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.
p-HYDROXYBENZOATE, ETHYL, METHYL, PROPYL ESTERS
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
Chemical methods are available for the determination of
p-hydroxybenzoic acid and its methyl ester in tissues and body fluids.
The alkyl esters of p-hydroxybenzoic acid are well absorbed after oral
administration to dogs. Dogs were given either the free acid or the
methyl ester in doses of 1000 mg/kg bw orally or 50 mg/kg bw i.v. and
excretion in the urine was measured. The total material recovered
(i.e., p-hydroxybenzoic acid, p-hydroxybenzoates and metabolic
conjugates) represented from 60% to 95% of that ingested. Plasma ester
concentrations rarely reached measurable levels, but high plasma
levels and high urinary output of p-hydroxybenzoic acid and conjugated
products indicated hydrolysis of the ester linkage. Enzymatic
hydrolysis of the ester was demonstrated in vitro using preparations
of liver and kidney. Studies on one man given 70 mg/kg orally
suggested that metabolism in man is similar to that in the dog (Jones
et al., 1956). In dogs the diglucuronide of p-hydroxybenzoic acid has
been shown to be the main metabolite excreted in the urine. Man
excreted free p-hydroxybenzoic acid and p-hydroxyhippuric acid in
approximately equal proportions (Quick, 1932). The urine of rats
receiving p-hydroxybenzoic acid or its methyl, ethyl or propyl esters
contained the following metabolites: p-hydroxybenzoic acid 40%,
p-hydroxyhippuric acid 23.5%, ether sulfate 5%, ester glucuronides
23%, ether glucuronides 1.2% and 5% of an unidentified substance
(Derache & Gourdon, 1963). Dogs given 50 mg/kg bw i.v. or orally
excreted 80-89% in the urine within 48 hours. After i.v. injection
significant levels were found in the plasma only immediately
afterwards. When dogs were infused at the rate of 2 mg/kg/min until a
total of 100 mg/kg bw was given, the levels in most organs were below
the plasma level. Appreciable amounts were only present in liver and
kidneys. No accumulation was noted in a dog given 1 mg/kg bw of the
methyl ester orally every day for a period of one year. This dog
excreted 96% of the daily dose within 24 hours in the urine (Sokol,
1952).
Similar metabolic experiments in the dog to those described with
the methyl ester have been reported for the propyl ester with
essentially similar results (Jones et al., 1956).
Dogs given 1000 mg/kg bw ethyl ester orally 50 mg/kg i.v.
excreted 66-70% in the urine. Very low plasma levels, just detectable
were found two hours after oral administration or at five and 15
minutes after i.v. injection (Sokol, 1952).
Results of further experiments in the dog were essentially
similar to those using the methyl ester except that detectable but
very low levels of the ethyl ester were found in the plasma two hours
after oral administration, and at five and 15 minutes after i.v.
administration (Jones et al., 1956).
The metabolism of various alkyl-p-hydroxybenzoates have been
investigated in rabbits and in rats.
It was noted that metabolism in the rat of p-hydroxybenzoic acid
and its methyl, ethyl and propyl esters resulted in the appearance in
the urine first of free p-hydroxybenzoic acid, followed by the
glucuronide and p-hydroxyhippuric acid, the concentration of which
increased as that of the free p-hydroxybenzoic acid fell out (Derache
& Gourdon, 1963).
In the rat the propyl ester was eliminated as p-hydroxybenzoic
acid, p-hydroxyhippuric acid, as ester and ether glucuronides, as an
ethereal sulfate and an unidentified compound. No unhydrolyzed ester
was detected in the urine (Derache & Gourdon, 1963).
