WORLD HEALTH ORGANIZATION
Toxicological evaluation of some food colours, thickening
agents, and certain other substancse
WHO FOOD ADDITIVES SERIES NO. 8
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
MISCELLANEOUS FOOD ADDITIVES
DICHLORODIFLUOROMETHANE
BIOLOGICAL DATA
BIOCHEMICAL ASPECTS
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,
1974).
TOXICOLOGICAL STUDIES
Special studies on reproduction
Rat
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
Rat
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.,
1973).
Special studies on mutagenicity
Rat
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
Rat
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.,
1966).
Dog
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
Rat
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).
Comments:
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
freezant.
EVALUATION
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
REFERENCES
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 &
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Arch. environ. Hlth, 22, (2), 265-279
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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).
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Taylor, G. J., Harris, W. G. & Bogdonoff, M. D. (1971) Ventricular
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induced arrhythmias in cats exposed to dichlorodifluoromethane,
Toxicol. appl. Pharmacol., 29, 242-248
See Also:
Toxicological Abbreviations
Dichlorodifluoromethane (ICSC)
DICHLORODIFLUOROMETHANE (JECFA Evaluation)