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
RED 2G
EVALUATION FOR ACCEPTABLE DAILY INTAKE
BIOLOGICAL DATA
BIOCHEMICAL ASPECTS
Rats were injected intravenously with Red 2G. Bile was collected
for six hours and analysed. The recovery of the colour was on average
64% of the administered quantity (Priestley and O'Reilly, 1966). Biliary
excretion of i.v. administered Red 2G was also reported (Ryan and
Wright, 1961), Red 2G (purity 72%) was orally administered in aqueous
solution to rats and rabbits, the urine was collected free from faeces
over periods of 24 hours and the daily excretion of Red 2G and its
metabolites measured. No intact dye could be found in the urine or any
hydroxylated derivatives thereof. P-aminophenol was found without
preliminary acid hydrolysis. P-aminophenol was also found as the only
component after acid hydrolysis of the glucuronic acid fraction. The
total amount of p-aminophenol was estimated after hydrolysis with N and
5N HCl. The former hydrolysis both N-acetyl-
p-aminophenol and p-amino-phenylsulfate; the latter, in addition,
hydrolysis p-aminophenol glucuronide. Forty-eight hours after oral
administration of 216 mg of the dye to rats 49% of the dye excreted was,
after hydrolysis, found as p-aminophenol; this was 45 and 55% after oral
feeding of 720 mg of the dye to rabbits. 5-5.6% and 2% of the dye was
excreted as o-aminophenol and aniline, respectively 24 hours after
administration of 1 g Red 2G to rabbits. After administration of 1 g Red
20 to rats 0.26% of the dye was excreted as o-aminophenol (Daniel,
1958).
When rabbits were fed 0.5 g per kg bodyweight of Red 2G the
following metabolites could be identified in urine over a period of
48 hours: total p-aminophenol 46%, p-aminophenylglucuronide 37% and
aniline 0.6% and o-aminophenol 9% (Daniel, 1962). The ratio of
o-aminophenol to p-aminophenol was the same for rabbits fed Red 2G and
rabbits fed aniline previously examined (Parke, 1960) indicating that
hydroxylation does not necessarily precede fission of the azolinkage.
In another experiment 250 mg/kg body weight of Red 2G was
administered by gastric intubation to five male and five female rats. On
average the males excreted 61.8% of the dose in the urine and the
females 71.5%. 42.2% of the dose was excreted in the urine as
p-aminophenol in 48 hours, 9.2% as aniline in 24 hours and 3% as
unreduced dye in 24 hours. The corresponding faecal excretion was 6.3%,
1.0% and 0.1%. For females urinary excretion amounted to 56.4% as
p-aminophenol in 48 hours, 2% as aniline in 24 hours and 2.6% as
unreduced dye in 24 hours. The corresponding faecal excretion was
8.6%, 0.3% and 1.6% (Walker, 1971).
In vitro studies were conducted on samples of mouse, rat and
human blood with 100 and 200 mg% concentration of Red 2G or
acetylphenyl-hydrazine as a positive control. No Heinz bodies were seen
with Red 2G (Rofe, 1955). No binding of Red 2G to serum protein occurs
(Jenkins et al., 1966a).
The contents of a rat caecum were incubated at 37°C with a solution
of Red 2G in isotonic saline. At one hour intervals a sample of the
incubate was filtered and Red 2G was estimated in the filtrate by
measuring the optical density. Two metabolites of Red 2G were detected
in the incubation mixture after separation by thin-layer chromatography
on silica plates. One was 2-amino-8-acetamido-1-naphthol-3,6-disulfonic
acid, the other, aniline, was detected using two different solvent
systems. When Red 2G was incubated at 37°C with liver homogenate the
same two metabolites were detected (Jenkins et al., 1966b).
When a mixture of Red 2G and caecal contents were incubated at 37°C
darkening at the surface was observed. This was attributed to oxidation
of a metabolite of Red 2G (Jenkins et al., 1966b).
Two groups of 12 rats were fed purified diet and a purified diet
containing 0.51% Red 2G respectively. Faeces were collected for four
days from day 16 and it was calculated that 48.2% of the sulfur derived
from Red 2G was excreted in the faeces (Jenkins et al., 1966b).
TOXICOLOGICAL STUDIES
Special studies on reproduction
Two groups of 46 male and 46 female rats were fed 0.2% Red 2G in
their diet for 18 weeks and then mated for 10 days. The progeny were
weaned on the same diet and mated at 16 weeks and the F2 generation was
also weaned on the same diet. No adverse effects were seen on litter
size, litter weight and weaning weight nor were there any abnormalities
at autopsy (Robinson et al., 1971).
