INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY
WORLD HEALTH ORGANIZATION
TOXICOLOGICAL EVALUATION OF SOME
FOOD COLOURS, ENZYMES, FLAVOUR
ENHANCERS, THICKENING AGENTS, AND
CERTAIN FOOD ADDITIVES
WHO FOOD ADDITIVES SERIES 6
The evaluations contained in this publication were prepared by the
Joint FAO/WHO Expert Committee on Food Additives which met in Rome,
4-13 June 19741
World Health Organization Geneva 1975
1 Eighteenth Report of the Joint FAO/WHO Expert Committee on
Food Additives, Wld Hlth Org. techn. Rep. Ser., 1974, No. 557.
FAO Nutrition Meetings Report Series, 1974, No. 54.
ORANGE RN
BIOLOGICAL DATA
BIOCHEMICAL ASPECTS
The dye was administered in aqueous suspension to male rabbits
(2-3 kg) by stomach tube at a level of 0.5 g/kg. The urine was
analysed every 24 hours for three days after dosing. The identified
metabolites and their proportions in 24 hours expressed as a
percentage of the amount of dye administered were as follows (Daniel,
1959):
p-aminophenol 3%
aniline 0.9%
1-amino-2-naphthol-6 sulfonic acid 42%
When rabbits were fed 0.5 g per kg bw of the colour the following
metabolites could be identified in 48 hours urine; total p-aminophenol
(63%), p-aminophenylglucuronide (by difference 40%), aniline (0.9%),
O-aminophenol (3%), 1-amino-2-naphthol-6 sulfonic acid (42% - 24
hours).
Reduction of the colouring occurred in vitro with bacteria
(Streptococcus faecalis) isolated from intestinal contents (Walker,
1968). This like any other azo dye is probably reduced in the gut by
bacterial azo reductases (Walker, 1970).
TOXICOLOGICAL STUDIES
Special studies on the effect of erythrocytes
Administration of 160 and 320 mg/kg bw/day to pigs for 10 days
induced the formation of methaemoglobin and decreased the red cell
life span from 54 days to 24 and 15 days respectively. In the order
of decreasing potency nitrosobenzene, O-aminophenol, 1-amino-2-
naphthol-6 sulfonic acid and p-aminophenol induced the formation of
methaemoglobin when incubated with erythrocytes from pigs and humans.
Pig erythrocytes were slightly more sensitive than erythrocytes
obtained from humans, except to the action of nitrosobenzene, where
human erythrocytes were more sensitive. 1-amino-2-naphthol-6 sulfonic
acid was about three times as potent as p-aminophenol (Würtzen et al.,
1972).
Special studies on metabolites
After intravenous injection of 7.8 mg Orange RN per kg bw to
female pigs the following metabolites were identified in 24-hour
urine: Orange RN (31%), 4'-hydroxy-l-phenylazo-2-naphthol-6 sulfonic
acid (3%), total p-aminophenol (34%), total O-aminophenol (4%).
1-amino-2-naphthol-6 sulfonic acid was present, but not determined.
When the urine collection was expanded to 72 hours the excretion of
p-aminophenol accounted for the rest of the dye. The excretion pattern
for p-aminophenol suggests that Orange RN is partly excreted in the
bile and thereafter undergoes azoreduction in the gut. After
administration of the Orange RN (78 mg per kg bw) to female pigs by
stomach tube the following metabolites were identified in the urine:
total coloured metabolies (Orange RN and 4'-hydroxy-l-phenylazo-2-
naphthol-6 sulfonic acid) (0.4%), total p-aminophenol (52%), total
O-aminophenol (6%), aniline (0.3%). 1-amino-2-naphthol-6 sulfonic acid
was present, but not determined (Larsen & Tarding, 1974).
