868. Benzyl acetate (WHO Food Additives Series 37)

BENZYL ACETATE, BENZYL ALCOHOL, BENZALDEHYDE, AND BENZOIC ACID
AND ITS SALTS

First draft prepared by E. Vavasour, Chemical Health Hazard Assessment
Division, Bureau of Chemical Safety, Food Directorate, Health
Protection Branch, Health Canada, Ottawa, Ontario, Canada

Explanation

Biological data

Biochemical aspects
Absorption, distribution, and excretion
Biotransformation
Effects on enzymes and other biochemical parameters

Toxicological studies
Acute toxicity
Short-term toxicity
Long-term toxicity and carcinogenicity
Reproductive toxicity
Developmental toxicity
Genotoxicity
Special studies: Effects on the pancreas

Observations in humans

Comments

Evaluation

References

1. EXPLANATION

All of these substances were evaluated previously by the
Committee, most of them individually. Benzyl acetate was evaluated at
the eleventh, twenty-seventh, twenty-ninth, thirty-first, thirty-
fifth, and forty-first meetings (Annex 1, references 14, 62, 70, 77,
88, and 107); monographs or monograph addenda were prepared at the
eleventh, thirty-fifth, and forty-first meetings (Annex 1, references
15, 89, and 108). Benzyl alcohol was evaluated at the twenty-third
meeting (Annex 1, reference 50). Benzaldehyde was evaluated at the
eleventh meeting, when a monograph was prepared (Annex 1, references
14 and 15). Benzoic acid and its salts were evaluated at the sixth,
ninth, seventeenth and twenty-seventh meetings (Annex 1, references 6,
11, 32, and 62).

With the exception of sodium benzoate, which is used as a food
preservative, all of the other compounds are used in foods as
flavouring ingredients (benzyl alcohol and benzyl benzoate are also
used as carrier solvents in foods). In addition, further human
exposure occurs due to the natural occurrence of these compounds in
foods, endogenous formation of benzoate through the phenylalanine-
tyrosine pathway, and a variety of uses such in soaps and cosmetics,
pharmaceuticals, insect repellents, pesticides, and other industrial
uses. Since these compounds are metabolized along a common pathway,
they have been grouped under the same ADI of 0-5 mg/kg bw.

At its forty-first meeting, the Committee recommended that a full
review of these substances be conducted in order to determine whether
additional studies would be required. In particular, the absence of
studies of reproductive and developmental toxicity was noted. At the
present meeting, studies on disposition and metabolism, short- and
long-term treatment, genotoxicity, reproductive and developmental
toxicity, and human observations were reviewed and evaluated. While
few data pertaining specifically to benzyl benzoate were located, it
was considered to be adequately represented by the existing database
for the other compounds, since it is hydrolysed to benzyl alcohol and
benzoic acid. The following monograph is a compilation of studies from
the previous monographs and monograph addenda and those reviewed for
the first time at the present meeting.

2. BIOLOGICAL DATA

2.1 Biochemical aspects

2.1.1 Absorption, distribution, and excretion

Benzyl acetate

Benzyl acetate is absorbed from the gastrointestinal tract,
through the lungs, and through the intact skin. It is hydrolysed in
man to benzyl alcohol and acetate; the benzyl radical is oxidized to
benzoic acid and excreted as hippuric acid (Snapper et al., 1925).

Benzyl acetate was readily hydrolysed in vitro by a pancreatin
preparation (Grundschoser, 1977). Benzylmercapturic acid and hippuric
acid were isolated from the urine of rats that had been injected
subcutaneously with benzyl acetate (Clapp & Young, 1970).

Male B6C3F1 mice and Fischer 344 rats were treated either
intravenously or orally with ¹⁴C-benzyl acetate. The intravenous
dose was equivalent to 10 mg/kg bw for mice and 5 mg/kg bw for rats.
For oral administration, benzyl acetate was dissolved in corn oil and
administered at doses equivalent to 10, 100, or 1000 mg/kg bw for mice
and 5, 50, or 500 mg/kg bw for rats. The compound was readily absorbed
from the gastrointestinal tract of both species, and about 90% of the
total dose was recovered as urinary metabolites after 24h. A small
proportion (0.3-1.3%) of the total dose was excreted in the faeces
after both intravenous and oral administration. Elimination of benzyl
acetate as carbon dioxide or volatile substances was minimal after
intravenous treatment and consequently was not determined after oral
treatment. Analysis of tissues of animals sacrificed 24 h after
intravenous or oral administration of labelled compound showed no
¹⁴C activity, indicating that elimination of the label was virtually
complete by this time. This clearance pattern indicates that benzyl
acetate is readily absorbed and excreted after oral administration.
The relative amounts of benzyl acetate absorbed, metabolized, and
excreted were apparently unaffected by the size or number of doses
administered. Repeated treatment of rats with benzyl acetate at
500 mg/kg bw per day for 14 days, followed by a single dose of
labelled compound did not change the clearance pattern. More than 90%
of the radiolabel in the urine was present as hippuric acid, with
minor amounts as benzyl alcohol and benzylmercapturic acid (up to 4%);
no unchanged benzyl acetate was found, and the levels of benzoyl
glucuronide were not measured. There was no evidence to suggest
saturation or reduction of metabolic capacity in either species over
the dose range tested (Matthews & Burka, 1984; Abdo et al., 1985; US
National Toxicology Program, 1986).

Male Fischer 344 rats received [methylene-¹⁴C]benzyl acetate by
gavage as the pure substance or in corn oil or propylene glycol at
doses of 5, 250, or 500 mg/kg bw, and radiolabel was measured in
faeces, urine, plasma, and tissues. Metabolites were identified in
urine and plasma by high-performance liquid and thin-layer
chromatography. Absorption of benzyl acetate was more rapid at the
lower doses and in the absence of a vehicle. The bulk of the
administered dose (70-89%) was excreted in the urine over the initial
24-h period, and very little (about 4%) was found in the faeces after
72 h. The elimination of benzyl acetate and metabolites was
essentially complete by three days, as negligible residues were found
in tissue, regardless of whether benzyl acetate was given pure or in
corn oil. Since benzyl acetate was not found in the plasma or urine at
any time after administration, the authors concluded that it was
readily hydrolysed. Small amounts of benzyl alcohol, the initial
product of ester cleavage, were detected only in plasma samples. At
the higher doses, benzoic acid was the major metabolite in plasma,
while at 5 mg/kg bw, hippuric acid (the glycine conjugate of benzoic
acid) predominated. Hippuric acid was the major metabolite in urine,
and the proportion of the original dose it represented was not
significantly affected by dose. The proportion of the dose present as
benzoyl glucuronide increased with dose, interpreted by the authors as
a limited capacity for glycine conjugation. A small proportion of the
total dose (1.0-3.6%) was excreted in the urine as benzoic acid and
benzylmercapturic acid, and the proportion was not affected by dose or
vehicle (Chidgey & Caldwell, 1986).

[methylene-¹⁴C]Benzyl acetate was applied dermally to the
shaved backs of male Fischer 344 rats at a dose of 100, 250, or
500 mg/kg bw in order to study the metabolism and disposition of
benzyl acetate applied by this route. After administration of the pure
compound, 28-48% of the dose was recovered at the application site and
26-46% was absorbed and excreted in the 0-24-h urine. Smaller amounts
of the administered dose were recovered in the 24-48-h urine (< 2%),
the 48-72-h urine (< 1%), the 0-72-h faeces (< 3%), and the carcass
after 72 h (< 2%). The proportion absorbed was unaffected by dose,
area of application, or use of ethanol as a vehicle. Excretion in the
urine during the first 24 h after treatment accounted for about 95% of
the absorbed dose. At all doses, the major urinary metabolite was
hippuric acid (about 95%); benzoyl glucuronide, benzoic acid, and
benzylmercapturic acid each accounted for 1-2% of the urinary
radiolabel. Benzoyl glucuronide accounted for a larger proportion of
the metabolites at 500 mg/kg bw than at 250 mg/kg bw (Chidgey
et al., 1987).

The effect of aging on the disposition of benzyl acetate was
studied in male Fischer 344 rats and C57Bl/6N mice. Rats aged 3-4, 9,
and 25 months, received a single oral dose of 5 or 500 mg/kg bw
¹⁴C-benzyl acetate in corn oil or a single intravenous dose of
5 mg/kg bw. The mice, aged 2, 13, or 25 months, received a single oral

dose of 10 mg/kg bw in corn oil. Urine and faeces were collected for
96 h. The major route of elimination in the rats was the urine, most
of the radiolabel being excreted within 24 h in rats of all ages
tested. More than 90% of the radiolabel in the urine was identified as
hippuric acid and minor amounts as benzylmercapturic acid, the only
other urinary metabolite. There was no dose- or age-related difference
in the percentage excreted as hippuric acid at any time; however, the
excretion of benzylmercapturic acid was affected by both dose and age:
at the low dose, the proportion excreted in 25-month-old rats (2%) was
significantly greater than that in 3-4-month and 9-month-old rats
(1%); at the high dose, benzylmercapturic acid comprised 2% of
metabolites in both 3-4-month- and 25-month-old rats. The percentage
of the total dose excreted in the faeces was slightly affected by age,
with a small but statistically significant decrease in the 25-month-
old group, but not by dose. After intravenous administration of
5 mg/kg bw benzyl acetate, four metabolites were identified in the
plasma: benzyl alcohol, benzoic acid, hippuric acid, and benzyl
glucuronide, and the plasma levels of hippuric acid and benzoyl
glucuronide were significantly higher in 25-month-old rats than those
at other ages. Most of the radiolabel in bile was associated with
benzyl alcohol (90%), with minor amounts in benzoic acid (8%) and
hippuric acid (2%). Comparison of the percentage of radioactivity
excreted in bile with that retrieved in the faeces suggested that
enterohepatic recirculation of benzyl acetate-derived radiolabel had
occurred; the percentage of the total dose excreted in the bile was
significantly lower in aged than young rats, confirming the decrease
in faecal excretion and the higher plasma levels of benzyl acetate-
derived radiolabel.

The major route of excretion in mice receiving an oral dose of
10 mg/kg bw benzyl acetate was the urine. Significantly less
radiolabel was excreted in the 24-h urine of 25-month-old mice than in
that of 2- or 13-month-old animals. Hippuric acid was the main urinary
metabolite detected in all groups, constituting 93-96% of the total
radiolabel. This was the only metabolite detected in the urine of
2- and 25-month-old mice, while in 13-month-old mice, small amounts of
benzaldehyde (3%) and benzylmercapturic acid (2%) were occasionally
detected. Faecal excretion was a minor route and the amount was
similar in all age groups, comprising about 1.0-1.5% of the total
administered dose. The authors concluded that aging changes minor
routes of metabolism and excretion of benzyl acetate occur but not the
formation of hippuric acid from benzyl acetate (McMahon et al.,
1989).

The effects of gavage and dietary administration on the
toxicokinetics of benzyl acetate were compared in B6C3F1 mice and
Fischer 344 rats. The plasma concentrations of benzoic acid, benzyl
alcohol, and hippuric acid were measured at intervals of 24 h in
groups of six rats and 12 mice given 500 and 1000 mg/kg bw benzyl
acetate, respectively, in corn oil by gavage, and at intervals over a

15-h period in 10 rats and 10 mice fed 10 800 ppm and 2700 ppm benzyl
acetate, respectively, in the feed ad libitum for seven days. The
authors calculated that the latter doses represented about 615 and
648 mg/kg bw in rats and 850 and 900 mg/kg bw in mice, which
approximated the doses administered by gavage. [The calculation for
the mouse appears to be incorrect, since, using a standard conversion
factor, the dose would appear to be closer to 400 mg/kg bw.] The rate
of hydrolysis of benzyl acetate in plasma was measured by adding pure
compound to control rat plasma at room temperature. Hydrolysis was
rapid under these conditions, with a half-life of about 4 min. Since
partial inhibition was noted when sodium fluoride, an esterase
inhibitor, was added, it was concluded that plasma esterases
contributed to the rapid hydrolysis. After administration by gavage,
benzyl acetate was not detected in plasma samples taken at any time,
and benzyl alcohol was not detected in plasma after 5 min in mice and
10min in rats. Benzoic acid and hippuric acid levels in the plasma
rose rapidly after gavage, increasing to peak concentrations after
about 3 h. The plasma concentrations of benzoic acid after
administration in the feed were much lower than those after gavage--
about 40-fold less in the rat and 300-fold less in the mouse--while
the plasma concentrations of hippuric acid were similar after both
routes of administration. The prolonged presence of hippuric acid in
the plasma in both species after both routes of administration
indicates a zero-order formation mechanism. The authors suggested that
the difference in the plasma levels of metabolic products of benzyl
acetate were the cause of the different carcinogenic responses after
gavage and dietary administration in the long-term studies in rats and
mice conducted by the US National Toxicology Program (see sections
2.2.3.1 and 2.2.3.2) (Yuan et al., 1993).

Benzyl alcohol

Blood and urine samples were collected from 14 term and nine
pre-term infants who received similar intravenous doses of benzyl
alcohol in medications, in order to estimate the levels of benzoic and
hippuric acids. The mean peak concentrations of benzoic acid in the
plasma of pre-term babies were almost 10times higher than those in the
term newborns. The normalized area under the concentration-time curve
(AUC) for benzoic acid in plasma was also significantly higher in the
pre-term babies. In addition, larger percentages of benzyl alcohol
doses were found as benzoic acid in the urine of pre-term babies and
less as hippuric acid. These results suggest that hippuric acid
formation is deficient in pre-term newborms (Lebel et al., 1988).

Benzoic acid and benzoate salts

The liver is the main site of conjugation with glycine in man and
in most experimental animals, except the dog, in which the kidney is
the main site of biosyntheses (Friedmann & Tachau, 1911; Snapper
et al., 1925). Sheep appear to have a reduced capacity to conjugate
benzoic acid with glycine. Infusion of increasing amounts of benzoic

acid into the rumen at levels up to 1.8 g/kg bw led to a progressive
decrease in conjugation and increasing excretion of free benzoic acid
in the urine. Doses of 1.1 and 1.8 g/kg bw were toxic, leading to
death. Potassium deficiency also occurred, seen as the usual symptoms
of severe muscular weakness and tremors (Martin, 1966). Benzoates
exist in the free state in blood and are not bound to proteins. In the
dog, kidney clearance was estimated to be 0.90-1.89% (Knoefel & Huang,
1956).

Since for many years a liver function test was used in man that
was based on the urinary excretion of hippuric acid after a test dose
of benzoic acid (6 g orally or 1.5-2.0 g intravenously), much
information exists on the excretion of benzoic and hippuric acids in
man. Normal urinary excretion of hippuric acid was estimated to be
1.0-1.25 g/day, equivalent to 0.7-1.7 g of benzoic acid (Stein
et al., 1954). Other determinations of the normal excretion in man
and rat yielded values of 1-3 mg/kg bw (Armstrong et al., 1955). The
maximal rate of excretion after ingestion of benzoic acid was
17 mg/min for hippuric acid and 0.67 mg/min for benzoylglucuronic
acid, equivalent to 24 g/day as benzoic acid (Schachter, 1957). Up to
10 g of benzoate are excreted quantitatively (Barnes, 1959). After
high intakes of sodium benzoate, up to 3% is conjugated with
glucuronic acid, and all metabolites are eliminated completely within
14 h (Schachter, 1957). Up to 75-80% of an administered dose of
benzoic acid is eliminated within 6 h. Sodium benzoate also decreases
excretion of uric acid (Quick, 1931), urea, and ammonia in man (Lewis,
1914).