Special studies on the metabolites
In rats fed purified diets containing 0.1%, 0.2% and 0.3% Red 2G
for two weeks there was a linear relationship between intake of Red 2G
and relative spleen weight. In rats fed purified diets containing
0.004%, 0.008% and 0.012% phenylhydroxylamine for two weeks there was
also a linear relationship between intake of phenylhydroxylamine and
relative spleen weight. For individual samples of rat blood and human
blood the amount of oxidation of haemoglobin to methaemoglobin was
linearly related to the logarithm of phenylhydroxylamine concentration.
From the dose-response curve it has been estimated
that for rat blood the no-effect dose is between 0.5 and 1 µg
phenylhydroxylamine/ml blood. The response of human blood to
phenylhydroxylamine was more variable than the response of rat blood.
The no-effect concentration of phenylhydroxylamine in vitro for human
blood ranged from 0.46 to 4.1 µg/ml. At all levels of Red 2G fed to rats
the proportion of Red 2G metabolized to phenyl-hydroxylamine was
constant (Jenkins et al., 1966e, 1967; Gellatly and Burrough, 1966,
1967).
Acute toxicity
LD50
Animal Route (g/kg bw) Reference
Mouse Oral 7.35 Anonymous, 1974
i.p. 4.76 (4.13-5.85) Anonymous, 1974
i.p. 3.0-4.0 Anonymous, 1965
Rat i.p. 6.35 (5.62-7.17) Anonymous, 1974
Oral >5.0 Anonymous, 1974
Guinea-pig Oral 4.81 (3.16-7.35) Anonymous, 1974
i.p. 3.00 (1.83-4.91) Anonymous, 1974
Rabbit Oral >5.0 Anonymous, 1974
Chicken Oral >10.0 Anonymous, 1974
A dose of 5 g/kg body weight was administered on each of two
successive days to a rabbit weighing 3.8 kg and a dose of 25 g/kg body
weight was administered on each of two successive days to a rabbit
weighing 4.3 kg. No signs of toxicity were observed and their red cells
contained no Heinz bodies (Anonymous, 1974).
Histological studies in rats, rabbits and guinea-pigs dosed as
given above showed extensive renal necrosis. In mice dosed orally with
Red 2G there was gross leptomeningeal vascular engorgement and focal
sub-arachnoid haemorrhage (Anonymous, 1974).
Short-term studies
Mouse
Red 2G was fed for six weeks to five groups of 10 mice at dietary
levels of 0, 0.02, 0.1, 0.5 and 1.0%, The toxic effects observed were
development of Heinz bodies, methaemoglobinaemia, splenic enlargement,
accelerated splenic erythropoiesis and increased levels of haemosiderin
in splenic macrophages. No toxic effects were observed in mice fed the
diet containing 0.02% (Gellatly et al., 1968).
Groups of 15 male and 15 female mice were given diets containing
0.0, 0.01, 0.1, 1.0 and 2.0% Red 2G for three months. Five mice of each
sex at each dose level were killed at 26, 55 and 96 days. No adverse
effects on growth or food consumption were found. At 0.1, 1.0 and 2.0%
a dose-related decrease in Hb, RBC and PCV was seen, with some
compensation for red cell loss at three months. A dose-related increase
in reticulocytes, Heinz bodies, anisocytosis, polychromasia,
poikilocytosis and marrow reaction was seen. Considerable amounts of
colouring were found in the upper gastrointestinal tract at both 1.0 and
2.0% in both sexes and at the 0.1% level in males: the bile duct
contained a dark brown fluid. Enlargement and congestion of the spleen
was observed in both sexes at the 2.0% level and at the 1.0% level in
females throughout the test. A temporary effect on the relative spleen
weight was seen at 0.1 and 1.0% in both sexes at 26 and 55 days. The
relative liver weights were constantly significantly increased in
females at 2% and scattered, significant increases in relative liver
weights affected both males and females at 0.1 and 1.0%. Significant
increases in kidney weight were also noted, being temporary at days 26
and 55 at the doses of 0.01, 0.1 and 1.0% but in females present at 2.0%
at 96 days. Hyperplasia and excessive haemosiderin were found in the
spleen of both sexes, together with an increase in round cell
infiltration and pigment in Kupffer's cells in the liver (Anonymous,
1965).
Rat
Groups of five rats (Carworth Farm E) were given diets containing
0.0, 1.0, 2.5 and 5.0% Red 2G for 90 days. Retarded growth associated
with reduced food consumption was seen at 2.5 and 5.0%. A reduction of
both Hb and PCV and macrocytosis, reticulocytosis and polychromasia were
evident at 5.0% with circulating normoblasts and a normoblastic marrow.
