Registration Dossier

Toxicological information

Specific investigations: other studies

Currently viewing:

Administrative data

Endpoint:
specific investigations: other studies
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From Feb. 14, 2003 to Mar. 22, 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods
Remarks:
Well documented study, followed basic scientific principles/standards, GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: Guidance for Cosmetic Safety Evaluation" (Japan Cosmetic Industry Association, ed., March 30, 2001)
Qualifier:
according to
Guideline:
other: Guidelines for toxicity studies required for application for manufacture (import) approval of drugs" of Ministry of Health, Labour and Welfare (Yakushin-1 No. 24, September 11, 1989)
Principles of method if other than guideline:
The study was performed to determine the photosensitisation potential of test substance in guinea pigs. Animals were exposed to test substance (2% w/v) and the test sites were exposed to UV light daily for 5 consecutive days during the induction phase. Animals were challenged with test substance (2% w/v) after 2 weeks of rest period followed by exposure to UV radiation. Animals were observed 24 and 48 h after each application for skin irritation. The test was conducted in accordance with Guidelines for toxicity studies required for application for manufacture (import) approval of drugs" of Ministry of Health, Labour and Welfare (Yakushin-1 No. 24, September 11, 1989), and "Guidance for Cosmetic Safety Evaluation" (Japan Cosmetic Industry Association, ed., March 30, 2001)
GLP compliance:
yes
Remarks:
according to Ordinance No. 21 of Ministry of Health and Welfare, March 26, 1997
Type of method:
in vivo
Endpoint addressed:
other: Photosensitisation

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material: 2-Amino-6-chloro-4-nitrophenol; Chlororange base
- Substance type: Pure active substance
- Physical state: Yellow needle-shape crystals or orange-yellow fine powder
- Solubility: Soluble in water and in alcohol
- Storage: In a cold and dark place under tightly sealed conditions
- Stability: Stability of the test substance under the storing conditions in the administration period was confirmed
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Wella Japan ; n°batch R0067439
- Expiration date of the lot/batch: 3 years from the date of production (december 2002)
- Purity test date: No data
- Purity : >97%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in a cold and dark place under tightly sealed conditions
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/vehicle: stability of the test substance under the storing conditions in the administration period was confirmed (results in report)
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING : No

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Ichikawa Laboratory Co., Ltd, 5-35-21 Takenotsuka, Adachi-ku, Tokyo (bred by Japan SLC, Inc.)
- Age at study initiation: 4 weeks
- Weight at study initiation: 224 - 254 g
- Fasting period before study: Not reported
- Housing: One or two animals were housed in each aluminium cage (300 W x 400 D x 230 H mm) equipped with automatic water-wash system
- Diet: CG-7 pellet food; ad libitum
- Water: Private tap water; ad libitum
Food and water were analyzed for the quality and found to be within standard ranges.
- Acclimation period: 5 + 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22±3°C (observed values: 20.7 - 22.8°C)
- Relative humidity: 50 ± 20% (observed values: 49.0 - 51.2%)
- Air changes: 10 times or more/hour (by the all fresh air procedure)
- Photoperiod: 12 hours light/day (6 a.m. - 6 p.m., 150 - 300 lux)

EXPERIMENT INITIATION DATE: Feb. 14, 2003

EXPERIMENT COMPLETION DATE : Mar. 22, 2003

Administration / exposure

Route of administration:
dermal
Vehicle:
other: Distilled water for injection
Details on exposure:
TEST MATERIAL
Preparation: The following preparation was carried out shortly before use.
A. At the time of induction:
1) Distilled water for injection-FCA emulsion: Distilled water for injection was mixed with an equal volume of FCA.
2) Test substance: Distilled water for injection was added to the test substance, and 2.0% (w/v) test-substance solution was prepared
3) Positive substance: Acetone was added to TBS, and 1.0% (w/v) TBS solution was prepared.
B. At the time of challenge
1) Test substance: Distilled water for injection was added to the test substance, and 2.0% (w/v) test-substance solution was prepared.
2) Positive substance: Acetone was added to TBS, and 0.5% (w/v) TBS solution was prepared.
Analyses of stability and homogeneity:
Stability: Examination with the eye was performed at the time of preparation and after administration, and neither heat generation nor foaming was found.
Homogeneity: Examination with the eye was performed at the time of preparation, and the color was homogenous and dispersion was uniform.