Benzoic acid is rapidly absorbed (Schanker et al., 1958) and
rapidly and completely excreted in the urine (Schachter, 1957; Barnes,
1959). One healthy man given 6, 9, 13.9, 34.7, and 69.3 mmol sodium
benzoate showed complete elimination of the drug within 10-14 h
(Schachter, 1957). Sodium benzoate does not accumulate, as shown by
experiments on the distribution and elimination of 1-¹⁴C-labelled
compound administered intraperitoneally, orally, or subcutaneously to
rats. Virtually quantitative excretion occurs in the urine within one
to two days; less than 1% of radiolabel appears in the faeces and a
few parts per million in organs. All of the radiolabel was identified
as benzoic acid (Lang & Lang, 1956). Of an oral or subcutaneous dose
of labelled benzoic acid, 90% appeared in the urine as hippuric acid,
0.1% occurred in expired carbon dioxide, and 2% remained in the
carcass (Bernard et al., 1955).

The maximal urinary excretory rate achieved depends on the dose
of benzoate given. Limiting values of hippuric acid excretion were
approached in man at a dose of 14 mmol (Schachter, 1957), due to
limitations on the availability of glycine (Quick, 1933). In rats, the
tolerance of large doses of benzoic acid depends on the addition of
adequate amounts of glycine to the diet, leaving sufficient glycine
for protein synthesis. Preformed glycine is usually used, although

some is synthesized (Quick, 1931; Barnes, 1959). When rats were fed
1.5% benzoic acid (as the sodium salt) in the diet, they excreted
> 95% of the drug as hippuric acid in the urine. As the benzoate in
the diet was increased to 3.75%, the ratio of hippuric acid to total
benzoic acid in the urine decreased. Additional glycine increased
elimination to 86-99%. The only other derivative found in significant
amounts in the urine was benzoyl glucuronide (Griffith, 1929). Dogs
and rabbits excrete hippuric acid independently of the route of
administration of benzoic acid (Quick, 1931).

The pharmacokinetics and metabolism of orally administered sodium
benzoate in human subjects were studied by simultaneous measurement of
concentration-time data for benzoic and hippuric acids in plasma and
urine. After administration of doses of 40, 80, and 160 mg/kg bw, the
AUC for benzoic acid increased disproportionately with dose, while
that for hippuric acid increased proportionately with dose. While the
peak concentrations of benzoic acid in plasma increased with
increasing dose, the peak concentrations of its metabolite, hippuric
acid, did not change. These results suggest that transformation of
orally administered benzoic acid to hippuric acid is a saturable
process in humans (Kubota et al., 1988; Kubota & Ishizaki, 1991).

The rate of excretion of hippuric acid in urine was studied in a
person who received an oral dose of 250 mg (4.2 mg/kg bw) benzoic
acid. The concentration of hippuric acid in the urine was maximal
0.5-1 h after dosing, and excretion was almost complete within 4 h
(Akira et al., 1994). When the study was repeated with an oral dose
of 10 mg (0.16 mg/kg bw), most of the hippuric acid was excreted
within 0.5-1 h after dosing (Baba et al., 1995).

2.1.2 Biotransformation

Benzyl acetate

A study was conducted to ascertain the pathway by which benzyl
acetate is metabolized to benzylmercapturic acid in the rat. Male
Fischer 344 rats received 500 mg/kg bw benzyl acetate in corn oil by
gavage, either alone or with intraperitoneal doses of 200 mg/kg bw
pyrazole (an inhibitor of alcohol dehydrogenase), 10 mg/kg bw
pentachlorophenol (an inhibitor of sulfotransferase activity), or
both. Within 24 h, 92% of the dose was retrieved in the urine; a small
proportion (3%) was recovered in the faeces over 72 h, and less than
1% remained in the carcass after this time. Treatment with pyrazole or
pentachlorophenol did not affect the rate or route of excretion, but
treatment with both agents delayed the excretion of metabolites in the
urine. Four urinary metabolites of benzyl acetate were identified:
hippuric acid (74.6%), benzoyl glucuronide (12.9%, benzoic acid
(3.0%), and benzylmercapturic acid (1.1%). Inhibition of the
transformation of benzyl alcohol to benzaldehyde by co-administration
of benzyl acetate and pyrazole increased the proportion of the dose

excreted as benzylmercapturic acid from 1.1 to 12.0%, and the
percentage of the dose excreted as benzoyl glucuronide was
concomitantly reduced from 12.9 to 6.3%. Inhibition of the formation
of the sulfate conjugate of benzyl alcohol by pentachlorophenol
inhibited the formation of benzylmercapturic acid but had no effect on
the other metabolites. The authors concluded that the formation of
benzylmercapturic acid involves the formation of benzyl sulfate as an
obligatory intermediate and that therefore it is unlikely that the
formation of benzylmercapturic acid from benzyl acetate is associated
with the formation of a reactive metabolite of toxicological
significance (Chidgey et al., 1986).

Benzyl alcohol

Benzyl alcohol and benzaldehyde were detected within 5 min in
the plasma of CD-1 mice given single intraperitoneal doses of
700-1100 mg/kg bw benzyl alcohol in peanut oil. Prior administration
of pyrazole, an inhibitor of alcohol dehydrogenase, resulted in
increased plasma levels of benzyl alcohol (203%), and prior
administration of disulfiram, an inhibitor of aldehyde dehydrogenase,
resulted in increased plasma levels of benzaldehyde (368%) (McCloskey
et al., 1986).

Benzyl alcohol generated by the metabolism of toluene in human
and rat liver microsomes was further metabolized to benzoic acid and
benzaldehyde by human, but not rat, microsomes. Human liver microsomes
metabolized benzyl alcohol to benzaldehyde at a rate of 16.0 µmol/min
per mg protein. The reaction was inhibited by the addition of carbon
monoxide, and decreased by raising the pH of the incubation mixture
from 7.4 to 10, the optimum for alcohol dehydrogenase. The addition
of sodium azide, a catalase inhibitor, or ADP-ribose, an alcohol
dehydrogenase inhibitor, had no effect on the reaction. These
results suggest that cytochrome P450, but not catalase or alcohol
dehydrogenase, is responsible for the metabolism of benzyl alcohol to
benzaldehyde in human liver microsomes. Benzyl alcohol does not appear
to be metabolized by microsomal enzymes in the rat (Chapman et al.,
1990).

Benzaldehyde

Benzaldehyde is hydrogenated in rabbits to benzoyl alcohol, which
is further oxidized to benzoic acid and excreted as hippuric acid
(Bray et al., 1951).

In a study of the effects of disulfiram on the oxidation of
benzaldehyde in rat liver samples in vitro, the K_m values for
aldehyde dehydrogenase activity were higher in microsomes than in
mitochondria or cytosol, indicating that microsomal oxidation of
benzaldehyde is unlikely to be important in the rat. Disulfiram was
found to inhibit oxidation of benzaldehyde by aldehyde dehydrogenase

in rat liver slices, by 24% at a benzaldehyde concentration of
25 µmol/litre and by 13% at a concentration of 250 µmol/litre. At the
latter concentration, only 12-16% of the benzaldehyde that disappeared
was converted to benzyl alcohol by the action of aldehyde reductase
(Hellstrom-Lindahl & Weiner, 1985).

Evidence for the metabolism of benzaldehyde to benzylmercapturic
acid was obtained in an experiment in which pure benzaldehyde was
administered to groups of five male Sprague-Dawley rats at a dose of
400, 750, or 1000 mg/kg bw per day by gavage for 13 consecutive days.
Control rats received tap water. Urine was collected and analysed for
the presence of benzylmercapturic acid after the second, eighth, and
thirteenth doses; the metabolite was found in the urine of all treated
animals but none of the controls. Since this metabolite is derived
from conjugation of glutathione with the sulfate ester of benzyl
alcohol, these results suggest that benzaldehyde can undergo reduction
to form benzyl alcohol (Laham & Potvin, 1987).

The urinary metabolites of benzaldehyde were identified and
quantified in New Zealand white rabbits after administration of a
single dose of 350 or 750 mg/kg bw by gavage; a control group received
water. Urine was collected from all animals for 15 consecutive days.
About 83% of the administered dose was excreted in the urine of both
groups. The main urinary metabolite was hippuric acid, comprising 70%
of the dose in the group receiving the low dose and 67% in the group
at the high dose, indicating that benzaldehyde is predominantly
oxidized to benzoic acid (of which hippuric acid is the glycine
conjugate) in the rabbit. Small amounts of free benzoic acid were also
detected (1.6 and 1.4%), as was the conjugate with glucuronic acid,
benzoylglucuronic acid (8.8 and 11.2%). Other metabolites detected
were benzyl glucuronide (2.9 and 3.0%) and traces of benzylmercapturic
acid. Endogenously derived hippuric acid and free benzoic acid were
detected in control rabbits at previously established baseline levels
(Laham et al., 1988).

Benzoic acid and benzoate salts

Phenylalanine and tyrosine are precursors of endogenous benzoate.
Experiments with labelled phenylalanine showed that 1-2% is
metabolized by this pathway. In a study in which humans were given
14-28 mg/kg bw per day of deuterio-phenylalanine for 4-6 days, rabbits
were given 50-400 mg/kg bw per day for 6-12 days, and guinea-pigs were
given 300 mg/kg bw per day for 12 days, no labelled benzoic acid
was seen in rabbits or guinea-pigs (Bernard et al., 1955).
[3-¹⁴C]-Phenylalanine given intraperitoneally to rats resulted in
0.6-1% of the activity as urinary hippuric acid (Haberland et al.,
1954).

1,3,4,5-Tetrahydroxycyclohexanoic acid (quinic acid) may also
serve as a precursor of benzoic acid in intermediary metabolism
(Dickens & Pearson, 1951). Several human subjects given 6 g quinic
acid orally or 250 g of prunes excreted increased amounts of hippuric
acid during the following 24 h (Quick, 1931). When deuterio-benzoic
acid was administered to man and rats, it was excreted with its
deuterium content unchanged. Feeding of hydro-aromatic compounds to
guinea-pigs with body fluids enriched with deuterium led to urinary
excretion of deuterio-benzoic acid with a high deuterium content. A
similarly prepared rat fed 750 mg hydroxybenzoic acid over five days
excreted urinary benzoic acid enriched in deuterium. When human
subjects and guinea-pigs were given quinic acid over several days,
47-72% was converted to benzoic acid and excreted in the urine
(Bernard et al., 1955). Four rats irradiated with 700R and four
controls were given [carboxyl-¹⁴C]sodium benzoate intra-peritoneally
and fasted. Irradiation had no effect on conjugating ability, but the
irradiated rats excreted less labelled hippuric acid, owing to
dilution by endogenously produced benzoic acid (Schreier et al.,
1954).

The two known urinary metabolites of benzoic acid are hippuric
and benzoylglucuronic acidc. Conjugation with glycine and glucuronic
acid occurs in preference to oxidation because benzoic acid strongly
inhibits fatty oxidation in the liver. In man, rabbit, and rat,
benzoic acid is almost entirely excreted as hippuric acid, whereas
dogs excrete more conjugated glucuronic acid (Williams, 1959). Sheep
are less able to excrete hippuric acid and excrete large quantities of
free benzoic acid in their urine (Martin, 1966). The urine of man,
pig, rabbit, and sheep contains up to 10% benzoylglucuronic acid.

Male volunteers received oral doses of 2 or 5 g sodium benzoate
in aqueous solution; the 5-g dose was also given in conjunction with
5 g glycine in aqueous solution 1 h before treatment with benzoate
and with 2 g given every hour thereafter. Benzoate was excreted
quantitatively in the urine as hippurate, with a minor fraction
excreted as benzoyl glucuronide. No free benzoic acid was detected in
the urine. Less benzoyl glucuronide was detected after the 2-g dose
(1.8%) than the 5-g dose (3.3%), and concurrent administration of
glycine with the 5-g dose reduced the excretion of benzoyl glucuronide
(0.6%). Hippuric acid was excreted in the urine at an essentially
constant rate from about 1 to 3 h after administration.
Co-administration of glycine with benzoate increased the rate of
excretion of hippurate. These results indicate that, in humans, the
availability of glycine is rate-limiting for the formation of hippuric
acid. Urinary excretion of hippuric acid in untreated individuals was
estimated to be 15-30 mg benzoic acid equivalent per hour (Amsel &
Levy, 1969).

The urinary metabolites of benzoic acid were determined after
oral administration of ¹⁴C-benzoic acid, mostly at a dose of
50 mg/kg bw, to a large variety of species, including primates,
rodents, carnivores, reptiles, and birds. Excretion of the label was
rapid in all species except reptiles. The metabolites in most species
were hippuric acid and benzoyl glucuronide. In most of the herbivorous
and omnivorous species, including man, monkey, pig, and rodent,
benzoic acid was excreted in the urine almost entirely as hippuric
acid. Substantial amounts of the glucuronide of benzoic acid, in
addition to hippuric acid, were detected in the urine of carnivores,
except cats (Bridges et al., 1970).

The rate of conjugation of benzoic acid with glycine was measured
in homogenates of 110 samples of human liver and 67 samples of human
renal cortex. The rates of reaction were 254 ± 90 nmol/min per g liver
and 321 ± 99 nmol/min per g kidney. The rate of conjugation in the
liver decreased with age. While the rate of conjugation of benzoic
acid was greater in the renal cortex than in the liver, the larger
mass and strategic anatomical position of the liver were considered to
make it quantitatively the more important organ with respect to
glycine conjugation (Temellini et al., 1993).

2.1.3 Effects on enzymes and other biochemical parameters

Benzyl acetate

The possible role of glycine depletion in the toxicity induced by
large doses of glycine was investigated in groups of male Fischer 344
rats fed diets containing 0, 20 000, 35 000, or 50 000 ppm benzyl
acetate for up to 28 days. Two additional groups received diets
containing 50 000 ppm benzyl acetate and 27000 ppm glycine or
50 000 ppm benzyl acetate and 32 000 ppm alanine. A third group
received a diet containing 32 000 ppm alanine and served as an
isonitrogenous control. Mortality was 0, 0, 10, and 100% in rats fed
0, 20 000, 35000, and 50 000 ppm benzyl acetate alone. Supplementation
of the high dose of benzyl acetate with glycine reduced the mortality
rate from 100 to 10%, but supplementation with alanine had no effect.
Administration of benzyl acetate induced a dose-related body-weight
loss, which was alleviated by glycine but not alanine. Neuro-
behavioural signs (ataxia or convulsions), which were observed in
all groups receiving benzyl acetate alone or in combination with
alanine, were not seen in the group receiving benzyl acetate and
glycine. The authors concluded that supplementation with adequate
levels of glycine protects against the toxicity of benzyl acetate and
related compounds that are normally detoxified by conjugation with
glycine (Abdo & Wenk, 1995).

