BENZYL ACETATE
First draft prepared by
Dr P. Olsen
Institute of Toxicology, National Food Agency of Denmark
Ministry of Health, Soborg, Denmark
1. EXPLANATION
This compound was previously evaluated at the eleventh,
twenty-seventh, twenty-ninth, thirty-first, and thirty-fifth
meetings (Annex 1, references 14, 62, 70, 77, and 88). During some
of these meetings the Committee also considered related substances,
including benzyl alcohol, benzaldehyde, benzoic acid, and the
benzoate salts.
Benzyl acetate was first evaluated by the Committee at its
eleventh meeting (Annex 1, reference 14), when an ADI of 0-5 mg per
kg of body weight was allocated in terms of benzoic acid,
representing total benzoate from all food additive sources. At the
twenty-seventh meeting (Annex 1, reference 62) the ADI for benzyl
acetate was retained but made temporary because of concern raised by
preliminary findings from screening tests for carcinogenicity. At
its twenty-ninth meeting (Annex 1, reference 70) the Committee
considered new data on the metabolism of benzyl acetate and on the
occurrence of tumours in rats and mice given benzyl acetate by
gavage. The Committee extended the temporary ADI of 0-5 mg per kg
of body weight pending results from carcinogenicity studies with
benzyl alcohol. The temporary ADI was again extended at the thirty-
first meeting of the Committee (Annex 1, reference 77), as the
expected data were not available. At its thirty-fifth meeting
(Annex 1, reference 89) the Committee reviewed a lifetime gavage
studies with benzyl alcohol and found no evidence of a tumorigenic
effect. The Committee extended the temporary ADI of 0-5 mg per kg
of body weight for benzyl acetate until 1993, pending the evaluation
of results from ongoing long-term studies with benzyl acetate
incorporated into the diet of mice and rats and requested data from
and in vivo test for chromosomal damage to bone marrow.
Since the previous evaluation, additional toxicological data
have become available and are summarized and discussed in the
following monograph addendum.
2. BIOLOGICAL DATA
2.1 Toxicological studies
2.2.2 Short-term toxicity studies
2.2.2.1 Mice
Groups of 10 male and 10 female mice (B6C3F1, average age at
exposure 42 days; 13 days quarantine prior to test) received 0,
3 130, 6 250, 12 500, 25 000, or 50 000 mg/kg feed (equivalent to
0, 450, 900, 1 800, 3 600, or 7 200 mg/kg bw/day) of benzyl acetate
(benzyl acetate, properties consistent with structure and literature
references, purity: 99%; stability monitored periodically; no
degradation of bulk chemical observed) in their diet for a period of
13 weeks. Feed [feed prepared weekly and stored in dark; dose
formulation analyzed 4 times during study for benzyl acetate
concentrations, stability, and homogeneity; contained low and
biologically insignificant levels of aflatoxins, pesticides and
heavy metals] and water were provided ad libitum. The feed
consumption was recorded daily and the animals were weighed weekly.
Haematology, clinical chemistry (cholesterol and triglycerides), and
pancreatic enzymes (amylase, lipase, carboxypetidase, chymotrypsin,
ribonuclease), were performed at termination of the study.
Histopathology examinations were performed on all control, on 25 000
mg/kg feed female, and all 50 000 mg/kg feed animals.
Statistically significant (p<0.01) dose-related decreases in
final body weights were observed in all treated animals compared to
controls. The mean feed consumption of all exposed mice was lower,
but not statistically significantly lower, than that of the control
groups. Tremor was observed in female mice at a dose level of
12 500 mg/kg feed and above. At the dose level of 50 000 mg/kg feed
one male died and one female mice was killed in extremis. The
absolute and relative organ weights in treated animals were
influenced by the lowered terminal body weight and all significant
differences between treated and control groups were attributed to
treatment. No dose-related effects in haematology, clinical
chemistry, or pancreatic enzyme parameters were observed in treated
animals.
Histopathological examination revealed hippocampal necrosis,
cerebellar haemorrhage of the brain and hepatocellular necrosis in 1
male mouse receiving 50 000 mg/kg feed after 11 weeks of treatment.
At termination three female mice receiving 50 000 mg/kg feed showed
hippocampal necrosis and depletion of the pyramidal layer cells in
the brain (NTP, 1992).
