ALLYL ESTERS (ALLYL HEXANOATE, ALLYL HEPTANOATE, ALLYL ISOVALERATE) First draft prepared by Dr R. Walker, Professor of Food Science, Department of Biochemistry, University of Surrey, England 1. EXPLANATION Allyl hexanoate (allyl caproate, 2-propenyl hexanoate) is an artificial flavour; it has also been reported to occur naturally in pineapples. A temporary specification for this substance was issued by the twenty-fourth meeting of the Joint FAO/WHO Expert Committee on Food Additives (Annex 1, reference 53) but it has not previously been evaluated by the Committee for an ADI. Allyl heptanoate and allyl isovalerate have not been identified in nature; these flavours have not previously been considered by the Committee. In view of the similarities in metabolism between these compounds and the fact that they all give rise to allyl alcohol on hydrolysis, they are evaluated together in the following monograph for a Group ADI. 2. BIOLOGICAL DATA 2.1 Biochemical Aspects 2.1.1 Biotransformation Allyl hexanoate was hydrolysed slowly by artificial gastric juice in vitro (t´ 1120 min) but more rapidly by simulated pancreatic juice (t´ 1.98 min). This compound was also hydrolysed very rapidly by rat small intestinal mucosa preparations in vitro (t´9.6 x 10-2 sec) and by liver homogenates (t´ 3.96 sec) (Longland et al., 1977). Similarly, allyl hexanoate was reported to be completely hydrolysed within 2 hours by pancreatin when incubated at a concentration of 60 µl/l in the incubation mixture (Grundschober, 1977). Hydrolysis of allyl isovalerate by liver homogenates in vitro proceeds at a slower rate than allyl esters of straight chain acids (Butterworth et al., 1975; Drake, 1975) and the hepatotoxicity of a number of allyl esters was correlated to the rate of hydrolysis to allyl alcohol (see also Short-term Studies). Following hydrolysis, the allyl alcohol liberated is metabolized via two alternative oxidative pathways leading to the formation of acrolein or the epoxide, glycidol, as shown in Figure 1 (Patel et al., 1980). The epoxide may then be converted to glycerol by epoxide hydrolase. The conversion of allyl alcohol to acrolein is mediated by alcohol dehydrogenase (ADH), a step which is blocked in the ADH- deficient rat lung (Patel et al., 1980), in genetically-deficient deermouse liver (Belinsky, 1985) or by ADH inhibitors (Reid, 1972; Serafini-Cessi, 1972; Diluzio & Hoffman, 1973; Jaeschke et al., 1987; Pentilla et al., 1987). The acrolein may then be further oxidized to acrylic acid by NAD- or NADP-dependent enzymes in the liver cytosol or microsomes (Jaeschke et al., 1987) or to glycidaldehyde by a microsomal enzyme with subsequent conversion to glyceraldehyde by epoxide hydrolase (Patel et al., 1980). Alternatively, acrolein may react directly both enzymically and non-enzymically to form stable adducts with glutathione or other low molecular weight thiol compounds (Ohno et al., 1985). Both glycidol and glycidaldehyde are substrates for lung and liver cytosolic glutathione-S-transferases (Patel et al., 1980). When rats were dosed with allyl esters of weak acids, 3- hydroxypropylmercapturic acid was detected as a glutathione-derived metabolite in the urine and bile (Clapp et al., 1969; Kaye & Young, 1970; Kaye & Young, 1972; Kaye, 1973). The same metabolite was identified after dosing with allyl alcohol or acrolein and, by comparison of the percentage conversion after administration of these compounds or allyl esters of weak acids, it may be concluded that the esters were completely hydrolysed to allyl alcohol and that most of the alcohol was converted to acrolein (Kaye, 1973).2.2 Toxicological studies 2.2.1 Acute toxicity Species Sex Route LD50 Reference mg/kg b.w. Allyl hexanoate Rat both oral 218 (186-255) Jenner et al., 1964 Taylor et al., 1964 both oral 327 (277-386) Meisel, 1982 male oral 393 female oral 276 (215-352) Guinea both oral 280 (246-319) Jenner et al., pig 1964 Rabbit ? dermal 300 (200-600) Shelanski & Moldovan, 1971 ? dermal 820 (700-940) Moreno, 1974 Allyl heptanoate Mouse both oral 630 (514-772) Jenner et al., 1964 Rat both oral 500 (392-638) Jenner et al., 1964 Rabbit ? dermal 810 (440-1180) Moreno, 1974a Guinea both oral 444 (363-541) Jenner et al., pig 1964 Allyl isovalerate Mouse both oral >500 NTP, 1983 Rat both oral >250 <500 NTP, 1983 ? oral 230 (216-290) Moreno, 1977 Rabbit ? dermal 560 (290-1060) Moreno, 1977 2.2.2 Short-term studies 2.2.2.1 Mouse - allyl isovalerate Groups of 5 male and 5 female B6C3F1 mice were given allyl isovalerate by gavage in corn oil for 14 consecutive days at daily doses of 0, 31, 62, 125, 250 or 500 mg/kg b.w. All animals of both sexes that received 500 mg/kg b.w. died within 48 h of commencement of the study; all other animals survived to termination. Inactivity and piloerection were seen in animals receiving 250 and 500 mg/kg b.w. and male mice receiving 250 mg/kg gained no weight; body weights in other dose groups were comparable to controls at termination of the study (NTP, 1983). Groups of 10 male and 10 female B6C3F1 mice, 6 weeks of age at commencement of the study were dosed with allyl isovalerate by gavage in corn oil at doses of 0, 15, 31, 62, 125 or 250 mg/kg b.w. (five day/week) for 13 weeks. At the end of the study, autopsies were performed on all survivors. Animals from control and highest dose groups were subjected to detailed histological examination. In addition the liver was examined histologically for the 62 and 125 mg/kg b.w. groups and in the latter group stomachs were also examined microscopically. Five out of 10 males and six of 10 females in the highest dose groups died, all but one of the deaths (a female) being compound related; deaths in other groups were caused by gavage error. Compound related effects noted at necropsy or histologically were limited to the 125 and 250 mg/kg b.w. dose groups and included "thickening" of the urinary bladder wall and gastric mucosa and small intestine (both groups) ulcerative inflammation of the stomach, coagulative necrosis of the liver and cytoplasmic vacuolation of the liver (top dose group only). No compound related effects were seen in the liver, stomach or bladder of mice from other groups (NTP, 1983). 