Toxicological evaluation of some food colours, thickening
    agents, and certain other substancse


    The evaluations contained in this publication were prepared
    by the Joint FAO/WHO Expert Committee on Food Additives which
    met in Geneva, 14-23 April 19751

    World Health Organization, Geneva 1975

    1 Nineteenth Report of the Joint FAO/WHO Expert Committee on Food
    Additives, Wld Hlth Org. techn. Rep. Ser., 1975, No. 576;
    FAO Nutrition Meetings Report Series, 1975, No. 55.

    The monographs contained in the present volume are
    also issued by the Food and Agriculture Organization
    of the United Nations, Rome, as
    FAO Nutrition Meetings Report Series, No. 55A

    ISBN 92 4 166008 2

    (C) FAO and WHO 1975





         Five groups, each of 10 young male rats, were given
    intragastrically daily, five times a week, for a total of 43 doses,
    either 2.5 ml or 5 ml corn oil, 2.5 ml corn oil + 56 mg Freon(R) 12,
    5 ml corn oil + 112 mg Freon(R) 12 and 2.5 ml corn oil + 56 mg (R)
    12 + 0.02 mg Freon(R) 11 + 0.03 mg Freon(R) 21 + 0.03 mg Freon(R) 22.
    The F concentration in the urine was determined after one, two, three,
    four, six and eight weeks and the plasma alkaline phosphatase activity
    was determined after two, four and eight weeks. The control group
    (2.5 ml corn oil) and the group receiving mixed Freons were kept for a
    further four weeks on control diet and urinary fluoride determined
    after two, three and four weeks. Plasma alkaline phosphatase was
    determined after a further four weeks on control diet. A sample of
    fibial bones was examined for fluoride at the end of the respective
    administration periods and again after a further four weeks on control
    diet. Weight gain was similar in all groups except that receiving 5 ml
    corn oil + 112 mg Freon(R) 12, in which it was slightly reduced
    compared with controls. All animals remained healthy. The plasma
    alkaline phosphatase of animals receiving 5 ml corn oil + 112 mg
    Freon(R) 12 was higher than controls at eight weeks and only slightly
    raised at eight weeks in animals receiving 2.5 ml corn oil + 56 mg
    Freon(R) 12. Eight weeks administration to rats of Freon(R) 12 had no
    effect on urinary fluoride excretion and raised bone fluoride only
    very slightly at the highest dose level administered (Barnes &
    Sherman, 1966).

         14C-labelled Freon(R) 12 dissolved in corn oil was administered
    as single dose to 15 rats by gavage. 0.5% of the total dose was
    excreted in the urine as at least three metabolites (20-30% of these
    were a glucuronide). 1.9% of the total dose was metabolised to CO2.
    No residual radioactivity was found in the tissues after 30 hours.
    Most of the ingested Freon(R) 12 was excreted as such through the
    lungs with a half-life of 15 minutes, all being eliminated after
    24 hours (Griffith, 1969). After six to 20 minutes inhalation of
    14C-labelled Freon(R) 12 at 8000-12 000 ppm v/v by four males and two
    females (beagles) eventually all inhaled Freon(R) 12 was recovered in
    the exhaled air within one hour. Only traces of radioactivity were
    found in urine or as exhaled 14C-CO2. All tissues contained
    measurable concentrations (total >1% of dose) of non-volatile
    radioactivity 24 hours after exposure. These residues could not be
    identified. Pretreatment for three days with 60 mg phenobarbital/day
    or 50-90 minutes exposure did not change the results (Blake & Mergner,


    Special studies on reproduction


         Eighty-four female and 44 male weanling albino rats were given 0,
    0.2 and 2% Freon(R) 12 in corn oil by gavage for three months and were
    then mated. Dosage continued for the males but ceased during day
    18 - day 5 of lactation for the females. The F1a generation was used
    for long-term studies. The F0 animals were mated a second time to
    initiate an F1b litter. Rats of these groups were also bred to
    produce F2a and F3a litters. No effect due to treatment was seen on
    fertility, viability of pups and lactation in the three generation
    study above (Sherman et al., 1974).

    Special studies on teratogenicity


         Three groups of rats (total 78 rats) were dosed with 0, 0.2 and
    2% Freon(R) 12 by gavage from days 6-16 of pregnancy with sacrifice on
    day 21. No effects on parents or offspring were noted (Culik et al.,

    Special studies on mutagenicity


         The F0 animals of the reproduction study were mated to initiate
    an F1b litter. Animals were kept on 0, 0.2 and 2% Freon(R) 12 by
    gavage until pregnancies were terminated in mid-term to give a
    dominant lethal assay. This showed no evidence of adverse effects
    (Scherman et al., 1974).