Benzyl alcohol

In a study to evaluate the effects of short-term intake of benzyl
alcohol on alcohol and aldehyde dehydrogenase activities, groups of
four male and four female Sprague-Dawley rats received either a 2%
(v/v) solution of benzyl alcohol as the sole drinking fluid ad
libitum or deionized water, for 12 consecutive days. Enzymatic
activities were determined in the cytoplasmic and mitochondrial
fractions of liver tissue obtained from the animals after treatment.
Pretreatment with benzyl alcohol decreased cytoplasmic alcohol and
aldehyde dehydrogenase activities when ethanol and acetaldehyde,
respectively, were the substrates, but had no effect on alcohol
dehydrogenase activity when benzyl alcohol was the substrate. The
effect on alcohol dehydrogenase activity was seen only in female rats.
Similarly, benzyl alcohol pretreatment resulted in decreased activity
of aldehyde dehydrogenase in female rats when acetaldehyde was the
substrate, but not when benzaldehyde was the substrate (Messiha 1991).

Benzoic acid and benzoate salts

Benzoic acid inhibits pepsin digestion, and sodium benzoate
inhibits trypsin digestion of fibrin, but they have no effect on
amylase or lipase. Trypsin digestion of casein is only initially
depressed by sodium benzoate (Kluge, 1933). Benzoic acid is
a specific, powerful inhibitor of D-amino acid oxidase, a
10^-4 mmol/litre solution resulting in 50% inhibition (Klein & Kamin,
1964). Concentrations in the range of 10^-3 mmol/litre have
non-specific inhibitory effects on the metabolism of fatty acids,
including acetoacetate formation (Avigan et al., 1955).

Mice fed 3 g sodium benzoate daily for 10 days had a 10%
reduction in creatine output, probably due to depletion of the glycine
pool (Polonowski & Boy, 1941).

Large oral doses of benzoic acid used therapeutically to
alleviate the effects of inherited urea cycle disorders were studied
with respect to their effect on intermediary metabolism. Documented
effects on the urea cycle, gluconeogenesis, fatty acid metabolism,
carnitine status, and the tricarboxylic acid cycle were attributed to
sequestering of coenzyme A by benzoate as a result of glycine
depletion required for the formation of hippuric acid from benzoyl
coenzyme A. In addition, binding of benzoate to albumin results in
toxic reactions due to the displacement of other metabolites from
albumin-binding sites; e.g. bilirubin toxicity and appetite
suppression result from increased serum tryptophanlevels, which
increase the turnover of brain serotonin (Tremblay & Qureshi, 1993).

2.2 Toxicological studies

2.2.1 Acute toxicity

The results of studies of the acute toxicity of these compounds
are summarized in Table 1.

Table 1. Acute toxicity of benzyl acetate, benzyl alcohol, benzaldehyde,
benzoic acid, benzoate salts, and benzyl benzoate

Species Route LD₅₀ Reference
(mg/kg bw)

Benzyl acetate
Rat Oral 2490-3690 Jenner et al.( 1964);
von Oettingen (1960)
Rabbit Oral 640 von Oettingen (1960)

Benzyl alcohol
Mouse Intraperitoneal 650 (7 days) McCloskey et al. (1986)
Rabbit Oral 1040 Graham & Kuizenga, (1945)

Benzaldehye
Mouse Intraperitoneal 1150 McCloskey et al. (1986)
Rat Subcutaneous 5000 Macht (1922)
Rat Oral 1300 Taylor et al. (1964)
Guinea-pig Oral 1000 Jenner et al. (1964)

Benzoic acid and benzoate salts
Rat Oral (Na salt) 2700 Deuel et al. (1954)
Rat Intravenous (Na salt) 1714 ± 124 Spector (1956)
Rat Oral (acid) 2000-2500 Ignat'ev (1965)
Rabbit Oral (Na salt) 2000 Spector (1956)
Rabbit Subcutaneous (Na salt) 2000 Spector (1956)
Dog Oral (Na salt) 2000 Spector (1956)

Benzyl acetate

Groups of five B6C3F1 mice or Fischer 344 rats of each sex were
administered single doses of benzyl acetate (purity, 96.0-101.3%) at
doses of 250, 500, 1000, 2000, or 4000 mg/kg bw in corn oil by gavage
and were observed for 15 days. All mice receiving 4000 mg/kg bw and
females receiving 2000 mg/kg bw became inactive immediately after

treatment. All mice at the highest dose and one male and two females
at 2000 mg/kg bw died on day 2 of the study. All rats receiving the
highest dose became inactive 2 h after treatment, and four males and
two females at this dose died on day 2. No other compound-related
effects were reported (US National Toxicology Program, 1986).

Benzyl alcohol

Groups of 12-30 male CD-1 mice, weighing 23-28 g, were given
benzyl alcohol as a 10% solution in peanut oil at intraperitoneal
doses of 500-1500 mg/kg bw and were observed for up to two weeks for
clinical signs. In animals at all doses between 500 and 1000 mg/kg bw,
sedation, loss of motor function, and laboured respiration were
observed. The lethal effect of benzyl alcohol occurred within two
periods: an initial acute phase, with an LD₅₀ of 1000 mg/kg bw, and
a delayed lethal effect up to seven days after treatment with an
LD₅₀ of 650 mg/kg bw. Inhibition of the metabolism of benzyl alcohol
to benzaldehyde by pretreatment with pyrazole markedly increased
mortality (McCloskey et al., 1986).

Single intravenous doses of pure benzyl alcohol were administered
to CD2F1, B6D2F1, and C57Bl/6 mice at doses of 0.05-0.2, 0.05-0.4, and
0.025-0.1 ml/kg bw, respectively. The mice were observed for two
weeks. Within the first 24 h of observation, convulsions were seen at
the highest doses, and dyspnoea and reduced motility were seen at all
doses except the lowest. A decrease in body-weight gain or a slight
decrease in body weight was noted in B6D2F1 and C57Bl/6 mice at all
doses except the lowest. Assessment of haemolytic and precipitation
potential in blood samples from the mice indicated strong activity of
benzyl alcohol at concentrations of 0.4-3.0 µl/ml (Montaguti et al.,
1994).

Benzaldehyde

Groups of three male and three female rats were given one-third
of the LD₅₀ intragastrically for four days and were then killed and
the livers examined for gross changes. None were detected (Taylor
et al., 1964).

The toxicity of benzaldehyde was determined in a study similar to
that described for benzyl alcohol above (McCloskey et al., 1986).
All deaths were observed within 4 h after treatment, with an LD₅₀ of
1150 mg/kg bw. The most prominent signs of toxicity were tremors,
sedation, respiratory distress, and, in animals monitored for seven
days, weight loss, averaging 2.25 g per animal.

Benzoic acid and benzoate salts

No LD₅₀ values were available for potassium benzoate. Benzoic
acid is not acutely toxic to man (Lehman, 1908) or to experimental
animals at moderate doses (Rost et al., 1913; Smyth & Carpenter,
1948).

An outbreak of poisoning affected 28 cats that had eaten meat
containing 2.39% benzoic acid. The effects were nervousness,
excitability, and loss of balance and vision. Convulsions occurred,
and 17 cats died or were killed. Autopsies showed damage to the
intestinal mucosa and liver. The sensitivity of cats may be due to
their failure to form benzoyl glucuronide, and toxic effects may
develop after ingestion of quantities > 0.45 g/kg bw as single doses
or 0.2 g/kg bw as repeated doses (Bedford & Clarke, 1971).

2.2.2 Short-term toxicity

2.2.2.1 Mice

Benzyl acetate

Groups of five B63F/N mice of each sex, aged six weeks, received
0, 125, 250, 500, 1000, or 2000 mg/kg bw benzyl acetate (purity, 96%)
in corn oil by gavage daily for 14 days. On day 16, all surviving
animals were killed and autopsied. All male mice at the highest dose
had died by day 3 of the study. Weight changes were not dose-related.
The only effect reported at autopsy was a roughening of the mucosa of
the stomach in the cardiac region in two males and in all females at
the highest dose and in one female at 1000 mg/kg bw per day (US
National Toxicology Program, 1986).

Groups of 10 B6C3F1 mice of each sex, about eight weeks of age,
were given 0, 62.5, 125, 250, 500, or 1000 mg/kg bw per day (males) or
0, 125, 250, 500, 1000, or 2000 mg/kg bw per day (females) of benzyl
acetate (purity, 96%) in corn oil by gavage, five times a week for 13
weeks. The animals were observed twice daily for signs of toxicity,
and clinical examinations were conducted weekly. Necropsies were
performed on all animals, and 35 tissues and organs, including brain,
liver, kidney, and stomach, from mice in the control and high-dose
groups were examined histologically. Seven of the females at the
highest dose died. Compound-related clinical effects observed at the
highest dose included trembling, inactivity, laboured breathing, and
depressed body temperature. At autopsy, no gross or microscopic
effects were seen (US National Toxicology Program, 1986). Subsequent
histopathological examination of brain tissue revealed hippocampal
necrosis in one female at 1000 mg/kg bw per day (US National
Toxicology Program, 1993).

Groups of 10 male and 10 female B6C3F1 mice with an average age
at exposure of 42 days (13 days' quarantine before the test) received
benzyl acetate (properties consistent with structure and literature
references; purity, 99%; stability monitored periodically; no
degradation of bulk chemical observed) in their diet at doses of
0, 3130, 6250, 12 500, 25 000, or 50 000 mg/kg feed, equal to 0, 425,
1000, 2000, 3700, and 7900 mg/kg bw per day for males and 0, 650,
1280, 2980, 4300, and 9400 mg/kg bw per day for females, for 13 weeks.
Feed was prepared weekly and stored in the dark, and the formulations
were analysed four times during the study for benzyl acetate
concentrations, stability, and homogeneity; it contained low,
biologically insignificant levels of aflatoxins, pesticides, and
heavy metals. Feed and water were provided ad libitum; feed
consumption was recorded daily, and the animals were weighed weekly.
Haematological and clinical chemical tests (cholesterol and
triglycerides) and assays for pancreatic enzymes (amylase, lipase,
carboxypetidase, chymotrypsin, ribonuclease) were performed at
termination of the study. Tissues from all control animals, females at
25 000 mg/kg, and all animals at 50000 mg/kg feed were examined
histologically.

Statistically significant (P < 0.01), dose-related decreases in
final body weights were observed in all treated animals in comparison
with controls. The body-weight decrement exceeded 10% of control
values in all treated groups, with the exception of males receiving
the lowest dose. The mean feed consumption of all exposed mice was
nonsignificantly lower than that of the control groups. Tremor was
observed in female mice at > 12500 mg/kg feed. At 50000 mg/kg feed,
one male died and one female mice was killed in extremis. The
absolute and relative organ weights of treated animals were influenced
by the lowered terminal body weight, and all significant differences
between treated and control groups were attributable to treatment. No
dose-related effects were observed in haematological, clinical
chemical, or pancreatic enzyme parameters.

Histopathological examination revealed hippocampal necrosis,
cerebellar haemorrhage of the brain, and hepatocellular necrosis in
one male mouse receiving 50 000 mg/kg feed after 11 weeks of
treatment. At termination, three female mice receiving 50000 mg/kg
feed had hippocampal necrosis and depletion of cells of the pyramidal
layer in the brain (US National Toxicology Program, 1993).

Benzyl alcohol

Benzyl alcohol (purity, 99%) was administered in corn oil to
groups of five male and five female B6C3F1 mice by gavage at doses of
0, 125, 250, 500, 1000, and 2000 mg/kg bw on five days a week for
12 doses over a 16-day period. All mice receiving 2000 mg/kg bw and
one male and two females receiving 1000 mg/kg bw died before the end
of the study. Lethargy and rough coats were observed in male mice at

the three highest doses and in female mice at the two highest doses.
Mice at 1000 and 2000 mg/kg bw had blood in the urinary bladder at
necropsy (US National Toxicology Program, 1989).

Doses of 0, 50, 100, 200, 400, and 800 mg/kg bw benzyl alcohol
(purity, 99%) in corn oil were administered to groups of 10 male and
10 female B6C3F1 mice by gavage on five days a week for 13 weeks. The
animals were observed twice daily, and their body weights were
recorded at the beginning and end of the study. Necropsy was performed
on all animals, and unspecified tissues from all vehicle controls and
those at the highest dose were examined histologically. In addition,
the brains of animals at 400 mg/kg bw per day and of all animals that
died during the test were examined. Deaths occurred in most groups,
with the exception of vehicle controls, but all except one of the
deaths was attributable to the gavage procedure. The final mean body
weight of males at 800 mg/kg bw per day was 5% lower than that of
controls; the final mean body weight of female mice at this dose was
8% lower and that of females at 400 mg/kg bw was 5% lower. Both male
and female mice at the high dose showed staggering during the first
and second weeks of the study. No treatment-related histopathological
effects were observed (US National Toxicology Program, 1989).

Benzaldehyde

Benzaldehyde (purity, 99%) was administered by gavage in corn oil
to groups of five male and five female B6C3F1 mice, aged eight weeks,
at a dose of 0, 200, 400, 800, 1600, or 3200 mg/kg bw per day on five
days per week for 16days (12 doses). The mice were observed twice
daily, and their body weights were recorded weekly. Gross necropsy was
performed on all animals. All mice at 1600 and 3200 mg/kg bw had died
by day 3, and one male receiving 800 mg/kg bw had died by day 10. The
mean body weights of the treated mice that survived to termination
were comparable to those of the respective vehicle controls. No
treatment-related gross lesions were observed (Kluwe et al., 1983;
US National Toxicology Program, 1990).

In a 13-week study, benzaldehyde (purity, 99%) was administered
to groups of 10 male and 10 female B6C3F1 mice, seven to eight weeks
old, in corn oil by gavage at a dose of 0, 75, 150, 300, 600, or
1200 mg/kg bw per day on five days per week. The animals were observed
twice daily, and their body weights were recorded weekly. Gross
necropsy was performed on all animals, and 35 tissues and organs from
all control and high-dose animals and all male mice receiving
600 mg/kg bw per day, including brain, liver, kidney, and stomach,
were retained for histopathological examination. In addition, the
spleen, stomach, and kidneys of all female mice at 600 mg/kg bw per
day and the kidneys and livers of all males at 300 mg/kg bw per day
were examined histologically. During the first week of the study, nine
males and one female at the high dose died. By the end of the study,
the mean body weight of male mice in the next highest dose group

(600 mg/kg bw per day) was 9% less than that of the vehicle controls,
while the mean body weight of females at the high dose was comparable
to that of the respective controls. The only treatment-related effect
reported was mild-to-moderate renal tubular degeneration in all male
mice at 1200 mg/kg bw and in one male at 600 mg/kg bw per day. These
lesions were not observed in males receiving lower doses of
benzaldehyde or in the female mice (Kluwe et al., 1983; US National
Toxicology Program, 1990).