At 1% the red cells were of mixed size with increased polychromasia.
Heinz bodies were present at all concentrations. Further there was a
significant increase in relative weight of spleen in all dome groups and
of kidney at 2.5 and 5% (Anonymous, 1965).
Groups of five male and five female rats were given diets
containing 0.0, 0.05, 0.1, 0.5, 1.0 and 2,0% Red 2G for three weeks.
Signs of increased erythropoiesis and enlargement and darkening of the
spleen could be seen in animals at all levels down to 0.1%, and even at
0.05% some of the adverse effects could be noticed. The spleen was
significantly enlarged at all dietary levels except 0.05% and a
significant increase in relative weight of kidneys was seen at 0.5, 1.0
and 2.0%. Increased haemosiderin was found in the liver, spleen and
kidneys (Anonymous, 1965).
Groups of 10 male and 10 female rats were given diets containing
0.0, 0.01, 0.05 and 1% Red 2G for two months. Signs of erythropoiesis
were seen at 0.1% and splenomegaly with congestion in some animals at
0.05 and 0.1% (Anonymous, 1965).
Rats were fed 1-1.5 g/kg/day Red 2G for 75 days. The mean peak
Heinz body level was 80% falling to a maintained level of 30%. Internal
changes included a moderate though well controlled anaemia, pronounced
reticulocytosis, and splenomegaly (Rofe, 1957).
Three groups o£ 12 rats received Red 2G in the drinking-water at
levels of 0%, 0.1% and 0.5% for 100 days. Heinz bodies were seen after
10 days in the red cells of rats fed 0.5% Red 2G in the drinking-water,
fewer were seen after 18 days and none or very few on later occasions.
A few Heinz bodies were occasionally seen in the red cells of some of
the rats fed 0.1% Red 2G in the drinking-water. The spleens of rats fed
0.1% Red 2G were slightly larger than controls and the spleens of rats
fed 0.5% Red 2G were very much larger than controls, Histological
examination of livers of rats fed 0.5% Red 2G revealed an increase in
haemosiderin present in Kupffer's cells and increased erythropoietic
activity. Histological examination of the spleens of rats fed 0.5% Red
2G also revealed increased erythropoiesis and red pulp engorgement.
There was no effect of Red 2G on urine specific gravity (Jenkins et al.,
1966d; Gellatly et al., 1966).
Four groups of 24 male and 24 female rats were fed a diet
containing purified diet, cooked sausage without colour, cooked sausage
with 30 and 80 ppm Red 2G. The cooked sausage was mixed so that the
levels of Red 2G were equivalent to 0, 24 and 114 ppm. The feeding was
continued for 13 weeks. Food intake and body weight gain were recorded
weekly. After nine weeks a liver function test (bromosulfthalein
retention test) was carried out. After 12 weeks the refractive index of
the urine was measured. Just before the end of the experiment a detailed
haematological investigation was done. In the beginning of the
experiment after 10-11 days the presence of Heinz bodies was controlled.
At post-mortem the weights of the spleen, kidneys, liver, heart and
testes were determined. The diets containing Red 2G in sausage meat had
no effect on growth, organ function or organ weights. Blood tests also
revealed no evidence of toxicity. Histological examination revealed
that, in the spleens of rats fed
144 ppm Red 2G with sausage meat, there was increased erythropoiesis,
increased splenic red pulp haemosiderin and increased splenic red pulp
reticular impregnation with iron. No effects on spleen were seen in rats
fed 24 ppm Red 2G with sausage meat. Red 2G at 24 ppm and at
144 ppm in sausage meat had no detectable histological effect on liver.
There was some evidence that the diet provided in this experiment was
not totally adequate. There was thyroid hyperplasia and loss of hair in
many rats. In this experiment 144 ppm of Red 2G had still an effect
(Jenkins, 1966c; Gellatly and Burrough, 1968).
Long-term studies
Mouse
Groups of 40 male and 40 female mice (Colworth C57B1) were fed
diets containing 0, 0.005, 0.025, 0.125 and 0.625% Red 2G for 80 weeks.
Growth, food intake and survival were recorded. Blood tests were
performed after three, seven and 80 weeks for the presence of Heinz
bodies and at the end of the test also for values of Hb, Ret, WBC,
differential cell counts and methaemoglobin. Serum and urine
biochemistry were investigated (for urine refractive index Ph, glucose,
protein and urea nitrogen). For liver function SGOT, SGPT and SLDH were
estimated. The absolute organ weight and the histology of the brain,
heart, liver, spleen, kidneys and testes were determined. Heinz bodies,
increased methaemoglobin, splenic darkening and enlargement, increased
splenic haemosiderin content and accelerated splenic erythropoiesis were
seen in mice fed dietary levels of 0.125 and 0.625% of Red 2G. There was
no evidence of carcinogenicity attributable to the feeding of Red 2G to
mice. More than three-quarters of the animals in each group survived for
two years (Jenkins et al., 1971a; Gellatly et al., 1970a, b; Gellatly
and Marlow, 1970a).
