BRILLIANT BLACK PN
Explanation
This food colourant was evaluated by JECFA in 1974 and allocated
a temporary ADI of 0-2.5 mg/kg bw; further work required included
metabolic studies and adequate reproductive and embryotoxicity
including teratological studies. The Committee reviewed the position
again in 1978 and the required data was not available. In addition,
the Committee felt that the etiology and pathology of intestinal cysts
observed in a 90-day study in pigs should be determined.
Since the previous review further data have become available and
is incorporated into this monograph.
BIOLOGICAL DATA
BIOCHEMICAL ASPECTS
At concentrations of 2 to 400 mg/l the colour inhibits pepsin but
not lipase (Diemair & Mausser, 1951) and at 12.5 mg/l it inhibits
trypsin inconsistently (Diemair & Boeckhoff, 1953). Intravenous
injection into rabbits and dogs of 50 mg/kg produced only small
amounts in the urine (Hecht, 1980). Heated in the presence of reducing
sugars the colour is partially decomposed and gives an orange
derivative, isolated by paper chromatography, which is the disodium
salt of 4'-(4-sulfo-1-phenylazo)-1'-amino-7'-sulfonaphthalene (Saenz
Lascano Ruiz & Laroche, 1960).
Rats were given Brilliant Black PN. During the experimental work,
the faeces and urine of the animals were collected and the presence of
the dye was noted in faeces and not in the urine. Quantitative
determination of the dye in the faeces indicated that, from the total
amount of the dye administered to the rats, 0.6% was excreted in the
faeces (Piekarski, 1960).
Three male and two female Wistar rats were orally intubated with
the colourant labelled on the Cleve's Acid moiety with 14C. One male
and one female rat were accustomed to a diet containing 3% unlabelled
colour prior to dosing. The dose administered varied from 1-6 mg/kg bw
for the nonhabituated animals and 8-10 mg/kg for animals pretreated
with the colour in the diet. Radioactivity was excreted mainly in the
faeces (94-98%), with less than 5% in urine, within 40 hours. After 40
hours the gastrointestinal tract contained 0-0.6% of the administered
activity; trace amounts were detected in kidneys and residual
carcasses and no measurable activity was found in liver and blood. TLC
analysis showed the presence of one major and several minor
metabolites in the faeces and two major and at least four minor
metabolites in urine. It was concluded that Brilliant Black PN was
virtually completely degraded by the gut flora of the rat and
metabolites containing the Cleve's Acid moiety were poorly absorbed
from the gastrointestinal tract. Habituation to Black PN did not alter
excretion or metabolite patterns (Anon., 1980).
The metabolism of Black PN was investigated qualitatively and
quantitatively in rat and man. After oral administration of 20-100 mg
of the colourant to rats (body weight 300-400 g), sulfanilic acid (SA)
and 4-acetamido-1-naphthylamine-6- or 7-sulfonic acid (ANSA) were
detected in the urine; both of these metabolites together with
unchanged Black PN (traces), 1,4-diaminonaphthalene-6-sulfonic acid
(DSA) and 8-acetamido-1-hydroxy-2-naphthylamine-3,5-disulfonic acid
(AHNDA) were detected in the faeces. After oral doses of 240 mg of
Black PN in man, SA was the only metabolite identified in urine;
faecal examination was not carried out.
Following i.p. administration of 10-100 mg to rats, unchanged
Black PN (traces), 1-(4'-sulfophenylazo)-4-naphthylamine-6-sulfonic
acid (SNSA), SA, DSA, ANSA and AHNDA were excreted in the urine and
all but Black PN and SNSA in the faeces. The excretion of Black PN and
SNSA in the bile followed oral or intravenous administration of the
colourant and ANSA was also excreted in the bile following intravenous
dosing. Differences in metabolism between the oral and i.p.
routes coupled with the finding of Black PN, SNSA and SA in the
gastrointestinal tract of rats dosed orally with Black PN indicated
that the gut microflora cleaved both azo links in Black PN whereas
rat-liver azo reductase preferentially attacks the azo group linking
the two naphthalene rings.
Quantitative estimations were limited to SA, ANSA, SNSA and Black
PN. The amount of SA excreted in urine and faeces indicated that the
reduction of the azo-link between the benzene and naphthalene rings
was virtually complete, the remaining intact azo group being accounted
for by SNSA. The amount of SA in the urine was similar in the rat and
man (Ryan & Welling, 1970).
TOXICOLOGICAL STUDIES
Special studies on haematology
Five rats were given 1.5 g/kg bw per day orally for 22 days and a
cat was fed 0.1 g/kg bw per day for seven days. No Heinz bodies were
produced in either species (Deutsche Forschungsgemeinschaft, 1957).
Special studies on mutagenicity
The colour was tested for mutagenic effect in a concentration of
0.5 g/100 ml in cultures of Escherichia coli. No mutagenic effect
was found (Luck & Richerl, 1960).
