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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 21,1993 - November 24,1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report date:
1993

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
March 22, 1990
Deviations:
no
Remarks:
Urinalysis not performed
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Calcium 3-hydroxy-4-[(4-methyl-2-sulphonatophenyl)azo]-2-naphthoate
EC Number:
226-109-5
EC Name:
Calcium 3-hydroxy-4-[(4-methyl-2-sulphonatophenyl)azo]-2-naphthoate
Cas Number:
5281-04-9
Molecular formula:
C18H14N2O6S.Ca
IUPAC Name:
calcium 3-hydroxy-4-[(4-methyl-2-sulfonatophenyl)diazenyl]-2-naphthoate
Test material form:
solid: nanoform
Details on test material:
- D&C Red 7
- Analytical purity: 98% w/w
- R-92-214

Test materials used in this dossier are all considered to fall under the definition of nano-materials according to the European Commission Recommendation 2011/696/EU as the synthesis and manufacturing of this pigment always yields particulate material with a fine particle size distribution.
Specific details on test material used for the study:
-Analytical purity : 98 % w/w

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
- Sources:Charles River Laboratories Japan (Atsugi Breeding Center)
- Age at study initiation : 7 weeks
- Weight at study initiation : females 196.5-227.4 g, males: 235.5-299.6 g
- Housing : Individually housed in stainlelss suspended cages
- Diet : CA-1 (CLEA Japan, Inc.) ad libitum
- Water : Tap water, ad libitum
- Acclimation period : 1 week for quarantine and acclimaition

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 5% gum arabic solution
Details on oral exposure:
- Amount of vehicleL 10 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of test substance in the dosing formulation was analytically confirmed to attain the required level.
Duration of treatment / exposure:
Males : Total of 42 days ( 14 days before mating, 14 days for mating, and 14 days after mating)
Females : Total of 41-50 days (14 days before mating, during mating, pregnancy and up to lactation day 3)
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
13/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: doses were selected on the basis of a 14-day preliminary repeat dose study at 1000 mg/kg bw/day. No deaths and clinical signs observed. 3 cases of necrosis or regeneration of tubular epithelium were seen in males.

Examinations

Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes (see table 1&2)
- Time schedule: Males and Females - on each administration day.

BODY WEIGHT: Yes
- Time schedule for examinations: Males - measured once a week on administration days 0, 7, 14, 21, 28, 35, and 42; Females - measured once a week on administration days 0, 7, 14, and 21, during pregnancy on days 0, 7, 14, and 20, during lactation on day 0 and 4.


FOOD CONSUMPTION AND COMPOUND INTAKE :
- Food consumption for each animal determined: Yes
- Time schedule for examinations : Males - measured once a week on administration days 0, 7, 14, 21, 28, 35, and 42; Females - measured once a week on administration days 0, 7, 14, and 21, during pregnancy on days 0, 7, 14, and 20, during lactation on day 0 and 4.


OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:


HAEMATOLOGY: Yes
- Time schedule for collection of blood: Males - day after final administration day
- Anaesthetic used for blood collection: Yes (pentobarbital)
- Animals fasted: No data
- How many animals: 13 animals per male per group
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Males - day after final administration day
- Anaesthetic used for blood collection: Yes (pentobarbital)
- Animals fasted: No data
- How many animals : 13 animals per male per group
- Parameters checked in table 2 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 3 under 'Any other information on results incl. tables')
HISTOPATHOLOGY: Yes (see table 4 under 'Any other information on results incl. tables')
Statistics:
x squared test was conducted for copulation and fertility indices. For all the other parameters, Bartlett's test was used to examine uniformity of distribution in each group, and one-way statistical analysis was performed when the distribution is uniform. Where significant differences were observed, Dunnett's test or Scheff's test was performed to examine differences in averages between each test group and control groups. Kruskal-Wallis's test was conducted when the distribution was not uniform. Significant testing was carried out at the 5% and the 1% levels.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
- Males: red stained feces at all doses. One animal with crust formation during whole treatment period at 100 mg/kg bw/day.
- Females: red stained feces at all doses. One animal with crust formation from day 22 to sacrifice at 1000 mg/kg bw/day.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Males - 1 day after termination of administration period: Significantly lower values of MCH (p<0.05) were seen in the 300 and 1000 mg/kg bw/day groups compared to the controls. Dose-dependent decrease in WBC was seen in the the 300 and 1000 mg/kg bw/day groups, but the effects were not significant. In the absence of historical control data, no corresponding histopathology findings and other affected parameters, the findings are considered of no biological relevance.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Males - 1 day after termination of administration period: Significantly lower levels of inorganic phosphorus and Calcium were seen in the 300 mg/kg bw/day group. Significant low levels of Total cholesterol and pottasium and significant high values of Chloride and GOT were noted in the 1000 mg/kg bw/day group.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Males - 1 day after termination of administration period: Significant high values of relative weight of kidney were observed in the 1000 mg/kg bw/daygroup.

Females - Day 4 of lactation: Significant low values of absolute and relative weight of thymus were noted in the 100 mg/kg kw/day group (p< 0.01 for absolute weight, p<0.05 for relative weight). Significant low values of absolute weight of thymus were noted in the 1000 mg/kg bw/day group (p <0.05).

Females sacrificed at pregnancy Day 25: Non-significant low values of absolute and relative weight of thymus were noted in one animal with total litter loss in the 1000 mg/kg bw/day group. In the absence of significant effects on WBC and lymphocytes, thymus findings are probably of little biological relevance. Historical control incidence is not available for comparison.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Males: No dose-related effects were observed.