2.2.2.2 Rats
Groups of 10 male and 10 female rats (F344, average age at
exposure 43 days; 13 days quarantine prior to test) received 0,
3 130, 6 250, 12 500, 25 000, or 50 000 mg/kg feed (equivalent to
0, 210, 420, 840, 1 680, or 3 360 mg/kg bw/day) of benzyl acetate
(benzyl acetate, properties consistent with structure and literature
references, purity: 99%; stability monitored periodically, and no
degradation of bulk chemical observed) in their diet for a period of
13 weeks. Feed: [feed prepared weekly and stored in dark; analyzed
during study for benzyl acetate concentrations, stability, and
homogeneity; contained low and biologically insignificant levels of
aflatoxins, pesticides and heavy metals] and water provided ad
libitum. The feed consumption was recorded daily and the animals
were weighed weekly. After 11 weeks of treatment haematological and
clinical chemical (cholesterol and triglycerides) parameters were
determined. Pancreatic enzymes (amylase, lipase, carboxypetidase,
chymotrypsin, and ribonuclease) were determined in all treated male
and female rats except the 50 000 mg/kg feed group. At termination
liver peroxisomes morphometry were performed on female rats given 0,
25 000, or 50 000 mg/kg feed benzyl acetate. Histopathology
examinations were performed on all control, 25 000 mg/kg feed and
50 000 mg/kg feed rats.
Nine male and female rats died or were killed moribund in the
50 000 mg/kg feed group between weeks 2 and 8 of the study. Final
mean body weights of treated male and female rats were similar to or
slightly lower than those of the controls. Male rats given 25 000
mg/kg feed showed 10% decreased (p<0.01) terminal mean body weight.
At the 50 000 mg/kg feed level the body weight of the one surviving
male and female rats was less than half of the controls. The
average feed consumption was reduced in 25 000 mg/kg feed male rats
and 50 000 mg/kg feed male and female rats. Tremor, ataxia, and
urine stains were observed in the 50 000 mg/kg feed group. Serum
cholesterol was significantly decreased in females in the 12 500
mg/kg feed (p<0.01), 25 000 mg/kg feed (p<0.001), and 50 000 mg/kg
feed (only one female rat alive after 11 weeks) groups. No other
dose-related effects were seen of the haematological, clinical
chemical, or pancreatic enzyme parameters in treated rats. The
volume, surface, and numerical density of hepatic peroxisomes in
female rats (25 000 mg/kg feed) were significantly (p<0.001)
increased. No differences in organ weights attributive to treatment
were observed. Histopathological examination of male and female
rats receiving 50 000 mg/kg feed benzyl acetate revealed
degeneration and necrosis of neurons and glia cells in cerebellum
and hippocampus of the brain, renal tubular degeneration, and
degeneration and sarcolemma nuclear hyperplasia in skeletal thigh
muscles. Testicular tubular atrophy was seen in a few male rats
receiving 12 500 mg/kg feed benzyl acetate or more (NTP, 1992).
2.2.3 Long-term toxicity/carcinogenicity studies
2.2.3.1 Mice
Groups of 60 male and 60 female mice (B6C3F1, average age at
initial exposure 40 days; 11 days quarantine prior to test) received
0, 330, 1 000, or 3 000 mg/kg feed (equal to 0, 37, 112, or 346
mg/kg bw/day for males, equal to 0, 42, 132, or 382 mg/kg bw/day for
females) of benzyl acetate (benzyl acetate, properties consistent
with structure and literature references, purity: 98-9%; stability
monitored periodically, and no degradation of bulk chemical
observed) in their diet for a period of 103 weeks. Interim
sacrifice was carried out on 10 mice of each sex from all groups
after 15 months of exposure. Feed [feed prepared weekly and stored
in dark; analyzed during study for benzyl acetate concentrations,
stability, and homogeneity; contained low and biologically
insignificant levels of aflatoxins, pesticides and heavy metals] and
water provided ad libitum. Feed consumption was measured daily
per cage for 5 days once every 4 weeks. The animals were weighed
weekly during the first 13 weeks of the study and every 4 weeks
thereafter. Haematology and clinical chemistry (cholesterol
triglycerides, alkaline phosphatase, creatinine kinase, and sorbitol
dehydrogenase) were carried out on interim sacrifice mice after 15
months. Necropsy and a thorough histopathological examination were
performed on all animals. Organ weights included brain, right
kidney, and liver.