Brilliant Black PN was nonmutagenic in liquid fluctuation assays
using an E. coli strain sensitive to base substitutions and a
Salmonella typhimurium specific for frame shifts. The colour did not
produce DNA damage to a repair deficient strain of E. coli, with or
without microsomal activation (Haveland-Smith & Combes, 1980).
Special studies on reproductive toxicology
A multigeneration study was performed on Wistar rats in which the
colourant was administered in the diet at concentrations of 0, 0.1,
1.0 or 3% for three successive generations. One litter was reared from
each of the F0, F1 and F2 parents. Each generation started with 60
animals of each sex in the control group and 40 of each sex in the
test groups. After a nine-week test period, 24 males and 24 females
from the control group, and 14 males and 14 females from each test
group were used for teratogenicity studies; the remainder were used
for the reproduction study. No adverse effects were observed with
respect to fertility, litter size and weight, general condition,
male/female ratio, growth during lactation, survival or maturation.
Autopsy of parent rats and pups at weaning did not reveal any
treatment related changes in organ weights other than caecal
enlargement in the 3% dose group. Gross and microscopic examination of
the F3 generation at weaning did not reveal any abnormalities due to
treatment and no adverse effects were seen in the teratology study. It
was concluded that Brilliant Black PN did not exert any adverse
effects on reproductive function of Wistar rats when fed at dietary
levels up to 3% (1500 mg/kg bw per day) for three successive
generations (Koetner & Dreef-van der Meulen, 1980).
Special studies on sensitization
In an experiment with guinea-pigs it was found that Black PN had
no sensitizing activity (Bar & Griepentrog, 1960).
Special studies on teratogenicity
In a preliminary study, Brilliant Black PN was administered to
four groups of 15 pregnant SPF-derived Wistar rats by gavage at dose
levels of 0, 250, 500 or 2500 mg/kg bw from day 0-19 of pregnancy. In
a second study, involving four groups of 30 pregnant rats, a similar
protocol was used. On day 21, the animals were killed and ovaries and
uterus removed. The number of corpora lutea in each ovary was recorded
and the foetuses examined. Live foetuses, embryonic and foetal
resorptions and dead foetuses were counted and the number and position
of implantation sites were recorded. In the second study, half the
foetuses in the control and top dose groups were examined for skeletal
malformations and half for visceral defects. No abnormalities in
condition or behaviour of the dams were observed in either study. At
autopsy, no signs of embryo-toxicity or teratogenicity were observed.
One foetus in the top dose group showed a complexity of malformations
but this was considered to be a fortuitous finding. It was concluded
that ingestion of Brilliant Black PN at doses up to 2500 mg/kg bw was
without teratogenic effect in the rat (Koeter, 1979).
Acute toxicity
LD50
Animal Route (mg/kg bw) Reference
Mouse Oral >5 000 DFG, 1957
>2 000 Gaunt et al., 1967
i.p. 500-1 000 Gaunt et al., 1967
Rat Oral >5 000 Gaunt et al., 1967
i.p. 1 100 Gaunt et al., 1967
>2 000 DFG, 1957
i.v. 2 500 DFG, 1957
approx.
Short-term studies
Rat
Groups of 16 male and 16 female weanling rats were fed diets
containing 0, 0.3, 1.0 and 3.0% colour for 90 days. Growth retardation
associated with diminished food intake was evident only in males at
the 3% level. This was shown by a paired feeding test. Haematological
examination, liver and kidney function tests were normal. Organ weight
of testes and kidneys increased in males at the 3% level only. No
untoward histopathological findings were seen (Gaunt et al., 1967).
Pig
Groups of three male and three female 10-week-old pigs were dosed
orally at levels of 0, 100, 300 and 900 mg/kg per day for 90 days. The
colourant was administered mixed with a quantity of diet and syrup
prior to the main feed. No adverse effects were observed on growth,
haematology, urine analyses, serum transaminase activities or organ
weights. Cysts containing mucus and fibrin were found in the mucosa of
the ileum of four pigs given 900 mg/kg per day and one given 300 mg/kg
per day. It was suggested that this might have been due to an irritant
effect of local high concentrations of the colourant (Gaunt et al.,
1969).
Long-term studies
Rat
Sixteen rats were fed Black PN at 0.1% of the diet (average daily
intake 0.06 g/kg bw) for 410 days and were observed for 761 days. The
total dose per animal was 5.6 g. One rat died prematurely. No tumours
were observed (Hecht & Wingler, 1952; DFG, 1957).