    Special studies on inhalation

         The Committee was aware of much literature on the inhalation
    toxicity of dichlorodifluoromethane. The cardiotoxicity of the Freons
    as a class produced by inhalation has been clearly established.
    However, these occur at relatively high doses and were not
    considered relevant to the evaluation of the oral toxicity of
    dichlorodifluoromethane. For the purposes of the completeness of this
    monograph the Committee felt that pertinent references to the
    inhalation toxicity should be provided (Aviado & Belej, 1974; Aviado &
    Smith, 1974; Azar et al., 1971; Clayton, 1967; Paulet, 1971; Reinhardt
    et al., 1971; Taylor et al., 1971; Thompson & Harris, 1974).

    Acute toxicity

         Because of its limited solubility in oil (2.12% w/v) oral acute
    LD50 studies cannot be carried out. 4 ml sesame oil containing
    Freon(R) 12 at 8.3%, 6.5% and 5.1% were administered once daily for
    14 days to groups of 15 adult rats. None died but the rats developed
    diarrhoea and did not gain weight. Organ weights showed no significant
    changes. No unusual histopathological findings in stomach, liver,
    kidney, spleen and adrenals were noted. The doses administered were
    equivalent to 667, 527 or 395 mg/kg body weight (Imamichi, 1973).

    Short-term studies


         Fifty male and 50 female weanling rats were given by intragastric
    intubation corn oil containing 2.1% Freon(R) 12 at a dose rate of
    160-379 mg/kg/day five times a week for 18 weeks. Only one dose level
    was studied over 90 administrations and the five-day feeding method
    makes the study less valuable than one with continuous feeding. No
    differences between controls and test animals were noted with regard
    to weight gain, food intake, food efficiency, clinical observation,
    mortality (all deaths were due to accidental lung untubation), tibia
    length, haematology and urinalysis. Male rats excreted slightly more
    fluoride in their urine after two and three months, female rats after
    one and three months. Plasma alkaline phosphatase was slightly
    increased in the test group but SGOT values were similar to controls.
    No organ weight, pathological or histopathological changes were seen
    which could be attributed to the administration of Freon(R) 12
    (Quigley & Sherman, 1966). In another experiment two groups of 10 rats
    were given diets with or without extraction with Freon(R) 12 for
    42 days. No adverse effects were noted as regards body weight gain,
    food consumption, organ weights and histopathology (Sherman et al.,


         Three male and three female beagles were divided into two groups.
    The test group received orally a capsule containing 170 mg Freon(R)
    12 in corn oil apart from 528-792 Freon(R) 12 contained in
    Gainesburgers1 frozen in Freon(R) 12 giving an average of 88 mg
    Freon(R) 12/kg body weight. Again only one dose level was studied. No
    adverse effects were noted on body weight, food consumption, clinical
    observations, haematology, urinalysis, urinary fluoride, biochemical
    parameters, liver function tests, gross pathology and histopathology
    following administration of Freon(R) 12 (Sherman et al., 1966). Three
    groups of four male and four female beagles were fed 0, 300 and


    1    Commercial semi-dry dog food.

    3000 ppm Freon(R) 12 in the diet as frozen Gainesburgers1 for two
    years. No abnormalities were found as regards nutrition, haematology,
    urinalysis, biochemistry and histopathology. Urinary fluoride was not
    increased. Bone fluoride did not increase and Freon(R) 12 was found in
    the fatty tissue of two dogs at the high dose level only (also
    possibly in bone marrow). Adrenal function remained normal and no
    significant pathological change was seen (Sherman et al., 1966).

    Long-term studies


         The F1a rats of a multigeneration reproduction study were
    divided into groups of 50 males and 50 females and dosed for two
    years with 0, 0.2 and 2% Freon(R) 12 by gavage to give 0, 15 and
    150 mg/kg bw/day (0, 300 and 3000 ppm). An interim kill of six rats
    per group was made at one year. There was slightly decreased weight
    gain in high dose animals but no other abnormalities were found in
    clinical, biochemical, urinalysis, haematological, gross and
    histopathological parameters. There was no increase in fluoride
    excretion in dosed animals but higher fluoride content of bone was
    seen at one year but not at two years. The results for bone marrow and
    adrenals were equivocal but trace amounts could have been present. No
    evidence of carcinogenicity was seen (Sherman et al., 1974).


