Toxicological evaluation of some food
additives including anticaking agents,
antimicrobials, antioxidants, emulsifiers
and thickening agents
WHO FOOD ADDITIVES SERIES NO. 5
The evaluations contained in this publication
were prepared by the Joint FAO/WHO Expert
Committee on Food Additives which met in Geneva,
25 June - 4 July 19731
World Health Organization
Geneva
1974
1 Seventeenth Report of the Joint FAO/WHO Expert Committee on
Food Additives, Wld Hlth Org. techn. Rep. Ser., 1974, No. 539;
FAO Nutrition Meetings Report Series, 1974, No. 53.
BENZOIC ACID AND ITS POTASSIUM AND SODIUM SALTS
Explanation
These substances have been evaluated for acceptable daily intake
by the Joint FAO/WHO Expert Committee on Food Additives (see Annex 1,
Refs No. 6 and No. 13) in 1961 and 1965.
Since the previous evaluation, additional data have become
available and are summarized and discussed in the following monograph.
The previously published monographs have been expanded and are
reproduced in their entirety below.
BIOLOGICAL DATA
BIOCHEMICAL ASPECTS
The liver is the main site of conjugation with glycine in both
man and most experimental animals (rabbits, rats) with the exception
of the dog. Where the kidney is the main site of biosyntheses (Snapper
et al., 1924; Friedmann & Tachau, 1911), sheep appear to have reduced
capabilities of conjugating benzoic acid with glycine. Infusion of
increasing amounts of benzoic acid into the rumen at levels up to
1.8 g/kg led to progressive fall in conjugation and increasing
excretion of free benzoic acid in the urine. Doses of 1.1 and 1.8 g/kg
were toxic leading to death. Potassium deficiency also occurred as
shown by the usual symptoms of severe muscular weakness and tremors
(Martin, 1966). For many years a liver function test was used in man
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). Hence there
exists a large amount of experience on the excretion of benzoic acid
and hippuric acid in man. In the blood benzoates exist in the free
state and not bound to proteins (Knoefel & Huang, 1956). In the dog
the kidney clearance was estimated to be 0.90-1.89 (Knoefel & Huang,
1956).
Normal urinary excretion of hippuric acid in man 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 yield values lying between 1-3 mg/kg bw (Armstrong et al., 1955).
The maximum rate of the hippuric acid excretion after ingestion of
benzoic acid was observed to be 17 mg/min and for benzoyl glucuronic
acid 0.57 mg/min equivalent to 24 g/day calculated as benzoic acid
(Schachter, 1957). Up to 10 g of benzoate is quantitatively excreted
by man (Barnes, 1959). At high intakes of sodium benzoate up to 3% is
conjugated with glucuronic acid and all metabolites eliminated
completely within 14 hours (Schachter, 1957). 75 to 80% of
administered benzoic acid is eliminated by man in six hours (Quick,
1931). Sodium benzoate also decreases uric acid excretion (Quick,
1931), urea and ammonia excretion in man (Lewis, 1914).
Precursors of endogenous benzoate are phenylalanine and tyrosine.
Experiments with labelled phenylalanine showed that about 1 to 2% is
metabolized by this pathway. Rabbits given 50-400 mg/kg bw per day of
deuterio-phenylalanine for six to 12 days, humans given 14-28 mg/kg bw
per day for four to six days and guinea-pigs given 300 mg/kg bw per
day for 12 days were examined (Bernard et al., 1955). 1-14C acetate,
however, did not produce labelled benzoic acid in rabbits and guinea-
pigs (Bernard et al., 1955). 3-14C-dl phenylalanine given
intraperitoneally to rats produced 0.6 to 1% of activity as urinary
hippuric acid (Altman et al., 1954).