Acute toxicity
LD50
Animal Route mg/kg bw Reference
Rats Oral 7 500 Dacre, 1969
Mice Oral 7 500 Dacre, 1969
Short-term studies
Rat
Orange RN was fed to five groups of 15 male and 15 female rats at
dietary levels of 0 (0%), 60 (0.006%), 600 (0.06%), 1200 (0.12%), or
6000 (0.6%) ppm for over three months. At 6000 ppm (0.6%), there was
marked Heinz body production, methaemoglobinaemia and reticulocytosis
together with enlargement of the spleen and increased splenic iron. At
1200 ppm (0.12%), these effects were present but less severe and at
600 ppm (0.06%) the effects were of borderline significance. There was
also an increased water intake and decreased renal concentrating
ability in the rats at the highest dosage level (Gaunt et al., 1970).
Pig
Four groups of three male and three female Danish Landrace pigs
were given either 0, 10, 40 or 160 mg/kg bw/day of Orange RN in
their diet for 110 days. In the 160 mg group hepatosis with liver
enlargement, fibrosis and bile-duct proliferation was observed.
Proliferation of the cells of the bile-ductule epithelium was found
in all test groups and the intensity of proliferation was dose-
related. At the highest dose level macrotic anaemia, haemoglobinemia
and increase in ASAT and LD serum levels were observed. Heinz-body
formation was marked in the groups fed 160 and 40 mg/kg bw. There was
corresponding haemosiderosis of the spleen, liver and kidneys at these
levels together with significant increases in the relative weight of
the spleen and liver. In the highest dose group there was also focal
liver necrosis in half the pigs (Olsen et al., 1973 and 1973a).
Long-term studies
Mouse
Three groups of 20 male and 20 female mice were given in their
diets 0, 0.05% or 0.25% of the dye for 20 months. More than 50% of the
mice survived for this period after which they were sacrificed,
autopsies performed and histological examinations undertaken. There
were no significant differences in the mean organ weights of the
different groups except that the mice on the 0.25% dose level showed
elevated relative spleen weights. The food intake and growth rate of
all the animals fed at the 0.05% and 0.25% levels showed no marked
differences from the control animals. Mice on the 0.25% diet showed
increased eosinophiland monocyte counts after 15 months (Dacre, 1969).
Rat
Three groups of 20 male and 20 female rats were given in their
diets 0, 0.05% or 0.25% of the dye for two years. More than 50% of the
rats survived for this period after which they were sacrificed,
autopsies performed and histological examinations undertaken. There
were no significant differences in the mean organ weights of the
different groups except that the rats on the 0.25% dose level showed
elevated relative spleen weights. The female rats on the 0.05% diet
showed a decreased food consumption with a corresponding decrease in
growth rate. There were no significant haematological abnormalities,
no gross pathological changes and no consistent histopathological
changes (Dacre, 1969).
Comments:
Available short and long-term studies in pigs, rats and mice
demonstrate adverse effects in the haemopoietic system (Heinz bodies)
and the liver. Evidence of haemolytic anaemia in rats and pigs
reinforces the significance of the observations of splenomegaly
reported in the available long-term studies in the rat and mouse.
In addition bile duct proliferation has been recorded which was
present in a dose-related manner. More information is needed on the
metabolism of this colour, on reproduction, and possible embryotoxic
including teratological effects.
EVALUATION
Not possible on the data available.
REFERENCES
Dacre, J. C. (1969) Proc. Univ. Otago med. Sch., 47, 3
Daniel, J. W. (1962) Toxicol. appl. Pharmacol., 4, 572
Gaunt, I. F. et al. (1971) Fd. Cosmet Toxicol., 9, 619
Larsen, J. C. & Tarding, F. (1974) To be published in Acta
Pharmacologica et Toxicologica
Olsen, O. et al. (1973) Toxicology, 1, 249
Olsen, P. & Hansen, E. (1973a) Acta pharmacol, toxicol., 32, 314
Walker, R. (1968) Ph.D. Thesis, University of Reading
Walker, R. (1970) Fd. Cosmet Toxicol., 8, 659
Würtzen, G., Larsen, J. C. & Tarding, F. (1972) 8th International
Congress on Clinical Chemistry, Copenhagen