Females - Sacrificed at Day 4 of lactation: 2 cases of involution of thymus were seen in the 100 mg/kg bw/day group and 5 cases in the 1000 mg/kg bw/day group. A case of pale coloured area in kidney was seen in the 100 mg/kg bw/day and the 1000 mg/kg bw/day groups respectively. A case of pale coloured renal cortex was observed in another animal of the 1000 mg/kg bw/day group.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Males - 1 day after termination of administration period:
Thymus: Two cases of hemorrhage were seen in the 1000 mg/kg bw/day group, one case in the control group. In the absence of a dose-response relationship, this is considered incidental.
Heart: Small areas of myocardial degeneration were seen in 2 cases in the control group. No other abnormalities were observed.
Liver: No treatment-related findings were observed. Ten cases of very slight microgranuloma were seen respectively in the control and the 1000 mg/kg bw/day groups. Slight fatty changes in peripheral zone were seen in a case of the control group and in four cases of the 1000 mg/kg bw/day group. Therefore, there was no significant difference in severity and incidence of the findings between the control and the 1000 mg/kg bw/day groups. A case of very slight focal necrosis was seen in the 1000 mg/kg bw/day group.
Kidney: Large numbers of regenerating tubular epithelium were observed in three cases in the 300 mg/kg bw/day group and twelve cases in the 1000 mg/kg bw/day group compared to the control groups. The severity was augmented in the 1000 mg/kg bw/day group. The regenerating epithelial cells were found predominantly in convoluted proximal tubules, showing increased cell density, slightly enlarged nucleoli, and slightly bright or basophilic cytoplasm. In most cases, slightly-yellowish debris was noted in the tubular lumen. A single case of cast in tubular lumen was found in the 100 mg/kg bw/day group. In all groups, including the control group, eosinophilic bodies were observed with no significant differences in incidence and severity among the groups.
Adrenal cortex : Brown pigment deposits were observed both in the 1000 mg/kg bw/day and the control groups but there is no significant difference in incidence and severity.
Spleen: Brown pigment deposits and extramedullary hematopoiesis were found both in the 1000 mg/kg bw/day and the control groups, with no significant difference in incidence and severity.
Testis: Atrophy of tubule was found in 2 cases in the control group and 3 cases in the 1000 mg/kg bw/day group. One of the cases in the 1000 mg/kg bw/day group had calcification in the tubule.
Epididymis: Decreased numbers of sperm were noted in each one case in the control and the 1000 mg/kg bw/day groups. Both animals had atrophy of the tubule.

Females
Thymus: A case of very slight and a case of slight involution were found in the control group. In the 1000 mg/kg bw/day group, increased numbers of involution were found with a case rated "very slight", 3 cases rated "slight", and 2 cases rated "moderately slight".
Liver: Very slight microgranuloma was seen in a case of the control and 2 cases of the 1000 mg/kg bw/day group sacrificed at Day 4 of lactation, and one non-pregnant animal in the control group sacrificed at Day 25 of pregnancy. Very slight to slight fatty changes in peripheral zone were seen in 3 cases in the control group, and very slight to moderate fatty changes in 2 cases in the 1000 mg/kg bw/day group. There were no significant differences in severity and incidence between the control and the 1000 mg/kg bw/day groups.
Kidney: In all treatment groups sacrificed at Day 4 of lactation, increased numbers of incidences of regenerating tubular epithelium were observed. The findings are accompanied with foamy epithelial cells and vacuolar degeneration predominantly in convoluted proximal tubule. In many cases, necrotized epithelial cells were noted, and eosinophilic necrotized cells and yellowish debris were contained in the tubular lumen. In tubular basement of these animals, regenerating epithelial cells included large-sized basophilic cytoplasma. The incidence was similar in all dose groups; the severity of this lesion was slightly increased in the high-dose group.
In all dose groups, there were some animals completely free of kidney findings. Among those were a non-delivering animal (total implantation loss) in the 100 mg/kg bw/day group, 2 non-pregmant animals in the 300 mg/kg bw/day group, and a non-copulated animal in the 1000 mg/kg bw/day group.

Other kidney findings that are considered incidental included each one case of cast in the tubular lumen in the control and the 1000 mg/kg bw/day groups, each one case of slight vacuolar degeneration in the control and the 100 mg/kg bw/day groups, and each one case of focal dilatation of tubule in the 100 mg/kg bw/day and the 1000 mg/kg bw/day groups. The case of cast of the animal in the control group was considered be associated with the very slight incidence of chronic nephropathy.


Adrenal cortex: A case of very slight brown pigment deposit and a case of focal necrosis were observed in the 1000 mg/kg bw/day group.
Spleen: Brown pigment deposits and extramedullary hematopoiesis were observed in 11 animals in the control group and 12 animals in the 1000 mg/kg bw/day group. Two cases were also found in non-pregnant animals in the control group, sacrificed at Day 25 of pregnancy. There were no clear differences in incidence and severity between the treated and the control groups.
Ovary: No abnomarities were found with non-pregnant animals (two in the control and two in the 300 mg/kg bw/day groups) and in the non-copulated animal ( one in the 1000 mg/kg bw/day group).
Histopathological findings: neoplastic:
no effects observed

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOEL
Effect level:
< 100 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes

Applicant's summary and conclusion