LITHOL RUBINE BK
EXPLANATION
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
below.
BIOLOGICAL DATA
Biochemical aspects
No information available.
Toxicological studies
Acute studies
No information available.
Short-term studies
No information available.
Long-term studies
Mice
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).
Rats
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.
Comments
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
established.
EVALUATION
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.
REFERENCES
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)