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Toxicological information

Repeated dose toxicity: dermal

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Administrative data

Endpoint:
repeated dose toxicity: dermal
Remarks:
other: 15 and 64 applications
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1965
Report Date:
1965
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 1962-1973
GLP compliance:
no
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): D&C Blue 6

Test animals

Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 1.6 to 2.7 kg
- Fasting period before study: -
- Housing: single
- Diet: Purina Rabbit Chow ad libitum
- Water: tap water ad libitum

Administration / exposure

Type of coverage:
semiocclusive
Vehicle:
other: 1. white petrolatum USP XVI, 2. Ointment hydrophilic USP XVI
Details on exposure:
TEST SITE
- Area of exposure: abdominal skin - application of ointment with gentle inunction
- Pre-treatment of skin: 1. shaved
2. shaved and abraded
- Type of wrap if used: loose gauze and adhesive tape binder
- Time intervals for shavings or clipping: periodically
- Abrasion: weekly by hypodermic needle
- Removal of binders: 18 to 20 hours after application


TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 g/kg bw
- Concentration: 0.1% and 1% in ointments
- Constant volume or concentration used: constant concentration, variable volume


VEHICLE
- Justification for use and choice of vehicle: test item is insoluble in water
1. white petrolatum USP XVI
2. Ointment hydrophilic USP XVI
- Amount(s) applied (volume or weight with unit): 0.5 g/kg bw
- Concentration (if solution): -
- Lot/batch no. (if required): -
- Purity: USP


USE OF RESTRAINERS FOR PREVENTING INGESTION: yes
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
NA
Duration of treatment / exposure:
15 applications 5 days/week: 21 days
64 applications 5 days/week: 90 days
Frequency of treatment:
5 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
0.5 and 5 mg/kg bw
Basis:
nominal per unit body weight
No. of animals per sex per dose:
15 applications - shaved: 3 rabbits
15 applications - abraded: 3 rabbits
64 applications - shaved: 3 rabbits
Control animals:
yes, concurrent vehicle
Details on study design:
1. Formulation ointment based on White Petrolatum USP XVI
2. Formulation based on Ointment Hydrophilic USP XVI
Supplier of White Petrolatum USP XVI, Ointment Hydrophilic USP XVI, and the 0.1% and 1% test item formulations in both vehicles was E.R. Squibb and Sons.

3 or 4 days treatment free period after last application
Positive control:
NA

Examinations

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


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily


DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: daily


BODY WEIGHT: Yes
- Time schedule for examinations: prior to each application


HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior first dosing and
3-week phase: 24 hours after the 12th, 13th or 14th application
13-week phase: 24 hours after the 18th or 19th application and on the day of the 62nd or 63rd application
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: all
- Sampling site: marginal ear vein
- Parameters examined: erythrocyte counts, total and differential leukocyte counts, hematocrit, hemoglobin


URINALYSIS: Yes
- Time schedule for collection of blood: prior first dosing and
3-week phase: 24 hours after the 12th, 13th or 14th application
13-week phase: 24 hours after the 18th or 19th application and on the day of the 62nd or 63rd application
- Metabolism cages used for collection of urine: No - catheterization
- Animals fasted: No
- Parameters examined: appearence, pH, specific gravity, glucose, acetone, protein, bilirubin, occult blood, microscopic examination of sediment

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
- Organs preserved: brain, thyroid, lungs, heart, liver, kidneys, adrenals, skin, bone marrow, macroscopic lesions
- Microscopic investigation: all preserved tissues
- Animals: all

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Dermal irritation:
no effects observed
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:
no effects observed
Clinical biochemistry findings:
not examined
Urinalysis findings:
no effects observed
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
6 of the 54 rabbits dies during the study:
- Control group
- WP: after the 8th, 28th 35th application (3)
- HO: after the 8th application (1)
- Treatment groups
- 0.1% in HO: after the 29th application (1)
- 1% in WP: after the 34th application (1)

These deaths were attributed to coincidal mucoid colitis and not related to dermal application of the test substance.

DERMAL IRRITATION
There was no dermal irritation attributable to the color. The signs of irritation noted (slight erythema, atonia, slight desquamation) were considered the results of the vehicles and the technique of application.

The application sites were bluish stained by the test substance.

BODY WEIGHT AND WEIGHT GAIN
-

HAEMATOLOGY
-

URINALYSIS
-

GROSS PATHOLOGY
-

HISTOPATHOLOGY
-

Effect levels

Dose descriptor:
NOEL
Effect level:
5 mg/kg bw/day
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The repeated dermal administration of the 0.1% and 1% test substance formulations to New Zealand white rabbits at the rate of 500 mg/kg bw/day (5 days/week) to the intact and abraded skin of animals of both sexes, for up to 13 weeks, resulted in no test substance-related deaths, clinical signs or skin irritation. No adverse effects were noted in clinical laboratory investigations, macroscopical or microscopical evaluations. Consequently, the NOAEL for dermal application to rabbit skin was 5 mg/kg bw/day. 
Executive summary:

There are no repeated dose toxicity tests with the notified substance. However, Produkt 1998 is oxidized within 5 to 15 minutes to indigo in presence of moisture and oxygen. The present study was performed based on data from repeated dose toxicity tests with Indigo (CAS-Nr. 482-89-3), commercial name: D&C Blue No. 6, certified by U.S. FDA (for coloring surgical sutures). The test substance was applied at an amount of 500 mg/kg bw/day of an 0.1% and 1% ointment formulation, 5 days per week to the intact and abraded skin of male and female New Zealand white rabbits according to the following protocol matrix:

No. of applications

Test material

No. of animals

Skin Intact

Skin Abraded

(m/f)

(m/f)

(m/f)

15

WP

3/3

2/1

1/2

15

0.1% in WP

2/4

1/2

1/2

15

1% in WP

4/2

2/1

2/1

15

HO

4/2

2/1

2/1

15

0.1% in HO

2/4

1/2

1/2

15

1% in HO

4/2

2/1

2/1

64

WP

2/1

2/1

-

64

0.1% in WP

2/1

2/1

-

64

1% in WP

2/1

2/1

-

64

HO

1/2

1/2

-

64

0.1% in HO

2/1

2/1

-

64

1% in HO

1/2

1/2

-

m: male; f: female; WP: White petrolatum; HO: Hydrophilic ointment

 

Weight trends, mortality and signs of systemic toxicity and dermal irritation were recorded. Blood counts and urinalyses were performed on all animals at the start and termination of the study. Additional samples were taken during the fourth weeks from those animals tested for 13 weeks (64 applications). All animals were subjected at autopsy or death or terminal sacrifice. Tissues examined microscopically were: brain, thyroid, lung, heart, liver, kidneys, adrenals, skin, and bone marrow.

The repeated dermal administration of the 0.1% and 1% test substance formulations to New Zealand white rabbits at the rate of 500 mg/kg bw/day (5 days/week) to the intact and abraded skin of animals of both sexes, for up to 13 weeks, resulted in no test substance-related deaths, clinical signs or skin irritation. No adverse effects were noted in clinical laboratory investigations, macroscopical or microscopical evaluations. Consequently, the NOAEL for dermal application to rabbit skin was 5 mg/kg bw/day.