2.2.2.2 Rat - allyl heptanoate Allyl heptanoate was fed to weanling Osborne-Mendel rats of both sexes for 18 weeks at dietary levels of 0, 1000, 2500 and 10,000 mg/kg diet. There was a dose-related growth depression which was severe and associated with a poor food efficiency at the highest dose level only. Gross liver enlargement was observed at all dose levels; in addition, at the highest dose level kidneys were enlarged in both sexes and hearts were enlarged in males only. Males in the 2500 and 10 000 mg/kg diet groups were reported to have enlarged testes (it is not clear from the report whether this "enlargement" relates to absolute or relative organ weights; if the latter, the increased relative organ weights may relate to the growth depression rather than to organ specific effects). Microscopic changes reported included hydropic degeneration in the periportal areas of the liver ranging from moderate at the highest dose level to lesser degrees at lower dose levels. The extent of bile duct proliferation correlated with the degree of hydropic degeneration and hepatocyte enlargement also was seen in some groups (Hagan et al., 1965). Groups of weanling Charles River albino rats of both sexes were given allyl heptanoate in the diet daily at doses of 0, 49.6, 157 and 496 mg/kg b.w./day for 13 weeks. Weight gain and food intake were recorded weekly, and urinalysis, haematological and clinical chemical examinations were carried out at weeks 6 and 12. At termination, autopsies were performed on all animals and detailed histological examinations were carried out on all rats from the top dose group and on half of the control animals. In the rest of the controls and the other two dose groups, histological examination was limited to liver and kidney, and to tissues showing gross abnormalities at autopsy. There was a reduced food intake in the treated groups, which was statistically significant in the high and mid-dose groups, and an associated deficit in body weight gain. At week 6 there was a small but significant depression in leucocyte count in males of the top dose group only; no such effect was seen in females and other haematological parameters were normal in animals of both sexes. At week 12, no significant haematological differences were seen other than a small non-dose-related decrease in leucocyte count in the males of the 49.6 mg/kg dose group. Clinical chemical examinations at weeks 6 and 12 revealed a decrease in some parameters such as blood glucose, total serum protein and albumin which appeared to be due to the reduced food intake and not to specific toxic effects. Urine composition was unaffected by treatment. At necropsy no gross nor microscopic lesions related to treatment were observed and elevated relative organ weights in high- dose males were related to reduced food intake and body weight gain. It was concluded that daily treatment with allyl heptanoate in this study did not result in any signs of toxicity; the reasons for the reduced food intake could not be determined but might have been due to unpalatability of diet (Damske et al., 1980). Allyl hexanoate Groups of 10 male and 10 female Osborne-Mendel rats were given allyl hexanoate by gavage in corn oil at dose levels of 0, 15, 65 and 100 mg/kg b.w. daily for 18 weeks. Weight gain, food intake and general condition were recorded weekly. At termination, the animals were exsanguinated and haematological and gross pathological examinations were performed. Detailed histological examinations were performed only on eight rats from the control and high dose groups; based on observations in the high dose group, livers from eight animals in the 15 and 65 mg/kg b.w. dose groups were also examined microscopically. In the high dose group, gross appearance of the liver was described as nodular and wrinkled with granular or rough surface. Microscopically the high dose group showed slight to moderate bile duct proliferation, some "lobular architectural disarrangement", slight fibrosis and pigment deposition in macrophages; necrotic foci were seen in 2 of 8 animals examined. In the 65 mg/kg b.w. dose group, very slight bile-duct proliferation was observed in 2 of eight animals studied. The livers of the 15 mg/kg b.w. dose group were unaffected by treatment (Hagan et al., 1967). In a companion study to the foregoing, 5 rats of each sex received allyl hexanoate in the diet at a concentration of 1000 mg/kg diet (equivalent to 50mg/kg b.w.) for 28 weeks. No adverse effects were observed (Hagan et al., 1967). Groups of 15 male and 15 female Wistar rats were given daily oral doses of 0, 35 or 100 mg allyl hexanoate/kg b.w. as a solution in corn oil; a further group of 10 animals of each sex were similarly dosed with 12 mg/kg b.w./day for 13 weeks. Food and water intake and body weights were recorded weekly, urinalysis was performed during week 2, week 5 or 6, and in the final week of treatment, and renal function tests were also carried out. At termination, autopsies were performed and organ weights recorded, and detailed histopathological examinations were