Rat
Groups of 40 male and 40 female rats (Colworth wistar) were fed
diets containing 0, 0.004, 0.016, 0.064 and 0.16% Red 2G for two years.
Growth, food intake and survival were recorded. Blood tests were
performed after 11 and 40 days for packed cell volumes and the presence
of Heinz bodies and after two years' feeding also for values of Hb, Ret,
WBC and differential cell counts. Serum biochemistry
(Na, K, Cl, urea nitrogen, creatinine, SGPT, SGOT, SLDH and alkaline
phosphatase) was investigated. The absolute organ weight and the
histology of the brain, heart, liver, kidneys, spleen, pituitary and
adrenals was determined. Splenic darkening and enlargement, with
degeneration of splenic elastica, were seen in rats fed dietary levels
of 0.064 and 0.16% Red 2G. More than half the animals survived for two
years (Jenkins et al., 1971a, b; Gellatly et al., 1970b, c; Gellatly and
Marlow, 1970b; Billing and Jenkins, 1968; Robinson, 1968; Groger and
Robinson, 1969).
REFERENCES
Anonymous (1965) Acute and short-term feeding studies on Red 2G with
associated haematological investigations, British Industrial Biological
Research Association, Research Report 3/1965. Unpublished report
submitted to WHO
Anonymous (1974) Acute toxicity by oral intubation, intraperitoneal
injection of Red 2G in several animal species. Summary of toxicological
data prepared by J. M. Philp, Research Division, Unilever Ltd, submitted
to WHO by Unilever Ltd
Billing, C. C. and Jenkins, F. P. (1968) Chronic toxicity of Red 2G;
2-year rat feeding trial (0 and 0.5% Red 2G), biochemical studies after
feeding for 180 days. Unpublished report from Unilever Research
Laboratories, submitted to WHO by Unilever Ltd
Daniel, J. W. (1958) The metabolism of azo dyes. V. Azo-geramine
(benzeneazo-8-acetamide-l-naphthol 3; 6-disulphonic), October 1958,
Report No. IHR/126. Unpublished report from Industrial Hygiene Research
Laboratories, submitted to WHO by Imperial Chemical Industries (ICI)
Daniel. J. W. (1962) The excretion and metabolism of edible colours,
Toxic Appl. Pharmac., 4, 572
Gellatly, J. B. M., Salmond, G. and Burrough, R. (1966) Sub-acute
toxicity of Red 2G; pathology. Unpublished report from Unilever Research
Laboratories, submitted to WHO by Unilever Ltd
Gellatly, J. B. M., Jenkins, F. P., Salmond, G., Robinson, J. and
Marlow, K. (1968) Sub-acute toxicity of Red 2G in mice. Unpublished
report from Unilever Research Laboratories, submitted to WHO by Unilever
Ltd
Gellatly, J. B. M., Conniff, R. A. and Lovell, M. J. (1970a) Chronic
toxicity of Red 2G; 2-year rat feeding trial (0-0.16% Red 2G),
macroscopic pathology of rats killed at termination of the trial,
Unpublished report from Unilever Research Laboratories, submitted to WHO
by Unilever Ltd
Gellatly, J. B. M., Lovell, M. J. and Conniff, R. A. (1970b) Chronic
toxicity of Red 2G: 80 week mouse feeding trial, macroscopic pathology
of mice killed at termination of the trial. Unpublished report from
Unilever Research Laboratories, submitted to WHO by Unilever Ltd
Gellatly, J. B. M., Marlow, K. and Salmond, G. (1970c) Chronic toxicity
of Red 2G; 80 week mouse feeding trial, pathology of mice dying or
killed during the trial. Unpublished report from Unilever Research
Laboratories, submitted to WHO by Unilever Ltd
Gellatly, J. B. M., Marlow, K. and Salmond, G. (1970d) Chronic toxicity
of Red 2G; 2-year rat feeding trial (0-0.16% Red 2G), pathology of rats
dying or killed during the trial. Unpublished report from Unilever
Research Laboratories, submitted to WHO by Unilever Ltd
Gellatly, J. B. M. and Burrough, R. (1966) Effect of diets containing
aniline and metabolites of aniline on spleen weights; pathology.