VEHICLE
- Justification for use and choice of vehicle: Since the test substance is soluble in water, distilled water for injection was chosen.

Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
30 minutes
Frequency of treatment:
Induction exposure: Once daily from Day 0 to Day 4, for 5 consecutive days
Challenge exposure: On Day 18
Doses / concentrations
Dose / conc.:
2 other: %
Remarks:
Basis: nominal conc.
for Induction exposure and challenge exposures
No. of animals per sex per dose:
Test group: 10 animals
Positive control: 5 animals
Test substance control: 5 animals
Control animals:
yes
Details on study design:
EXPOSURE OF TEST SUBSTANCE
A. INDUCTION EXPOSURE
- No. of exposures: 5
- Test groups: One
- Area of exposure: The day before start of the administration, fur on the neck and back of each animal was removed with an electric hair clipper (using 0.05 mm blade, Daito Denkikogyo) and an electric shaver (Braun), and an area of about 2 x 4 cm was prepared for induction
- Type of covering: Open
- Treatment with Freund's Complete Adjuvant: On Day 0, 0.1 mL of distilled water FCA emulsion was subcutaneously injected at each of 4 corners of the induction area with a 1.0 mL disposable syringe with 23-gauge needle. The skin of the induction area was stripped with adhesive cellophane tape (Sekisui Chemical Co., Ltd.) 5 or 6 times until mild erythemas developed.
- Application: Using micropipettes, 0.1 mL of 2% test substance solution, distilled water for injection, 1% TBS were applied dermally in the treatment group, control group and positive control group respectively.
- Exposure to UV Light during Induction: After 30 minutes of application, each animal was restricted in the positioners and irradiated with long-wave ultraviolet light at about 4 mW/cm 2 from about 10-cm distance for 45 minutes (irradiation energy: about 10 J/cm2 ) using ultraviolet light irradiation apparatus with glass filter (2 mm thick) attached. The intensity of ultraviolet light was measured with an ultraviolet photometer
- Frequency of applications: Stripping, open application of each substance and UV irradiation were performed once daily from Day 0 to Day 4, for 5 consecutive days.

B. CHALLENGE EXPOSURE:
- Challenge exposure was performed two weeks after the last induction exposure.
- Test groups: One
- Area of exposure: On the day before challenge, dorsal fur in a region lower than the induction area of each animal was removed with an electric hair clipper and an electric shaver. Two sections of 1.5 x 1.5 cm each were set bilaterally symmetrical to the median line for challenge
- Type of covering: Open
- Application: Using micropipettes, 0.02 mL of 2.0% test-substance solution was applied on each site in the test-substance group and in the test-substance control group, and 0.02 mL of 0.5% TBS solution was applied in the positive substance group.
- Exposure to UV Light during Challenge: After 30 minutes of administration, each animal was restricted in the positioners. The left site was used as irradiation site and the right site as non-irradiation site. Non-irradiation site was covered with aluminium foil, and the irradiation sites were exposed to ultraviolet light using the ultraviolet irradiation apparatus similarly to induction.

Examinations

Examinations:
OBSERVATION: At 24 and 48 hours after challenge (Day 19, 20), challenge sites were observed for signs of erytherma and edema
Positive control:
- Positive control: 3,5,4' -Tribromosalicylanilide (TBS)
- Lot # AP01
- Vehicle: Acetone (As TBS is soluble in acetone, acetone is selected as vehicle)
- Concentration: 1% in induction phase and 0.5% in challenge phase

Results and discussion

Details on results:
- Test substance: All animals (10/10) treated with test substance revealed no skin reaction, neither erythema nor edema in the photo-irradiation or non-irradiation site after 24 and 48 h of challenge
- Test substance control: All animals (5/5) in the control group did not show signs of erythema or edema
- Positive control:
a. Irradiation site: All the 5 animals showed strong skin reaction after 24 and 48 hours of challenge.
b. Non-irradiation sites: Very slight erythema was observed in 1 animal after 24 hours and in 3 animals after 48 hours of challenge
Details of the scores of erythema and edema are provided in table 1 in ‘Any other information on results incl. Tables’

Any other information on results incl. tables

Table 1: Results of photosensitization of Chlororange after challenge exposure (study# 91793)