conducted. No differences were noted between treated and control animals in body weight, water intake, haematological parameters, serum chemistry, urine composition or in renal concentration tests. There were slight increases in food intake in the highest dose group. Liver weights were increased in the 35 and 100 mg/kg b.w. dose groups and all treated animals showed evidence of periportal vacuolation which was dose related in incidence and severity and which in the 100 mg/kg b.w. group was accompanied by enlarged hepatocytes, focal periportal necrosis and bile-duct proliferation. Weights of spleen, kidneys, stomach and small intestine were increased in both sexes in the highest dose group and small intestine weight was also increased in females of the 35 mg/kg b.w. group. It was not possible to determine a no observed adverse effect level for allyl hexanoate in this study (Clode et al., 1978). When allyl hexanoate was administered to rats by gavage at a daily dose level of 15 mg/kg b.w. for 18 weeks there were no observed adverse effects (this study was reported in summary only) (Bär & Griepentrog, 1967). Allyl isovalerate In similar studies to those described for mice, groups of 5 male and 5 female Fischer F344/N rats were given allyl isovalerate by gavage for 14 days and groups of 10 animals of each sex were dosed (5 day/week) for 13 weeks at levels of 0, 31, 62, 125, 250 and 500 mg/kg bw. In the fourteen days study, all rats given 500 mg/kg b.w. and two rats of each sex given 250 mg/kg b.w. died. At termination, mean body weights in the 250 mg/kg b.w. group were depressed relative to weights in the control group by 23% and 13% in males and females, respectively. Inactivity, laboured breathing, diarrhoea and piloerection were observed in both sexes in the two highest dose groups and in necropsy gross dark red areas were seen in the stomachs of three animals of each sex at the top dose level. In the thirteen week study, all ten males and 4/10 females that received 250 mg/kg b.w. died and body weight gain was significantly depressed in males of the 125 mg/kg b.w. group and females of the 250 mg/kg group. Dose-related effects seen at necropsy were thickening of the intestinal wall and redness of the mucosal surfaces of the intestines and urinary bladder. Enlargement of internal lymph nodes and adrenals was reported but was unaccompanied by histological lesions. Histopathological examination revealed multifocal coagulative necrosis, cholangiofibrosis, and bile duct hyperplasia at the 125 and 250 mg/kg b.w. dose levels. The effects were dose related in incidence and no such lesions were observed in the 31 and 62 mg/kg b.w. dose groups (NTP, 1983). Combinations of esters Groups of 10 male rats were given by gavage 21 consecutive daily doses of allyl alcohol and a series of allyl esters (acetate, propionate, hexanoate, isobutyrate, isovalerate and 2- ethylhexanoate) at equimolecular doses corresponding to 5, 25 or 60 mg/kg b.w. of allyl alcohol. After 21 days the animals were sacrificed and the livers examined histologically. The severity of the liver lesions was classified according to the scheme: periportal cell enlargement, followed by necrosis and subsequent fibrosis with bile duct hyperplasia. The severity of the hepatic lesions from the straight chain esters was similar to that produced by the corresponding dose of allyl alcohol and more marked than that produced by the esters of the branched chain acids. The differences were attributed to differences in the rate of hydrolysis since the straight chain esters were hydrolysed in vitro approximately 100 times faster than the branched chain esters (Butterworth et al., 1975). 2.2.3 Long-term/carcinogenicity studies 2.2.3.1 Mouse - allyl isovalerate Groups of 50 male and 50 female B6C3F1 mice, initially 50 days old, received allyl isovalerate at doses of 0, 31 or 62 mg/kg b.w. by gavage in corn oil (10 ml/kg b.w.) five times per week for 103 weeks. Survivors were killed at 112-114 weeks of age. No significant differences were observed in survival rates in males; reduced mean body weight gain and significantly lower survival rate in the lower dose group females was attributed to a high incidence of a genital tract infection. An increase in the incidence of epithelial hyperplasia and squamous cell papillomas was observed in the non-glandular forestomach in male mice; the observed incidences for hyperplasia were 1/50, 1/50 and 7/48, and for papillomas 0/50, 1/50 and 3/48 in the control, low and high dose groups respectively. In females, the corresponding incidences for forestomach epithelial hyperplasia were 0/50, 2/50 and 3/50, and for squamous cell papillomas were 1/50, 0/50 and 2/50 respectively. The incidence of lymphomas was slightly elevated in males (5/50, 6/50 and 8/50 in the respective dose groups) but the increase was not significant by the trend test nor by the incidental tumour test; in females the corresponding incidences were 11/50, 11/50 and 18/50 which gave a dose-response trend, the high dose tumour incidence being significantly higher (P<0.05) than controls. Significant reductions in tumour incidence were observed in treated male mice in respect of hepatocellular carcinomas (18/50, 6/50, 9/50), alveolar/bronchiolar adenomas or carcinomas (13/50, 6/50 5/49) and for thyroid follicular cell adenomas (5/47, 0/46, 1/49). No treatment-related non-neoplastic lesions were observed in mice of either sex (NTP, 1983). 