         Ingested Freon(R) 12 is probably mostly excreted via the lungs,
    only 2% being metabolized to CO2 and 0.5% to several unidentified
    urinary metabolites. No tissue accumulation or increase in urinary
    fluoride occurs but bone fluoride may increase slightly. Exposure for
    six to 12 weeks caused hepatic damage only in guinea-pigs at doses of
    4000 mg/m3. Human exposure to 1000 ppm for 2.5 hours produced no
    adverse effects. Short-term studies in rats and dogs were only at one
    dose level. The long-term studies in rats and dogs showed no adverse
    effects apart from some reduction in weight gain of rats at the high
    dose level. Some Freon(R) 12 is retained in fat and liver. The
    reproduction and dominant lethal studies showed no deleterious
    effects, nor was there any teratogenicity seen in rats, the only
    species studied. Tests in dogs, cats and monkeys show that exposure to
    centrations above 4% predispose to ventricular tachyarrhythmia on
    challenge with adrenaline but these effects are probably of no
    significance in relation to the safety of Freon(R) 12 as food


    Level causing no toxicological effect in the rat

         3000 ppm in the diet equivalent to 150 mg/kg body weight

    Estimate of acceptable daily intake for man

         0-1.5 mg/kg body weight


    Aviado, D. M. & Belej, M. A. (1974) Toxicity of aerosol propellants on
         the respiratory and circulatory systems. Cardiac arrhythmia in
         the mouse, Toxicology, 2, (1), 31-34

    Aviado, D. M. & Smith, D. G. (1974) Toxicity of aerosol propellants in
         the respiratory and circulatory systems. Respiration and
         circulation in primates, Toxicology, 3, (2), 241-252

    Azar, A., Reinhardt, C. F., Maxfield, M. E., Smith, P. E. & Mullin, L.
         S. (1971) Cardiac toxicity of aerosol propellants, J. Am. Med.
         Ass., 215, (9), 1501-1502

    Barnes, J. R. & Sherman, H. (1966) Metabolism studies with
         dichlorodifluoromethane (Freon-12(R) Food Freezant). Unpublished
         report from Haskell Laboratory submitted to the World Health
         Organization by E. I. du Pont de Nemours & Co.

    Blake, D. A. & Mergner, G. W. (1974) Inhalation studies on the
         biotransformation and elimination of 14C-trichlorofluoromethane
         and 14C-dichlorodifluoromethane in beagles, Toxicol. appl.
         Pharmacol., 30, 396-407

    Clayton, J. W., jr (1967) Fluorocarbon toxicity and biological action,
         Fluorine Chem. Rev., 1, 197-242

    Culik, R., Sherman, H., Aftosmis, J. G. & Reinhardt, C. F. (1973)
         Teratogenic study in rats with dichlorodifluoromethane (Freon(R)
         12). Unpublished report from Haskell Laboratory submitted to the
         World Health Organization by E. I. du Pont de Nemours & Co.

    Griffith, F. D. (1969) Metabolism of Freon(R) 12 by rats. Unpublished
         report from Haskell Laboratory submitted to the World Health
         Organization by E. I. du Pont de Nemours & Co.

    Imamichi, T. (1971) Acute toxicity study in rats with Freon(R) 12 Food
         Freezant. Unpublished report from the Nippon Veterinary and Zoo
         Technical College submitted to the World Health Organization

    Paulet, G. (1971) Action of fluorochloro hydrocarbons used in
         aerosols. Problems of their retention by the organism after
         inhalation. (Trib. Cent. Belg. Etude Doc. Eaux, 23, 487-497),
         Chem. Abstr., 74, (19), 97243

    Reinhardt, C. F., Azar, A., Maxfield, M. E., Smith, P. E., jr &
         Mullin, L. S. (1971) Cardiac arrhythmias and aerosol sniffing,
         Arch. environ. Hlth, 22, (2), 265-279

    Sherman, H., Barnes, J. R., Stula, E. F. & Clayton, J. W., jr (1966)
         Feeding studies with dichlorodifluoromethane. Unpublished report
         from Haskell Laboratory submitted by E. I. du Pont de Nemours &

    Sherman, H., Barnes, J. R., Aftosmis, J. G. & Zapp, J. A.,
         jr (1974) Long-term feeding studies in rats and dogs with
         dichlorodifluoromethane (Freon(R) - 12 Food Freezant).
         Unpublished report from Haskell Laboratory submitted to the World
         Health Organization by E. I. du Pont de Nemours & Co.

    Taylor, G. J., Harris, W. G. & Bogdonoff, M. D. (1971) Ventricular
         arrhythmias induced in monkeys by the inhalation of aerosol
         propellants, J. Clin. Invest., 50, 1546-1550

    Thompson, E. B. & Harris, W. S. (1974) Time course of epinephrine-
         induced arrhythmias in cats exposed to dichlorodifluoromethane,
         Toxicol. appl. Pharmacol., 29, 242-248

    See Also:
       Toxicological Abbreviations
       Dichlorodifluoromethane (ICSC)