Unpublished report from Unilever Research Laboratories, submitted to WHO
by Unilever Ltd
Gellatly, J. B. M. and Burrough, R. (1967) Pathology of Red 2G and
phenylhydroxylamine in rat diets. Unpublished report from Unilever
Research Laboratories, submitted to WHO by Unilever Ltd
Gellatly, J. B. M. and Burrough, R. (1968) Toxicity of Red 2G; effect of
feeding to rats in a sausage meat diet for 90 days; pathology.
Unpublished report from Unilever Research Laboratories, submitted to WHO
by Unilever Ltd
Gellatly, J. B. M. and Marlow, K. (1970a) Chronic toxicity of Red 2G; 80
week mouse feeding trial, histopathology of mice killed at termination
of the trial. Unpublished report from Unilever Research Laboratories,
submitted to WHO by Unilever Ltd
Gellatly, J. B. M. and Marlow, K. (1970b) Chronic toxicity of Red 2G;
2-year rat feeding trial (0-0.16% Red 2G), histopathology of rats
killed at termination of the trial. Unpublished report from Unilever
Research Laboratories, submitted to WHO by Unilever Ltd
Jenkins, F. P., Campbell, P. J., Robinson, J. and Salmond, G. (1966a)
Metabolism of Red 20 (protein binding capacity). Unpublished report from
Unilever Research Laboratories, submitted to WHO by Unilever Ltd
Jenkins, F. P., Salmond, G., Campbell, P. J,, Hardy, J. and Robinson, J.
(1966b) Metabolism of Red 20 by rat liver homogenate. Unpublished report
from Unilever Research Laboratories, submitted to WHO by Unilever Ltd
Jenkins, F. P., Salmond, G. and Gellatly, J. B. M. (1966c) Sub-acute
toxicity of Red 20 in sausage meat. Unpublished report from Unilever
Research Laboratories, submitted to WHO by Unilever Ltd
Jenkins, F. P., Salmond, G., Robinson, J., Campbell, P. J. and Gellatly,
J. B. M. (1966d) Sub-acute toxicity of Red 20. Unpublished report from
Unilever Research Laboratories, submitted to WHO by Unilever Ltd
Jenkins, F. P., Robinson, J., Gellatly, J. B. M., Salmond, G. and
Campbell, P. J. (1966e) Effect of diets containing aniline and
metabolites of aniline on spleen weights. Unpublished report from
Unilever Research Laboratories, submitted to WHO by Unilever Ltd
Jenkins, F. P., Robinson, J., Gellatly, J. B. M. and Salmond, G. (1967)
Effect of Red 20 and phenylhydroxylamine on spleen weight. Unpublished
report from Unilever Research Laboratories, submitted to WHO by Unilever
Ltd
Jenkins, F. P., Robinson, J., Salmond, G. and Groger, W. (1971a) Chronic
toxicity of Red 2G; 80 week mouse feeding trial, living animal studies.
Unpublished report from Unilever Research Laboratories, submitted to WHO
by Unilever Ltd
Jenkins, F. P., Robinson, J., Salmond, G. and Groger, W. (1971b) Chronic
toxicity of Red 20; 2-year rat feeding trial (0-0.16% Red 20), living
animal studies. Unpublished report from Unilever Research Laboratories,
submitted to WHO by Unilever Ltd
Parke, D. V. (1960) Biochem. J., 77, 494-503
Priestley, G. and O'Reilly, W. J. (1966) Protein binding and the
excretion of some azo dyes in rat bile, J. Pharm. Pharmacol., 18,
41-45
Robinson, J. (1968) Chronic toxicity of Red 20; 2-year rat feeding trial
(0 and 0.5% Red 2G), biochemical studies after feeding for
200 days. Unpublished report from Unilever Research Laboratories,
submitted to WHO by Unilever Ltd
Robinson, J. and Paragreen, G. (1971) Reproduction in rats fed Red 2G.
Unpublished report from Unilever Research Laboratories, submitted to WHO
by Unilever Ltd
Rofe, P. (1955) Induction of Heinz bodies by edible azo dyes in the rat.
Unpublished report from Industrial Hygiene Research Laboratories,
submitted to WHO by Imperial Chemical Industries (ICI)
Rofe, P. (1957) Azo dyes and Heinz bodies, Brit. J. industr. Med.,
14, 275-280
Ryan, A. J. and Wright, S. E. (1961) The excretion of some azo dyes in
rat bile, J. Pharm. Lond., 13, 492
Walker, R. (1971) Private communication to E. M. den Tonkelaar, National
Institute of Public Health, Bilthoven, Netherlands