TOXICOLOGICAL EVALUATION OF CERTAIN FOOD ADDITIVES
WHO FOOD ADDITIVES SERIES 10
The evaluations contained in this document were prepared by the
Joint FAO/WHO Expert Committee on Food Additives*
Rome, 21-29 April 1976
Food and Agriculture Organization of the United Nations
World Health Organization
*Twentieth Report of the Joint FAO/WHO Expert Committee on Food
Additives, Geneva, 1976, WHO Technical Report Series No. 599, FAO Food
and Nutrition Series No. 1.
GALLATES, DODECYL, OCTYL AND PROPYL
Explanation
These compounds have been evaluated for acceptable daily intake
for men by the Joint FAO/WHO Expert Committee on Food Additives in
1961, 1964, 1972 and 1973 (see Annex I, Refs No. 6, p. 60; No. 8,
pp. 22-23; No. 31, p. 75; No. 33, p. 183).
Since the previous evaluation, additional data have become
available and are summarized and discussed in the following monograph.
The previously published monograph has been expanded and is reproduced
in its entirety below.
BIOLOGICAL DATA
BIOCHEMICAL ASPECTS
The available evidence indicates that the esters are hydrolysed
in the body. Most of the gallic acid is converted into 4-O-methyl
gallic acid. Free gallic acid or a conjugated derivative of 4-O-methyl
gallic acid is excreted in the urine. Conjugation of the 4-O-methyl
gallic acid with glucuronic acid was demonstrated (Booth et al.,
1959).
Rats fed a low-methionine, low-choline diet containing 1% gallic
acid developed fatty livers which could be prevented by added choline
or methionine (Booth et al., 1961).
The detailed metabolic pathways for propyl gallate have been
described (Dacre, 1960). There is no evidence to suggest that other
esters of gallic acid differ greatly in their metabolism from the
pattern described for propyl gallate.
TOXICOLOGICAL STUDIES
Special studies on mutagenicity
(a) Cytogenetics
Propyl gallate was investigated in vitro at concentrations of
0.5, 5.0 and 50 µg/ml employing WI-38 human embryonic lung cells for
anaphase abnormalities. It was also investigated in vivo by the
cytogenetic analysis of metaphase cells from the bone marrow of rats
(Sprague Dawley C-D strain). The dosages employed were 5.0, 50.0 and
500 mg/kg. Propyl gallate was non-mutagenic in both assays (Weir and
Brusick, 1974).
(b) Host-mediated assay
In vitro - Salmonella TA-1530 and G-46 together with
Saccharomyces D-3 were employed. A 0.25% concentration was tested.
Propyl gallate was non-mutagenic to all organisms.
In vivo - Propyl gallate was tested at 5.0, 50.0 and 500 and
2000 mg/kg in ICR Swiss mice employing as indicator organisms,
Salmonella G-46 and TA-1530 and Saccharomyces D-3. Propyl gallate
was non-mutagenic to all organisms (Weir and Brusick, 1974).
(c) Dominant lethal test
Sprague Dawley C-D strain male rats were used. Dosages of 5.0,
50.0 and 500 mg/kg and 5000 mg/kg were employed.
Acute study - a single dose was administered with subsequent
mating for each of eight weeks. Propyl gallate did not produce any
significant dominant lethalityś
Subacute - five daily doses were administered (5 × 5.0, 5 × 50.0,
5 × 500 and 5 × 5000 mg/kg). The additional dose was investigated in
an attempt to obtain some degree of toxicity at the highest level.
Males were subsequently mated for each of seven weeks. No dominant
lethal effects were noted (Weir and Brusick, 1974).
Special studies on reproduction
Rat
n-Octyl gallate was fed in the diet to groups of eight male and
16 female rats for two successive generations at levels of 0, 0.1 or
0.3% (and 0.6% for one generation). Rats were mated to produce two
litters per generation with the next generation selected from
weanlings of the second litter. A dietary level of 0.1% (1000 ppm) had
no effect on reproduction performance or the offspring. At 0.3 and
0.6% dietary octyl gallate, there was no significant effect on the rat
fetuses during pregnancy, but a marked effect was observed on survival
through weaning. In the case of the 0.6% group, return to normal diet
for six weeks, prior to a third breeding, did not result in increased
survival of offspring through weaning (Plank et al., 1971).