Benzoic acid and benzoate salts

Groups of 50 male and 50 female mice were given benzoic acid at a
dose of 80 mg/kg bw per day, sodium bisulfite at 160 mg/kg bw per day,
or a mixture of the two at the same levels by gavage. The highest
mortality rate was observed in mice given the combination. A five-day
period of food restriction at 2.5 months induced 85% mortality in both
groups (Shtenberg & Ignat'ev, 1970).

2.2.2.2 Rats

Benzyl acetate

Groups of 15 male and 15 female rats were fed a mixture of
aromatic esters, including 15.8 mg/kg bw per day benzyl acetate, for
12 weeks. No adverse effects were noted (Oser, 1967).

Groups of five male and five female Fischer 344 rats, about six
weeks old, were given 0, 250, 500, 1000, 2000, or 4000 mg/kg bw per
day benzyl acetate (purity, 96%) by gavage in corn oil, daily for
14 days. The surviving animals were killed and autopsied on day 16.
None of the rats at 4000 mg/kg bw per day survived beyond two days,
and all rats at 2000 mg/kg bw per day had died within five days. No
other deaths were reported. Mean body-weight gain was > 10% lower
than that of controls in males at 500 and 1000 mg/kg bw per day and in
females at 1000 mg/kg bw per day. The only effect reported at autopsy
was caecums that were redder than normal in three animals at
4000 mg/kg bw per day (US National Toxicology Program, 1986).

Groups of 10 Fischer 344/N rats of each sex, about eight weeks
old, were given 0, 62.5, 125, 250, 500, or 1000 mg/kg bw per day
benzyl acetate (purity, 96%) in corn oil on five days per week for
13 weeks. The animals were observed twice daily for signs of toxicity,
and clinical examinations were conducted weekly. Necropsies were
performed on all animals, and 34 tissues and organs from rats in the
control and high-dose groups, including brain, skeletal muscle,
kidney, and pancreas, were examined histologically. Male rats at
1000 mg/kg bw per day and females at 500 and 1000 mg/kg bw per
day showed clinical symptoms including trembling, ataxia, and
sluggishness. Two males and one female at the highest dose had died by
day 86. Only male rats at 1000 mg/kg bw per day showed depressed mean

body weight relative to controls (21%). At autopsy, thickened stomach
walls were observed in two of nine surviving males and four females at
the high dose (US National Toxicology Program, 1986). A subsequent
microscopic re-examination of the brains revealed hippocampal necrosis
in eight of eight surviving males and four females at 1000 mg/kg bw
per day, the severity of the lesions being greater in the males than
the females (US National Toxicology Program, 1993).

Groups of 10 male and 10 female Fischer 344 rats, with an average
age at exposure of 43 days (13 days' quarantine before the test),
received benzyl acetate (properties consistent with structure and
literature references; purity, 99%; stability monitored periodically,
with no degradation of bulk chemical observed) in the diet at doses of
0, 3130, 6250, 12 500, 25 000, or 50 000 mg/kg feed, equal to 0, 230,
460, 900, 1750, or 3900 mg/kg bw per day for males and 0, 240, 480,
930, 1870, or 4500 mg/kg bw per day for females, for 13 weeks. Feed
was prepared weekly, stored in the dark, and analysed during the study
for benzyl acetate concentrations, stability, and homogeneity; it
contained low, biologically insignificant levels of aflatoxins,
pesticides, and heavy metals. Feed and water were 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 those at 50 000 mg/kg feed. At termination,
liver peroxisomes were examined morphometrically in female rats given
0, 25000, or 50000 mg/kg feed. Histopathological examinations were
performed on all control animals and those at 25 000 and 50 000 mg/kg
feed.

Nine male and female rats at 50 000 mg/kg feed died or were
killed when moribund between weeks 2 and 8 of the study. Male rats
given 25000 mg/kg feed showed a 10% decrease (P < 0.01) in mean
terminal body weight. The body weights of the one surviving male and
the one surviving female at 50000 mg/kg feed were less than half that
of the controls. The final mean body weights of treated male and
female rats in the other groups were similar to or slightly lower than
those of the controls. The average feed consumption of males at
25 000 mg/kg feed and males and females at 50000 mg/kg feed was
reduced. Tremor, ataxia, and urine stains were observed in rats at
50000 mg/kg feed. Serum cholesterol was significantly decreased in
females at 12 500 mg/kg feed (P < 0.01), 25 000 mg/kg feed
(P < 0.001), and 50000 mg/kg feed (only one female rat alive after
11 weeks). No other dose-related effects were seen on haematological,
clinical chemical, or pancreatic enzyme parameters in treated rats.
The volume, surface, and numerical density of hepatic peroxisomes in
female rats at 25 000 mg/kg feed were significantly (P < 0.001)
increased. No differences in organ weights attributable to treatment
were observed. Histopathological examination of male and female rats
receiving 50 000 mg/kg benzyl acetate revealed degeneration and

necrosis of neurons and glial cells in the cerebellum and hippocampus,
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 (US National Toxicology Program, 1993).

Benzyl alcohol

Benzyl alcohol (purity, 99%) was administered to groups of five
male and five female Fischer 344 rats in corn oil by gavage at a dose
of 0, 125, 250, 500, 1000, or 2000 mg/kg bw on five days per week for
12 doses over a 16-day period. All rats receiving 2000 mg/kg bw and
two males and three females receiving 1000 mg/kg bw died before the
end of the study. The final body weights of male rats at 1000 mg/kg bw
were 18% lower than those of controls, while the females at this dose
had a body-weight decrement of < 5%. Lethargy was observed in rats at
the two highest doses, which also had blood around the mouth and
nose, subcutaneous haemorrhages, and blood in the urinary and
gastrointestinal tracts (US National Toxicology Program, 1989).

Doses of 0, 50, 100, 200, 400, or 800 mg/kg bw benzyl alcohol
(purity, 99%) in corn oil were administered to groups of 10 male and
10 female Fischer 344 rats by gavage on five days per week for 13
weeks. The animals were observed twice daily, and their body weights
were recorded at the beginning and end of the study. Necropsy was
performed on all animals, and histological examination (tissues
unspecified) was performed on all vehicle controls and those at the
highest dose; the brains of animals at 400 mg/kg bw per day dose group
were also examined. Eight males and two females at 800 mg/kg bw per
day, one female at 400 mg/kg bw per day, one male at 200 mg/kg bw per
day, and one female in the vehicle control group died after treatment.
Four of the deaths in males and one in females at the high dose group
were attributed to gavage errors. Males and females at 800 mg/kg bw
per day exhibited signs of neurotoxicity, which included staggering,
laboured breathing, and lethargy. After eight weeks, eight males at
800 mg/kg bw per day had blood around the nose and mouth. After 13
weeks, the body weights of male and female rats at the high dose were
7 and 5% lower than those of the respective controls. Treatment-
related histopathological effects observed in rats at the high dose
were: necrosis of the dentate gyrus of the hippocampus in nine of nine
surviving males and seven of seven surviving females; skeletal muscle
necrosis in five males; thymic congestion, haemorrhage, and atrophy in
eight males; and nephrosis of the kidney in six of nine surviving
males, consisting of degeneration and regeneration of the tubular
epithelium (US National Toxicology Program, 1989).

Benzaldehyde

No tissue damage was seen grossly or histopathologically in
groups of five male and five female rats given 0 or 0.1% benzaldehyde
in their diet for 27-28 weeks or 1% for 16 weeks, (Hagan et al.,
1967).

Benzaldehyde (purity, 99%) was administered by gavage in corn oil
to groups of five male and five female Fischer 344 rats at a dose of
0, 100, 200, 400, 800, or 1600 mg/kg bw per day on five days per week
over 16 days (12 doses). The rats were observed twice daily, and their
body weights were recorded weekly. Gross necropsy was performed on all
animals. All rats that received 1600 mg/kg bw per day died on day 2 of
the study, and two males and two females receiving 800 mg/kg bw per
day died before the end of the study. The mean body weights of male
rats at 800 mg/kg bw were 14% lower than those of controls, and those
of females were 11% lower, at the end of the study. No treatment-
related gross lesions were observed (Kluwe et al., 1983; US National
Toxicology Program, 1990).

In a 13-week study, benzaldehyde (purity, 99%) was administered
to groups of 10 male and 10 female Fischer 344 rats at a dose of 0,
50, 100, 200, 400, or 800 mg/kg bw per day in corn oil by gavage on
five days per week. The animals were observed twice daily, and their
body weights were recorded weekly. Gross necropsy was performed on all
animals, and 35 tissues and organs from all control animals and those
at 400 and 800 mg/kg bw, including brain, kidney, and pancreas
(skeletal muscle not mentioned), were retained for histopathological
examination. Deaths occurred in six males and three females at the
high dose and one female at 400 mg/kg bw before the end of the study.
The terminal body weights of males at 800 mg/kg bw per day were 26%
lower than those of controls, whereas the terminal body weights of
treated female rats were comparable to those of controls. Treatment-
related lesions of the brain, forestomach, liver, and kidney were
observed in both male and female rats at 800 mg/kg bw per day, which
included: degeneration and necrosis of the cerebellum and necrosis of
the neurons of the hippocampus, hyperplasia and/or hyperkeratosis of
the squamous epithelium of the forestomach, degeneration and necrosis
(males only) of the liver, and degeneration or necrosis of the tubular
epithelium of the kidney (Kluwe et al., 1983; US National Toxicology
Program, 1990).

Benzoic acid and benzoate salts

Groups of five male and five female rats were fed sodium benzoate
for 30 days at levels of 16-1090 mg/kg bw. There were no effects on
body weight, appetite, or mortality and no histological changes in
organs (Smyth & Carpenter, 1948).

Groups of three male and three female rats were fed diets
containing 0, 2, or 5% sodium benzoate for 28 days. All animals
at the 5% level died during the first two weeks after showing
hyperexcitability, urinary incontinence, and convulsions. Male rats at
the 2% level had a significant decrease in body weight, and the food
intake of male and female animals at this dose was decreased in
comparison with controls (Fanelli & Halliday, 1963). Four groups of
15 rats were given sodium benzoate at a dietary level of 0 or 5%
benzoate or 5% benzoate plus 1% glycine for three weeks. The body
weights of animals fed benzoate were reduced, but less so when 1%
glycine was added. The total cholesterol content of the liver was
unaffected by treatment with benzoate, but that of phospholipids was
significantly reduced. The potassium concentration of skeletal muscle
was also low at this level. Supplementary glycine corrected the
potassium and phospholipid deficiencies (Kowalewski, 1960). Twenty-
eight young rats were given a diet containing 5% sodium benzoate for
three weeks. Nineteen animals died within two weeks. Food consumption
was significantly reduced, and most animals developed severe
diarrhoea. The changes seen at autopsy were haemorrhage in the gut and
nasal blood crust. Five adult rats on a similar diet died within five
weeks with severe weight loss (Kieckebusch & Lang, 1960). Groups of
four to 19 male rats were fed diets containing sodium benzoate at a
level of 0, 1.5, 2.0, 2.5, 3, 3.25, or 3.75% for 40 days. The average
growth of animals at all levels was reduced in comparison with that of
controls, and mortality was high, food efficiency poor, and growth
severely depressed at levels > 3%. Addition of glycine reduced the
toxic effects. Animals died after showing incoordination, tremor, or
convulsions and severe eye inflammation. Groups of 10-15 young male
rats fed restricted diets containing 0, 1.5, 2.0, 2.5, or 3% sodium
benzoate had no difference in weight gain, even at the 3% level.
Supplementary glycine addition again alleviated the weight loss
(Griffith, 1929).

Groups of 10 male and 10 female rats, four to five weeks old and
weighing 110-150 g, were fed diets containing sodium benzoate at a
level of 0, 0.5, 1, 2, 4, or 8% for six weeks. All rats at 8% and 19
at the 4% level died within four weeks, but 19 animals at 2%, 18 at
1%, and 17 at 0.5% survived for six weeks. Significant reductions in
body-weight gain were seen only in animals at 4 and 8%. The only acute
toxic effect observed was hypersensitivity. No morphological change
was seen at autopsy, except for atrophy of the spleen and lymph nodes
in rats at 4 and 8% (Sodemoto & Enomoto, 1980).

Groups of 8-10 rats were fed diets containing 1, 2, 4, or 8%
sodium benzoate for 90 days. Four rats on the 8% diet died within an
average of 13 days. The weight gain of the four survivors was two-
thirds that of controls with an identical food intake. Kidney and
liver weights were significantly higher than those of the control
group (Deuel et al., 1954).

2.2.2.3 Guinea-pigs

Benzoic acid and benzoate salts

Groups of four guinea-pigs given 150 mg/kg bw benzoate plus
benzoic acid daily for 65 days had no adverse effects. Scorbutic
animals fed the same dose had a reduced lifespan (Kluge, 1933).

2.2.2.4 Dogs

Benzoic acid and benzoate salts

Seventeen dogs given diets containing sodium benzoate or benzoic
acid at 1000 mg/kg bw for 250 days showed no effect on growth,
appetite, or well-being; but at higher doses ataxia, epileptic
convulsions, and death occurred (Rost et al., 1913).

2.2.3 Long-term toxicity and carcinogenicity

2.2.3.1 Mice

Benzyl acetate

Groups of 50 B6C3F1 mice of each sex, aged eight weeks, were
given benzyl acetate (purity, 99-101%) in corn oil by gavage at a dose
of 500 or 1000 mg/kg bw per day on five days per week for 103 weeks.
The mice were observed twice daily for signs of toxicity, and body
weights were recorded weekly for the first 13 weeks, monthly until
week 91, and every two weeks until the end of the study. Vehicle
control groups of 50 animals of each sex were given corn oil by
gavage. Complete gross necropsies and histopathological examinations
were performed on 40 tissues and organs, including brain, liver,
kidney, and stomach, from animals found dead and those sacrificed at
the end of the study.

The mean body-weight gains of treated and control male mice were
comparable throughout the study. Treated females had slightly higher
mean body weights than controls after week 20. The survival of female
mice was markedly lower in the control (30%) and low-dose groups (36%)
in comparison with the high-dose group (60%), and high mortality was
associated with infection, resulting in suppurative inflammation or
abscesses of the ovaries, uterus, mesentery, peritoneum, or multiple
organs (26/35 control, 14/32 low dose, and 8/20 high dose). In males,
there were no significant differences in survival rates between
controls and treated groups, although a greater number of control than
treated males died before week 45.

Hepatocellular adenomas occurred in 0/50 control, 5/49 low-dose,
and 13/50 high-dose males and 0/50 control, 0/50 low-dose, and 6/50
high-dose females. The incidence at the high dose was statistically
significantly increased in both males (P < 0.001) and females

(P < 0.05) and was also higher than that of historical controls
treated by gavage: The cumulative historical incidences of
hepatocellular adenomas in controls treated with corn oil by gavage in
six studies conducted at the contract laboratory before 3 August 1984
were 36/298 for male and 11/300 for female B6C3F1 mice. Hepatocellular
carcinomas were observed in male mice and in females at the high dose,
but the incidence did not increase with dose: 10/50 control 10/50,
14/49 low-dose, and 12/50 high-dose males and 1/50 control, 0/50
low-dose, and 4/50 high-dose females.