Rabbits fed methyl p-hydroxybenzoate (0.8 g/kg bw) for three
days excreted p-carboxyphenyl-ß-O-glucopyranosiduronic acid,
p-hydroxybenzoic acid, and p-hydroxyhippuric acid as major urinary
metabolites with minor amounts of the ester-type glucuronide and
p-carboxyphenylsulfate (Tsukamoto & Terada, 1962). In another study
where esters of p-hydroxybenzoate (methyl, ethyl, propyl, butyl,
isopropyl, isobutyl, and secbutyl-p-hydroxybenzoate) were administered
by stomach tube to rabbits at dose rates equivalent to 0.4 or
0.8 g/kg, the alkyl esters were excreted in the urine as free acid
(25-39%), glycine conjugate (15-20%), ester-type glucuronide (5-8%),
ether-type glucuronide (10-18%), and sulfate (7-12%). The rate of
excretion was decreased with increasing of chain length of the alkyl
group (Tsukamoto & Terada, 1964).
At high doses, propyl p-hydroxybenzoate was found to be a useful
anaesthetic for frogs and tadpoles (Kopsch, 1949).
TOXICOLOGICAL STUDIES
Special studies on carcinogenicity
Mouse
Groups each of 20 weanling female mice were administered 0.1 ml
of a 1% methyl p-hydroxybenzoate solution in polyethylene glycol, or
polyethylene glycol alone, twice weekly into the vagina, for a period
of 18 months. Of the control animals 11 to 20 survived 12 months, and
7 to 20 the 18 months. Of the test animals 16 to 20 survived 12 months
and 8 to 20 the 18 months. No carcinomas were observed in the test
animals (Boyland et al., 1961).
One hundred male mice (C57BL/6), seven weeks old were injected
with 2.5 mg of methyl p-hydroxybenzoate, and five weeks after
injection, injection site was excised and minced tissue injected into
secondary host. Twenty-three weeks after injection, no carcinogenic
response was observed (Homberger, 1968). In another study, female mice
(C57BL/6) were injected i.v. with either methyl p-hydroxybenzoate, or
methyl-p-hydroxybenzoate plus 3, 4, 9, 10-dibenzpyrene. The methyl
p-hydroxybenzoate did not cause an acceleration of appearance of lung
adenomas (Homberger, 1968).
Special studies on neurotoxicity
The blocking effect of a 0.1% solution of the methyl ester on
nervous conduction when applied directly to the spinal roots or to the
cervical vagus and sympathetics was found to be similar to that of a
0.05% solution of procaine (Nathan & Sears, 1961). The local
anaesthetic effect of the esters rose with increasing number of
C-atoms and the toxicity decreased (Alder-Hradecky & Kelentey, 1960).
Special studies on teratogenicity
See butyl p-hydroxybenzoate.
Special studies on chick embryo
When femora from 10-day-old chick embryos were cultured for two
days in a medium containing 10-5 or 10-6 M methyl p-hydroxybenzoate
or concentrations of propyl p-hydroxybenzoate in the 10-5-10-7M range
their dry weight increased in comparison in each case with that of the
control femur from the same embryo. A mixture of the two parabens did
not produce an additive effect. The favoured hypothesis is that
parabens stabilize lysosymes and so prevent the partial autolysis that
occurs in their absence during the early stages of culture (White,
1967).
Acute toxicity
ETHYL p-HYDROXYBENZOATE
LD50 References
Animal Route (mg/kg bw)
Mouse oral 8 000 Sokol, 1952
Mouse oral Na salt: approx. 2 500 Matthews et al., 1956
Mouse i.p. Na salt: 520 Matthews et al., 1956
Guinea- oral 2 000 - 2 400 Anon, 1939
pig
Rabbit oral 5 000 Sabalitschka &
Neufeld-Crzellitzer,
1954
Dog oral 5 000 Sabalitschka &
Neufeld-Crzellitzer,
1954
Doses of 5 g/kg were lethal in dogs and rabbits and 4 g/kg caused
harmful effects (Schübel & Manger, 1929).