Groups each of 10 male and 20 female rats were maintained on
diets containing 0, 1000 or 5000 ppm (0%, 0.1% or 0.5%) of octyl
gallate. The animals were bred twice for the first generation, and
three times for the second generation. At the time of weaning of the
B1B litters the 5000 ppm (0.5%) level was replaced by a 2500 ppm
(0.25%) level for the second generation. In the case of the second
generation approximately 24 hours after birth, selected litters were
redistributed to female parents so that control females nursed pups
from test animals, and test animals nursed pups from control and other
test groups. One half P2 females bred for the third time (F2c) were
examined by caesarean section at time of delivery and the number of
implantation sites, corpora lutea and fetuses determined. One half of
the pups from each litter were examined for skeletal abnormalities,
and the other half for visceral abnormalities. The other parameters
measured in this study were appearance, behaviour, growth of pups
during the nursing and weaning process, fertility index, gestation
index, live birth index, weaning survival index. Autopsies were
carried out on F2b weanling pups, (control suckled by control, each
groups suckled by respective group parent), as well as a microscopic
examination of pituitary, thyroid, liver, spleen, kidneys, adrenals,
stomach, pancreas, small intestine, large intestine and any unusual
lesion of five males and five females, high level and control groups.
Weaning survival index, and body weight at weaning was considerably
reduced in the 5000 ppm (0.5%) group of the F1A and F1B generation.
Reduction of indices was still apparent in the F2A and F2B
generations, when the dietary level was reduced to 2500 ppm (0.25%).
At the 1000 ppm (0.1%), the indices were similar to control.
Redistribution of F2B pups to females of control groups, resulted in
similar growth of all pups to weaning. Allowing pups from high level
group to be nursed by control dams resulted in a marked increase in
survival indices, whereas when control pups were nursed by high level
dams, there was a marked decrease in survival indices. Examination of
P2 parents following third breeding indicated a dose-dependent
reduction in implantation sites, as well as a reduction in number of
corpora lutea. Fertility index of high level P2 females was depressed
at the F2C stage. Skeletal evaluation of F2C litters showed
incomplete skull ossification in some pups in the test groups, but
this was not considered remarkable for the size of the fetuses.
Necropsy of the pups indicated a higher incidence of gross kidney
alterations than that observed in controls. No compound-related
histology was reported (Olson and Voelker, 1970).
Other special studies
Groups each of 60 rats (Wistar) equally divided by sex were
maintained on diets containing 0, 0.002 or 0.004% propyl gallate for
13 weeks, and then placed on fat-free diets having 20% of the
calorific food value of the normal mean daily food intake for male
rats for 9 weeks. Surviving animals were returned to ad lib normal
diets for 2 to 4 weeks. During the first phase of the study, there
were no differences in weight gain, blood composition and relative
organ/body weight of test and control animals. During the starvation
period, there was no difference in survival time of test and control
groups. Chemical determination of water, protein and fat content of
animals that were sacrificed or died, showed that there was no
difference in protein or fat utilization chemicals on the control and
propyl gallate groups (Tonkelaar et al., 1968).
Gallates have been shown to cause contact dermatitis in bakers
and other workers handling gallates. Patch tests with lauryl gallate
at 0.2% showed a weak positive response in one sensitized individual.
Other individuals have suffered recurring episodes of dermatitis
presumably caused by gallates in food products (Brun, 1970).
Repeated insult patch test with 0.1% n-octyl gallate solution
showed an overall incidence of reaction in 13/445 or 2.9% individuals.
Oral mucosa irritation/sensitization tests were conducted with beer
containing 20 ppm (0.002%) n-octyl gallate and showed that the
incidence and severity of erythema were greater with beer containing
n-octyl gallate, than with untreated beer. Oedema was also greater
with treated beer. Individuals that had previously shown reactions
indicative of sensitization in the patch test were more susceptible to
irritation (incidence and severity) than other individuals (Palazzolo
and Fancher, 1971a, b, c).
Acute toxicity
DODECYL GALLATE
Animal Route LD50 mg/kg bw References
Rat oral 6 500 Sluis, 1951
Rat i.p. 100-120 Esch, 1955
OCTYL GALLATE
Animal Route LD50 mg/kg bw References
Rat oral 4 700 Sluis, 1951
Rat i.p. 60-80 Esch, 1955
Rat (male) oral 2 710 Brun, 1970:
Rat (female) oral 1 960 Brun, 1970
Rat (male) oral 2 710 Brun, 1970
Rat (female) oral 2 330 Brun, 1970
PROPYL GALLATE
Animal Route LD50 mg/kg bw References
Rat oral 3 800 Orten et al., 1948
Rat oral 3 600 Lehman et al., 1951
Rat i.p. 380 Orten et al., 1948
Mouse oral 2 000-3 500 Lehman et al., 1951
Short-term studies
Rat
Levels of propyl gallate of 1.2% and 2.3% in the diet caused
interference with weight gain, the bitter taste of the gallate
apparently making the diet unpalatable. The higher dose level caused
some deaths (about 40%) during the first month; the survivors
continued to eat the diet for 10 to 16 months and showed retarded
growth, but no pathological lesions. The animals that died exhibited
renal damage (Orten et al., 1948).