Another group of 10 rats was given 0.5% Black PN in their
drinking-water (average daily intake 0.5 g/kg bw) for 384 days and
observed for 545 days. Total intake per animal was 20 g. No tumours
were seen (DFG, 1957). In a second experiment a group of 10 rats was
again given 0.5% Black PN in their drinking-water (average daily
intake 0.46 g/kg bw) for 502 days and observed for 923 days. Total
intake per animal was 50 g. No tumours were noted (DFG, 1957).
A group of 10 rats received twice weekly subcutaneously 0.5 ml of
a 1% solution (= 5 mg) for 365 days and was observed for 653 days. The
total amount per animal was 0.5 g. Two animals died prematurely but no
tumours were noted (DFG, 1957).
Groups of 24 male and 24 female weanling rats were fed for two
years on diets containing 0, 1000, 5000 or 10 000 ppm (0, 0.1, 0.5 or
1%) Brilliant Black PN. No effects attributable to treatment were
found in respect to mortality, food intake, body weight gain,
haematology, blood serum, chemistry, renal concentration tests, organ
weights or incidence of pathological findings, including tumours
(Gaunt et al., 1972).
Comments
The teratogenicity and multigeneration studies did not reveal any
toxicologically significant effects.
No further work was available on the etiology and pathology of
intestinal cysts which occurred in high dose level feeding studies in
pigs, therefore the ADI has been allocated on the basis of the no-
effect level in the pig.
EVALUATION
Level causing no toxicological effect
Mouse: 1% in the diet, equivalent to 1300 mg/kg bw
Rat : 1% in the diet, equivalent to 500 mg/kg bw
Pig : 100 mg/kg/day p.o.
Estimate of acceptable daily intake for man
0-1.0 mg/kg bw.
REFERENCES
Anon. (1980) Summary of report of TNO, Zeist submitted to WHO by the
EEC Colours Group
Bar, F. & Griepentrog, G. (1960) Die Allergenwirkung von freinden
Stoffen in den Lebensmitteln, Med. u. Ernahr., 1, 99-104
Deutsche Forschungsgemeinschaft-Farbstoff-Kommission (1957) Mitteilung
6, 2. Auflage. Toxikologische Daten von Farbstoffen und ihre
Zulassung für Lebensmittel in verschiedenen Landern, Wiesbaden,
Franz Steiner Verlag GmbH, p. 58
Diemair, W. & Hausser, H. (1951) Synthetic dyes and enzyme reactions,
Z. Lebensmittelunters. u. Forsch., 92, 165-170
Diemair, W. & Boeckhoff, K. (1953) Artificial colours and enzyme
reactions. III. Effect on trypsin and the intestinal juices,
Z. anal. Chem., 139, 35-42
Gaunt, I. F. et al. (1967) Acute (mouse and rat) and short-term (rat)
toxicity studies on Black PN, Fd. Cosmet. Toxicol., 5,
171-177
Gaunt, I. F. et al. (1969) Short-term toxicity of Black PN in pigs,
Fd. Cosmet. Toxicol., 7, 557-563
Gaunt, I. F. et al. (1972) Long-term feeding study on Black PN in
rats, Fd. Cosmet. Toxicol., 10, 17-27
Haveland-Smith, R. B. & Combes, R. D. (1980) Screening of food dyes
for genotoxic activity, Fd. Cosmet. Toxicol., 18, 215-221
Hecht, G. & Wingler, A. (1952) Biological study and suitable
chemical constitution of some azo dyes for food colouring,
Arzneimittel-Forsch., 2, 192-196
Koeter, H. B. W. M. (1979) Teratogenicity study with Brilliant Black
BN (E151) in rats. Rapport Nr. R6106. Unpublished report of TNO
Zeist, submitted to WHO by EEC Colours Group
Koeter, H. B. W. M. & Dreef-van der Meulen, H. C. (1980) Multi-
generation diet study with Brilliant Black BN (E151) in rats.
Rapport Nr. R6417. Unpublished report of TNO Zeist, submitted to
WHO by EEC Colours Group
Luck, H. & Richerl, E. (1960) Food additives and mutagenic effects
- 6th Report. Examination of food dyes allowed and first
suggested in West Germany for mutagenic effects on Escherichia
coli, Z. Lebensmittelunters. u.-Forsch., 112, 157
Piekarski, L. (1960) Absorption of brilliant black by the alimentary
canal of the rat, Roczn. panst. Zakl. Hig., 128, 351-356
Ryan, A. J. & Welling, P. G. (1970) The metabolism and excretion of
Black PN in the rat and man, Fd. Cosmet. Toxicol., 8, 487-497
Saenz Lascano Ruiz, I. & Laroche, C. (1960) The reduction of Brilliant
Black BN and the formation of monoazo dyes, Ann. Fals. Exp.
Chim., 53, 581-592
See Also:
Toxicological Abbreviations
Brilliant black PN (WHO Food Additives Series 6)
Brilliant black PN (WHO Food Additives Series 13)
BRILLIANT BLACK PN (JECFA Evaluation)