Challenge exposure Hours after challenge Group Dose level No. with + reactions Total no. in group Erythema Score(reaction in no. of animals) Edema Score(reaction in no. of animals) Mean evaluation score
Irradiation site (left) At 24 and 48 h Test group 2% 0 10 0(10) 0(10) 0
Non- Irradiation site (right) At 24 and 48 h Test group 2% 0 10 0(10) 0(10) 0
Irradiation site (left) At 24 and 48 h Test control group Water for injection 0 5 0(5) 0(5) 0
Non- Irradiation site (right) At 24 and 48 h Test control group Water for injection 0 5 0(5) 0(5) 0
Irradiation site (left) 24 positive control (TBS) 0.50% 5 5 2(1) and 3(4) 1(3) and 2(2) 4.2
Non- Irradiation site (right) 24 positive control (TBS) 0.50% 1 5 1(1) and 0(4) 0(5) 0.2
Irradiation site (left) 48 positive control (TBS) 0.50% 5 5 3(5) 1(1) and 2(4) 4.8
Non- Irradiation site (right) 48 positive control (TBS) 0.50% 3 5 1(3) and 0(2) 0(5) 0.6

General condition: No changes in general conditions were found in any animal m any group throughout the study period.

Body weight: Every animal in each group showed normal body weight gain throughout the study period. The mean body weight gain was 152.4 g in the test-substance group, 137.6 g in the test-substance control group and 150.8 g in the positive substance group.

Applicant's summary and conclusion

Conclusions:
Under the conditions of the test, dermal application of 2% Chlororange (2-amino-6-chloro-4-nitrophenol) to guinea pigs exhibited no skin reaction. Therefore, cholrorange does not have photosensitization potential.
Executive summary:

The purpose of study was to determine the photosensitization potential of 2% Chlororange (2-amino-6-chloro-4-nitrophenol) in guinea pigs.

Male Hartley guinea pigs (224 - 254 g) obtained from Ichikawa Laboratory Co., Ltd (bred by Japan SLC, Inc.) were used in the study. The animals were assigned to a total of 3 groups; a test-substance group (10 animals), a test-substance control group (5 animals) and a positive substance group (5 animals). The test substance solution was prepared in distilled water for injection. The positive control 3, 5, 4’ -Tribromosalicylanilide (TBS) was prepared in acetone.

At the start of the induction phase (Day 0), water/ FCA emulsion was subcutaneously injected at each of 4 corners of the induction area (2 x 4 cm) followed by stripping with adhesive cellophane 5 or 6 times until mild erythemas developed. Using micropipettes, 0.1 mL of 2.0% test-substance solution was applied in the test-substance group, 0.1 mL of distilled water for injection was applied in the test-substance control group, and 0.1 mL of 1.0% TBS solution was applied in the positive substance group. Each applied area was kept uncovered. After 30 minutes of induction exposure, animals were irradiated with long-wave ultraviolet light at about 4 mW/cm2 from about 10-cm for 45 minutes (irradiation energy: about 10J/cm2).Stripping, open application of each substance and UV irradiation were performed once daily from Day 0 to Day 4, for 5 consecutive days.

After 2 weeks, sections of 1.5 x 1.5 cm each were set bilaterally for challenge exposure. 0.02 mL of 2.0% test-substance solution was applied on each site in the test-substance group and in the test-substance control group, and 0.02 mL of 0.5% TBS solution was applied in the positive substance group. Each application site was kept uncovered. After 30 minutes of challenge exposure, the right site was covered with aluminium foil and left site exposed to ultraviolet light similarly to induction in all groups.

At 24 and 48 hours after challenge, challenge sites were evaluated for signs of erythema and edema reactions.

All animals in the test and vehicle control groups revealed no skin reaction; neither erythema nor edema was observed in the photo-irradiation or non-irradiation site after 24 and 48 h of challenge.

In the positive control group, astrong skin reaction was found at the photo-irradiation site in all animals at every observation point. However, at the non-irradiation sites, slight erythema was observed in 1 animal at 24 h and in 3 animals at 48 h. This confirmed that the guinea pigs used in this study responded normally to a known photo-sensitizer, TBS.

Under the conditions of the test, dermal application of 2% Chlororange (2-amino-6-chloro-4-nitrophenol) to guinea pig exhibited no skin reaction. Therefore, cholrorange does not have photosensitization potential.