METHYL p-HYDROXYBENZOATE
LD50
Animal Route (mg/kg bw) References
Mouse oral 8 000 Sokol, 1952
Mouse oral free acid: >8 000 Matthews et al., 1956
Na salt: 2 000 Matthews et al., 1956
i.p. free acid: 960 Matthews et al., 1956
Na salt: 760 Matthews et al., 1956
i.v. Na salt: 170 Matthews et al., 1956
METHYL p-HYDROXYBENZOATE (con't)
LD50
Animal Route (mg/kg bw) References
Guinea- oral 3 000 -3 600 Anon, 1939
pig
Rabbit oral 6 000 Sabalitschka &
Neufeld-Crzellitzer,
1954
Dog oral 6 000 Sabalitschka &
Neufeld-Crzellitzer,
1954
Oral doses of 3000 mg/kg bw are reported to be lethal in the dog
and rabbit, and doses of 2000 mg/kg bw caused harmful effects (Schubel
& Manger, 1929).
PROPYL p-HYDROXYBENZOATE
LD50
Animal Route (mg/kg bw) References
Mouse oral 8 000 Sokol, 1952
Mouse i.p. 400 Sokol, 1952
(free
ester)
Mouse oral free acid: >8 000 Matthews et al., 1956
Mouse oral Na salt: 3 700 Matthews et al., 1956
Mouse i.p. free acid: 640 Matthews et al., 1956
Na salt: 490 Matthews et al., 1956
Mouse i.v. Na salt: 180 Matthews et al., 1956
Doses of 6 g/kg were lethal in dogs and rabbits, and 3-4 g/kg
caused harmful effects (Schuebel & Manger, 1929).
Short-term studies
Guinea-pig
Daily doses of 11-100 mg methyl p-hydroxybenzoate for 120 days
showed no effect. Forty animals were given i.d. injections of 0.1%
solution in physiological saline, three times weekly up to 10
injections. There was no sensitivity reaction two weeks later
(Matthews et al., 1956).
Mixed propyl and methyl esters were fed to animals on scorbutic
diets. No additional pathological effects were noted (Cremer, 1935).
For details see the methyl ester. Similar experiments on skin
sensitivity to those done with the methyl ester revealed no skin
sensitivity (Matthews et al., 1956).
Rat
0.5-5.0 mg methyl p-hydroxybenzoate daily for 80 days had no
effect. The blood picture was not influenced (Cremer, 1935). Groups of
10 animals on a vitamin A deficient diet given 7.5-75 mg/kg bw of
mixed methyl and propyl ester for 30 days showed no additional
pathological changes (Cremer, 1935).
Feeding experiments similar to those reported for the methyl and
propyl esters were done at the 2% and 8% levels, but the duration was
only 12 weeks. Concentrations of 2% were without effect, but there was
reduced growth rate and evidence of toxicity at 8% (Matthews et al.,
1956).
Rabbit
Suspensions of 0.5 and 7.5% ethyl p-hydroxybenzoate had the same
local anaesthetic effect on the cornea as 0.12 and 0.27% solutions of
cocaine hydrochloride. Therefore, the local anaesthetic activity of
the ester is three to four times less than that of cocaine and twice
that of procaine (Truhaut, 1962a). Using the same method (Regner &
Quevauviller, 1939; Alder-Hradecky & Kelentey, 1960) found no local
anaesthetic effect on the cornea by 0.25-0.30% solutions of methyl,
ethyl, propyl or butyl p-hydroxybenzoates.
Dog
Dogs were fed 0.7 g/kg bw propyl p-hydroxybenzoate for 90 days
without ill effects or macroscopic changes (Ghirardi, 1940).
Three mongrel puppies were fed 1000 mg/kg bw methyl
p-hydroxybenzoate daily and two were fed 500 mg/kg daily for six days
a week. Those in the lower dose group were on test for 313 days, and
those on higher dose levels for periods longer than a year. At the end
of the experimental period all the animals appeared in excellent
condition with reasonable gains in weight. One animal receiving
500 mg/kg produced a normal litter of puppies near the end of the
experimental period. At a late stage of the experiment, blood samples
were analysed for presence of the drug and for metabolic end-products,
but there was no evidence of cumulation. Blood counts and urine
examination were normal. All dogs were killed and autopsied; no
abnormalities were found on microscopic or macroscopic examination of
the organs (Matthews et al., 1956).
