Lithol rubine BK was considered at the twenty-first and
    twenty-sixth meetings of the Committee (Annex 1, references 44 and
    59), but insufficient data were available for evaluation for an ADI.
    Since that time further data have become available and are summarized


    Biochemical aspects

         No information available.

    Toxicological studies

    Acute studies

         No information available.

    Short-term studies

         No information available.

    Long-term studies


         Groups of 60 male and 60 female Charles River CD1 mice were fed
    diets containing 0.05, 1.0, or 5.0% lithol rubine BK for 104 weeks;
    two groups of 60 animals of each sex were used as controls. Individual
    body weights were recorded weekly for the first 14 weeks, biweekly
    during the next 12 weeks, and monthly thereafter. Haematological
    examinations (haemoglobin, haematocrit, RBCs, total and differential
    leucocyte counts, and reticulocyte counts) were carried out on 10
    mice/sex/group at 3, 6, 12, and 18 months. At termination of the
    study, all animals were necropsied and the weights of the brain,
    kidneys, liver, and spleen were recorded. Complete histopathological
    examination was carried out on the control and high-dose groups only.

         No significant differences were observed in food consumption,
    body-weight gain, or haematological parameters except for a depressed
    reticulocyte count in the high-dose groups relative to controls after
    18 months, although the counts were within the expected range for mice
    of that age.

         Beginning at week 64, there was a treatment-related increase in
    mortality in males, and survival was significantly reduced in the 5%
    dose group at 91 and 104 weeks; increased mortality was not observed
    in females.

         No toxicologically-significant dose-related differences in organ
    weights or gross morphology were observed. Histopathological
    examination of animals dying on test and of animals examined at
    termination revealed a variety of degenerative, inflammatory,
    proliferative, or neoplastic lesions commonly associated with aging
    mice that occurred with similar frequency or sporadic distribution in
    controls and high-dose groups. Exceptions were degenerative renal

    changes, which occurred with higher incidence among treated males
    dying on test or terminally sacrificed, and alveolar adenomas, which
    were the most common tumours occurring in the study.  Statistically-
    significant increases in the unadjusted incidence of alveolar adenomas
    were seen in high-dose males, but these were considered of dubious
    toxicological significance because of unequal sampling of the low-
    and mid-dose groups and because of earlier diagnosis associated with
    the increased mortality in the high-dose group. No significant
    treatment-related increases were seen in tumours or non-neoplastic
    lesions at other sites, the occurrence of which were considered
    incidental or common for aged mice (IRDC, 1981a).


         In a long-term study with an in utero phase, lithol rubine BK was
    administered to Charles River CD rats at dietary concentrations of 0,
    0.05, 0.3, or 2%. In the in utero segment of the study, 60 rats of
    each sex were assigned to each treatment group and then mated after
    receiving the appropriate diet for 60 days. A minimum of 35 litters
    per dosage level was used to select 70 rats of each sex per group for
    the long-term segment of the study. In the reproductive phase,
    observations were made of the fertility index, gestation anomalies,
    and effects on parturition and lactation; indices for live births and
    survival to weaning were calculated. In the long-term phase, the pups
    were weaned onto their respective diets at 21 days and maintained on
    these diets throughout the remainder of the experiment.

         Individual body weights and food consumption measurements were
    recorded weekly throughout the in utero phase and the first 14 weeks
    of the long-term phase, biweekly for the next 12 weeks, and monthly
    thereafter. Ophthalmoscopic examinations, haematology (haemoglobin,
    haematocrit, total and differential leucocyte counts, RBCs, and
    reticulocyte counts), biochemical examinations (fasting glucose, BUN,
    SGOT, SGPT, serum alkaline phosphatase, total serum protein, and
    creatinine) and urinalysis (colour, pH, SG, qualitative tests for
    protein, glucose, bilirubin, ketones, occult blood, and microscopy on
    the sediment) were performed on 10 rats/sex/group at 3, 6, 12, 18, and
    21 months (males) and at 3, 6, 12, 18, and 24 months (females) in the
    long-term phase. Additional haemato logical (5 rats/sex/group),
    virological (2 rats/sex/ group), and microbiological (3 rats)
    evaluations were conducted at 20 months. An interim sacrifice and
    necropsy of 10 rats/sex/group was conducted after 12 months of
    treatment. For animals killed at the interim or terminal sacrifices,
    organ weights were recorded of the brain, kidney, liver, spleen,
    testes, thyroid, heart, adrenals, uterus, and ovaries. Complete
    histopathological examination was carried out on the control and
    high-dose groups only.

