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.



         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).


    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

    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

    Short-term studies


         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).


         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.,

    Long-term studies


         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).


         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.


    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.


    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,

    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)