2.2.3.2 Rat - allyl hexanoate A group of 5 male and 5 female Osborne-Mendel rats were given allyl hexanoate in the diet at a concentration of 2500 mg/kg (equivalent to 125 mg/kg b.w.) for 1 year. Body weight and food intake were recorded weekly and haematological examinations were carried out at 3, 6, and 12 months. At termination, detailed histological examinations were performed. No adverse effects were reported, in contrast with the short-term (18 week) study where minimal effects (very slight bile duct proliferation) were reported at a dose level of 65 mg/kg b.w. (Hagan et al., 1967). When allyl hexanoate was fed to rats at a dietary concentration of 0.5% for 1.5 years, 2/25 animals developed multiple bile duct adenomas and proliferative changes of the small bile ducts. An additional animal was reported to have adenomas (location not specified) after 8.5 months. The authors concluded that the small number of animals and tumour incidence were insufficient to allow a firm conclusion to be reached on the significance (Bär & Griepentrog, 1967) (This report was in summary only). Allyl isovalerate Groups of 50 male and 50 female Fischer 344/N rats, initially 46 days old, were given allyl isovalerate at doses of 0, 31 or 62 mg/kg b.w. by gavage in corn oil (5 ml/kg b.w.) five times per week for 103 weeks. Survivors were killed at 112-114 weeks of age when for males the numbers of survivors were 34 controls, 30 low dose and 28 high dose; the corresponding numbers of female survivors were 38, 36 and 29 respectively. There was a dose related increase in mononuclear-cell leukaemia, the incidences observed were 1/50, 4/50 and 7/50 in males of the control, low- and high dose groups; in females the corresponding incidences were 4/50, 6/50 and 9/49. In both sexes there was a significant dose-response trend (p<0.05), while the incidence in high-dose males was significantly higher than controls (p<0.05). Increased frequencies of non-neoplastic lesions (cholangiofibrosis, nodular regeneration, cirrhosis, focal periportal necrosis, fatty changes and cytoplasmic vacuolation) were observed in the livers of animals of both sexes in the high dose group but there was no increase in liver neoplasms (NTP, 1983). The authors of the NTP report concluded that allyl isovalerate was carcinogenic, causing increased incidence of haematopoietic system neoplasms (mononuclear cell leukaemias in male rats and lymphoma in female mice). In reviewing this and other relevant biological data, the International Agency for Research on Cancer concluded that there was limited evidence for the carcinogenicity of allyl isovalerate to experimental animals (IARC, 1985). 2.2.3.3 Dog - allyl heptanoate Four groups of 3 male and 3 female beagle dogs were given daily doses of allyl heptanoate of 0, 5, 25 and 75 mg/kg b.w. by capsule for up to 18 months. All the dogs in the top dose group died within 3 - 7 months; dogs in the two lower treatment groups were reported as surviving after 18 months. Administration of 75 mg/kg b.w. caused depressed growth and macroscopic changes in the appearance of the liver and haemorrhagic changes in the gastric mucosa. Less consistent changes were reported in the form of cysts in the urinary bladder and congestion in the lungs, digestive tract, kidneys, spleen and lymph nodes. Microscopically the livers showed a slight to moderate periportal fibrosis associated with slight to moderate proliferation of the bile duct epithelium. Slight fatty changes were also observed. The stomachs showed diffuse haemorrhage and necrosis of the mucosae with instances of focal sub-mucosal haemorrhage (Hagan et al., 1965). 2.2.4 Special studies on skin irritation During an acute dermal toxicity study on allyl heptanoate in rabbits, skin irritancy was evaluated on day 1. At dermal doses of 313-1250 mg/kg slight to moderate redness and oedema were reported (5000 mg/kg was a lethal dose) (Moreno, 1974). Similar results were obtained with allyl heptanoate (Moreno, 1974a). Allyl isovalerate applied undiluted to intact or abraded rabbit skin under occlusion for 24h was moderately irritating (Moreno, 1977). In a preliminary to a maximization test, 48 hour closed patch tests were carried out on the forearms of 5 volunteers with allyl hexanoate and four subjects displayed grade 1 irritation (Kligman, 1971). Conversely, in a later study in which 5 volunteers were subjected to patch tests, no signs of irritation were observed with allyl hexanoate nor with allyl heptanoate (Kligman, 1975; 1975a). Similarly, allyl isovalerate was without irritant effect in a closed patch study on the backs of 28 subjects (Epstein, 1976). 2.2.5 Special studies on contact sensitization In a maximization test on 25 healthy volunteers, allyl hexanoate was reported to produce 13 cases of sensitization and was considered a moderate sensitizer (Kligman, 1975); this contrasts with an earlier maximization test on a similar number of volunteers in which no cases of sensitization were detected (Kligman 1971). No sensitization was observed with allyl heptanoate using a similar protocol (Kligman, 1975a) nor with allyl isovalerate in 28 volunteers (Epstein, 1976). 