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.
POLYGLYCEROL ESTERS OF INTERESTERIFIED RICINOLEIC ACID
Explanation
These emulsifiers have been evaluated for acceptable daily intake
by the Joint FAO/WHO Expert Committee on Food Additives (see Annex 1,
Ref, No. 20) in 1969.
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 substance does not affect the hydrolysis of ground-nut oil by
pancreatic lipase in vitro.
The clearing of alimentary lipaemia by heparin was not affected
in rats fed with 9% of the substance in the diet for 45 weeks.
Ingestion of a diet containing 9% of the substance did not induce
an increase in the chylomicron count in rats, whereas ingestion of a
diet containing 10% of GNO (ground-nut oil) did. It was concluded that
the substance was not absorbed into the intestinal lymphatic vessels,
but was absorbed into blood capillaries and was cleared from the blood
in the liver.
A sample of the substance labelled with 14C in the polyglycerol
moiety was mixed with ground rat food (approx. or equal 10%) and
administered in a slurry by gastric intubation to three rats.
Radioactivity was recovered in faeces (54%), urine (31%) and CO2
(8%). The faecal radioactivity was not in the lipid soluble fraction,
suggesting that it was present as free polyglycerols.
Tracer amounts of 14C-(1)-oleic and 14C-(1)-stearic acids were
condensed with ricinoleic acid and coupled with polyglycerol. The
products were administered to rats as a 50% aqueous emulsion by
gastric intubation. With the 14C-stearyl marker, radioactivity was
recovered in expired CO2 (18%), faeces (52%) and urine (1%): with
14C-oleyl marker, recoveries were: CO2 (30%), faeces (51%), urine
(1%). 14C-stearyl marked material was also fed as a dietary component
with recoveries in expired CO2 (11%), faeces (10%) and urine (1%);
when intubated as a dietary slurry recoveries were: expired CO2
(27%), faeces (16%) and urine (2%). Although different modes of
administration affect the pattern of turnover the findings indicate
that the markers were hydrolyzed from esters with ricinoleic hydroxyl
groups and were then utilized normally in metabolism.
The substance was prepared with 3H-(12)-ricinoleic acid as a
marker, and administered to rats by intubation in a dietary slurry
containing a dose of 2 ml (14 µCi). After 24 hours, 5% of administered
tritium was found in the faeces, and 1% was present in the remainder
of the intestinal contents. The urine contained 10%. Epididymal fat
contained 5% of which about one-third was incorporated in hydroxy
fatty acids. Similar findings were reported with 3H-(9:10)-ricinoleic
acid.
Digestive enzymes in (i) the juice from duodenum and jejunum,
(ii) mucosa scrapings, and (iii) pancreas homogenates prepared from
rats liberated some free ricinoleic acid from the substance
incorporating 3H-(12)-ricinoleic acid as a marker.
The digestibility of the substance with 9% in diet (plus 1% of
ground-nut oil) was 98.0% compared with 99.8% for 10% ground-nut oil,
as calculated from intake and faecal excretion of fat (Howes & James,
1968; Jenkins & Philp, 1961, 1968; Rutherford & Jones, 1967; Watson &
Gordon, 1962).
TOXICOLOGICAL STUDIES
Special studies on liver enlargement
Mouse
Groups of mice were fed diets containing 0%, 5%, 10% or 15% of
the substance; control groups were fed corresponding levels of GNO.
Each group was divided into two feeding regimes: ad libitum or
restricted to seven hours per day. There were 10 male and 10 female
mice on each regime. The trial continued for 14 days. Liver
enlargement occurred in all groups fed with the substance, but the
effect was reversible with return to normal after two further weeks on
a normal diet. Kidney weights were not significantly affected.
In a further similar trial the substance (or GNO) was fed at
levels of 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7% and 8%. Compared to diets
containing equal levels of GNO, 3% or more of the substance
significantly increased liver weight, but the degree of liver
enlargement was not proportional to the feeding level (Wilson et al.,
1967a, b).