Weanling rats were given diets containing 2.5% and 5% dodecyl
gallate. All animals fed the smaller quantity were dead within 10
days, and all animals fed the larger quantity died within seven days
(Allen & De Eds, 1951).
Rats fed for 70 days on a diet containing 7% fat and 0.2% dodecyl
gallate showed no effect on body weight (Tollenaar, 1957).
Weanling rats were fed diets which contained 20% lard and 0, 0.1,
0.2, 0.3, 0.4 and 0.5% propyl gallate for six weeks. There was no
effect on body weight, liver weight, liver weight to body weight
ratio, left adrenal weight, total liver lipid, composition of liver
poly-unsaturated fatty acids, liver cholesterol, adrenal cholesterol
or serum sodium (Johnson & Hewgill, 1961).
Propyl gallate was added to the dietary fat of weanling rats at
levels of 0.02% in the fat for 13 weeks. The fat content of the diet
accounted for 30% of its calorific value. There was a very slight
inhibition of growth. The same rats were then placed on a partial
starvation diet and kept until they died. The survival time of the
animals which had received the propyl gallate was considerably reduced
and the reduction in their total body protein was greater (Buckman,
1962).
Groups each of 20 rats (equally divided by sex) were maintained
on diets containing 0, 1000, 2500 and 5000 ppm (0%, 0.1%, 0.25% and
0.5%) of octyl gallate for 13 weeks. All groups showed normal weight
gains, food consumption. Haematologic and blood chemistry and urine
analyses were normal. A complete gross and histopathologic examination
showed no compound-related effects (Blackmore and Voelker, 1969a).
Guinea-pig
Propyl gallate fed to guinea-pigs in groups of 20 at a level of
0.0117% in the diet for 14 months caused no detectable ill effects
(Orten et al., 1948).
Dog
Groups each of eight dogs (equally divided by sex) were fed diets
containing 0, 0.1, 0.3% n-octyl gallate for 90 days, and 1.0% for four
weeks; the 1.0% level has been then reduced to 0.6% for the rest of
the study. All groups showed normal weight gains, food consumption
(except 1.0% group). Haematologic and blood chemistry and urine
analyses were normal. A complete gross and histopathologic examination
showed no compound-related effects (Lindberg et al., 1970). In another
study, groups each of eight dogs (equally divided by sex) were
maintained on diets containing 0, 0.1, 0.25 or 0.5% octyl gallate for
13 weeks. All animals showed normal food consumption and weight gain.
Haematologic and urine analyses were similar for test and control
animals. SGOT was slightly elevated in the 0.5% group. Gross and
histopathologic examination of tissues and organs showed no compound-
related effects (Blackmore and Voelker, 1969b).
A level of 0.0117% of propyl gallate in the diet was well
tolerated by a group of seven dogs over a period of 14 months (Orten
et al., 1948).
Pig
Diets containing 0.2% of propyl, octyl or dodecyl gallate were
fed to pigs without demonstrable ill effect; mo anaemia was observed
(Esch, 1955).
Long-term studies
Mouse
Groups each of 50 mice (University Animal Breeding Station closed
strain colony) equally divided by sex were maintained on diets
containing 0, 0.5 or 1.0% n-propyl gallate for a period of 21 months.
Water intake and food consumption and growth of test animals was
comparable to controls. Treated male mice showed a greater percentage
survival than control mice at termination. Haematologic measurements
(haemoglobin, packed cell volume differential white cell count) were
similar for test and control animals. At autopsy, a comparison of
relative organ/body weight showed a reduction in the relative spleen
weight of males on the 1% diet. No compound related histopathological
changes were observed (Dacre, 1974).
Mouse and rat
A level of 5% propyl gallate in the diet in a two-year chronic
toxicity test on rats and mice gave rise to patchy hyperplasia in the
proventiculus. At a level of 1% no difference was noted between test
and control animals (Lehman et al., 1951).
Rat
Rats in groups of 10 males and 10 females were fed for two years
on diets containing 0%, 0.00117%, 0.0117%, 0.117%, 1.17% and 2.34% of
propyl gallate. The groups receiving 1.17% and 2.34% of propyl gallate
showed stunted growth and evidence of renal damage. In the other
groups, there was no detectable effect on haemoglobin, erythrocyte or
leucocyte levels in the blood, or on the histological appearance of
the organs examined (Orten et al., 1948).