Squamous-cell papillomas or carcinomas of the forestomach
occurred in male mice and in females at the high dose. The incidence
in males was 4/49 controls, 4/48 at the low dose, and 11/49 at the
high dose, and that in female mice was 0/50 control, 0/50 at the low
dose, and 4/48 at the high dose. Most of the tumours were papillomas.
The incidence of combined papillomas and carcinomas in animals at the
high dose, while not statistically significant, exceeded the values
for historical controls gavaged with corn oil in six studies at
the contract laboratory: 2/296 for males and 2/297 for females.
Forestomach hyperplasia was also reported at a significantly higher
incidence in mice at the high dose (P < 0.05) than in controls: 1/49
control, 7/48 low-dose, and 22/49 high-dose males and 1/50 control,
6/50 low-dose, and 17/48 high-dose females (Abdo et al., 1985; US
National Toxicology Program, 1986).

Groups of 60 male and 60 female B6C3F1 mice, with an average age
at initial exposure of 40 days (11 days of quarantine before test),
received diets containing benzyl acetate (properties consistent with
structure and literature references; purity, > 98%; stability
monitored periodically, and no degradation of bulk chemical observed)
at a dose of 0, 330, 1000, or 3000 mg/kg feed, equal to 0, 37, 112,
and 346 mg/kg bw per day for males and 0, 42, 132, and 382 mg/kg bw
per day for females, for 103 weeks. Feed was prepared weekly, stored
in the dark, and analysed during the study for benzyl acetate
concentrations, stability, and homogeneity; it contained low,
biologically insignificant levels of aflatoxins, pesticides, and heavy
metals. Feed and water were provided ad libitum; feed consumption
was measured daily per cage for five days once every four weeks. The
animals were weighed weekly during the first 13 weeks of the study and
every four weeks thereafter. Ten mice of each sex from each group were
sacrificed after 15 months of exposure. Haematological and clinical
chemical determinations (cholesterol, triglycerides, alkaline
phosphatase, creatinine kinase, and sorbitol dehydrogenase) were
carried out on mice killed at the interim sacrifice, and necropsy and
a thorough histopathological examination were performed on all
animals. The brain, right kidney, and liver were weighed.

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 those at 3000 mg/kg
feed. Almost all of the deaths occurred during the last nine months of
the study. The average feed consumption of treated mice was similar to
that of the control groups. All treated mice except females at
330 mg/kg feed had decreased mean body weights in comparison with
controls, with weights 13 and 9% lower at termination in males and
females, respectively (statistics not reported). The observation of
decreased weight gain at such low levels of intake, with no apparent
effect on food palatability, is inconsistent with the observations in
the previous study (Abdo et al., 1985; US National Toxicology
Program, 1986), perhaps due to the unreliability of data on food
consumption in studies conducted with organoleptic test materials.
Slight, inconsistent, dose-related decreases (significant at the
P < 0.05 level) in cholesterol, triglyceride, and (females only)
alkaline phosphatase levels were observed; no dose-related effects
were seen at haematology. Statistically significant (P < 0.05 or
lower), dose-related increases in the incidence and severity of non-
neoplastic lesions of the nasal mucosa and glands were found in all
treated mice. 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 seen in male and female mice at interim sacrifice. No
neoplasms or dose-related preneoplastic lesions occurred in the nose.
A dose-related, negative trend in the incidences of hepatocellular
carcinoma and hepatocellular adenoma, which was statistically
significant (P < 0.01) for hepatocellular adenomas in animals at
3000 mg/kg feed, was seen in male but not female mice (US National
Toxicology Program, 1993).

Benzyl alcohol

Benzyl alcohol (purity, 99%) was given to groups of 50 B6C3F1
mice of each sex, eight to nine weeks of age, at a dose of 0, 100, or
200 mg/kg bw per day in corn oil by gavage on five days a week for 103
weeks. The doses were selected on the basis of those found to induce
neurotoxic effects (lethargy and staggering) in short-term studies.
The mice were observed twice daily, and their body weights were
recorded weekly for the first 12 weeks and once a month thereafter.
Gross necropsy was performed on all animals, and 50 tissues and
organs, including brain, liver, kidney, and stomach, from all vehicle
controls, animals at the high dose, and animals at the other doses
that died before 22 months or had gross lesions were examined
histologically.

The mean body weights of treated and control mice were comparable
throughout the study. The survival of control females was
significantly lower than that of animals at the high dose after week
74, but no other differences in survival were seen: 68% of control,
66% of low-dose, and 70% of high-dose males; and 50% of control, 62%
of low-dose, and 72% of high-dose females. No significant treatment-
related effects were noted at gross necropsy or histopathological
examination. No increase was seen in the incidence of hepatocellular
or forestomach neoplasia (US National Toxicology Program, 1989).

Benzaldehyde

Groups of 50 male and 50 female B6C3F1 mice, aged eight (male) or
nine (female) weeks, received benzaldehyde (purity, 99%) at doses of
0, 200, or 400 mg/kg bw per day (males) or 0, 300, or 600 mg/kg bw per
day (females) in corn oil by gavage on five days per week for 103
weeks. The mice were observed twice daily, and their body weights were
recorded weekly for 13 weeks and monthly thereafter. Gross necropsy
was performed on all animals, and 38 tissues and organs, including
brain, kidney, stomach, liver, and tissues with gross lesions, from
all mice at the high dose, vehicle controls, and all mice that died
before the end of the study were examined histologically, as were
gross lesions and stomachs from all mice at the low dose.

Treatment had no effect on the body weights or survival of male
or female mice, and no unusual clinical signs were observed. The
incidence of focal hyperplasia of the forestomach was significantly
increased in males at the high dose (P < 0.01) and in females at the
low (P < 0.05) and high ( P < 0.01) doses (males: 7/50, 8/50, 16/50;
females: 12/50, 23/50, 39/50), while the incidence of squamous-
cell papillomas of the forestomach was increased significantly only in
female mice (P < 0.05) (males: 1/50, 2/50, 5/50; females: 0/50, 5/50,
6/50). Although the incidence of papillomas in male and female mice
exceeded the historical control values for studies of the US National
Toxicology Program (1.6% and 1.9%, respectively, for papillomas and
carcinomas combined), they only slightly exceeded the highest
incidence in the controls. No squamous-cell carcinomas were considered
to have been identified in any of the groups. No treatment-related
effects on the liver were detected (US National Toxicology Program,
1990).

Benzoic acid and benzoate salts

Parenteral administration of benzoic acid has not been shown to
cause tumours (Hosino, 1951). Groups of 25 male and 25 female mice
were given benzoic acid at a dose of 40 mg/kg bw per day, sodium
bisulfite at 80 mg/kg bw per day, or a mixture of the two for 17
months. Mortality was greater in the groups receiving the mixture
(62%) than in those receiving the individual substances (32%) at eight
months. Mortality at 17 months and pathology were not reported
(Shtenberg & Ignat'ev, 1970).

Doses of 0.5, 1, 2, 4, and 8% sodium benzoate were administered
in drinking-water to groups of four male and four female albino Swiss
mice for 35 days. All of the mice receiving 8% died within three weeks
of administration, three males and three females at the 4% level died
during the study period, and the body weights of the surviving animals
were substantially reduced. On the basis of survival, body weights,
intake of test material, and histological changes, 2% sodium benzoate
was chosen as the dose for a long-term study.

In the main study, a 2% solution of sodium benzoate (purity, 99%)
was administered in the drinking-water to groups of 50 male and 50
female five-week-old mice for their lifetime. Groups of 100 males and
100 females were used as untreated controls. Both treated and control
animals were 'carefully checked'; their body weights were measured
weekly, and gross pathological changes were recorded. The animals were
either allowed to die or were sacrificed when moribund. Complete
necropsies were performed on all animals, and the liver, spleen,
kidneys, bladder, thyroid, heart, pancreas, testes, ovaries, brain,
nasal turbinates, at least four lobes of the lungs, and organs with
gross pathological changes were examined histologically. The average
daily intake of sodium benzoate was 124.0 mg for males and 119.2 mg
for females on the basis of daily water consumption of 6.2 and 5.9 ml,
respectively. The dose of sodium benzoate was equivalent to 6200 mg/kg
bw per day for males and 5960 mg/kg bw per day for females. Treatment
had no effect on survival or the incidence of tumours in the
limited numbers of tissues investigated. It is uncertain whether
histopathological end-points other than neoplasmas were investigated
(Toth, 1984).

2.2.3.2 Rats

Benzyl acetate

Groups of 50 Fischer 344/N rats of each sex, seven weeks old,
were given benzyl acetate (purity, 99-101%) in corn oil by gavage at
a dose of 250 or 500 mg/kg bw per day on five days per week for 103
weeks. The rats were observed twice daily for signs of toxicity, and
body weights were recorded weekly for the first 13 weeks, monthly
until week 91, and every two weeks until the end of the study. Vehicle
control groups of 50 animals per sex were given corn oil by gavage.
Complete gross necropsies and histopathological examinations of 39
tissues and organs, including brain, kidney, pancreas, and skeletal
muscle, were performed on animals found dead and on those sacrificed
at the end of the study.

Mean body-weight gains were comparable between treated and
control groups throughout most of the study. No significant
differences were found in the survival of treated rats as compared
with controls. In male rats, 76% of the controls, 92% of the low-dose
group, and 80% of the high-dose group survived to 104-106 weeks; the

survival rates for females were 80% of the controls, 72% of the
low-dose group, and 72% of the high-dose group.

The incidence of all malignant epithelial tumours in the
preputial gland (cystadenocarcinoma, adenocarcinoma, and carcinoma)
was elevated in males at the high dose (control 1/50, low dose 1/50,
high dose 6/50), but the increase was not statistically significant.
The incidence of acinar-cell adenomas in the pancreas of male rats was
22/50 in controls, 27/50 at the low dose, and 37/49 at the high dose,
the last incidence being significantly greater than that in the
vehicle controls (P = 0.001). Acinar-cell hyperplasia was also
observed in male rats, but the incidence did not increase with dose
(37/50 control, 34/50 low dose, and 36/49 high dose). As these
incidences were calculated after examination of additional pancreatic
tissue, the historical incidences of this lesion in vehicle controls
were not relevant for comparison. When the slides of the pancreas were
read initially, the incidences of acinar-cell adenomas in males were
found to be 3/50, 8/50, and 8/49, which are within the range of the
historical control values. No acinar-cell hyperplasia or adenoma of
the pancreas was observed in females, and no acinar-cell carcinoma
were observed in male or female rats. There were increased incidences
of retinopathy (not specified) and cataracts in males at the high dose
and females at the low dose, but the authors attributed this effect to
the proximity of these rats to fluorescent light (Abdo et al., 1985;
US National Toxicology Program, 1986).

Groups of 60 male and 60 female Fischer 344 rats, of an average
of 41 days at initial exposure (12 days' quarantine before the test),
received benzyl acetate (properties consistent with structure and
literature references; purity, >98%; stability monitored
periodically, and no degradation of bulk chemical observed) at doses
of 0, 3000, 6000, or 12 000 mg/kg feed, equal to 0, 130, 260, and
510 mg/kg bw per day for males and 0, 145, 290, and 575 mg/kg bw per
day for females, for 103 weeks. Ten rats of each sex from each group
were sacrificed after 15 months. Feed was prepared weekly, stored
in the dark, and analysed during the study for benzyl acetate
concentration, stability, and homogeneity; it contained low,
biologically insignificant levels of aflatoxins, pesticides, and heavy
metals. Feed and water were provided ad libitum. The feed
consumption was measured daily per cage for five days once every four
weeks. The animals were weighed weekly during the first 13 weeks of
the study and every four weeks thereafter. Haematological and clinical
chemical determinations (cholesterol, triglycerides, alkaline
phosphatase, creatinine kinase, and sorbitol dehydrogenase) and (in
males only) assays for pancreatic enzymes (amylase, lipase, and
carboxypeptidase) were carried out on rats at the interim sacrifice.
Necropsy and a thorough histopathological examination were performed
on all animals, and the brain, right kidney, and liver were weighed.

The mean body weights of animals at 12 000 mg/kg feed were about
5% lower than those of the control groups for most of the study. No
significant differences in survival rate, average feed consumption,
clinical findings, the results of clinical chemistry and haematology,
pancreatic enzyme assays, or the incidences of neoplasms and
non-neoplastic lesions were observed in treated rats in comparison
with controls (US National Toxicology Program, 1993).

Benzyl alcohol

Benzyl alcohol was administered in corn oil by gavage to groups
of 50 Fischer 344/N rats of each sex at a dose of 0, 200, or 400 mg/kg
bw per day on five days a week for 103 weeks. The rats were observed
twice daily, and body weights were recorded weekly for the first 12
weeks and once a month thereafter. Gross necropsy was performed on all
animals; and 49 tissues and organs, including brain, kidney, pancreas,
and skeletal muscle, from all female rats and from male rats in the
vehicle control and high-dose groups and those in the other groups
that died before 22 months or which had gross lesions were examined
histologically.

The mean body weights of treated and control animals were
comparable throughout the study. No compound-related clinical signs
were observed, although a sialodacryoadenitis viral infection was
widespread among the study animals in the third month. The survival of
treated females was significantly lower than that of vehicle controls:
70% of controls, 34% of low-dose females, and 34% of high-dose
females; this was due to a much higher incidence of accidental deaths
related to the gavage process. Survival among the male rats was
comparable in all groups: 56% of controls, 54% at the low dose, and
48% at the high dose.

Cataracts and retinal atrophy were observed at increased
incidences in rats at the high dose. The authors attributed this
effect to the proximity of this group of animals to fluorescent light
for most of the study. An increased incidence of hyperplasia of the
forestomach epithelium was seen (not statistically significant) in
male rats: control, 0/48; low dose, 0/19; high dose, 4/50. Haemorrhage
and foreign material in the respiratory tract seen in treated rats
that died before the end of the study were suggested by the authors to
have been the result of either direct deposition of material into the
lung during gavage 'accidents' or the anaesthetic properties of benzyl
alcohol resulting in reflux of gavage material and aspiration into the
lungs. No pancreatic acinar-cell adenomas were reported, and no other
effects of treatment were seen at gross necropsy or histopathological
examination (US National Toxicology Program, 1989).

Benzaldehyde

Groups of 50 male and 50 female Fischer 344 rats received a dose
of 0, 200, or 400 mg/kg bw per day benzaldehyde (purity, 99%) in corn
oil by gavage on five days per week for 103 weeks. The rats were
observed twice daily, and their body weights were recorded weekly for
13 weeks and monthly thereafter. Gross necropsy was performed on all
animals, and 43 tissues and organs, including kidney, skeletal muscle,
pancreas, and brain, from all male rats, vehicle controls, all females
at the high dose, and all rats that died before the end of the study,
were examined histologically, as were 13 tissues and organs from
females at the low dose.

