First draft prepared by Dr R. Walker,
    Professor of Food Science, Department of Biochemistry,
    University of Surrey, England


         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. 


    2.1  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
    (t9.6 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 l/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).

    FIGURE 1

    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., 
                                                      Taylor et al., 
              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 &
              ?         dermal    820 (700-940)       Moreno, 1974
                        Allyl heptanoate

    Mouse     both      oral      630 (514-772)       Jenner et al.,

    Rat       both      oral      500 (392-638)       Jenner et al.,

    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  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).  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.,

         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.,

         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)
    (Br & 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.,

    2.2.3  Long-term/carcinogenicity studies  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).  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 (Br &
    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).  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  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,

    Ames test1        S. typhimurium        10.5 g/plate       -              Oda et al., 1978
                      TA98, TA100

    Rec assay         B. subtilis           0-18 g/disc        +              Oda et al., 1978
                      H17 vs M45

    Rec assay         B. subtilis           0-20g/disc         -              Yoo, 1986
                      H17 vs M45

    Basic test        Drosophila            0.5mM in feed       -              Wild et al., 1983
    (sex linked       melanogaster

    Micronucleus      Mouse                 2 x 156 mg/kg       -              Wild et al., 1983
    test                                                                       bw i.p.

                                            ALLYL ISOVALERATE

    Ames test1,2      S. typhimurium        0 - 1000 g/        -              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

    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
  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
                        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.  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.


         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).  Special studies on genotoxicity

         The genotoxicity of allyl alcohol, acrolein and glycidol are
    shown in Table 3.

    Test system       Test object           Compound &          Result         Reference

    Ames test1        S. typhimurium        Allyl alcohol       Positive2      Lutz et al., 1982
    (liquid           TA100                 Acrolein            Positive 
    suspension)                             Glycidol            Weak 

    Ames test1        S. typhimurium        Allyl alcohol       Negative       Principe et al., 
                      TA98, TA100,          0.025-0.1                          1981
                      TA1535, TA1537,       l/plate

    Forward           S. coelicolor         Allyl alcohol       Negative       Principe et al., 
    mutation                                2-100l/plate                      1981

    8-azaguanine      Aspergillus           Allyl alcohol       Negative       Principe et al., 
    resistance        nidulans              10-40 l/plate                     1981

    Ames test1        S. typhimurium        Allyl alcohol       Negative       Bignami et al., 
                      TA98, TA100,          Acrolein            Positive       1977
                      TA1535, TA1538                            (TA1538, 

    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
  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 l/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.   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.,


         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

         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.


         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.


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    See Also:
       Toxicological Abbreviations