Propyl, octyl and dodecyl gallate were fed to rats at
concentrations of 0.035%, 0.2% and 0.5% in the diet. Growth was
affected only at the 0.5% level of dodecyl gallate; there was
significant retardation, particularly in the second generation. At
this level of dodecyl gallate, some litters were lost in the second
generation because they were not sufficiently fed by the mothers. A
slight hypochromic anaemia was noticed in the groups on diets
containing 0.2% octyl and dodecyl gallate. No abnormalities were
observed in the organs or tissues of the rats at autopsy (Esch, 1955).
Young rats in groups of 12 males and 12 females were fed diets
containing 7% fat and 0.2% octyl or dodecyl gallate. There was no
significant difference between test and control animals over three
generations (Sluis, 1951).
Comments
Mice fed propyl gallate at levels of 0%, 0.5% and 1% for 21
months did not differ from controls with respect to food consumption,
growth and haematology. Survival of treated males was better than
their controls. At autopsy, there was reduced spleen/body weight ratio
in male mice fed 1% propyl gallate. Histopathology was negative. This
author calculates a no effect level to be 1500 mg/kg.
The mutagenic potential of propyl gallate has been investigated
in a number of test systems. Propyl gallate was found to be non-
mutagenic.
In a special test designed to mobilize the fat stores of propyl
gallate-loaded rats, there was no indication of adverse effects due to
such treatment.
The previously stated requirement, for studies of the effect on
reproduction of mixtures of BHA, BHT and propyl gallate, was no longer
considered necessary by the Committee.
The Committee was made aware of studies carried out in the USSR
with propyl gallate. They will be evaluated when they are received.
EVALUATION
Estimate of acceptable daily intake for man
0-0.2a mg/kg bw.b
FURTHER WORK OR INFORMATION
Submission of the results of studies in progress in the USSR.
REFERENCES
Allen, C. S. & De Eds, F. D. (1951) J. Amer. Oil Chem. Soc., 28, 304
Blackmore, R. H. & Voelker, R. W. (1969a) 13-week dietary
administration - Rats. Octyl gallate. Final report (Project No.
458-115). Unpublished report from Hazleton Labs., Inc., Falls
Church, Va. USA, submitted to the World Health organization by F.
& M. Schaefer Brewing Co., Brooklyn, N.Y. USA
Blackmore, R. H. & Voelker, R. W. (1969b) 13-week dietary feedings.
Dogs. Octyl gallate. Final report (Project No. 458-117).
Unpublished report from Hazleton Labs., Inc., Falls Church, Va.,
USA, submitted to the World Health Organization by F. & M.
Schaefer Brewing Co., Brooklyn, N.Y., USA
Booth, A. N., Masri, M. S., Robbins, D. J., Emerson, O. H., Jones, F.
T. & De Eds, F. (1959) J. Biol. Chem., 234, 3014
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Brun, R. (1970) Dermatologica, 140, 390
a As sum of dodecyl, octyl, and propyl gallate n-octyl gallate
should not be used in beverages.
b Temporary.
Buckman, N. D. (1962) Vop. Pitan., 21, 68
Dacre, J. C. (1960) J. N. Z. Inst. Chem., 24, 161
Dacre, J. C. (1974) Fd. Cosmet. Toxicol., 12, 125
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subacute oral toxicity study with Cold-Pro, GA-8 in Beagle dogs
(IBT No. C8472). Unpublished report from Industrial Bio-Test
Labs., Inc., Northbrook, Ill., USA, submitted to the World Health
Organization by Nutrico, Inc., Milwaukee, Wisconsin, USA
Olson, W. A. & Voelker, R. W. (1970) Modified two-generation
reproduction study - Rats. Octyl Gallate. Final report (Project
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test with n-octyl gallate (IBT No. F9309). Unpublished report
from Industrial Bio-Test Labs., Inc., Northbrook, Ill., USA,
submitted to the World Health Organization by Nutrico, Inc.,
Milwaukee, Wisconsin, USA
Palazzolo, R. J. & Fancher, O. E. (1971c) Oral mucosa irritation/
sensitization test with treated beer (IBT No. F9655). Unpublished
report from Industrial Bio-Test Labs., Inc., Northbrook, Ill.,
USA, submitted to the World Health Organization by Nutrico, Inc.,
Milwaukee, Wisconsin, USA
Palazzolo, R. J. & Fancher, O. E. (1971b) Oral mucosa irritation/
sensitization test with treated beer (IBT No. F9310). Unpublished
report from Industrial Bio-Test Labs., Inc., Northbrook, Ill.,
USA, submitted to the World Health Organization by Nutrico, Inc.,
Milwaukee, Wisconsin, USA
Plank, J. B., Wright, P. L., Keplinger, M. L.& Fancher, O. E. (1971)
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rats (IBT No. P84). Unpublished report from Industrial Bio-Test
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Organization by the US Food and Drug Administration