Treatment had no effect on the body weights of male or female
rats throughout the study. The survival of males at the high dose was
significantly decreased in comparison with controls, and there was a
significant, dose-related trend to reduced survival in the treated
groups. The rate of survival among males at the end of the study was
74% of controls, 58% at the low dose, and 42% at the high dose. There
was no statistically significant difference in the survival rate of
treated female rats in comparison with controls: 66% of controls, 66%
at the low dose, and 58% at the high dose; however, survival tended to
decrease at an earlier stage in animals at the high dose than in the
other groups.

An increased incidence of pancreatic acinar-cell nodular
hyperplasia and adenomas was noted in males at the high dose
(hyperplasia: 6/49, 6/49, 12/48; adenomas: 3/49, 2/49, 7/48 in
control, low-dose, and high-dose animals, respectively). Although the
incidence of pancreatic adenomas at the high dose was statistically
significant (P = 0.038), it was within the range of the historical
control incidences of pancreatic acinar-cell neoplasms at the study
laboratory (0/49-11/50). No acinar-cell carcinomas were detected in
any group. A number of other tumours were observed in treated groups,
at statistically significantly increased incidences in comparison
with controls, but these either were not dose-related (malignant
mesotheliomas of the tunica vaginalis and/or mesentery in male rats)
or were due to increases in early-stage but not advanced-stage
neoplasms (mononuclear-cell leukaemia in males) (US National
Toxicology Program, 1990).

Benzoic acid and benzoate salts

Three groups of 20 male and 20 female rats were pair-fed for
eight weeks on diets containing 0, 0.5, or 1% benzoic acid and then
fed ad libitum over four generations. Two generations were fed for
their lifespan, and the third and fourth generations were autopsied
after 16 weeks. No effects were observed on growth, fertility,
lactation, or lifespan. Examination post mortem showed no
abnormalities (Kieckebusch & Lang, 1960). In another experiment, 20

male and 30 female rats were fed a diet containing 1.5% benzoic acid
for 18 months; 13male and 12 female rats served as controls. Fifteen
animals in the test group and three controls died, and the remaining
treated animals had reduced body weight and food intake. Experiments
with groups of 20 treated and 10 control animals of another strain
gave similar findings (Marquardt, 1960).

Groups of 10 male and female rats received benzoic acid at
40 mg/kg bw per day, or sodium bisulfite at 80 mg/kg bw per day, or a
mixture of the two in the diet for 18 months. Growth was slightly
reduced, and the erythrocyte sedimentation rate was increased. Rats
fed benzoic acid developed some tolerance to a lethal dose of the
compound given terminally. No pathological analysis was reported
(Shtenberg & Ignat'ev, 1970).

Groups of 50 male and 52 female Fischer 344 rats, four to five
weeks old, received diets containing 1% (500 mg/kg bw per day) or 2%
(1000 mg/kg bw per day) sodium benzoate for 18-24 months. Controls,
consisting of 25 male and 43 female rats, received basal diet. Food
intake was adequately controlled to avoid an excess; tap water was
available ad libitum. Survival was very poor in all groups, due to
intercurrent sialodacryoadenitis and mycoplasma infections. All
surviving animals were sacrificed between 18 and 25 months, all were
autopsied, and various tissues were examined histopathologically. No
adverse clinical signs directly attributable to treatment were
observed, and only negligible differences in average body weight and
mortality rate were seen between the treated and control groups.
Although a variety of tumours occurred among treated and control rats
of each sex, they were of similar type and incidence (Sodemoto &
Enomoto, 1980).

2.2.4 Reproductive toxicity

2.2.4.1 Mice

Benzyl acetate

The potential reproductive toxicity of benzyl acetate was
assessed by examining sperm morphology, vaginal cytology, and the
weights of male reproductive organs at the end of the 13-week feeding
study (US National Toxicology Program, 1993). Dietary levels of
3130-50 000 ppm benzyl acetate had no effect on the weights of the
epididymis, cauda epididymis, or testis or on sperm motility or
density or the percent of abnormal sperm. The mean length of the
estrous cycle of mice at the high dose was significantly greater than
that of the control group. This effect was associated with a
significant decrease in body weight (Morrissey et al., 1988).

Benzyl alcohol

Benzyl alcohol was one of several chemicals tested in a
preliminary screening study in CD-1 mice. The study consisted of
three phases: two to determine dose and the third phase to assess
developmental toxicity. In the first phase, benzyl alcohol was
administered by gavage at a dose of 10, 100, or 1000 mg/kg bw per day
to groups of three virgin female mice for five consecutive days,
followed by a seven-day observation period. All animals were observed
daily for signs of toxicity and mortality; body weights were recorded
on treatment days 1 and 5 and on days 3 and 7 after treatment. At each
dose tested, signs of clinical toxicity, including salivation, stained
fur, and rough coat, were reported. At 1000 mg/kg bw per day, one
animal had a hunched posture, and another was sacrificed on day 4 of
treatment after convulsing. The body weights did not appear to be
affected by treatment. On the basis of the one death at the high dose,
doses of 200, 380, 720, 1370, and 2605 mg/kg bw per day were selected
for the second phase of the study.

The aim of the second phase was to estimate the LD₁₀ of benzyl
alcohol in pregnant mice. Groups of four pregnant mice were given a
dose of 200, 380, 720, 1370, or 2605 mgl/kg bw per day on days 6-15 of
gestation. All surviving animals were sacrificed on day 17 of
gestation and examined for the presence or absence of viable fetuses.
All animals were observed daily for signs of clinical toxicity and
mortality, and body weights were measured. Clinical signs were
observed intermittently at the three lower doses but were not
attributable to treatment; a single animal at 380 mg/kg bw per day was
sacrificed after showing unsteady gait, languid behavior, and pale
eyes, but similar symptoms were not observed at the next highest dose.
At 1370 mg/kg bw per day, clinical signs of toxicity affecting all
animals included unsteady gait, languid behavior, hunched posture,
tremors, rapid respiration, squinted eyes, convulsions, hyperactivity,
prostration, and laboured breathing, and two animals died (one was
sacrificed and one was found dead) on day 4 of treatment. At
2605 mg/kg bw per day, all animals died before the second day of
treatment. Body-weight gains of 17.5, 11.1, 6.5, and 7.1 g were
reported for the animals at 200, 380, 720, and 1370 mg/kg bw per day,
respectively, between day 1 of treatment and day 2 after treatment,
indicating a dose-dependent decrease (no statistical analysis was
presented). All dams at 200 mg/kg bw per day, two of three at
380 mg/kg bw per day, and two of four at 720 mg/kg bw per day had
viable fetuses; the other surviving dams at these doses resorbed their
litters. One dam at 1370 mg/kg bw per day had live fetuses, but the
other showed no evidence of live pups or resorptions. The LD₁₀ for
benzyl alcohol was calculated to be 550 mg/kg bw per day, and this
dose was used in the third phase of the study.

In the phase aimed to study developmental toxicity, 50 female
mice were given benzyl alcohol at 550 mg/kg bw per day by gavage on
days 6-15 of gestation; a further 50 mice received the corn oil
vehicle. All dams were allowed to deliver naturally, and pups and dams
were observed until day 3 post partum, when the experiment was
terminated. Body weight, clinical observations, and mortality were
recorded daily throughout treatment and up to day 3 post partum.
Mortality was not significantly increased in animals given benzyl
alcohol over that in the control group. One treated mouse showing
languid behaviour, laboured breathing, and a rough coat died, but no
other deaths or clinical signs were reported. Maternal body weight and
body-weight gain during treatment and up to day 3 post partum were
virtually identical for treated and control animals. All other
parameters examined, including gestation index, average number of live
pups per litter, and postnatal survival and pup body weight on days 0
and 3 post partum, were not significantly different from the control
values. The authors concluded that, at the predicted LD₁₀, benzyl
alcohol had no significant effects on the development of CD-1 mice.
The NOAEL was 550 mg/kg bw per day (York et al., 1986).

In another preliminary screening test for the reproductive hazard
of benzyl alcohol in CD-1 mice, an initial eight-day evaluation was
used to determine the maximum tolerated dose, which was used in the
phase on developmental toxicity. Groups of 10 female mice were given
benzyl alcohol at 0, 160, 325, 645, 1300, or 2595 mg/kg bw per day by
gavage on eight consecutive days, followed by an eight-day observation
period. Distilled water was used as the vehicle. The end-points
evaluated included clinical toxicity, body-weight change, and
mortality. During treatment, one animal in the control group and one
each at 160 and 325 mg/kg bw per day died; no deaths occurred at
645 mg/kg bw per day, but eight deaths occurred at 1300 mg/kg bw per
day evenly over the 10-day treatment period. All animals at
2595 mg/kg bw per day died on the first day of treatment. Clinical
signs of toxicity, including hunched posture, tremors, piloerection,
prostration, ataxia, and dyspnoea, were observed in the animals at
1300 and 2595 mg/kg bw per day. Clinical signs were reported in at
least one mouse at 645 mg/kg bw per day, with piloerection in four
mice and all other signs in one mouse, suggesting that this dose
approached the threshold for such effects. Body-weight gain was
comparable to that of controls in animals at 160 and 320 mg/kg bw per
day during both the treatment and observation periods. In animals at
645 and 1300 mg/kg bw per day, body-weight gain during treatment was
reduced by about 4% in comparison with that of the control group and
remained at about 7% below control values until the end of the
observation period. No statistical evaluation of the data was
presented. The authors concluded that the maximum tolerated dose for
benzyl alcohol was between 645 and 1300 mg/kg bw per day and chose
750 mg/kg bw per day as the dose for use in the remainder of the
study.

Benzyl alcohol dissolved in distilled water was administered by
gavage at a dose of 750 mg/kg bw per day to 50 mice on days 7-14 of
gestation; evidence of copulation was considered the first day of
gestation. A control group of 50 animals received distilled water
only. All animals were allowed to deliver their litters and nurse
their pups for three days, at which time necropsies were performed.
Maternal body-weight gain and mortality, mating, gestation, numbers of
live and dead pups per litter, total litter weight on days 1 and 2
post partum, litter weight change between days 1 and 3 post partum,
and pup survival on days 1 and 3 post partum were recorded.
During the treatment period, 18 deaths were reported, all of
which were attributed to treatment; a further death was reported on
day 15 of gestation, the day after treatment was terminated. Clinical
signs of toxicity, including hunched posture, tremors, inactivity,
prostration, hypothermia, ataxia, dyspnoea, swollen or cyanotic
abdomen, and piloerection, were reported in up to 20 mice during
treatment. Piloerection was also reported in some animals up to day 3
post partum, but no other clinical signs were seen after the period
of administration. No differences were observed in the mating or
gestation indices, the total number of resorptions, the mean length of
gestation, or the number of live pups per litter between treated and
control groups. Maternal body weight, measured on days 4 and 7 of
gestation, was not significantly different from control values;
however, statistically significant reductions were reported on day 18
of gestation (P < 0.001) and on day 3 post partum (P < 0.05).
Maternal body-weight gain during days 7-18 of gestation was
significantly lower than that of controls (P < 0.001). Significant
reductions in pup body weight were reported, including a lower mean
pup weight per litter on days 1 (P < 0.01) and 3 post partum
(P < 0.001), a mean litter weight change between day 1 and day 3
post partum (P < 0.05), and a mean pup weight change between days 1
and 3 post partum (P < 0.001). No differences in pup survival were
observed by day 3 post partum. The authors concluded that benzyl
alcohol may be a reproductive hazard, apparently on the basis of the
reductions in pup body weights, an effect that was observed in
conjunction with maternal toxicity evidenced by increased mortality,
reduced body weights, and clinical toxicity during the period of
administration. As effects were seen on the dams and fetuses at the
only dose used in this study, there was no NOAEL. The LOAEL was
750 mg/kg bw per day (US National Institute of Occupational Safety and
Health, 1983; Hardin et al., 1987).

2.2.4.2 Rats

Benzyl acetate

3130-50 000 ppm benzyl acetate had no effect on the weights of the
epididymis, cauda epididymis, or testis, on sperm motility, or on the
density or percent of abnormal sperm.

Benzaldehyde

A single study was conducted to examine the potential
reproductive toxicity of benzaldehyde, and the report was available as
a translation from Romanian. A group of 10 rats of breeding age were
given 2 mg benzaldehyde in oil (type not specified) by gavage every
other day for 32 weeks, equivalent to about 5 mg/kg bw per day. Ten
controls were used. Two pregnancies in each rat, one at 75 days and
one at 180 days, were studied. The end-points examined included the
number of pregnant females, number of offspring born, pup body weight
at days 7 and 21 post partum, and pup viability. At the end of
treatment, the body weights of control and treated rats were similar:
265 g and 260 g, respectively. It was reported that fewer females in
the group given benzaldehyde than in the control group became
pregnant; however, no data or statistical analyses were presented. The
authors concluded that treatment did not significantly modify any of
the parameters studied. No further details were available. The NOAEL
was about 5 mg/kg bw per day (Sporn et al., 1967).

2.2.5 Developmental toxicity

2.2.5.1 Rodents

Benzyl acetate

A study of developmental toxicity was conducted with benzyl
acetate in Wistar rats, 10-15 weeks old, which included an initial
dose-finding phase. Groups of six pregnant rats were given 0, 250, or
500 mg/kg bw per day on days 6-15 of gestation; the finding of sperm
in a vaginal smear was considered to be day 0 of gestation. On day 20
of gestation, all animals were examined and the state of the fetuses
was determined. No significant effects on maternal weight gain or the
fetuses were reported in this portion of the study; no other data were
presented. On the basis of these results, doses of 0, 10, 100, 500,
and 1000 mg/kg bw per day were selected for the next phase.

In the study of developmental toxicity, groups of 20 rats were
given benzyl acetate at 0, 10, 100, 500, or 1000 mg/kg bw per day in
olive oil by gavage on days 6-15 of gestation; the finding of sperm in
a vaginal smear was considered to be day 0 of pregnancy. A second,
untreated control group was also included. During pregnancy, all
animals were examined daily, and food intake and maternal body weights
were recorded every other day. All pregnancies were terminated on day
20 of gestation, and all maternal animals were examined for gross
internal changes, and the numbers of implantations, corpora lutea,
resorptions, and live and dead fetuses were recorded. All fetuses were

examined for external malformations, sexed, and weighed. One-half of
the fetuses from each litter were examined for skeletal malformations
with alizarin red S and one-half for malformations of the internal
organs by Wilson's method.