Long-term studies
Rat
Forty rats were fed 15 mg/kg bw, 20 animals 150 mg/kg bw and 20
animals 1500 mg/kg bw of a mixture of 40% ethyl ester and 60% propyl
ester as the sodium salts for 18 months in the diet. The rate of
weight gain showed some growth stimulation at the 15 and 150 mg/kg bw
levels. The group on 1500 mg/kg bw showed initial retardation followed
later by normal growth. Mortality rate and pathological examination of
the major organs in all treated groups showed no significant
difference from the control group (Anon, 1940; Anon, 1942).
When the methyl and propyl esters were fed to rats over an
18-month period at a level of 150 mg/kg bw, no ill effects were
observed. There was some evidence of growth stimulation. When fed at
1600 mg/kg bw, there was a decrease in growth rate, but no
pathological changes could be found (Sokol, 1952).
Groups of 12 male and 12 female rats received 2% and 8% methyl
p-hydroxybenzoate in the diet respectively and were compared with
equal numbers of control animals over a period of 96 weeks. At the 2%
level, the animals did not show significant differences from the
controls, but at the 8% level there was a reduction in growth rate
during the earlier part of the experiment, with a tendency to return
to normal later. Food intake remained fairly constant throughout the
experiment. All animals dying during the course of the experiment or
killed at the end were autopsied and examined macroscopically and
microscopically; no significant changes were found in the organs
(Matthews et al., 1956).
Experiments were done with groups of 12 animals, similar to those
done with the methyl ester. No harmful effect was observed with a diet
containing 2% of propyl p-hydroxybenzoate, but the growth rate was
depressed at the 8% level. The feeding lasted for 96 weeks. There were
no significant pathological findings (Matthews et al., 1956).
A group of 65 rats (35 males and 30 females) was fed a diet
containing 2% of the ethyl ester for life span, with 50 animals as the
control group. All animals were autopsied on death. No adverse effects
could be detected on weight gain, except a small retardation during
the first month, and the mortality rate, haematology, tumour incidence
and histopathology of major organs did not differ from the controls
(Truhaut, 1962b).
A group of 39 rats (19 males and 20 females) was injected weekly
with 1 ml of an aqueous solution of 10% sodium ethyl p-hydroxy-
benzoate for their life span. The 27 controls (16 males and 11
females) were injected with 1 ml of a 3% sodium chloride solution.
Because of irritation by the high pH of the ester solution, the
frequency of injection had to be reduced to one in two weeks from the
fourth to the tenth month and later to one injection a month up to the
end of the experiment. No effect on mortality and tumour incidence
could be detected (Truhaut, 1962).
OBSERVATIONS IN MAN
Sensitivity: see under the butyl ester (Observations in man).
Solutions of methyl p-hydroxybenzoate in propylene glycol, in
concentrations up to 5%, were applied to the skin of 50 human subjects
for four to eight hours every other day up to 10 applications without
evidence of irritation. Higher concentrations than this produced some
irritation. There was no evidence of development of sensitization
(Matthews et al., 1956).
Tests for skin irritation and sensitivity were made similar to
those done with the methyl ester. Ethyl p-hydroxybenzoate at a
concentration of 7%, no evidence of irritation or sensitivity was
observed, but irritation occurred at higher concentrations. Ingestion
of 0.05% aqueous solutions caused local anaesthesia of the buccal
mucosa (Bubnoff et al., 1957).
A local anaesthetic effect on the buccal mucosa has been reported
after ingestion of 0.1% aqueous solution of methyl p-hydroxybenzoate
(Bubnoff et al., 1957). Two grams of the ester taken daily for one
month produced no ill effects (Sabalitschka & Dietrich, 1924).
The propyl ester has been used therapeutically at doses of
800 mg/kg bw over three days for the treatment of moniliasis (Rossier
& Wegmann, 1953). Tests for skin irritation and sensitivity were made
similar to those done with the methyl ester. At a concentration of
12%, no evidence of irritation or sensitivity was observed, but
irritation occurred at higher concentrations (Matthews et al., 1956).
Ingestion of 0.03% aqueous solutions caused local anaesthesia of the
buccal mucosa (Bubnoff et al., 1957).
