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
BENZYL VIOLET 4B
Explanation
This food colour has been evaluated for acceptable daily intake
by the Joint FAO/WHO Expert Committee on Food Additives (see Annex I,
Refs 8 and 10) in 1964.
EVALUATION FOR ACCEPTABLE DAILY INTAKE
BIOLOGICAL DATA
BIOCHEMICAL ASPECTS
Rats received orally 20 or 200 mg benzyl violet dissolved in
10 ml of water. Urine and faeces were collected during 36 hours. With
the faeces respectively 90 and 98% of the administered doses was
excreted. In the urine no colouring could be found (Hess and Fitzhugh,
1955).
Less than 5% of the administered benzyl violet was found in the
bile of dogs after an oral administration of 200 mg per animal (Hess
and Fitzhugh, 1955). Other aspects are discussed by Minegishi and
Yamaha (1977).
Despite low degree of absorption, unlike some colourings, violet
6B does not have a cathartic effect (Radomski and Deichmann, 1956).
Protein-binding
Nature of interaction between serum proteins and violet 6B was
investigated by electrophoresis.
Electropherogram on cellulose acetate showed migration to the
anode in a single band. The complex between serum proteins and violet
6B were not separated by electrophoresis on cellulose acetate or
polyacrylamide gel, but could be separated by filtration on Sephadex
gel (Gangolli et al., 1972).
TOXICOLOGICAL STUDIES
Acute toxicity
Species Route LD50 mg/kg bw References
Mouse oral 4 000 Tullar, 1947
Mouse i.p. 432 Tullar, 1947
Mouse i.v. 141 Tullar, 1947
Rat oral 2 000 Lu et al., 1964
Rabbit i.p. 500 Tullar, 1947
Rabbit i.v. 150 Tullar, 1947
Short-term studies
Rat Groups of 15 male and 15 female rats (strain Wistar) were
given diets containing 0 (control), 0.5, 1.0 or 3% violet 6B (purity
min. 85%) for 13 weeks. There was marked excretion of the colour in
the faeces indicating a low level of absorption. No statistically
significant effects were noted in body weight gain, haematologieal
values, serum or urine analysis or renal function tests. There were no
adverse histopathological findings attributable to the feeding of the
colouring. The weights of heart, liver and coecum were lower in the
rats given 3% of the dye than the controls and the relative kidney
weights were higher. The no-effect level in this study was 1% (Gaunt
et al., 1974).
Dog
Groups of two male and two female beagles were fed at levels of
0.5, 1.0 and 2.0% of the colour in the diet for two years. Of each
group one dog died during the experiment. No pathological changes to
account for death could be found. Dose-related changes were a tan
non-icteric tinge of the subcutaneous tissue and (microscopically
confirmed) hyperkeratosis of the ears (USFDA, 1964) (no details
available).
Long-term studies
Mouse
Groups of 48 male and 50 female mice (strain ASH-CS1) were fed
diets containing 0, 70, 700 or 3500 ppm violet 6B (purity min. 85%)
for 80 weeks. The faeces of all treated mice were stained but the
urine appeared normal suggesting little absorption from the GI tract.
There were no effects on mortality, rate of body weight gain or
haematology at 12, 28, 52 and 80 weeks. No adverse findings including
tumours were seen in treated compared with control animals. Except in
the females with 700 and 3500 ppm a slight increase in lymphosarcoma
in the thymus was found. Whether this finding is relating with the
administration of the dye is not known. It was concluded that feeding
up to 3500 ppm did not exert carcinogenic effect (Grasso et al., 1971,
1974).
Rat
Eighteen young rats were given 20 mg of the colour (2% aqueous
solution) by subcutaneous injections once a week for two years; 14
fibrosarcomas were induced (Nelson and Davidow, 1957).
Four groups of 15 male and 15 female rats (strain Wistar)
received during 75 weeks respectively 0 (control), 0.03, 0.3 and 3.0%
of benzyl violet (purity not given) in their diet. No depression of
growth, food consumption or food efficiency was found. Haematology did
not show abnormalities. Seven males and four females of the 0.03%, one
male and three females of 0.3% and four males and seven females of 3%
level died (in control group six males and one female), 3/7 females on
3% level that died during the experiment showed tumours (none in the
controls). Five male and five female rats per group were examined
histologically after 75 weeks. 1/5 male and 4/5 female rats of 3%
level showed malignant tumours (one male at controls). The incidence
of the skin tumours in rats of the 3% level was 20% (possibly from
contact of the fur and skin of the rats with the food) (Mannell et
al., 1962).
Groups of 15 litter-mated male rats were fed the colour at 0.1%,
0.5% and 1.0% in the diet for two years. The only pathological effect
noted was slight dilation of the gastric glands at the 1.0% level
(FDA, 1964) (no details available).