         For the in utero phase, no compound-related effects were seen on
    body weights, food consumption, ophthalmoscopic examination,
    fertility, or gestation and lactation indices. In the long-term phase,
    mean food consumption values were similar for control and treated
    rats, but there was a treatment-related depression in body-weight
    gain, most marked in the high-dose group. Males showed a larger
    decrement of body-weight gain than females in the same dose group, the
    deficit compared to controls reaching about 19% for high-dose males by
    week 91 of the study. There was an accelerated mortality rate in male
    rats in the high-dose group and, for males, the study was terminated
    at week 95 when there were only 9 survivors in the high-dose group
    compared with 17 and 29 in the two control groups.

         No changes considered to be related to treatment were seen in the
    haematological and clinical biochemical examinations and, apart from
    the colour of the urine, no differences attributable to treatment were
    observed in urinalysis values. At 20 months, the additional
    haematological investigation revealed no treatment-related effect, but
    one low-dose male and one high-dose male had markedly elevated
    leucocyte counts. Virological investigations showed that titres for
    the viruses PVM and KRV were higher than expected for rats of that
    age, and microbiological examination of lung and tracheal samples from
    three rats revealed infection with Mycoplasma pulmonis; the most
    prominent organism in the tissues of these animals was Pseudomonas.

         No compound-related macroscopic changes were detected in rats
    which were sacrificed at 12 months or terminally. There were no
    statistically-significant variations in mean organ weights at the
    12-month interim sacrifice, and subsequent statistically-significant
    variations in the high-dose males were related to the decrement in
    mean body weight. Histopathological examinations revealed a higher
    incidence of chronic nephritis, renal tubular epithelial hyperplasia,
    myocardial fibrosis, reticular hyperplasia, and pigment deposition in
    the spleeen than in the controls. Atrophy/degeneration of testicular
    tubules in high-dose males that died on study from 12 months to
    termination was also higher than in the controls. These changes are
    common in aging rats and no specific compound effect was identified
    other than an acceleration of these changes. There was no significant
    increase in incidence of the above lesions at termination.

         For all malignant tumours in the males, the Z statistic was
    significant at the 0.01 level in the Kruskal-Wallis test for adjusted
    trend, but it was claimed that this single value was not
    toxicologically significant because of the small number of tumours
    present (unadjusted incidence was 4% in the controls and 9% in the
    high-dose group). There was an unusually high incidence of pituitary
    adenomas in one control group of males; the incidence in the high-dose
    group was not significantly increased over either control group. The
    unadjusted incidence of Leydig cell adenomas in males was 2% in

    controls and 6% in high-dose animals, not significant at the 0.05
    level, but tests for unadjusted trend and homogeneity of life table
    curves were significant at the 0.01 level. However, they were of
    doubtful toxicological significance because of the small number of
    tumours involved (IRDC, 1981b).

    Observations in man

         No information available.


         In the long-term mouse study, there was a dose-related increase
    in mortality and renal pathology, but detailed histopathology was not
    conducted on the low- and intermediate-dose groups. The long-term
    study in rats was complicated by high mortality rates, which led to
    premature termination of the study for males. In addition, only
    limited histopathological examinations were conducted. In view of
    these limitations, it was not possible to determine an unequivocal
    no-effect level in either study. Therefore, an ADI could not be


    Estimate of acceptable daily intake for man

         No ADI allocated.

    Further work or information

    Required for re-evaluation for an ADI

         1. Results of a complete histopathological examination of all
    dose groups in the long-term mouse study.

         2. Results of a new long-term study in rats.

         3. An adequate reproduction/teratology study.


    IRDC (1981a). D&C Red No. 6. Long-term feeding study in mice.
         Unpublished report No. 355-012 from International Research and
         Development Corporation. Submitted to WHO by the Public Health
         Administration, Leidschendam, The Netherlands.

    IRDC (1981b). D&C Red No. 6. Long-term feeding study in rats exposed
         in utero. Unpublished report No. 355-014 from International
         Research and Development Corporation. Submitted to WHO by the
         Public Health Administration, Leidschendam, The Netherlands.

    See Also:
       Toxicological Abbreviations
       LITHOL RUBINE BK (JECFA Evaluation)