carcinogenicity Evaluation, eds. G.M. Williams et al., pp. 253-267. Elsevier/North-Holland Biomedical Press, New York.
Kaul, K.L. and Novak, R.F. 1987. Inhibition and induction of rabbit liver microsomal cytochrome P-450 by pyridine. J. Pharmacol. Exp. Ther. 243(1): 384–390.
Kim, S.G., Williams, D.E., Schuetz, E.G., Guzelian, P.S., and Novak, R.F. 1988. Pyridine induction of cytochrome P-450 in the rat: role of P-450j (alcohol-inducible form) in pyridine N-oxidation. J. Pharmacol. Exp. Ther. 246(3): 1175–1182.
Kim, S.G., Reddy, S.L., States, J.C., and Novak, R.F. 1991. Pyridine effects on expression and molecular regulation of the cytochrome P450IA gene subfamily. Mol. Pharmacol. 40: 52-57.
Kim, H., Putt, D., Reddy, S., Hollenberg, P.F., and Novak, R.F. 1993. Enhanced expression of rat hepatic CYP2B1/2B2 and 2E1 by pyridine: differential induction kinetics and molecular basis of expression. J. Pharmacol. Exp. Ther. 267(2): 927–936.
Kim, H., Reddy, S., and Novak, R.F. 1995. 3-Methylcholanthrene and pyridine effects on CYP1A1 and CYP1A2 expression in rat renal tissue. Drug Metab. Dispos. 23(8): 818–824.
King-Herbert, A., and Thayer, K. 2006. NTP workshop: animal models for the NTP rodent cancer bioassay: stocks and strains—should we switch? Toxicol. Pathol. 34(6): 802-805.
King-Herbert, A.P., Sills, R.C., and Bucher, J.R. 2010. Commentary: update on animals models for NTP studies. Toxicol. Pathol. 38(1): 180-181.
MacGregor, J.A., Hamilton, C.M., Kubicek, J.E., and Mirsalis, J.C. 2000. Pyridine does not induce unscheduled DNA synthesis (UDS) in hepatocytes of male B6C3F1 mice treated in vivo. J. Appl. Toxicol. 20(5): 389-393.
Meek, M.E., Bucher, J.R., Cohen, S.M., Dellarco, V., Hill, R.N., Lehman-McKeeman, L.D., Longfellow, D.G., Pastoor, T., Seed, J., and Patton, D.E. 2003. A framework for human relevance analysis of information on carcinogenic modes of action. Crit. Rev. Toxicol. 33(6): 591-653.
Nijssen, L.M., Ingen-Visscher, C.A. van, and Donders, J.J.H., eds. 1963-2010. VCF Volatile Compounds in Food: Database, Version 12.3. TNO Quality of Life, Zeist, The Netherlands.
NTP. 2000. Toxicology and carcinogenesis studies of pyridine (CAS No. 110-86-1) in F344/N rats and B6C3F1 mice (drinking water studies). NTP-TR-470. NIH Publication. No. 00-3960. National Toxicology Program, Research Triangle Park, North Carolina.
Oser, B.L., Ford, R.A., and Bernard, B.K. 1984. Recent progress in the consideration of flavoring ingredients under the Food Additives Amendment. 13 GRAS Substances. Food Technol. 34(7): 66-89.
Pai, V., Bloomfield, S.F., Jones, J., and Gorrod, J.W. 1978. Mutagenicity testing of nitrogenous compounds and their N-oxidised products using trp+ reversion in E. coli, in Biological Oxidation of Nitrogen, Proceeding of the International Symposium 2nd (1977), ed. J.W. Gorrod. 375-382.
Rao, G.N., Haseman, J.K., Grumbein, S., Crawford, D.D., and Eustis, S.L. 1990. Growth, bodyweight, survival and tumor trends in F344/N rats during an eleven year period. Toxicol. Pathol. 18(1, Part 1): 61-70.
Riebe, M., Westphal, K., and Fortnagel, P. 1982. Mutagenicity testing, in bacterial test systems, of some constituents of tobacco. Mutat. Res. 101: 39-43.
Shibamoto, T., Kamiya, Y., and Mihara, S. 1981. Isolation and identification of volatile compounds in cooked meat: sukiyaki. J. Agric. Food Chem. 29(1): 57–63.
Smith, R.L., and Ford, R.A. 1993. Recent progress in the consideration of flavoring ingredients under the Food Additives Amendment. 16. GRAS substances. Food Technol. 47(6): 104-117.
Smith, R.L., Waddell, W.J., Cohen, S.M., Feron, V.J., Marnett, L.J., Portoghese, P.S., Rietjens, I.M.C.M., Adams, T.B., Lucas Gavin, C., McGowen, M.M., Taylor, S.V., and Williams, M.C. 2009. Recent progress in the consideration of flavoring ingredients under the Food Additives Amendment. 24. GRAS Flavoring Substances. Food Technol. 63(6): 46-62.
Stofberg, J. and Kirschman, J.C. 1985. The consumption ratio of flavoring materials: A mechanism for setting priorities for safety evaluation. Food Chem. Toxicol. 23(9): 857-860.
Stofberg, J. and Grundschober, F. 1987. Consumption ratio and food predominance of flavoring materials. Perfum. Flavorist 12(3): 27–68.
Tang, J., Jin, Q.Z., Shen, G.-H., Ho, C.-T., and Cheng, S.S. 1983. Isolation and identification of volatile compounds from fried chicken. J. Agric. Food Chem. 31(6): 1287–1292.
Turusov, V.S., Tori, M., Sills, R.C., Willson, G.A., Herbert, R.A., Hailey, J.R., Haseman, J.K., and Boorman, G.A. 2002. Hepatoblastomas in mice in the U.S. National Toxicology Program (NTP) studies. Toxicol. Pathol. 30(5): 580-591.
Vitzthum, O.G., Werkhoff, P., and Hubert, P. 1975. New volatile constituents of black tea aroma. J. Agric. Food Chem. 23(5): 999–1003.
Warren, G., Abbott, E., Schultz, P., Bennett, K., and Rogers, S. 1981. Mutagenicity of a series of hexacoordinate rhodium (III) compounds. Mutat. Res. 88: 165-173.
Wilke, T.J., Jondorf, W.R., and Powi, G. 1989. Oxidative metabolism of 14C-pyridine by human and rat tissue subcellular fractions. Xenobiotica 19(9): 1013-1022.