The survival rate of treated male mice was similar to that of
the control group, while survival of treated female mice increased
with dose, statistically significantly (p<0.01) in the 3 000 mg/kg
feed group. Almost all deaths occurred during the last 9 months of
the study. The average feed consumption of treated mice was similar
to that of the control groups. All treated mice, except 330 mg/kg
feed females, showed decreased mean body weights compared to those
of controls, weights were 13% and 9% lower at termination in males
and females, respectively (statistics not reported). A slight
decrease (inconsistent dose-related significance at the p<0.05
level) in cholesterol, triglyceride and (females only) alkaline
phosphatase levels was observed for treated mice compared to the
control groups. No dose-related effects were seen in haematology.
Statistically significant (p<0.05 or lower) dose-related increased
incidences and severities of non-neoplastic lesions of the nasal
mucosa and glands occurred in all treated male and female mice
compared to the control groups. The nasal lesions consisted of
atrophy and degeneration, primarily of the olfactory epithelium,
cystic hyperplasia of the nasal submucosal glands, and exudate and
pigmentation of the nasal mucosal epithelium. The lesions were most
pronounced in male mice and were already present in male and female
mice at interim sacrifice after 15 months of exposure. No neoplasms
nor pre-neoplastic dose-related lesions occurred in the nose. A
dose-related negative trend in the incidence of hepatocellular
carcinoma and hepatocellular adenoma, statistically significant
(p<0.01) for hepatocellular adenoma in the 3 000 mg/kg feed group,
occurred in male mice. This effect was not seen in female mice
(NTP, 1992).
2.2.3.2 Rats
Groups of 60 male and 60 female rats (F344; average age at
initial exposure 41 days; 12 days quarantine prior to test) received
0, 3 000, 6 000, or 12 000 mg/kg feed (equal to 0, 113, 225, and 550
mg/kg bw/day) of benzyl acetate (benzyl acetate, properties
consistent with structure and literature references, purity: 98-9%;
stability monitored periodically, no degradation of bulk chemical
was observed) in their diet for a period of 103 weeks. Interim
sacrifice was carried out on 10 rats of each sex from all groups
after 15 months of exposure. Feed [feed prepared weekly and stored
in dark; analyzed during study for benzyl acetate concentrations,
stability, and homogeneity; contained low and biologically
insignificant levels of aflatoxins, pesticides and heavy metals] and
water provided ad libitum. The feed consumption was measured
daily per cage for 5 days once every 4 weeks. The animals were
weighed weekly during the first 13 weeks of the study and every 4
weeks thereafter. Haematology, clinical chemistry (cholesterol
triglycerides, alkaline phosphatase, creatinine kinase, and sorbitol
dehydrogenase) and (in males only) analysis of pancreatic enzymes
(amylase, lipase, carboxypetidase) were carried out on interim-
sacrificed rats after 15 months. Necropsy and a thorough
histopathological examination were performed on all animals. Organ
weights included brain, right kidney, and liver.
No significant differences in the survival rate, average feed
consumption, clinical findings, clinical chemistry, haematology,
pancreatic enzyme assays, or incidences of neoplasms and non-
neoplastic lesions were observed in treated male and female rats
compared to those of the controls. The mean body weights of the
12 000 mg/kg feed males and treated females were approximately 5%
lower than those of the control groups throughout most of the study
(NTP, 1992).
2.2.8 Special studies on genotoxicity
Genotoxicity studies with benzyl acetate are summarized in
Table 1.
Table 1. Results of genotoxicity tests on benzyl acetate
Test system Test object Concentration of Result Reference
benzyl acetate
In vitro Bacterial S.typhimurium 33-10 000 µg/plate Negative NTP, 1992
mutagenicity assay (1) TA1535, TA1537,
TA100, TA98
Mammalian cell Mouse lymphoma 0.25-1.75 µl/ml Positive (2) NTP, 1992
mutation assay (1) cells (TK locus) (3), 700-1 700
µg/ml
Sister chromatid Chinese hamster 50-5 000 µg/ml Negative NTP, 1992
exchange assay (1) ovary(CHO) cells
Chromosomal aberration Chinese hamster 160-1 600 µg/ml Negative (4) NTP, 1992
assay (1) ovary(CHO) cells
In vivo Sex-linked Drosophilia 300 ppm & 20 000 Negative NTP, 1992
recessive lethal melanogaster ppm (5)
mutation test
Sister chromatid Mouse bone 312-1 250 mg/kg bw Negative NTP, 1992
exchange test marrow cells
Chromosomal Mouse bone 325-1 700 mg/kg bw Negative NTP, 1992
aberration test marrow cells
Micronucleus test Mouse bone 312-1 250 mg/kg bw Negative NTP, 1992
marrow cells
Test system Test object Concentration of Result Reference
benzyl acetate
Micronucleus test Mouse peripheral 3130-50 000 ppm Negative NTP, 1992
blood
(1) In presence and absence of metabolic activation
(2) Laboratory 1: positive only in presence of metabolic activation
Laboratory 2: positive in presence of metabolic activation, test
in absence of metabolic activation not conducted
(3) Concentration unit equivocal, (laboratory 1)
(4) Negative in presence of metabolic activation. Two out of three trials
in a test showed P values of less than 0.05
in absence of metabolic activation
(5) Feeding: 300 mg/kg feed, injection: 20 000 ppm
3. COMMENTS
At its present meeting, the Committee reviewed data from short-
term and long-term studies in rats and mice in which benzyl acetate
had been incorporated into the diet. These studies did not show any
increase in the incidence of either hepatocellular or forestomach
tumours in mice or of pancreatic tumours in rats, which had been
observed previously in studies with benzyl acetate administered by
gavage in corn oil.