1,3,4,5-tetrahydroxycyclohexanoic acid (quinic acid) may also
serve as precursor of benzoic acid in intermediary metabolism (Dickens
& Pearson, 1951). Several human subjects were given 6 g quinic acid
orally or 250 g prunes and excreted hippuric acid in increased amounts
during the following 24 hours (Quick, 1931). When deuterio-benzoic
acid was administered to man and rats it was excreted with its
deuterium content unchanged. Feeding guinea-pigs, with body fluids
enriched in D2O, with hydro-aromatic compounds led to urinary
excretion of deuterio-benzoic acid with high D content. A similarly
prepared rat, when fed 750 mg hydroxy benzoic acid over five days,
excreted urinary benzoic acid enriched in D. When human subjects and
guinea-pigs were given quinic acid over several days, 47 to 72% was
converted to benzoic acid and excreted in the urine (Bernard et al.,
1955). Four rats irradiated with 700 roentgens and four controls were
given intraperitoneally carboxyl-14C-labelled sodiumbenzoate and
fasted. Irradiation had no effect on the conjugating ability but the
irradiated rats excreted less labelled hippuric acid due to dilution
by endogenously produced benzoic acid (Schreier et al., 1954).
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 the D-amino acid oxidase (50% inhibition by
10-4M) (Klein & Kamin, 1941). Concentrations in the range of 10-3M
exert some unspecific inhibitory effects on the metabolism of fatty
acids, e.g. on acetoacetate formation (Avigan et al., 1955).
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 millimoles of
sodium benzoate showed a complete elimination of the drug within 10 to
14 hours (Schachter, 1957). Cumulation does not occur as shown by
experiments on the distribution and elimination of sodium
benzoate-l-C14 administered i.p., orally or s.c. to the rat.
Practically quantitative excretion occurs in the urine within one to
two days, less than 1% of radioactivity appears in the faeces, a few
ppm appear in organs. All radioactivity was identified as labelled
benzoic acid (Lang & Lang, 1956). Orally or s.c. administered labelled
benzoic acid appeared to 90% in the urine as hippuric acid, 0.1% of
radioactivity occurred in the expired CO2 and 2% remained in the
carcass (Bernard et al., 1955).
Two urinary metabolites of benzoic acid are known, namely
hippuric acid and benzoyl-glucuronic acid. 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 than hippuric
acid (Williams, 1959). Sheep are less able to excrete hippuric acid
and excrete much free benzoic acid in their urine (Martin, 1966). The
urine of man, pig, rabbit and sheep contains up to 10% of benzoyl-
glucuronic acid.
The maximum 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 13.9 millimoles (Schachter, 1957).
Limitations in availability of glycine account for this (Quick, 1933).
In the rat 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. Normally preformed glycine is used
though some is synthesized as well by the rat (Quick, 1931; Barnes,
1959). When rats were fed 1.5% benzoic acid (as the sodium salt) in
the diet, they excreted 95% and more of the drug as hippuric acid in
the urine. As the benzoate in the diet was increased to 3.75% so the
ratio of hippuric acid to total benzoic acid in the urine decreased.
Additional glycine raised elimination to 86 to 99%. The only other
derivative, found in significant amounts in the urine, was benzoyl
glucuronide (Griffith, 1929). Dogs and rabbits excrete hippuric acid
independent of the route of administration of benzoic acid (Quick,
1931).
TOXICOLOGICAL STUDIES
Special studies on carcinogenicity
See under long-term studies (Hosino, 1951).
Special studies on reproduction
Mouse
From the animals subjected to a 17-month study some were mated
and reproduction studied over five generations. Only body weights are
given in the results (Shtenberg & Ignat'ev, 1970).
Acute toxicity
LD50 Reference
Animal Route (mg/kg bw)
Rat Oral (Na salt) 2 700 Deuel et al., 1954
i.v. (Na salt) 1 714 ± 124 Spector, 1956
Rabbit Oral (Na salt) 2 000 Spector, 1956
s.c. (Na salt) 2 000 Spector, 1956
Dog Oral (Na salt) 2 000 Spector, 1956
Data on the LD50 of potassium benzoate are not available.
