INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY
WORLD HEALTH ORGANIZATION
SUMMARY OF TOXICOLOGICAL DATA OF CERTAIN FOOD ADDITIVES
WHO FOOD ADDITIVES SERIES NO. 12
The data contained in this document were examined by the
Joint FAO/WHO Expert Committee on Food Additives*
Geneva, 18-27 April 1977
Food and Agriculture Organization of the United Nations
World Health Organization
* Twenty-first Report of the Joint FAO/WHO Expert Committee on Food
Additives, Geneva, 1977, WHO Technical Report Series No. 617
ORANGE RN
EVALUATION FOR ACCEPTABLE DAILY INTAKE
BIOLOGICAL DATA
BIOCHEMICAL ASPECTS
Metabolism
The dye was administered in aqueous suspension to male rabbits
(2-3 kg weight) 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:
p-aminophenol, 3%, aniline, 0.9% and 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-aminophenyl-glucuronide (by difference 40%),
aniline (0.9%), O-aminophenol (3%), 1-amino-2-naphthol-6 sulfonic acid
(42% - 24 hours) (Daniel, 1962).
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 azo-reduction 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 metabolites (Orange RN and 4'-hydroxy-l-phenylazo-2-
naphthol-6 sulfonic acid) (0.4%), total p-aminophenol (52%), total
O-aminophenol (6%), aniline (O.3%). 1-amino-2-naphthol-6 sulfonic acid
was present, but not determined (Larsen and Tarding, 1976).
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).
After gavage of 66 mg 35S-labelled Orange RN (labelled in the
naphtholsulfonic acid moity) per kg bw to six male rats (weighing
approximately 300 g) 11.5% (8.6-14.1) of the radioactivity was
recovered in the urine in 48 hours, while 44% (33-55) was found in the
faeces. Of the radioactivity excreted in urine 1.5% (0.3-3.4) was
unreduced dye.
After i.v. administration of 6 mg 35S-Orange RN per kg bw to
four male rats 33% (24-50) of the radioactivity was found in urine
within 48 hours, of which 15% (11.2-18.2) was unreduced Orange RN.
Twenty-seven per cent. (24-33) of the activity was recovered in the
faeces (Larsen and Tarding, 1977).
After gavage of 80 mg 35S-labelled Orange RN (labelled in the
naphtholsulfonic acid moity) per kg bw to four male rabbits (weighing
3.5 kg) 52% (26-69) of the dose was recovered in the urine in 24
hours. Of this only 1.5% of the dose (0.5-2.2) was unreduced dye.
Three per cent. (1.2-4.9) of the dose was found in the faeces after 24
hours.
When 15 mg of 35S-labelled Orange RN per kg bw was given i.v. to
two rabbits, 60% (68 and 51% respectively) of the dose was recovered
in the urine within 24 hours, of which 37% was unreduced Orange RN.
9.5% (10.0 and 9.0% respectively) of the dose was found in the faeces.
The presence of radioactivity in the bile at sacrifice after 24
hours indicated entero-hepatic circulation both after oral and i.v.
administration (Larsen and Tarding, 1977).
Using 35S-labelled ANSA* the absorption and excretion as well as
the localization in the liver cell of ANSA was investigated in rats
and rabbits. The results after p.o. administration shows that the
rabbit absorbs and excretes into the urine a significant larger amount
of ANSA (30-40%) than the rat (6-13%). The results after i.v.
administration shows that some 60% of the injected dose is excreted in
the urine in both species. In the rat 15-20% are recovered from the
faeces, while only traces (0.1-0.5%) are found in faeces of rabbits. A
significant amount of ANSA was retained in the blood and the liver of
both species 24 and 48 hours after i.v. administration. After
differential centrifugation of liver homogenates the majority of ANSA
was found in the 9000 g supernatant (Larsen and Tarding, 1977).
TOXICOLOGICAL STUDIES
Special studies of the effect on 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.
* 1-amino-2-naphthol-6-sulfonic acid.