2.2.6 Special studies on the haematopoietic and immunologic systems Following the observation, in carcinogenicity studies on allyl isovalerate, of marginal increases in mononuclear cell leukaemia in rats and of malignant lymphoma in mice (NTP, 1983) and in view of the fact that isovaleric acidaemia has been associated with pancytopenia in humans, the effects of allyl isovalerate on the haematopoietic and immune systems of female B6C3F1 mice and Fischer 344/N rats of both sexes were investigated in a short-term (14 day) study. The animals were dosed by gavage for 5 days per week for 2 weeks with allyl isovalerate in corn oil at dose levels of 0, 31, 62 or 125 mg/kg b.w. (rats and mice) or 250 mg/kg b.w. (rats only). Haematological, immunological and histological studies were performed 48-72 h after the final treatment. In addition, bone marrow slides from female mice from the NTP 13-week study (see short-term studies) were also examined. The body weights of rats of both sexes were reduced at the 250mg/kg b.w. dose level and of males at the 125 mg/kg b.w. level. No changes in the body weights of the female mice were observed but there was a 20% increase in mean spleen weight and the splenic follicles were large with prominent germinal centres. No treatment-related effects were seen in haematological parameters nor in bone-marrow cellularity in mice or rats. However, there were significant decreases in pluripotent haematopoietic stem cells (CFU-S) in the spleen and in granulocyte- macrophage progenitors (CFU-GM) in the bone marrow of treated mice. In vitro enzyme assays of these cells showed that haematopoietic suppression was correlated with a depression of hexose monophosphate shunt metabolism but that enzymes of the Embden-Meyerhof and tricarboxylic acid pathways were unaffected. Examination of host resistance in mice following challenge with Plasmodium yoelii or Listeria monocytogenes showed no significant differences between control and treated animals, nor were there other effects on the immune system. The authors concluded that the myelotoxic effects were minimal and of a degree that did not alter host resistance (Hong et al., 1988). Minimal to moderate hypocellularity of the bone marrow was observed in the 125 mg/kg b.w. group of mice, both sexes, from the 13-week NTP study and was most striking for megakaryocytes. The degree of hypocellularity was never severe. 2.2.7 Special studies on genotoxicity The genotoxicity of allyl hexanoate and allyl isovalerate are shown on the next page. 2.2.8 Special studies on metabolites 2.2.8.1 Biochemical aspects For pathways of metabolism of allyl alcohol see Biochemical Aspects section of allyl esters. Test system Test object Concentration Result Reference ALLYL HEXANOATE Ames test1 S. typhimurium 0-3.5mg/plate - Wild et al., 1983 TA98, TA100, TA1535, TA1537, TA1538 Ames test1 S. typhimurium 10.5 µg/plate - Oda et al., 1978 TA98, TA100 Rec assay B. subtilis 0-18 µg/disc + Oda et al., 1978 H17 vs M45 Rec assay B. subtilis 0-20µg/disc - Yoo, 1986 H17 vs M45 Basic test Drosophila 0.5mM in feed - Wild et al., 1983 (sex linked melanogaster recessive) Micronucleus Mouse 2 x 156 mg/kg - Wild et al., 1983 test bw i.p. ALLYL ISOVALERATE Ames test1,2 S. typhimurium 0 - 1000 µg/ - NTP, 1983; TA98, TA100 plate Mortelmans et al., TA1535, TA1537 1986 Basic test Drosophila 1200 - 2000 - Woodruff et al., melanogaster mg/l in feed 1985 4500mg/l injected injected (contd) Test system Test object Concentration Result Reference ALLYL HEPTANOATE No mutagenicity data were available for allyl heptanoate 1 Both with and without rat liver S9 fraction 2 Using a preincubation protocol 2.2.8.2 Acute toxicity Species Sex Route LD50 Reference (mg/kg b.w.) Mouse male oral 96(84-110) Dunlap et al., 1958 i.p. 60 Rat ? oral 64(56-74) Smyth et al., 1951 Rat both oral 70(63-79) Taylor et al., 1964 Rat male oral 105(79-140)* Dunlap et al., 1958 99(75-130)** i.p. 42(32-55) Rabbit male oral 71(42-125) Dunlap et al., 1958 percut. 89(40-250) * Rats 111-143 g b.w. ** Rats 170-252 g b.w. 2.2.8.3 Short-term studies Following acute or short-term exposure to allyl alcohol, the main target organ is the liver in which typical periportal changes are observed, ranging from fatty changes to cell necrosis (Piazza, 1915; Dunlap & Hine, 1955; Dunlap et al., 1958; Torkelson et al., 1959; Rees & Tarlow, 1967; Serafini-Cessi, 1972). The kidney may also be affected, changes reported include necrosis of the epithelium of the convoluted tubules and proliferation of interstitial tissue. Rat Groups of 10 rats (strain and sex not specified) received allyl alcohol in the drinking water at doses of 1.3-1.97 mg/kg b.w. The top dose level was associated with reduced appetite and increased mortality. The highest no effect level was 4 mg/kg b.w. The corresponding no effect level for acrolein was 0.17 mg/kg b.w. (Smyth et al., 1951). Groups of six male and six female rats received allyl alcohol in drinking water at concentrations of 0, 1, 5, 50, 100, 250, 500 or 1000 mg/L for 13 weeks, corresponding to daily intakes of 0.13, 0.62, 5.9, 11.6, 25.5, 41.0 or 72 mg/kg b.w. for males and 0.17, 0.94, 7.34, 13.2, 34.0, 43.7 or 67.4 mg/kg b.w. for females in the respective dose groups. At termination, histological examination (12 organs) was performed on half of the animals in each group. Few gross abnormalities were seen at autopsy; peritoneal fat was decreased in the 500 mg/L group and absent at 1000 mg/L. The no effect level reported from this study was approx. 