Rat
The liver enlargement produced by the substance was studied in
rats fed with 18% (+ 2% GNO) or 20% GNO for one week and was
attributed to cytoplasmic hypertrophy of liver parenchymal cells
rather than to hyperplasia since the liver weight:nucleus count ratio
was increased and there was no increase in mitotic activity. There
were no changes in the structural organization of the liver and no
histopathological evidence of toxicity. The hypertrophy was regarded
as a normal functional response to an increased hepatic work load. In
another similar feeding trial, total liver DNA was not affected while
liver RNA was increased by the substance, confirming that the
increased weight was due to hypertrophy and not to hyperplasia
(Gellatly & Jenkings, 1968; Wilson et al., 1968).
Acute toxicity
Animal Route LD50, mg/kg bw Reference
Rat oral > 18 500 Jenkins & Philp, 1961
Rat i.p. > 12 000 Jenkins & Philp, 1961
Rabbit oral > 25 000 Jenkins & Edwards, 1967
Guinea-pig oral > 30 000 Jenkins & Edwards, 1967
Mouse oral > 100 000 Jenkins & Philp, 1961
Chicken oral > 30 000 Jenkins & Edwards, 1967
Slight diarrhoea was the only effect seen in 42 rats out of
55 given 20 ml/kg by gastric intubation and observed for 21 days
subsequently; diarrhoea was seen in two of 22 control rats given
ground-nut oil (Jenkins & Edwards, 1967).
Twelve rats were given 10 ml/kg (approx. or equal 10 000 mg/kg)
daily for five days without significant effect on weight gain or
food intake and without note of toxicity during the period of
administration or 14 days later at autopsy (Jenkins & Edwards, 1967).
Short-term studies
Rat
Littermate trios of rats were fed on a diet containing 10%
ground-nut oil, 1% ground-nut oil, or 1% ground-nut oil + 9% of the
substance. Each trio was of the same sex and of similar weight, and
its members were distributed to the three groups at random. Treatment
of one lot of 16 trios was continued for 30 weeks and of another lot
for 45 weeks during which there were no signs of toxicity and no
significant effects of the substance on food or water intake or weight
gain.
The digestibility of the substance with 9% in diet (plus 1% of
ground-nut oil) was 98.0% compared with 99.8% for 10% ground-nut oil,
as calculated from intake and faecal excretion of fat.
After 11 weeks, there were no significant differences in body fat
weight, fatty acids, iodine value or hydroxyl value, or in liver
content of vitamin A, fat or free fatty acids, but liver phosphorus
was low. After 45 weeks, there were no differences in the
digestibility of fat, protein or whole diet between the groups.
Plasma clearance of bromsulphthalein was somewhat more rapid in
rats treated with 9% of the substance in the diet for 17, 21 and 45
weeks than in rats on control diets containing ground-nut oil: there
is no evidence, therefore, of impairment of liver function by the
substance.
The urine-concentrating power of the kidneys was not affected in
rats treated with 9% of the substance in the diet for 21 and 45 weeks.
There were no significant effects of 9% of the substance in the
diet for 45 weeks on haemoglobin, erythrocyte count, haematocrit
value, or prothrombin time. Erythrocyte fragility examined at 15 weeks
was not affected (Jenkins & Philp, 1961).
In the 30-week and 45-week trials, there were no significant
effects on weight of kidney, spleen, testes or adrenals and all organs
were macroscopically normal. The livers were significantly larger than
control in rats of the 45-week trial, but not in those of the 20-week
trial. Similar effects were seen in rats fed castor oil (Wilson &
Jenkins, 1968),.
The no-effect level for increased liver weight in a 13-week trial
was determined by feeding a diet containing 0%, 1%, 2%, 4% and 8% of
the substance using 10 rats in each group: there were significant
increases in liver weight in the 4% and 8% groups, the effect being
greater in females than males. In these rats (4% and 8% groups),
acetone bodies were detected in the urine during the first six weeks
of the trial, but the reaction was apparently due to a metabolite of
the substance rather than to the usual acetone bodies of ketotic
states (Wilson & Jenkins, 1968).