During administration of benzyl acetate, no mortality or changes
in maternal health were reported. Weight gain was comparable in all
treated and control groups for the intervals days 0-6 of gestation
(before treatment) and days 6-16 of gestation. Weight gain in dams
given 500 or 1000 mg/kg bw per day was slightly but not significantly
reduced on days 16-20 of gestation, but food intake was not
significantly different from that of controls at any time during or
after the period of administration. At termination of the pregnancies,
no abnormalities were observed in the internal organs of the dams, nor
were there any differences in the numbers of corpora lutea,
implantations, live or dead fetuses, or resorptions, the implantation
ratio, the sex ratio, or placental weight between treated and control
groups. Fetal body weights were significantly reduced (P < 0.05) at
the high dose and significantly elevated (P < 0.05) at 10 and
100 mg/kg bw per day. Neither external nor internal malformations were
reported in treated or control groups; however, the combined incidence
of internal organ variations, which included slight dilatation of the
lateral ventricle and renal pelvis, and the presence of a laevo-
umbilical artery was significantly increased at 500 and 1000 mg/kg bw
per day. When the incidence of individual variations was analysed,
however, only dilatation of the renal pelvis at the high dose was
significantly increased. Skeletal malformations were limited to a
single fetus at the high dose, which had fused ribs; however, the
incidence of fused ribs in this group was not statistically
significantly increased over control values. The incidences of total
and individual skeletal variations, including wavy ribs, dumb-bell-
shaped vertebrae, the absence or splitting of thoracic vertebrae, the
presence of lumbar ribs, and delayed ossification, in fetuses at the
high dose were significantly increased over those in the control
group. These variations were seen in multiple litters. The incidences
of skeletal variations in the remaining groups treated with benzyl
acetate were within the range seen in the control group. The authors
concluded that benzyl acetate is not teratogenic and suggested that
the increased incidences of skeletal and internal variations were due
to slight maternal toxicity and to the significant decrease in fetal
body weight. The NOAEL was 500 mg/kg bw per day on the basis of
fetotoxic effects at the highest dose tested (Ishiguro et al.,
1993).

Benzoic acid and benzoate salts

Groups of Sprague-Dawley rats (number per group unspecified) were
injected intraperitoneally with sodium benzoate at a dose of 100, 315,
or 1000 mg/kg bw on days 9-11 or 12-14 of gestation; evidence of
mating was considered day 1 of gestation. Control animals were treated

with sodium chloride at doses of 90 or 100 mg/kg on the same days as
the treated groups. Fetal body weight was reported to be reduced in
groups given sodium benzoate at 1000 mg/kg bw per day on gestation
days 12-14 and 9-11. The incidence of deaths in utero was 12% in the
group given the high dose on days 12-14 of gestation and 16% in the
group given the high dose on days 9-11. These rates were reported to
be greater than those in the sodium chloride control group; however,
neither data on the number of deaths in utero in the sodium chloride
group nor statistical analyses were presented. No gross anomalies were
observed in the groups given sodium benzoate on days 12-14 of
gestation, whereas an increase in gross anomalies (unspecified) was
reported in the animals given 1000 mg/kg bw per day on days 9-11 of
gestation. The apparent NOAEL in this study was 315 mg/kg bw per day
(Minor & Becker, 1971).

A study of the developmental toxicity of sodium benzoate
administered by gavage to multiple species was conducted by Food and
Drug Research Labs, Inc. (1972). The species tested and the treatment
protocol are presented in Table 2. The rationale for the choice of
doses was not indicated. A positive control group was included for
each species: control groups of rats and mice received aspirin at
150 mg/kg bw per day, hamsters received aspirin at 250 mg/kg bw per
day, and rabbits received 6-aminonicotinamide at 2.5 mg/kg bw per day;
a vehicle control group treated by gavage was also available. Day 0 of
gestation was taken as the day sperm was observed in a vaginal smear
or, for rabbits, the day of artificial insemination. All animals were
observed daily for adverse effects, and maternal body weights were
recorded throughout pregnancy. The maternal and fetal end-points
examined for each dose group in each study included the total number
of pregnancies, maternal survival, body weight, total number of
corpora lutea and implantation sites per pregnant female, total number
of live litters, total number of resorptions, number of live fetuses
per litter, sex ratio, total number of dead fetuses, number of dams
with one or more dead fetuses, average fetal weights, and incidences
of gross, skeletal, and soft-tissue abnormalities. No significant
deviations from the control values and no dose-related effects were
reported. There were no teratogenic effects of significance, and none
occurred in excess over those seen in the negative control groups, but
effects were seen in the respective positive control groups. As no
adverse effects occurred with any of the treatment schedules, the
NOAEL values were 175 mg/kg bw per day for mice and rats, 300 mg/kg bw
per day for hamsters, and 250 mg/kg bw per day for rabbits.

Table 2. Species tested and protocol for study of the potential developmental
toxicity of sodium benzoate

Species No. of rats Doses Treatment Time of
per dose (mg/kg bw) period necropsy

CD-1 mice 20 1.75, 8, 38, 175 Days 6-15 Day 17
Wistar rats 24 1.75, 8, 38, 175 Days 6-15 Day 20
Golden hamsters 20 3, 14, 65, 300 Days 6-10 Day 14
Dutch belted rabbits 10 2.5, 12, 54, 250 Days 6-18 Day 29

From Food and Drug Research Labs, Inc. (1972)

Groups of 27-30 pregnant Wistar rats were fed diets containing 0,
1, 2, 4, or 8% sodium benzoate on days 1-20 of gestation; the
appearance of sperm in a vaginal smear was considered day 0 of
gestation. All but five animals in each group were sacrificed on day
20 of gestation, and the numbers of viable fetuses, dead fetuses,
early and late resortions, and fetal, placental, and ovarian weights
were measured; abnormalities of maternal organs and fetal appearance
were also recorded. About 75% of the fetuses from treated animals were
stained with alizarin red S for skeletal examination, and the
remainder were fixed with Bouin's solution and examined for visceral
anomalies by Wilson's method. The remaining five dams in each group
were allowed to deliver naturally, and the number of offspring,
survival, body weight, and abnormalities were recorded. Three weeks
after birth, all surviving pups were weaned and examined for gross
abnormalities, and one-half of the pups and all of the dams were
necropsied. The remaining pups were necropsied at eight weeks of age,
body weight and food intake being measured weekly until necropsy.

During administration of sodium benzoate, maternal body weight
and body-weight gain were comparable in the controls and in animals at
1 and 2% in the diet; animals at the 4% level did not gain weight and
those at 8% lost weight (statistical comparisons not presented). Feed
intake was also comparable in the controls and animals at 1 and 2%
dietary levels but was markedly reduced in those at 4 and 8%. The
total intake of the compound over the period of administration was
reported to be 14 g/kg bw, corresponding to 700 mg/kg bw per day for
rats at the 1% level; 26.2 g/kg bw, corresponding to 1310 mg/kg bw per
day for rats at the 2% level; 37.5 g/kg bw, corresponding to
1875 mg/kg bw per day for rats at the 1% level; and 19.3 g/kg bw,
corresponding to 965 mg/kg bw per day for the rats at 8%. Two dams at
4% and three at 8% died after convulsions and depressed motor

activity. No differences in the average number of implants per female,
the numbers of dead, resorbed, or viable fetuses, or the average
weight of viable fetuses were reported in the animals at 1 or 2%
during examination on day 20 of gestation; in the groups at 4 and 8%,
the number of dead or resorbed fetuses was significantly increased,
and the average body weight of viable fetuses was significantly lower
than that of controls. Significant abnormalities and pathological
findings were seen only in the fetuses at 4 and 8%; these included
mild systemic oedema, anophthalmia, pyelectasis, microphthalmia,
hydrocephalus, pyelectasis, hydroplasia, and cerebral hypoplasia.
Delayed ossification, lumbar or cervical ribs, and varied sternebrae
were reported in animals in both the control and treated groups. The
percentage of animals with these findings was comparable to that in
controls among animals at 1 and 2% (each about 37%) but was increased
in the groups at 4% (96.5%) and 8% (100%). Additional anomalies seen
in the treated groups included a higher incidence of wavy ribs and
abnormal vertebrae in the rats at 4%, but not at 8%. As the number of
live fetuses examined was not reported, the toxicological significance
of these findings is not clear.

Among pups that were delivered naturally, no differences in the
delivery rate, number of perinatal deaths, lactation rate, or survival
up to week 8 were reported at the 1 and 2% dietary levels, but the
groups at 4 and 8% were reported to have delivery rates reduced by
50 and 8.2%, respectively, with complete loss of litters after
parturition. The surviving pups in the control group and at 1 and 2%
showed no significant differences in birth weight, weight at week 3 or
week 8, incidence of abnormalities at week 3 or 8, or organ weights at
week 8. The authors suggested that the effects on the dams and fetuses
at the 4 and 8% dietary levels were due to reduced maternal feed
intake in these groups, leading to malnutrition, since the actual
compound intake of the animals at 8% was lower than that of the group
at 2%, in which no adverse effects were seen. The authors concluded
that the effects seen at 4 and 8% in the diet were not relevant to the
experimental outcome. The NOAEL was 1310 mg/kg bw per day (Onodera
et al., 1978).

2.2.5.2 Chickens

Sodium benzoate had no teratogenic effect on chicken embryos
after injection into the air cell of eggs on day 4 of incubation at
levels as high as 5 mg per egg (Verrett et al., 1980).

2.2.6 Genotoxicity

The results of studies of the genotoxicity of benzyl acetate,
benzyl alcohol, benzaldehyde, benzoic acid and benzoate salts, the
metabolite, and hippuric acid are presented in Table 3.

Table 3. Results of tests for the genotoxicity of benzyl compounds

End-point Test object Concentration Result Reference

Benzyl acetate

In vitro

Reverse S. typhimurium 0.03-30 Negative^b Florin et al . (1980)
mutation TA98, TA100, µmol/plate^a
TA1535, TA1537

Reverse S. typhimurium 33-10 000 Negative^b US National
mutation TA98, TA100, µg/plate Toxicology Program
TA1535, TA1537 (1986, 1993);
Mortelmans et al.
(1986)

Gene mutation Mouse lymphoma 0.25-1.75 µl/ml Positive^b US National
cells (L5178Y), tk (260-1850 µg/ml); Toxicology Program
locus 600-1700 µg/ml (1986, 1993);
Caspary et al.
(1988); McGregor et
al. (1988)

Chromosomal Chinese hamster 160-5000 µg/ml Equivocal^c US National
aberration ovary cells Toxicology Program
(1986, 1993);
Galloway et al.
(1987)

Mitotic chromosome Saccharomyces 520-1817 µg/ml Positived Zimmerman et al.
loss cerevisiae D61.M (1989)

Table 3. (con't)

End-point Test object Concentration Result Reference

Benzyl acetate (con't)

Sister chromatid Chinese hamster 50-5000 µg/ml Negative^b US National
exchange ovary cells Toxicology Program
(1986, 1993);
Galloway et al.
(1987)

Unscheduled Fischer 344 rat NR Negative Mirsalis et al. (1983)
DNA repair hepatocytes

Unscheduled Fischer 344 rat 50, 200, 1000 Negative Mirsalis et al. (1983,
DNA repair hepatocytes mg/kg bw 1989)

Unscheduled Rat pancreatic 1000 mg/kg bw Negative Steinmetz &
DNA repair cells orally Mirsalis (1984)

In vivo

Sex-linked Drosophila 300 ppm (feed); Negative US National
recessive lethal melanogaster 20 000 ppm Toxicology Program
mutation (injection) (1993)

Chromosomal Mouse bone- 325-1700 Negative US National
aberration marrow cells mg/kg bw Toxicology Program
intraperitoneally (1993)
Micronucleus Mouse bone- 312-1250 Negative US National
formation marrow cells mg/kg bw Toxicology Program
intraperitoneally (1993)

Micronucleus Mouse peripheral 3130-50 000 Negative US National
ormation blood ppm in diet Toxicology Program
(1993)

Table 3. (con't)

End-point Test object Concentration Result Reference

Benzyl acetate (con't)

Sister chromatid Mouse bone- 325-1700 Negative US National
exchange marrow cells mg/kg bw Toxicology Program
intraperitoneally (1993)

Benzyl alcohol

In vitro

Reverse S. typhimurium 0.03-30 Negative^b Florin et al. (1980)
mutation TA98, TA100, µmol/plate^a
TA1535, TA1537

Reverse S. typhimurium 0.1-5.0 Negative Wissler et al. (1983)
mutation TA98, TA1535 µmol/plate^a

Reverse S. typhimurium < 10 000 Negative^b Ishidate et al. (1984)
mutation TA92, TA94, µg/plate
TA98, TA100,
TA1535, TA1537

Reverse S. typhimurium 100-6666 Negative^b Mortelmans et al.
mutation TA98, TA100, µg/plate (1986); US National
TA1535, TA1537 Toxicology Program
(1989); Zeiger
(1990)

Gene mutation Mouse lymphoma 156-5000 µg/ml Positive^e McGregor et al.
cells (L5178Y), tk (1988); US National
locus Toxicology Program
(1989)

Table 3. (con't)

End-point Test object Concentration Result Reference

Benzyl alcohol (con't)

Chromosomal Chinese hamster < 1000 µg/ml Negative Ishidate et al. (1984)
aberration lung cells

Chromosomal Chinese hamster 50-5000 µg/ml Positive^f US National
aberration ovary cells Toxicology Program
(1989); Anderson et
al. (1990)

Sister chromatid Chinese hamster 16-5000 µg/ml Equivocal^b US National
exchange ovary cells Toxicology Program
(1989); Anderson et
al. (1990)

In vivo

Micronucleus Mouse bone- 0, 50, 100, 200 Negative Hayashi et al.
formation marrow cells mg/kg bw (1988)
intraperitoneally

Benzaldeyde

In vitro

Reverse S. typhimurium 0.03-30 Negative^b Florin et al. (1980)
mutation TA98, TA100, µmol/plate^a
TA1535, TA1537

Reverse S. typhimurium 0.05-500 Negative^b Kasamaki et al.
mutation TA98, TA100 µg/plate^g (1982)

Table 3. (con't)

End-point Test object Concentration Result Reference

Benzaldeyde (con't)

Reverse S. typhimurium 10-1000 Negative^b Haworth et al.
mutation TA98, TA100, µg/plate (1983)
TA1535, TA1537
Reverse S. typhimurium 0.1-5.0 Negative Wiessler et al.
mutation TA98, TA1535 µmol/plate^a (1983)

Reverse S. typhimurium 33-3333 Negative^b US National
mutation TA98, TA100, µg/plate Toxicology Program
TA102, TA104, (1990)
TA1535, TA1537

Gene mutation Mouse lymphoma 50-800 µg/ml Positive^e US National
cells (L5178Y), tk Toxicology Program
locus (1990);
McGregor et al. (1991)

Chromosomal Chinese hamster < 50 nmol/litre Positive Kasamaki et al.
aberration B241 cells (1982)

Chromosomal Chinese hamster 50-1600 µg/ml Negative^b Galloway et al.
aberration ovary cells (1987); US National
Toxicology Program
(1990)

Sister chromatid Chinese hamster 5-1600 µg/ml Positive^b Galloway et al.
exchange ovary cells (1987!; US National
Toxicology Program
(1990)

Table 3. (con't)

End-point Test object Concentration Result Reference

Benzaldeyde (con't)

Sister chromatid Human < 2.0 mmol/litre Positive^e Jansson et al.
exchange lymphocytes (1988)