The Committee noted a documented association between the use of
corn oil as a vehicle control and an increased incidence of
pancreatic acinar cell hyperplasia and adenomas in male rats. In
addition, altered incidence of other site-specific neoplasms has
been observed after administration of corn oil by gavage (see
section 2.2.2).
Considering the use of both modes of administration in long-
term studies, the Committee concluded that the administration of
benzyl acetate in the diet was more relevant to its safety
assessment as a food additive than administration by gavage in corn
oil.
The Committee also reviewed data from new genotoxicity studies.
These studies showed no evidence of in vivo genotoxicity of benzyl
acetate when tested for induction of sister chromatid exchange,
chromosomal aberrations or micronuclei in mouse bone marrow cells.
The Committee noted the induction of necrosis of the brain involving
the cerebellum and/or hippocampus in rats and mice treated with very
high doses of benzyl acetate (50 000 mg/kg feed) for 13 weeks. No
such effect was observed in the long-term toxicity/carcinogenicity
studies in mice or rats at lower doses (3 130-25 000 mg/kg feed).
In the long-term toxicity/carcinogenicity study mentioned above, of
rats administered dietary benzyl acetate at levels up to 550 mg per
kg of body weight per day, no adverse effects were observed.
In the long-term toxicity/carcinogenicity study in mice given
330, 1 000, or 3 000 mg benzyl acetate per kg diet (equal to 37,
112, and 345 mg per kg body weight per day in males and 42, 132, and
382 mg per kg body weight per day in females), dose-related
degeneration and atrophy of the olfactory epithelium, cystic
hyperplasia of the nasal submucosal glands, and pigmentation of the
nasalmucosal epithelium were observed. The Committee considered the
changes of the nasal cavity to be a result of local irritant effects
of the test compound and not toxicologically relevant to the
assessment of food safety.
At the end of the study, treated male and female mice showed
lower mean body weights than controls.
4. EVALUATION
In the absence of associated pathological lesions in the
long-term toxicity/carcinogenicity study in mice and on the basis of
the NOEL of 550 mg per kg of body weight per day in the long-term
study in rats, the Committee included benzyl acetate in the group
ADI of 0-5 mg per kg of body weight with benzyl alcohol,
benzaldehyde and benzoic acid, and the benzoate salts.
The Committee noted the absence of reproduction/teratogenicity
studies for substances in this group, and recommended that a full
review of benzoic acid and benzoates, benzaldehyde, benzyl alcohol,
and benzyl acetate be performed in 1995 to determine whether these
or other studies are required.
5. REFERENCE
NTP (1992). NTP (National Toxicology Program) technical report on
the toxicology and carcinogenesis studies of benzyl acetate (CAS No.
140-11-4) in F344 rats and B6C3F1 mice, (Feed studies). NTP TR
431. Board Draft. NIH Publication 92-3162. U.S. Department of
Health and Human Services, Public Health Service, National Institute
of Health. Research Triangle Park, NC, USA.
See Also:
Toxicological Abbreviations
Benzyl acetate (ICSC)
Benzyl acetate (FAO Nutrition Meetings Report Series 44a)
Benzyl acetate (WHO Food Additives Series 26)
Benzyl acetate (WHO Food Additives Series 37)
BENZYL ACETATE (JECFA Evaluation)
Benzyl Acetate (IARC Summary & Evaluation, Volume 40, 1986)
Benzyl Acetate (IARC Summary & Evaluation, Volume 71, 1999)