12 mg/kg b.w./day while at an average 29 mg/kg b.w./day the only noticeable effect was an increased liver weight in males and kidney weight in females (Dunlap et al., 1958). Male Wistar albino rats were given daily intragastric doses of allyl alcohol of 0 or 30 mg/kg b.w. in corn oil for periods of 1, 10 or 28 days. The administration of a single dose produced marked periportal necrosis and associated losses of alcohol dehydrogenase and succinate dehydrogenase activities; hepatic cytochrome P450 concentrations and benzo[a]pyrene hydroxylase activities were reduced to about 60% of control values. Conversely, further daily dosing for 10 or 28 days led to a recovery both of histological appearance and of enzyme activities. It was concluded that metabolism of allyl alcohol becomes modified by repeated treatment (Lake et al., 1975). Similarly, no hepatic injury was observed following 28 daily oral doses of 25 mg/kg b.w., although direct infusion of acrolein caused typical necrotic changes (Butterworth et al., 1978). Groups of 15 male and 15 female Wistar rats were given allyl alcohol in drinking water at concentrations of 0, 50, 100, 200 or 800 mg/l for 15 weeks. No treatment-related effects were observed in results of haematological examinations or analysis of serum. There was a dose-related decrease in fluid intake at all treatment levels in both sexes while growth and food consumption were reduced in both sexes at 800 mg/l and males given 200 or 800 mg/L produced less urine than controls in a period following water deprivation or water loading. Increased relative weights of liver, spleen and kidney were observed at both sexes at the top dose level; relative kidney weights were also higher in the 200 mg/l group and in females given 100 mg/l. No effects due to treatment were seen at autopsy or histologically. The no observed adverse effect level was 50 mg/l, equal to 4.8-6.2 mg allyl alcohol/kg b.w./day (Carpanini et al., 1978). Although the effects of allyl alcohol are almost exclusively observed in the liver, changes in the pancreas described as acidophilia, vacuolation and necrosis of pancreatic acinar cells were reported following oral administration of a single dose of 50 mg/kg b.w. (Nizze et al., 1979). 2.2.8.4 Special studies on genotoxicity The genotoxicity of allyl alcohol, acrolein and glycidol are shown in Table 3. Test system Test object Compound & Result Reference concentration Ames test1 S. typhimurium Allyl alcohol Positive2 Lutz et al., 1982 (liquid TA100 Acrolein Positive suspension) Glycidol Weak positive2 Ames test1 S. typhimurium Allyl alcohol Negative Principe et al., TA98, TA100, 0.025-0.1 1981 TA1535, TA1537, µl/plate TA1538 Forward S. coelicolor Allyl alcohol Negative Principe et al., mutation 2-100µl/plate 1981 8-azaguanine Aspergillus Allyl alcohol Negative Principe et al., resistance nidulans 10-40 µl/plate 1981 (point mutation) Ames test1 S. typhimurium Allyl alcohol Negative Bignami et al., TA98, TA100, Acrolein Positive 1977 TA1535, TA1538 (TA1538, TA98) Ames test1 S. typhimurium Acrolein Negative Sasaki & Endo, TA98, TA100 TA98, TA100 1978 1 With and without rat liver S9-fraction 2 Lower in the presence of S9-fraction 2.2.8.5 Special studies on mechanisms of liver injury by metabolites The extent of damage to the liver by allyl alcohol was increased by the aldehyde dehydrogenase inhibitors disulfiram or cyanamide (Rikans, 1987; Jaeschke et al., 1987) or by phenobarbital induction, and was moderated by the alcohol dehydrogenase inhibitor, pyrazole (Diluzio & Hoffman, 1973). It was concluded that the toxicity is due to the formation of acrolein from allyl alcohol (Reid, 1972; Serafini-Cessi, 1972; Patel et al., 1980; Rikans, 1987). In agreement with this conclusion, co- administration of ethanol (3 g/kg b.w.) to rats inhibited the rate of allyl alcohol oxidation by more than 90% and the histological changes were completely prevented, despite glutathione levels being depressed (Penttila et al., 1987). As indicated by the results with disulfiram and cyanamide, oxidation of acrolein by aldehyde dehydrogenase is an important detoxication step for allyl alcohol- derived acrolein (Rikans, 1987; Jaeschke et al., 1987). In mice, allyl alcohol at a dose of 1 mmole/kg b.w. almost totally depleted hepatic glutathione with subsequent massive lipid peroxidation while enhanced glutathione levels protected against hepatotoxicity of allyl alcohol (Jaeschke et al., 1987). In vitro, allyl alcohol, acrolein and glycidol react with glutathione by a non-enzymic mechanism (Dore & Montaldo, 1984). The severity of liver damage 24h after i.p. administration of allyl alcohol (0.036 µl/kg b.w.) was evaluated in male rats at 4-5, 14-15 or 24-25 months of age. Allyl hepatotoxicity increased as a function of age but hepatic glutathione levels were unaffected indicating that the age-related susceptibility was not due to diminished availability of glutathione (Rikans & Kosanke, 1984). The location of allyl alcohol or acrolein-induced hepatic injury is usually periportal but centrilobular necrosis was induced by using retrograde infusion (Belinsky et al., 1983) and metabolism of allyl alcohol by alcohol dehydrogenase occurred at similar rates in both periportal and centrilobular regions. It was suggested that periportal necrosis seen after oral dosing is due to greater sensitivity of the mitochondrial respiratory chain to the toxic effects of acrolein in periportal cells. 2.2.8.6 In vitro studies on metabolite damage to kidney cells In freshly isolated renal epithelial cells from rats, allyl alcohol toxicity as assessed by glutathione depletion and loss of cell viability was more severe in cells from female animals. This correlated with higher alcohol dehydrogenase activity (Ohno et al., 1985). 