Two grams propyl ester was given daily to human volunteers for
50 days. No unhydrolyzed ester could be detected in the urine
(Sabalitschka & Neufeld-Crzellitzer, 1954).
Human studies on six subjects involved administration of 10 or
20 mg/kg bw of the propyl ester orally. After 60, 135 and 255 minutes,
p-hydroxybenzoate but not the ester, was detectable in serum. The
maximum serum level attained was 4.5 µg/ml (Heim, 1960-61).
Comments:
The long-term studies in rats are adequate for an assessment when
taken in conjunction with the evidence from the feeding experiments
lasting for a year with the dogs. Biochemical studies in animals have
provided detailed information on metabolism. However, additional
studies in man for methyl p-hydroxybenzoate and further biochemical
studies in man and animal for the ethyl ester would be desirable.
Allergic responses to the ethyl and methyl esters have been reported.
EVALUATION
Applicable to the methyl, ethyl and propyl esters.
Level causing no toxicological effect
Rat
20 000 ppm (2%) in the diet, equivalent to 1000 mg/kg bw.
Estimate of acceptable daily intake for man
0-10 mg/kg bw.*
FURTHER WORK OR INFORMATION
p-hydroxybenzoate, ethyl: further biochemical studies in man and
animals.
p-hydroxybenzoate, methyl: additional studies in man.
* Sum of ethyl, methyl and propyl esters of p-hydroxybenzoic acid.
REFERENCES
Alder-Hradecky, C. & Kelentey, B. (1960) Arch. int. Pharmacodyn.,
128, 135
Boyland, E., Charles, R. T. & Gowing, N. F. C. (1961) Brit. J. Cancer,
15, 252
Bubnoff, M. von., Schnell, D. & Vogt-Moykoff, J. (1957) Arzneimitt
Forsch., 7, 340
Cremer, H. (1935) Z. Lebensmitt. Untersuch., 70, 136
Derache, R. & Gourdon, J. (1963) Food Cosmet. Toxicol., 1, 189
Ghirardi, G. E. (1940) Arch. ital. Sci. farmacol., 9, 282
Heim, F. (1960-1961) Sitzber, Physik-Med. Soz. Erlangen, 81, 14
Anon. (1939) Unpublished report from Applied Research Inc. MX-124
Anon. (1940) Unpublished report from Applied Research Inc. MX-185
Anon. (1942) Unpublished report from Applied Research Inc. MX-185
Homberger, F. (1968) PB-183207
National Technical Information Service, Springfield, Va., USA.
Jones, P.S. et al. (1956) J. Amer. pharm. Ass., Sci. Ed., 45, 268
Kopsch, F. (1949) Anat. Anz., 97, 158
Matthew, C. et al. (1956) J. Amer. pharm. Ass., Sci. Ed., 45, 260
Nathan, P. W. & Sears, T. A. (1961) Nature (Lond.), 192, 668
Quick, A. J. (1932) J. biol. Chem., 97, 403
Regnier, M. & Quevauviller, A. (1939) Arch. Exp. Path. Pharm., 193, 48
Rossier, P. H. & Wegmann, T. (1953) Wien. Med. Wschr., 19/20, 358
Sabalitschka, T. & Dietrich, K. R. (1924) Pharmaz. Monatshefte, 5, 235
Sabalitschka, T. & Neufeld-Crzellitzer, R. (1954) Arzneimitt-Forsch,
4, 575
Schuebel, K. & Manger, J. (1929) Arch. exp. Path. Pharmak., 146, 208
Sokol, H. (1952) Drug Stand., 20, 89
Truhaut, R. (1962a) Estratto dai Rendiconti dell-Instituto Superiore
di Sanità (Document submitted to WHO in 1964)
Truhaut, R. (1962b) Ann. pharm, franz., 20
Tsukamoto, H. & Terada, S. (1962) Chem. Pharm. Bull. (Tokyo), 10, 86
Tsukamoto, H. & Terada, S. (1964) Chem. Pharm. Bull. (Tokyo), 12, 765
White, A. A. (1967) Proc. Soc. exp. Biol. Med., 126, 588
See Also:
Toxicological Abbreviations