Groups of 20 male and 20 female rats were kept in such a way that
the skin was in regular contact with the colour for 88-100 weeks.
Similar groups served as control. The experimental animals showed a
small number of ulcerative dermatitis of the scrotum, possibly as a
result of a chronic irritation caused by contact of the scrotal skin
with the floor of the cage and the colour. No malignant tumours of the
epidermis were found, but there was an increase in benign mammary
tumours (Mannell et al., 1964).
Twenty males and 20 females Sprague-Dawley rats were fed 5% of
acid violet 6B (purity 89.1%) for one year, 10 control males and 10
control females received basic diet only. Mortality was greater in
treated animals, five mammary carcinomas and four induced earduct
carcinomas were found in 18 female animals, one mammary fibroadenoma
occurred in female controls. No metastases were found. At one year
only one test male and one test female and five control females
survived. No tumours were found in the males (Uematsu and Miyaji,
1973).
Two groups of 35 female (Sprague-Dawley) rats received during 12
months respectively normal diet and a diet containing first 1%,
thereafter 3%, each for one week respectively and then maintained on
5% of benzyl violet 4B (purity 89.4%). The mortality in the test group
was increased significantly. Only two rats survived 12 months, in the
control group all rats survived. Growth of the rats of the test group
was reduced significantly, though food consumption was normal. In
22/35 rats in the experimental group, tumours developed externally.
Eleven rats with mammary gland carcinoma, four rats with squamous cell
carcinoma of the skin and seven rats with both types of tumours were
seen. Hyperplasia of sebaceous glands was observed in the skin at the
site of development of both types of tumours. The onset of tumour
development was earlier for the mammary gland carcinoma. The majority
of the squamous cell carcinomas developed in the earduct, but they
also appeared in the buccal and axillary regions. In the control group
no external tumours were noted. Macroscopy did not reveal nodules,
suggestive of metastasis of external tumours to internal organs (Ikeda
et al., 1974).
REFERENCES
Gangolli, S. D., Grasso, P., Golberg, L. and Hooson, J. (1972) Protein
binding by food colourings in relation to the production of
subcutaneous sarcoma, Food and Cosm. Tox., 10, 449-462
Gaunt, I. F., Hardy, J., Kiss, I. S. and Gangolli, S. D. (1974)
Shortterm toxicity of violet 6B (FD and C Violet no. 1) in the rat,
Food and Cosm. Tox., 12, 11-19
Grasso, P., Hardy, J., Gaunt, I. F., Mason, P. L. and Lloyd, A. G.
(1974) Longterm toxicity of violet 6B (FD and C Violet no. 1) in mice,
Food and Cosm. Toxicol., 12, 21-31
Grasso, P. et al. (1971) Food and Cosm. Toxicol., 9, 463-478
Hess, S. M. and Fitzhugh, O. G. (1955) Absorption and excretion of
certain triphenylmethane colours in rats and dogs, J. Phamac.
exp. Ther., 114, 38-42
Ikeda, Y., Horiuchi, S., Imoto, A., Kodama, Y., Aida, Y. and
Kobayashi, K. (1974) Induction of mammary gland and skin tumours in
female rats by the feeding of henzylviolet 4B, Toxicology, 2,
275-284
Lu, F. C. and Lavallee, A. (1964) Canad. pharm. J., 97, 30-
Mannell, W. A., Grice, H. C. and Allmark, M. G. (1962) Chronic
toxicity studies on food colours. V. Observations on the toxicity of
brilliant blue FCF, guinea green B and benzylviolet 4B in rats,
J. Pharm. Pharmacol., 14, 378-384
Mannell, W. A., Grice, H. C. and Dupuis, J. (1964) The effect on rats
of longterm exposure to guinea green B and benzylviolet 4B, Food and
Cosm. Tox., 2(3), 345-347
Minegishi, K. and Yamaha, T. (1977) Toxicology, 7, 367
Nelson, A. A. and Davidow, B. (1957) Fedn. Proc. Fedn. Am. Socs.
Exp. Biol., 16, 367
Radomski, J. L. and Deichmann, W. B. (1956) J. Pharmac. Exptl.
Ther., 118, 322
Tullar, P. E. (1947) Report dated 1.10.1947 from George Washington
University (unpublished report)
Uematsu, K. and Miyaji, T. (1973) Induction of tumours in rats by oral
administration of technical acid violet 6B, J. Nat. Canc. Inst.,
51, 1337-1338
United States Food and Drug Administration (1964) Unpublished report
submitted to WHO
See Also:
Toxicological Abbreviations
BENZYL VIOLET 4B (JECFA Evaluation)
Benzyl Violet 4B (IARC Summary & Evaluation, Volume 16, 1978)