Benzoic acid is not acutely toxic to man (Lehman, 1908) or to test
animals in moderate doses (Rost et al., 1913; Smyth & Carpenter,
1948).
Outbreaks of poisoning affecting 28 cats have followed ingestion
of 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 intestinal
mucosa and liver. The sensitivity of the cat may be due to its failure
to form benzoyl glucuronide and toxicity may develop with quantities
greater than 0.45 g/kg single doses or 0.2 g/kg repeated doses
(Bedford & Clarke, 1971).
Short-term studies
Mouse
Mice fed 3 g sodium benzoate daily for 10 days showed a 10%
reduction of their creatine output, probably due to depletion of the
glycine pool (Polonowski, 1941). Groups of 50 male and 50 female mice
were given benzoic acid at the rate of 80 mg/kg/day, sodium bisulfite
at 160 mg/kg/day and a mixture of the two at the same levels by
gavage. The highest mortality was observed in mice given the
combination (60% of 32% benzoic acid alone) and weight gain was
reduced. A five-day period of food restriction at 2.5 months produced
an 85% mortality in both groups (Shtenberg & Ignat'ev, 1970).
Rat
Groups of 10 rats (five males and five females) were fed sodium
benzoate for 30 days at levels ranging from 16-1090 mg/kg bw. There
were no observable effects on body weight, appetite or mortality nor
any histological changes in the organs (Smyth & Carpenter, 1948).
Groups of three male and three female rats were fed on 0%, 2% and 5%
sodium benzoate for 28 days. All animals on the 5% level died during
the first two weeks showing hyperexcitability, urinary incontinence
and convulsions. At the 2% level male rats showed a significant
decrease in body weight. The food intake of male and female animals
was decreased at the 2% level compared with controls (Fanelli &
Halliday, 1963). Four groups of 15 rats were given 0%, 5% sodium
benzoate and 5% benzoate + 1% glycine in their diet for three weeks.
Body weight was reduced at the 5% level but less so when 1% glycine
was added. Total cholesterol content of the liver was unaffected but
phospholipids significantly reduced in the liver at the 5% level.
Potassium concentration of skeletal muscle at the 5% level was also
low. Glycine corrected the potassium and phospho-lipid 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. Autopsy changes were gut
haemorrhage, nasal blood crust but normal urine. Five adult rats died
within five weeks with severe weight loss on a similar diet
(Kieckebusch & Lang, 1960). Groups of four to 19 male rats were fed
diets containing 0%, 1.5%, 2.0%, 2.5%, 3%, 3.25% and 3.75% sodium
benzoate for 40 days. Average growth was less than in controls at all
levels but above 3% mortality was high, food efficiency poor and
growth severely depressed. Addition of glycine reduced the toxic
effects. Animals died with incoordination, tremor or convulsions and
had severe eye inflammation. Feeding other groups of 10 to 15 young
male rats on restricted amounts of diet containing 0%, 1.5%, 2.0%,
2.5% and 3% sodium benzoate revealed no difference in weight gain at
the 3% level. Glycine addition again improved this weight loss
(Griffith, 1929).
Ninety-day feeding tests were carried out on groups of eight to
10 rats on diets containing 1%, 2%, 4% and 8% of sodium benzoate. In
the group on the 8% diet there were four deaths (average number of
days to death 13). The weight gain of the four survivors was two-
thirds of that of the controls on an identical food intake. Kidney and
liver weights were significantly higher than those of the control
group. At the lower levels no demonstrable effect was observed (Deuel
et al., 1954).
Guinea-pig
Experiments on groups of four animals showed that doses of
benzoate + benzoic acid of 150 mg/kg bw given daily up to 65 days had
no adverse effects. When the same dose was fed to scorbutic animals a
shortening of the life-span was observed (Kluge, 1933).
