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    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.


    See Also:
       Toxicological Abbreviations
       Polyglycerol esters of interesterified ricinoleic acid  (FAO Nutrition Meetings Report Series 46a)
       POLYGLYCEROL ESTERS OF INTERESTERIFIED RICINOLEIC ACID (JECFA Evaluation)