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.
GALLATES, DODECYL, OCTYL AND PROPYL
Explanation
These compounds have been evaluated for acceptable daily intake
by the Joint FAO/WHO Expert Committee on Food Additives (see Annex 1,
Refs Nos 6, 8 and 30) in 1961, 1964 and 1972.
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 dermatological effects
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
(Biotest Lab., 1971c). 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) Biotest Lab., 1971a, b, c).
Acute toxicity
DODECYL GALLATE
Animal Route LD50 mg/kg bw References
Rat oral 6 500 van Sluis, 1951
Rat i.p. 100-120 van Esch & van Genderen, 1954
OCTYL GALLATE
Animal Route LD50 mg/kg bw References
Rat oral 4 700 Van Sluts, 1951
Rat i.p. 60-80 van Esch & van Genderen, 1954
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, 1954).
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
polyunsaturated 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 (Hazleton Lab. Inc., 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 (Industrial Biotest Lab., 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 (Hazleton Lab. Inc., 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; no anaemia was observed
(van Esch & van Genderen, 1954).
Long-term studies
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 (Van Esch &
van Genderen, 1954).
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 (van Sluis, 1951).
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 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 (Industrial Biotest Lab., 1970a).
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. Ne compound-related
histology was reported (Hazleton Lab. Inc., 1970b).
Comments:
Reproduction studies in the rat indicate that the mortality of
suckled young produced by the higher levels was due to a factor
entering the mother's milk and was therefore a perinatal rather than a
prenatal effect. It has not been established whether the reported
decrease in implantation sites and number of corpora lutea that
occurred at the third breeding of the F2 parents was due to an effect
on viability of ova or sperm. The "no-effect" level in rats, based on
reproduction studies, is 1000 ppm (0.1%) of the diet, i.e. 50 mg/kg bw
per day. The n-octyl gallate can cause reactions indicative of
allergic sensitization in the buccal mucosa of individuals previously
sensitized by exposure to n-octyl gallate in beer or other beverages
consumed in large amounts, therefore, its use in such beverages is not
advisable.
Those occupationally exposed to gallate esters should be made
aware of the sensitizing potential of these substances.
With one exception, in long-term studies in rats gallates
caused no demonstrable ill effects when fed at a level of 0.2% in
the diet; in one investigation, however, this level resulted in
hypochromic anaemia. It seems likely that this may have been due to
interference with iron absorption, but the cause was not established.
Haematological effects were carefully examined in a number of other
investigations and no abnormalities were seen. It seems possible,
therefore, that this effect was related to the particular
circumstances of one study and can be properly disregarded in arriving
at the level that causes no significant effects in the rat. However,
the no-effect level in the reproduction study is used for this
evaluation.
EVALUATION
Estimate of acceptable daily intake for man
0-0.2* mg/kg bw.**
FURTHER WORK OR INFORMATION
Required by 1976. Studies on the effect on reproduction of
mixtures of BHA, BHT and propyl gallate.
REFERENCES
Allen, C. S. & De Eds, F. D. (1951) J. Amer. Oil Chem. Soc., 28, 304
Booth, A. N. et al. (1959) J. biol. Chem., 234, 3014
Booth, A. N., Robbins, D. J. & De Eds, F. (1961) J. Nutr., 75, 104
Brun, R. (1970) Dermatologica, 140, 390
Buckman, N. D. (1962) Vop. Pitan., 21, 68
Dacre, J. C. (1960) J. N.Z. Inst. Chem., 24, 161
Esch, G. J. van & Genderen, H. van (1954) Netherlands Institute of
Public Health, Report No. 481
Hazleton Lab. Inc. (1969a) Unpublished report. Project No. 458-117
Hazleton Lab. Inc. (1969b) Unpublished report. Project No. 458-115
Hazleton Lab. Inc. (1970a) Unpublished report. Project No. 458-116
Industrial Biotest Lab. (1970b) Unpublished report. IBT No. C8472
Industrial Biotest Lab. (1971a) Unpublished report. IBT No. P8473
Industrial Biotest Lab. (1971b) Unpublished report. IBT 8473
Industrial Biotest Lab. (1971c) Unpublished report. IBT No. F9655
* As sum of dodecyl, octyl, and propyl gallate n-octyl gallate
should not be used in beverages.
** Temporary.
Johnson, A. R. & Hewgill, F. R. (1961) Aust. J. exp. Biol. med. Sci.,
39, 353
Lehman, A. J. et al. (1951) Advanc. Food Res., 3, 197
Orten, J. M., Kuyper, A. C. & Smith, A. H. (1948) Food Techn., 2, 308
Sluis, K. J. H. van (1951) Food Manuf., 26, 99
Tollenaar, F. D. (1954) Fette u. Seifen, 56, 41