3. COMMENTS In evaluating these flavours, the Committee noted that they are rapidly hydrolysed to allyl alcohol and the corresponding acids by intestinal mucosal, pancreatic and hepatic esterases. The results of studies on the toxicity of the three esters indicated that the hepatotoxicity observed at high doses was due to the allyl alcohol and its metabolites. Accordingly the Committee considered supplementary toxicological data on allyl alcohol and concluded that the three esters should be evaluated for a group ADI on the basis of the allyl alcohol moiety. In its evaluation, the Committee also took account of the principles relating to food flavours outlined in Principles for the safety assessment of food additives and contaminants in food (Annex I, ref. 76). The hepatotoxicity of the esters and of allyl alcohol was less marked in repeated-dose short-term studies than in single-dose acute studies, although the mechanism of the acquired tolerance has not been fully elucidated. Mutagenicity studies on allyl hexanoate and allyl isovalerate yielded negative results, while most tests on allyl alcohol were negative. The Committee reviewed two long-term carcinogenicity studies in rats and mice in which allyl isovalerate was administered by gavage in corn oil at both the maximum tolerated dose and 50% of this dose. Epithelial hyperplasia and squamous-cell papillomas of the forestomach were observed in mice, but not rats, at the highest dose. There was no evidence of hepatic tumours in mice (the liver being the target organ for short-term toxicity). The Committee concluded that these results were not relevant to the low-dose, dietary exposure to allyl isovalerate as a food flavour but were probably due to the effects of the large bolus doses that were used. The Committee also noted the small increase in the incidence of leukaemia reported in the treated rats; however, the incidence was within the historical control range and no increase in the incidence of hepatic tumours occurred in rats. Since levels of dietary exposure to allyl isovalerate in food are much lower than the doses used in these studies, the Committee concluded that an ADI could be set. The evaluation was based on the no-observed-effect-level in short-term studies on allyl alcohol, with particular reference to hepatotoxicity; this provides a more conservative estimate than one based on the no-observed-effect level for the esters. The Committee noted that a number of other food flavours in use which are allyl esters and should be considered for inclusion in the group ADI on the basis of their hydrolysis to allyl alcohol. In addition, in view of evidence that allyl esters of such fatty acids as acetate, propionate, isobutyrate, and 2-ethylhexanoate are also rapidly hydrolysed, the Committee considered that their consumption should be taken into account since they could contribute to the total dietary load of allyl alcohol. 4. EVALUATION The Committee allocated an ADI of 0-0.05 mg/kg b.w. as allyl alcohol equivalent for allyl heptanoate, allyl hexanoate, and allyl isovalerate, which corresponds to 0-0.15 mg/kg b.w. allyl heptanoate, 0-0.13 mg/kg b.w. allyl hexanoate, 0-0.12 mg/kg b.w. allyl isovalerate, or combinations of these pro rata. 5. REFERENCES BAR, von F. & GRIEPENTROG, F. (1967) Die situation in der gesundheitlichen Beurteilung der Aromatisierungsmittel für Lebensmittel. Medizin und Ernahrung, 8, 244-251. BELINSKY, S.A., MATSUMURA, T., KAUFFMAN, F.C. & THURMAN, R.G. (1984) Rates of allyl alcohol metabolism in periportal and pericentral regions of the liver lobule. Mol. Pharmacol., 25, 158-164. BELINSKY, S.A., BRADFORD, B.U., FORMAN, D.T., GLASSMAN, E.B., FELDER, M.R. & THURMAN, R.G. (1985) Hepatotoxicity due to allyl alcohol in deermice depends on alcohol dehydrogenase. Hepatology, 5, 1179-1182. BUTTERWORTH, K.R., CARPANINI, F.M.B., DUNNINGTON, R., GRASSO, P. & PELLING, R. (1978) The production of periportal necrosis by allyl alcohol in the rat. Proc. Brit. Pharm. Soc., 57, 353P-354P. BUTTERWORTH, K.R., CARPANINI, F.M.B., GAUNT, I.F., GRASSO, P. & LLOYD, A.G. (1975) A new approach to the evaluation of the safety of flavouring esters. Brit. J. Pharm., 54, 268P. CARPANINI, F.M.B., GAUNT, I.F., HARDY, J., GANGOLLI, S.D., BUTTERWORTH, K.R. & LLOYD, A.G. (1978) Short-term toxicity of allyl alcohol in rats. Toxicology, 9, 29-45. CLAPP, J.J., KAYE, C.M. & YOUNG, L. (1969) Observations on the metabolism of allyl compounds in the rat. Biochem. J., 114, 6P. CLODE, S.A., BUTTERWORTH, K.R., GAUNT, I.F., GRASSO, P. & GANGOLLI, S.D. (1978) Short-term toxicity study of allyl caproate in rats. Fd. Cosmet. Toxicol., 16, 197-201. DAMSKE, D.R., MECLER, F.J., BELILES, R.P. & WEIR, R.J. (1980) 90- day toxicity study in rats: allyl heptanoate. Unpublished report of Litton Bionetics Inc., LBI project No. 21130-01 & -04. Submitted to WHO by FEMA. DILUZIO, N.R. & HOFFMAN, E.O. (1973). Protective influence of pyrazole on allyl formate induced injury. Gastroenterol., 64, 158. DORE, M. & MONTALDO, C. (1984) Studi sulla coniugazione in virto dell'alcool allilico e dei suoi metaboliti con il glutatione ridotto. Boll. Soc. It. Biol. Sper., 60, 1497-1501. DUNLAP, M.K. & HINE, C.H. (1955) Toxicity of allyl alcohol. Fed. Proc., 14, 335. DUNLAP, M.K., KODAMA, J.K., WELLINGTON, J.S., ANDERSON, H.H. & HINE, C.H. (1958) The toxicity of allyl alcohol I. Acute and chronic toxicity. Arch. Ind. Hlth., 18, 303-311. EPSTEIN, W.L. (1976) Unpublished report to RIFM dated 20th December 1976. Submitted to WHO by FEMA. GRUNDSCHOBER, F. (1977) Toxicological assessment of flavouring esters. Toxicology, 8, 387-390. HAGAN, E.C., JENNER, P.M., JONES, W.I., FITZHUGH, O.G., LONG, E.L., BROUWER, J.G. & WEBB, W.K. (1965) Toxic properties of compounds related to safrole. Toxicol. appl. Pharmacol., 7, 18-24. HAGAN, E.C., HANSEN, W.H., FITZHUGH, O.G., JENNER, P.M., JONES, W.I., TAYLOR, J.M., LONG, E.L., NELSON, A.A. & BROUWER, J.B. (1967) Food flavourings and compounds of related structure. II. Subacute and chronic toxicity. Fd. Cosmet. Toxicol., 5, 141-157. HONG, H.L., HUFF, J.E., LUSTER, M.I., MARONPOT, R.T., DIETER, M.P., HAYES, H.T. & BOORMAN, G.A. (1988) The effects of allyl isovalerate on the haematopoietic and immunologic systems in rodents. Fund. Appl. Toxicol., 10, 655-663. IARC (1985) IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Vol.36, Allyl