Chicken
Chickens in groups of 16 were used in a 90-day (13-week) trial.
Groups were fed basic diet (two groups), 15% of the substance (two
groups), 10%, 5% and 2% of the substance with 2% ground-nut oil (GNO)
(one group at each level) and 15%, 10% and 5% GNO (one group at each
level). Growth was adversely affected by the substances and by GNO at
the 10% and 15% dietary levels and was apparently due to dietary
imbalance and decreased food intake. Plasma levels of glutamate-
pyruvate aminotransferase and glutamate-oxaloacetate amino-transferase
were not significantly affected by any of the treatments. The
haematocrit value, haemoglobin and erythrocyte sedimentation rates
were not affected by the substance. Liver weight was significantly
increased by 15% of the substance, but not with 10% or less. Kidney
weight was significantly increased by 5%, 10% and 15% of the
substance. Spleen and heart weights were not selectively affected by
the substance, since GNO produced the same changes. Histological signs
of toxicity in various organs were absent except for lymphoid
infiltrations which were attributed to intercurrent subclinical virus
disease (Marek's disease) (Kirby et al., 1969).
Long-term studies
Mouse
Four groups of 25 male and 25 female mice were fed a diet with
10% ground-nut oil or 5% of the substance plus 5% GNO for 80 weeks.
A positive control group received 0.5 mg, 9, 10-dimethyl-1,
2-benzanthracene once per week for 16 weeks. After 50 weeks there was
no difference in growth between test and ground-nut oil groups but the
basic diet and positive control group showed reduced growth. Survival
was comparable for the three groups, also the haematological indices.
Liver and kidney of the test group were significantly heavier than the
controls. No specific histological lesion was seen. Carcass fat
contained no polyglycerol but only a small amount (0.1%) of ricinoleic
acid. Histopathology of all other major tissues showed no lesions
specifically associated with the substance nor was there any
significant difference in tumour incidence. The strain was sensitive
to the carcinogen used in the positive control (Wilson et al., 1967).
Groups of 20 male and 20 female mice were given repeated s.c.
injections of 0.5 ml of the substance weekly for five weeks with
milk or ground-nut oil as controls. After 80 weeks no adverse effects
were noted on growth, survival, haematology, organ weights and
histopathological findings. Tumour incidence was similar in test and
control groups (Wilson et al., 1967).
Seven groups of 20 male and 20 female mice were painted daily
with 20 mg of the substance for 60 weeks with or without a single
preceding application of 0.25 mg dimethylbenzanthracene and a positive
control group was included. After 80 weeks no adverse effects were
noted on growth, survival or tumour incidence in tests and controls
involving the substance. There was no promoting or cutaneous
carcinogenic effect (Wilson et al., 1967).
Rat
A three-generation test was carried out in two groups of 19
and 28 rats on diets containing 0% or 1.5% of the substance. No
significant differences were seen between the two groups as regards
fertility, pup weight, pup survival, litter number, etc. Each animal
was observed for over one year. No consistent abnormalities or
histopathological changes were seen in the third generation (Philp &
Jenkins, 1961).
In another experiment two groups of 30 male and 30 female rats
each were fed diets containing 5% of the substance plus 5% ground-nut
oil or 10% ground-nut oil for 104 weeks. No significant adverse
effects were seen on growth, food consumption, liver function tests at
weeks 89 and 103, specific gravity of the urine at weeks 89 and 103,
or survival. The kidneys of males and females at 5% of the substance
and the livers of females at 5% of the substance were significantly
enlarged. There was no polyglycerol or ricinoleic acid accumulation in
carcass fat. Histopathology of all organs showed no abnormalities
related to the substance administration nor was there a rise in tumour
incidence in the test group. The strain was sensitive to 20-methyl-
cholanthrene (Philp et al., 1961).