Dog
Feeding tests on 17 dogs over 250 days with sodium benzoate or
benzoic acid at the rate of 1000 mg/kg bw had no effect on growth,
appetite and wellbeing. Above this level ataxia, epileptic convulsions
and death occurred (Rost et al., 1913).
Long-term studies
Mouse
Parenteral administration of benzoic acid has been shown not to
cause tumour development (Hosino, 1951). Groups of 25 male and 25
female mice were given benzoic acid in doses of 40 mg/kg/day, sodium
bisulfite in doses of 80 mg/kg/day and a mixture of the two at the
same levels for 17 months. Mortality was increased in the groups
receiving the mixture (62%) compared with the individual substance
groups (32%) at eight months. Mortality at 17 months is not given and
pathology is not reported (Shtenberg & Ignat'ev, 1970).
Rat
Three groups of 20 male and 20 female rats were pair fed for
eight weeks on diets containing 0%, 0.5% and 1% benzoic acid and
thereafter fed ad libitum over four generations. Two generations
were fed for their whole life-span, the third and fourth generations
were autopsied after 16 weeks. No harmful effects were observed on
growth, fertility, lactation and life-span. The post-mortem
examination showed no abnormalities (Kieckebusch & Lang, 1960). In
another experiment 20 male and 30 female rats were fed on a diet
containing 1.5% benzoic acid with 13 male and 12 female rats as
controls for 18 months. Fifteen animals died in the test group, in the
control three only. The test animals showed reduced body weight and
food intake. Repeat experiments on groups of 20 test animals and 10
controls taken from another strain showed similar findings (Marquardt,
1960).
Groups of 10 rats, males and females, received benzoic acid at
40 mg/kg/day or sodium bisulfite at 80 mg/kg/day or a mixture of the
two in the diet for 18 months. The growth was slightly reduced and ESR
was increased. Rats fed benzoic acid developed some tolerance to a
lethal dose of the compound given terminally. No pathology is reported
(Shtenberg & Ignat'ev, 1970).
OBSERVATIONS IN MAN
In man tolerance appears to vary, 5.7 g sodium benzoate causing
marked gastrointestinal disturbances in some (Meissner & Shepard,
1866) others tolerating 25 g to 40 g (Bignami, 1924). Up to 12 g daily
have been given therapeutically without ill effects (Senator, 1879)
yet over five days have produced gastric burning and anorexia in 30%
of other subjects (Waldo et al., 1949). Toxic symptoms are local
gastrointestinal mucosal irritation or CNS effects with convulsions in
animals. Acute toxicity in man is readily reversible and probably due
to the disturbance in acid-base equilibrium rather than associated
with any tissue damage (Barnes, 1959).
Six men were given 0.3 g to 0.4 g of benzoic acid in their diet
for periods up to 62 days. No abnormalities were seen in blood
picture, urine composition, nitrogen balance and wellbeing (Chittenden
et al., 1909). Nine patients receiving penicillin treatment were given
1200 mg of benzoic acid daily divided into eight doses over a period
of five days in eight of the subjects and 14 days in one case. No
effect was observed, particularly, in no case did the endogenous
creatinine clearance show significant changes nor did routine urine
analysis show any abnormality (Waldo, 1949).
Comments:
Considering the metabolic and toxicological data the margin of
safety seems to be very large in animals and man. Benzoic acid is
effectively and rapidly metabolized and eliminated by the body without
apparent tissue injury. The rat seems closest to man as far as the
metabolism of benzoate is concerned. There is no reason to believe
that potassium benzoate differs toxicologically from benzoic acid and
sodium benzoate when these are used as food additives. The cat seems
to be much more sensitive than other species. Allergic responses to
benzoic acid have been reported.
EVALUATION
Level causing no toxicological effect
Rat: 10 000 ppm (1%) in the diet equivalent to 500 mg/kg bw.
Estimate of acceptable daily intake for man
0-5 mg/kg bw.*
* As sum of benzoic acid and Na and K benzoate (expressed as
benzoic acid).
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