compounds, aldehydes, epoxides and peroxides. International Agency for Research on Cancer; Lyon, pp. 69-74. JAESCHKE, H., KLEINWAECHTER, C. & WENDEL, A. (1987), The role of acrolein in allyl alcohol-induced lipid peroxidation and liver cell damage in mice. Biochem. Pharmacol., 36, 51-57. JENNER, P.M., HAGAN, E.C., TAYLOR, J.M., COOK, E.L. & FITZHUGH, O.G. (1964) Food flavourings and compounds of related structure I. Acute oral toxicity. Fd. Cosmet. Toxicol., 2, 327-343. KAYE, C.M. & YOUNG, L. (1970) Mercapturic acid formation from allyl compounds in the rat. Biochem. J., 119, 53P. KAYE, C.M. & YOUNG, L. (1972) The synthesis of mercapturic acids from allyl compounds in the rat. Biochem. J., 127, 87P. KAYE, C.M. (1973) Biosynthesis of mercapturic acids from allyl alcohol, allyl esters and acrolein. Biochem. J., 134, 1093-1101. KLIGMAN, A.M. (1971) Unpublished report dated 27th September, 1971 to RIFM. Submitted to WHO by FEMA. KLIGMAN, A.M. (1975a) Unpublished report dated 14th February, 1975 to RIFM. Submitted to WHO by FEMA. KLIGMAN, A.M. (1975b) Unpublished report dated 10th June, 1975 to RIFM. Submitted to WHO by FEMA. LAKE,, B.G., GANGOLLI, S.D., WRIGHT, M.G., GRASSO, P., CARPANINI, F.M.B. & BUTTERWORTH, K.R. (1975) The effect of repeated administration on allyl alcohol-induced hepatotoxicity in the rat. Biochem. Soc. Trans., 6, 145--146. LUTZ, D., EDER, E., NEUDECKER, T. & HENSCHLER, D. (1982) Structure-mutagenicity relationship in alpha, ß-unsaturated carbonylic compounds and their corresponding allylic alcohols. Mutat. Res., 93, 305-315. LONGLAND, R.C., SHILLING, W.H. & GANGOLLI, S.D. (1977) The hydrolysis of flavouring esters by artificial gastrointestinal juices and rat tissue preparations. Toxicology, 8, 197-204. MEISEL, M.L. (1982) Caproate allyl, Ro 81-3538/000: An acute oral toxicity study (LD50) in the rat. Unpublished report No. 100- 161/106 prepared by Hazleton Laboratories Deutschland GmbH. Submitted to WHO by FEMA. MORENO, O.M. (1974a) Unpublished report of M.B. Research Laboratories Inc. to RIFM, Project No. MB 74-676. Submitted to WHO by FEMA. MORENO, O.M. (1974b) Unpublished report of M.B. Research Laboratories Inc. to RIFM, Project No. MB 74-677. Submitted to WHO by FEMA. MORENO, O.M. (1977a) Unpublished report of M.B. Research Laboratories Inc. to RIFM, Project No. MB 76-1446. Submitted to WHO by FEMA. MORENO, O.M. (1977b) Unpublished report to RIFM dated 27th January, 1977. Submitted to WHO by FEMA. MORTELMANS, K., HAWORTH, S., LAWLOR, T., SPECK, W., TAINER, B. & ZEIGER, E. (1986) Salmonella Mutagenicity tests: II. Results from the testing of 270 chemicals. Environ. Mutagen., 8, Supp.7, 1-119. NATIONAL TOXICOLOGY PROGRAM (NTP) (1983) Carcinogenesis studies of allyl isovalerate. Report No. NTP-TR-253; PB-83-2509. NIZZE, E., LAPIS, K. & KOVACS, L. (1979) Allyl alcohol-induced changes in the rat exocrine pancreas. Digestion, 19, 359-369. ODA, Y., HAMONO, Y., INOUE, K., YAMAMOTO, H., NIIHARA, T. & KUNITA, N. (1978) Mutagenicity of food flavours in bacteria (1st report). Shouhin Eisei Hen, 9, 177-181. OHNO, Y., JONES, T.W. & ORMSTAD, K. (1985) Allyl alcohol toxicity in isolated renal epithelial cells: protective effects of low molecular weight thiols. Chem. biol. Interactions, 52, 289-299. PATEL, J.M., WOOD, J.C. & LEIBMAN, K.C. (1980) The biotransformation of allyl alcohol and acrolein in rat liver and lung preparation. Drug Metab. Disposition, 8, 305-308. PENTTILÄ, K.E., MÄKINEN, J. & LINDROS, K.O. (1987) Allyl alcohol liver injury: suppression by ethanol and relation to transient glutathione depletion. Pharmacol. Toxicol., 60, 340-344. PIAZZA, J.G. (1915) Zur kehntnis der Wirkung der Allylverbindungen Z. Exp. Path. Ther., 17, 318. PRINCIPE, P., DOGLIOTTI, E., BIGNAMI, M., CREBELLI, R., FALCONE, E., FABRIXI, M., CONTI, G. & COMBA, P. (1981) Mutagenicity of compounds of industrial and agricultural relevance in Salmonella, Streptomyces and Aspergillus. J. Sci. Fd. Agric. 32, 826-832. REES, K.R. & TARLOW, M.J. (1967) The hepatotoxic action of allyl formate. Biochem. J., 104, 757-761. REID, W.D. (1972) Mechanism of allyl alcohol-induced hepatic necrosis. Experientia, 28, 1058-1061. RIKANS, L.E. (1987) The oxidation of acrolein by rat liver aldehyde dehydrogenases: Relation to allyl alcohol hepatotoxicity. Drug Metab. Disp., 15, 356-362. RIKANS, L.E. & KOSANKE, S.D. (1984) Effect of aging on liver glutathione levels and hepatocellular injury from carbon tetrachloride, allyl alcohol or galactosamine. Drug and Chemical Toxicology, 7, 595-604 SERAFINI-CESSI, F. (1972) Conversion of allyl alcohol into acrolein by rat liver. Biochem. J., 128, 1103-1107. SHELANSKI, M.V. & MOLDOVAN, M. (1971) Unpublished report of Food and Drug Research Laboratories Inc. to RIFM. Submitted to WHO by FEMA. SMYTH, H.F., CARPENTER, C.P. & WEIL, C.S. (1951) Range finding toxicity data: List IV. Arch. Ind. Hyg. Occup. Med., 4, 119- 122. TAYLOR, J.M., JENNER, P.M. & JONES, W.I. (1964) A comparison of toxicity of some allyl, propenyl and propyl compounds in the rat. Toxicol. Appl. Pharmacol., 6, 378-387. TORKELSON, T.R., WOLF, M.A., OYEN, F. & ROWE, V.K. (1959) Vapor toxicity of allyl alcohol as determined on laboratory animals. J. Am. Ind. Hyg. Assoc., 20, 224-229. WILD, D., KING, M.T., GOCKE, E. & ECKHARDT, K. (1983) Study of artificial flavouring substances for mutagenicity in the Salmonella/microsome, Basc and micronucleus tests. Fd. Chem. Toxicol., 21, 707-719. YOO, Y.S. (1986) Mutagenic and antimutagenic activities of flavouring agents used in foodstuffs. J. Osaka City Med. Center, 34, 267-288.222.
See Also: Toxicological Abbreviations