Thirty male and 30 female rats were injected s.c. with 0.5 ml
twice weekly for 13 weeks at different sites, milk and ground-nut oil
were controls. After two years there were no adverse effects on growth
survival, haematological indices, organ weights or histopathology of
all tissues. No increase in tumour incidence was found but most rats
treated with the substances developed persistent nodules at injection
sites. Subcutaneous injections produced 2/60 fibrosarcomata as did
milk (2/60) but not ground-nut oil (Philp & Jenkins, 1961).
Six groups of 10 male and 10 female rats were painted with 50 mg
of the substance cutaneously daily for 60 weeks with or without a
single preceding application of 0.25 mg benzanthracene and observed
for two years. A positive control group was included. No deleterious
effects were noted on growth and survival. No skin tumours were seen
in the test group. The strain was sensitive to the carcinogen used as
positive control (Philp & Jenkins, 1961).
OBSERVATIONS IN MAN
Nineteen volunteers took the substance in soups, cakes and
toffees at the rate of 5 g/day for one week and 10 g/day for a second
week. Otherwise, the diet was balanced and constant for the two weeks
and an initial stabilization week. The group contained eight males and
11 females; except for two older females (64 and 66 years), all were
university students (19 to 24 years). There were no significant
adverse departures from control patterns attributable to the substance
in serum proteins, thymol turbidity, serum bilirubin, blood
cholesterol, serum glutamate-pyruvate animotransferase, serum
cholinesterase, creatinine clearance faecal fat or faecal nitrogen
(Groger et al., 1968).
Comments:
The metabolic fate of this material has been studied by a number
of indirect measurements. The long-term studies in rats and mice did
not show carcinogenic potential. The enlargement of liver and kidneys
observed in long-term tests was not accompanied by any lesions
detectable by histopathology. Only the rat study shows a no-effect
level for liver enlargement.
EVALUATION
Level causing no toxicological effect
Rat: 15,000 ppm (1.5%) in the diet equivalent to 750 mg/kg bw.
Estimate of acceptable daily intake for man
0-7.5 mg/kg bw.
REFERENCES
Gellatly, J. B. M. & Jenkins, F. P. (1968) Unpublished report of
Unilever Res. Labs. submitted by Unilever Ltd.
Groger, W., Philp, J. McL.& Wilson, R. (1968) Unpublished report of
Unilever Res. Labs. submitted by Unilever Ltd.
Howes, D. & James, C. T. (1968) Unpublished report of Unilever Res.
Labs. submitted by Unilever Ltd.
Jenkins, F. P. & Edwards, K. (1967) Unpublished report of Unilever
Res. Labs. submitted by Unilever Ltd.
Jenkins, F. P. & Philp, J. McL. (1961) Unpublished report of Unilever
Res. Labs. submitted by Unilever Ltd.
Kirkby, W. et al. (1969) Unpublished report of Unilever Res. Labs.
submitted by Unilever Ltd.
Philp, J. McL. & Jenkins, F. P. (1961) Unpublished report of Unilever
Res. Labs. submitted by Unilever Ltd.
Philp, J. McL. et al. (1961) Unpublished report of Unilever Res. Labs.
submitted by Unilever Ltd.
Rutherford, T. & Jones, P. (1967) Unpublished report of Unilever Res.
Labs. submitted by Unilever Ltd.
Watson, W. C. & Gordon, R. S. (1962) Biochem. Pharmac., 11, 229
Wilson, R., Ashmole, R. & Gellatly, J. B. M. (1968) Unpublished report
of Unilever Res. Labs. submitted by Unilever Ltd.
Wilson, R., Kirkby, W.& Ashmole, R. (1967a) Unpublished report of
Unilever Res. Labs. submitted by Unilever Ltd.
Wilson, R., Kirkby, W. & Ashmole, R. (1967b) Unpublished report of
Unilever Res. Labs. submitted by Unilever Ltd.
Wilson, R. & Jenkins, F. P. (1968) Unpublished report of Unilever Res.
Labs. submitted by Unilever Ltd.
Wilson, R., Kirkby, W. & Philp, J. McL. (1967) Unpublished report of
Unilever Res. Labs, submitted by Unilever Ltd.