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).
Acute toxicity
Animal Route LD50 mg/kg Reference
mg/kg bw
Rat 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, 60, 600, 1200, or 6000 ppm for over three
months. At 6000 ppm, there was marked Heinz body production,
methaemoglobinaemia and reticulocytosis together with enlargement of
the spleen and increased splenic iron. At 1200 ppm, these effects were
present but less severe and at 600 ppm 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., 1971).
Cat
A daily oral dose of 200 mg/kg for six days induced increase in
Heinz bodies (Wingler, 1953).
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-duct epithelium was found in
all test groups and the intensity of proliferation was dose-related.
At the highest dose level macrotic anaemia, haemoglobinaemia 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).
The induction and persistence of haematological changes and
hepatic lesions were studied in a time-sequence experiment where four
groups of SPF Danish Landrace pigs were given Orange RN (monosodium
salt of 1-phenylazo-2-naphthol-6-sulfonic acid) 160 mg/kg bw in a
balanced powdered diet for 7, 14, 28 and 56 days respectively,
followed by a restitution period of 82 days on normal diet. A fifth
group served as control. Serial liver biopsies were taken during the
experiment for histological evaluation. Anaemia with Heinz bodies was
induced after Orange RN for 28 days. Increased number of reticulocytes
and Heinz bodies already after seven days. A marked bile duct/ductule
proliferation was seen after Orange RN for 28 and especially 56 days.
Only slight proliferation after seven or 14 days. The decrease in RBC
disappeared after seven days recovery, but the increased value for
reticulocytes and Heinz bodies could still be observed after 21 and
30-40 days respectively. The hepatic lesions persisted after 82 days
of restitution but the intensity was reduced. Large individual
variations in reversibility could be noted among the pigs (Hasselager
and Hansen, 1977).
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 eosinophil 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).
REFERENCES
Dacre, J. C. (1969) Acute and chronic toxicity studies on Orange RN,
Proc. Univ. Otaga med. Sch., 47, 3
Daniel, J. W. (1962) The excretion and metabolism of edible food
colors, Toxicol. Appl. Pharmacol., 4, 572-594
Gaunt, I. F., Brantom, P. G., Kiss, I. S., Grasso, P and Gangolli,
S. D. (1971) Short-term toxicity of Orange RN in rats, Food Cosmet.
Toxicol., Vol. 9, iss. 5, pp. 619-630
Larsen, J. C. and Tarding, F. (1976) Acta pharmacol. et toxicol.,
39, 525-535
Larsen, J. C. and Tarding, F. (1977) Abstract. 19th Meeting of the
European Society of Toxicology, in Copenhagen, June 19-22
Hasselager, E. and Hansen, E. (1977) Abstract of data to be published
Olsen, P., Wuertzen, G., Hansen, E., Carstensen, J. and Poulsen, E.
(1973) Short-term peroral toxicity of the food color Orange RN in
pigs, Toxicology., 1 (3), 249-260
Olsen, P. and Hansen, E. (1973a) Bile duct proliferation in pigs fed
the food color Orange RN, Acta. Pharmacol. et Toxicol., 32, 314
Walker, R. (1968) Ph.D. Thesis, University of Reading
Walker, R. (1970) The metabolism of A20 compounds: A review of the
literature, Fd. Cosmet. Toxicol., Vol. 8, pp. 659-676
Wingler, A. (1953) Dyes in foodstuff dyeing. The problem of exposure
to cancer. 2. Krebsforsch., 59, 134-155
World Health Organization (1974) Food Additive Series, No. 6, Geneva
Switzerland
Wuertzen, G., Larsen, J. C. and Tarding, F. (1972) Formation of
hemiglobin in vivo and in vitro after administration of a food
additive Orange RN, Scand. J. Clin. Lab. Invest. Suppl., 29(126),
13-20
See Also:
Toxicological Abbreviations
Orange RN (WHO Food Additives Series 6)
ORANGE RN (JECFA Evaluation)