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Toxicological information

Repeated dose toxicity: dermal

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Administrative data

Endpoint:
repeated dose toxicity: dermal
Remarks:
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 August 1963 to 7 June 1965
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented publication/study report which meets basic scientific principles

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1967
Report Date:
1967
Reference Type:
publication
Title:
Toxicology of Indigo - a review
Author:
Ferber KH
Year:
1987
Bibliographic source:
J. Environ. Pathol. Toxicol. Oncol. 7, 73 -84 (1987)

Materials and methods

Principles of method if other than guideline:
U.S. FDA
GLP compliance:
no
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): D&C Blue 6
- Substance type: active ingredient
- Physical state: solid - dark blue powder
- Analytical purity: Sample A: 95%
Sample B + D: 97%
- Certified No.: Sample A: W4500
Sample B + D: W6182
- Lot/batch No.: K7024

Test animals

Species:
mouse
Strain:
Swiss Webster
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Pied Piper Farms
- Age at study initiation: -
- Weight at study initiation: -
- Fasting period before study: -
- Housing: 5/cage
- Diet: ad libitum
- Water: tap water ad libitum

Administration / exposure

Type of coverage:
open
Vehicle:
other: spectrograde benzene
Details on exposure:
TEST SITE
- Area of exposure: midscapular region
- Time intervals for shavings or clipplings: regular to keep skin relatively free of hair


REMOVAL OF TEST SUBSTANCE - No


TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL
- Concentration (if solution): 1%
- Constant volume or concentration used: yes

The dose levels wer corrected for purity of the dye content: for preparation of the 1% indigo formulation calculated for 100% indigo, a 1.05% or 1.03% formulation of the test substance as supplied had to be prepared for 95% and 97% purity of the delivered batches, respectively.

Sample A: Week 1 - 6
Sample B: Week 7 - 68
Sample D: Week 69 - 95


VEHICLE
- Justification for use and choice of vehicle (if other than water): TS is insoluble in water
- Amount(s) applied (volume or weight with unit): 0.1 mL
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
NA
Duration of treatment / exposure:
up to 95 weeks - application of formulation once a week
Frequency of treatment:
weekly
Doses / concentrations
Remarks:
Doses / Concentrations:
1 mg/animal
Basis:
other: calculated as 100% pure dye nominal per animal
No. of animals per sex per dose:
Negative Control: 100
Positive Control: 100
Dose Group: 50
Control animals:
yes, concurrent no treatment
yes, concurrent vehicle
Details on study design:
About 1 to 5 animals from each group, incl. moribund mice, were sacrificed at monthly intervals following 2 to 36 applications. These resulted in a total of 28 or 30 interim killed animals from each control group and 16 mice from the test substance group.
3 to 5 mice of the surviving animals of each sex from each group were sacrificed after 74 applications. The remaining surviving mice were sacrificed after 95 applications.
Positive control:
NA

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly


DERMAL IRRITATION (if dermal study): No data


BODY WEIGHT: Yes
- Time schedule for examinations: Week 1 - 16: weekly (except negative control)
Week 27 - 52: biweekly
Week 53 - end: monthly
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
- Preserved tissues: application site, brain, pituitary, thyroid, thymus, lungs, liver, spleen, kidneys, adrenals, stomach, pancreas, small and large intestines, urinary bladder, gonads, lymph nodes (axillary), gross lesions
- Microscopic examination:
- animals sacrified in first 9 months:: application site all animals + liver from 9 to 13 mice/group
- animals sacrified at 75 weeks: application site, liver, lung from 10 mice/group
- terminal sacrifice: application site, liver, lung from 29, 26, 13 mice from the negative, vehicle control and Blue 6 groups, respectively
- most tissue masses, macroscopic lesion

Statistics:
Survival by life-table technique

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Dermal irritation:
not specified
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
The appearence and behavior of the mice was generaly comparable with that of controls. Clinical signs observed were either incidental findings or due to the vehicle and also observed in the control group. Survival of animals was high during the first year.

BODY WEIGHT AND WEIGHT GAIN
-

GROSS PATHOLOGY
Macroscopic findings in the D&C Blue 6-treated group were also seen in the vehicle control group and either incidental findings or due to the benzene application.

HISTOPATHOLOGY
Dermal application of a 1% solution of D&C Blue 6 in benzene for 95 weeks did not produce any distinctive histologic alteration which exceeded the range established in the sectons from the vehicle control mice.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 other: mg/animal/day
Sex:
male/female
Basis for effect level:
other: clinical signs; mortality; survival; body weight; gross pathology; histopathology
Dose descriptor:
NOAEL
Effect level:
ca. 25 other: mg/kg bw/week
Sex:
male/female
Basis for effect level:
other: based on assumed mean body weight of 40 g

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
No evidence was found that in mice repeated dermal application of 1 mg/animal, once per week for up to 95 weeks produced any effect attributable to the test substance. Lesions and tumors seen in treated mice were comparable to those in vehicle control animals.
Hence the NOAEL for repeated dermal administration of indigo is about 25 mg/kg bw/week based on assumed mean body weight of 40 g.
Executive summary:

The test substance was applied as a 1% solution (W/V) in spectro-grade benzene at a dose of 1 mg of test material (once/week) for up to 95 weeks, to 50 male and 50 female Swiss-Webster mice. 100 males and 100 females were used as negative controls (no treatment). 100 males and 100 females received application of the vehicle (benzene).

Autopsies were performed on all died or sacrificed animals in the absence of marked autolysis. Microscopic examination of the skin (application site) were performed from all animals died or sacrificed in the first 9 months and of the liver from 9 to 13 mice/group. Microscopic examination of the lungs, liver and skin (application site) from 10 negative controls, 10 vehicle controls, and 10 compound-treated animals were performed at 75 weeks. At the terminal sacrifice (95 weeks), sections of lung, liver and skin from 29 negative controls, 26 vehicle controls and 13 compound-treated animals were examined microscopically. Histopathology was also performed on most tissues and on grossly abnormal organs of the animals.

 

No evidence was found that in mice repeated dermal application of 1 mg/animal, once per week for up to 95 weeks produced any effect attributable to the test substance. Lesions and tumors seen in treated mice were comparable to those in vehicle control animals.