In vivo

Sex-linked Drosophila 150 ppm (feed), Negative Woodruff et al.
recessive melanogaster 2500 ppm (1985); US National
lethal mutation (injection) Toxicology Program
(1990)

Benzoic acid and benzoate salts

In vitro

Reverse S. typhimurium < 10 000 Negative^b Ishidate et al. (1984)
mutation TA92, TA94, µg/plate (acid);
TA98, TA1535, < 3000 µg/plate
TA1537 (sodium salt)

Reverse S. typhimurium 33-10 000 Negative^b Zeiger et al. (1988)
mutation TA97, TA98, µg/plate
TA100, TA1535,
TA1537

Chromosomal Chinese hamster < 1500 µg/ml Equivocal Ishidate et al. (1984)
aberration lung cells (acid); < 2000 (acid);
µg/ml (sodium positive
salt) (sodium salt)^h

Table 3. (con't)

End-point Test object Concentration Result Reference

Benzoic acid and benzoate salts (con't)

Sister chromatid Chinese hamster 1-10 mmol/litre Equivocal Oikawa et al. (1980)
exchange ovary cells

Sister chromatid Human lymphoblastoid 1-30 mmol/litre Negative^b Tohda et al. (1980)
exchange cells

Sister chromatid Human lymphocytes < 2.0 mmol/litre Negative Jansson et al.
exchange (1988)

Hippuric acid

In vitro

Reverse mutation S. typhimurium 0.1-5.0 Negative Wiessler et al.
TA98, TA1535 µmol/plate^a (1983)

^a Qualitative test only; not tested to limits of cytotoxicity or solubility
^b In presence and absence of metabolic activation
^c Negative in presence of metabolic activation; two of three trials showed P values <0.05 in absence of metabolic activation.
^d Weak response at dose that induced about 80% toxicity
^e In absence of metabolic activation
^f In presence of metabolic activation
^g No rationale provided for highest dose tested
^h Tabulation of chromosomal aberrations included gaps
Benzoic acid and benzoate salts

Sodium benzoate at concentrations of 0.05 × 10² to 5 × 10⁴ ppm
induced an array of cytological effects in Vicia faba root
mitotic cells, involving all the stages of the mitotic cycle. The
most remarkable were inhibition of DNA synthesis and induction of
anaphase bridges and subsequent micronuclei (Njagi & Gopalan, 1982).
It induced chromosomal aberrations in rat cells in vitro and was
mutagenic in the recombination (rec) assay. Negative results were
obtained in an assay for reverse mutation in Salmonella typhimurium
(Kawachi, 1975, cited by Sodemoto & Enomoto, 1980).

A number of studies conducted in 1974 (Food and Drug Research
Labs., Inc., 1974) were reviewed but were not included in this
monograph, since they were considered not to contribute useful
information.

2.2.7 Special studies: Effects on the pancreas

Benzyl acetate

A series of experiments was conducted to assess the role of
benzyl acetate in the induction of tumours of the pancreas in rats, as
observed in the study of the US National Toxicology Program (1986) in
which animals were treated by gavage. Male Fischer 344 rats were
injected intraperitoneally with azaserine and fed diets containing
benzyl acetate dissolved in corn oil at levels of 0, 0.4, or 0.8%,
equivalent to 0, 200, and 400 mg/kg bw per day, respectively, for six
months. Azaserine-induced atypical acinar-cell foci in pancreatic
tissue were assessed for density, diameter, and volume percentage.
Fewer lesions per cubic centimetre were seen in the groups fed benzyl
acetate than in controls, but the mean diameter and volume of the foci
were greater than in controls.

In a second experiment, groups of 25 male rats were fed control
diet or diet containing 0.8% benzyl acetate from weaning until autopsy
at two years of age, at which time the pancreases were examined
histologically. Benzyl acetate had no effect on the growth or survival
of the rats, and the incidence of pancreatic acinar foci or adenomas
was not increased over that in controls. A marginal increase in the
number of carcinomas in situ was observed (3/38 compared with 0/49),
which was not statistically significant ( P = 0.0616; Fisher's exact
test). The historical control incidence of this lesion was not
provided. The authors in terpreted their result as indicating a weak
promoting activity of benzyl acetate.

A long-term study to assess the promoting effects of benzyl
acetate in azaserine-pretreated rats had to be terminated after one
year owing to poor survival, apparently due to chronic renal disease.
In this study, the incidence of pancreatic carcinoma was lower in rats

given dietary concentrations of 0.4 or 0.8% benzyl acetate than in
controls, and the combined incidences of acinar adenoma, carcinoma
in situ, and carcinoma were 89% in controls, 72% at 0.4% benzyl
acetate, and 73% at 0.8%.

Alkaline elution analysis of DNA from rats treated intra-
peritoneally with 150, 500, or 1500 mg/kg bw benzyl acetate
revealed no evidence of damage to DNA in pancreatic cells. The authors
concluded that the results of the first two experiments suggest
that benzyl acetate is a weak promoter of carcinogen-induced and
spontaneously occurring preneoplastic foci in the pancreas (Longnecker
et al., 1990).

2.3 Observations in humans

Benzyl alcohol

Gasping respiration was observed in premature infants who had
received benzyl alchohol in medications administered intravenously.
The syndrome was characterized by central nervous system dysfunction
with hypoactivity, hypotonia, and depression of the sensorium,
followed by apnoea, seizure activity, and coma; severe metabolic
acidosis; skin breakdown; haematological and hepatic disturbances,
including thrombocytopenia, leukopenia, direct hyperbilirubinaemia,
hyperammonaemia; hypotension and renal failure; cardiovascular
collapse; and death at 6-46 days of age. Infants with this syndrome
had received 99-234 mg/kg bw per day of benzyl alcohol, while a
matched control group of infants who had received benzyl alcohol but
who did not develop the syndrome had received doses of 27-99 mg/kg bw
per day. (The median intravenous lethal dose in the rat is 314 mg/kg
bw.) Benzyl alcohol was detected in the serum of these infants, and
benzoic and hippuric acids were identified in urine samples at
statistically significantly higher levels than in urine from infants
who had not received benzyl alcohol. It should be noted that some
endogenous formation of these compounds occurs. The authors suggested
that the accumulation of benzyl alcohol was probably due to the large
doses given relative to the size of the infants and/or to the reduced
capacity of their metabolic systems to detoxify it (Gershanik
et al., 1982).

Contact allergy was diagnosed in a metal-grinder who developed an
itchy, patchy rash on the fingers and hands after the introduction of
a new cutting oil. There was no recurrence of the rash when use of the
new oil was discontinued. Patch testing of the subject revealed that
he was sensitive to a number of fragrances, including and/or
containing benzaldehyde, and to benzyl alcohol, which was identified
as a component of the cutting oil (Mitchell & Beck, 1988).

A condition described as relapsing tinea affecting the anogenital
region and adjacent thigh area was observed in a man after treatment
with antimycotic lotions and ointments. The condition was resolved by
treatment with zinc oxide and topical corticosteriods and after
stopping use of perfumed soap and foam-bath preparations. Positive
reactions to a number of therapeutic ointments were seen in patch
testing, and benzyl alcohol was subsequently identified as the
sensitizing agent (Wurbach et al., 1993).

Benzaldehyde

The fatal dose in a case of acute poisoning was 50-60 ml (Dadlez,
1928).

Benzoic acid and benzoate salts

Human tolerance appears to vary: marked gastrointestinal
disturbances were induced by 5.7 g sodium benzoate (Meissner &
Shepard, 1866), while other people tolerated 25-40 g (Bignami, 1924).

Up to 12 g of sodium benzoate daily have been given
therapeutically without ill effects (Senator, 1879), but the same dose
of benzoic acid given over five days produced gastric burning and
anorexia in 30% of other subjects (Waldo et al., 1949). The toxic
symptoms are local gastrointestinal mucosal irritation or effects on
the central nervous system. Acute toxicity in man is readily
reversible and is probably due to disturbance of the acid-base balance
rather than to tissue damage (Barnes, 1959).

In six men given 0.3-0.4 g benzoic acid in their diet for up to
62 days, no abnormalities were seen in the blood picture, urine
composition, nitrogen balance, or well-being (Chittenden et al.,
1909). Nine patients on penicillin treatment received 1200 mg benzoic
acid daily, in eight doses, over five days for eight subjects and for
14 days in one case. No effect was observed; in particular, no
significant change was seen in endogenous creatinine clearance, and
routine urine analysis showed no abnormality (Waldo et al., 1949).

Some patients who suffer from asthma, rhinitis, or urticaria
experience exacerbation of symptoms after ingestion of foods or
beverages containing benzoates (Freedman, 1977).

Oral doses of 250 mg/kg bw sodium benzoate per day to infants,
given to counteract the effects of inherited urea cycle disorders,
were well tolerated, but boluses of the drug induced vomiting. Acute
benzoate poisoning was observed after accidental treatment with
800 mg/kg bw over 24 h. The clinical symptoms were similar to those
observed in salicylate poisoning, with vomiting, hyperpnoea, and
irritability. The plasma benzoate levels were elevated, and urinary
excretion of hippuric acid was increased; plasma glycine levels were

decreased. The plasma levels returned to normal and the symptoms were
resolved within 24 h of discontinuation of benzoate treatment
(Batshaw, 1983).

Sodium benzoate was used therapeutically in three infants to
alleviate the symptoms of non-ketotic hyperglycaemia, a metabolic
disorder, at doses of 125-1000 mg/kg bw per day, given orally in four
divided doses. All of them showed anorexia and vomiting while
receiving sodium benzoate at the higher doses, and repeated vomiting
was seen at 900 mg/kg bw day. In one patient, glycosuria,
hypocalcaemia, and metabolic acidosis were observed after doses of
1000 mg/kg bw per day, suggesting a causal relationship between
ingestion of benzoate and the apparent renal tubular dysfunction
(Wolff et al., 1986).

An episode of anaphylaxis in response to ingestion of sodium
benzoate present in foodstuffs was described in a 19-year-old woman.
The patient had experienced generalized itching one week after eating
cheese. The symptoms consisted of flush, angioedema, dyspnoea, and
severe hypotension; they were resolved by administration of adrenalin
and corticosteroids. Adherence to a benzoate-free diet prevented any
recurrence of the symptoms. An oral challenge with 20 mg sodium
benzoate induced localized itching on the arms and generalized
itching, suggesting intolerance to sodium benzoate. Resolution of an
occult maxillary sinusitis resulted in better tolerance to sodium
benzoate after a second oral challenge, with only mild localized
itching after a 160-g dose (Michils et al., 1991).

3. COMMENTS

On the basis of the present review, a number of issues were
identified that are important in the safety evaluation of benzyl
acetate, benzyl alcohol, benzaldehyde, and benzoic acid and its salts.
Since they are all metabolized to benzoic acid, it was considered
reasonable to assume that the results of studies on one member of the
group would apply to the others. The results of a number of studies in
humans and experimental animals indicate that formation of hippuric
acid from benzoic acid is a saturable process in which the
availability of glycine is the rate-limiting step. This observation is
particularly relevant to the interpretation of the toxic effects of
these compounds in experimental animals, since supplementation of the
diet with glycine was shown to alleviate the toxic effects induced by
high doses of benzyl acetate and benzoic acid, including body-weight
decrements and neurotoxic effects. Even with saturation of hippuric
acid formation, however, clearance of compounds in the benzyl group is
relatively rapid in experimental animals and humans. An extensive
review of the toxicity of benzoate showed that at high doses it
interferes with intermediary metabolism, including the urea cycle,
gluconeo-genesis, fatty acid metabolism, and the tricarboxylic acid
cycle, probably by sequestering coenzyme A before its conjugation with
glycine. This observation is consistent with similar effects
(metabolic acidosis, convulsions, and hyperpnoea) in experimental
animals and humans given very high doses. Administration of benzyl
acetate to rats by gavage was shown to result in higher peak plasma
levels of benzoic acid in comparison with ingestion of a similar daily
dose in the diet, while the plasma levels of hippuric acid were
similar regardless of the method of administration. This finding is
reflected in the greater toxicity of these compounds when administered
by gavage. Since depletion of glycine may be a major factor in the
effects observed at the high doses used in animals, the results of
studies by gavage would appear to be inappropriate for extrapolating
to human exposure. In addition, depletion of glycine might be of
concern with respect to the developing fetus and neonate. It should be
noted that benzoic and hippuric acids are generated as a result of
phenylalanine-tyrosine metabolism.

Long-term studies by feeding and gavage in mice and rats were
available for review by the Committee. No definitive conclusion could
be drawn from the results of carcinogenicity studies with sodium
benzoate in mice and rats, as insufficient detail was provided and the
survival in the study of rats was poor. The Committee reviewed the
studies evaluated in the previous monographs and an additional study
in which benzaldehyde was administered to rats and mice in corn oil by
gavage and concurred with the conclusions that neither benzyl acetate
nor benzyl alcohol is carcinogenic. As in the studies with mice and
rats given benzyl acetate by gavage in corn oil, increased incidences
of pancreatic acinar-cell adenomas in rats and of papillomas of the
forestomach in mice were noted after administration of benzaldehyde.

The Committee concluded that these results were of no relevance, as
noted at the forty-first meeting, because of the use of corn oil as
the vehicle and that the results of studies by dietary administration
were more relevant to the assessment of food additives.

Several studies addressing aspects of the potential developmental
and reproductive toxicity of benzyl acetate, benzyl alcohol,
benzaldehyde, and sodium benzoate were reviewed at the present
meeting. Developmental delays and reduced fetal and postnatal pup body
weights were observed only at doses that were maternally toxic. Doses
that induced extreme maternal toxicity were associated with
embryotoxic and fetotoxic effects and malformations in a study with
sodium benzoate. A multigeneration study with rats showed no effect on
growth, fertility, lactation, or survival.

The genotoxicity database was also reviewed. None of the four
compounds was mutagenic in assays for reverse mutation, either with or
without metabolic activation. The compounds all induced gene mutation
in the mouse lymphoma assay at the tklocus (benzoate was not tested in
this assay), although the requirement for metabolic activation varied.
Some weak clastogenic activity was noted in vitro but not in
in vivo when the latter assays were performed.

4. EVALUATION

The Committee was aware of reports of idiosyncratic human
intolerance to benzoate. These data were not considered relevant to
the establishment of an ADI for this group of compounds. The Committee
endorsed the view expressed in the report of the twenty-seventh
meeting that appropriate labelling is a feasible means of offering
protection to susceptible individuals.

The Committee was satisfied that the data reviewed on compounds
in this group are sufficient to demonstrate lack of carcinogenic,
developmental and reproductive potential. Consequently, it concluded
that further studies were not required, and the group ADI of 0-5 mg
per kg bw as benzoic acid equivalents was maintained.

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    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 32)
       BENZYL ACETATE (JECFA Evaluation)
       Benzyl Acetate  (IARC Summary & Evaluation, Volume 40, 1986)
       Benzyl Acetate  (IARC Summary & Evaluation, Volume 71, 1999)