Reaction mass of sodium 7-hydroxy-8-((E)-(4-(1-(4-((E)-(4-hydroxyphenyl)diazenyl)phenyl)cyclohexyl)phenyl)diazenyl)naphthalene-1,3-disulfonate and sodium 8,8'-(1E,1'E)-(4,4'-(cyclohexane-1,1-diyl)bis(4,1-phenylene))bis(diazene-2,1-diyl)bis(7-hydroxynaphthalene-1,3-disulfonate)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Remarks:

the tested substance is one of the two components of the substance under registration

Adequacy of study:

key study

Study period:

From January 24th to March 24th, 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Justification for type of information:

The tested substance is one of the two components of the substance under registration.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

adapted July 17, 1992

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

A LLNA study has not been conducted because adequate data from guinea pig Maximisation test study are already available.

Test material

Test material information

Constituent 1

Reference substance name:

Acid Orange 033_constituent 1

IUPAC Name:

Acid Orange 033_constituent 1

In vivo test system

Test animals

Species:

guinea pig

Strain:

Pirbright-Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Strain: white.
- Breeder: Ciba-Geigy Ltd. Animal Production Stein.
- Weight at study initiation: between 300 to 433 g.
- Housing: the animals were housed individually in Macrolon cages (Type 3).
- Diet: standard guinea pig pellets -NAFAG No. 845, ad libitum.
- Water: ad libitum.
- Acclimation: 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 to 70 %
- Photoperiod: 12 hours light cycle day.

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

other: physiological saline

Concentration / amount:

5 % of test item

Induction 2

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Remarks:

vaseline

Concentration / amount:

50 % of test item

Day(s)/duration:

48 hours

Adequacy of induction:

highest technically applicable concentration used

Challenge

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Remarks:

vaseline

Concentration / amount:

50 % of test item

Day(s)/duration:

24 hours

No. of animals per dose:

CONTROL GROUP: 5 males and 5 females
TEST GROUP: 10 males and 10 females main experiment.

Details on study design:

PRE TESTS
- Test groups: 5 male and 5 female guinea pigs.

Intradermal Induction
- Concentration selection: the concentration for the intradermal injections was selected on account of the solubility of the test article in standard vehicles and its local and systemic tolerability in a pretest.
- Concentration: 5 % in physiological saline (w/v). Since 5 % test item in physiological saline could be injected and was well tolerated, this concentration was used for the intradermal induction.

Epidermal Applications (induction and challenge)
- Concentration selection: the concentrations for the epidermal applications were selected on account of the primary irritation potential of the test article.
- Concentrations: 10, 20, 30, and 50 % in vaseline. 50 % in vaseline was the highest possible concentration of the test article.
- Results: erythema reactions were observed with 20, 30, and 50 % test item in vaseline.

MAIN STUDY
A1. INDUCTION EXPOSURE - intradermal injections
- Exposure period: performed on test day 0.
- No. of exposures: three pairs of intradermal injections (0.1 ml per injection) were made simultaneously into the left and right side of the shaved neck of the test and control group animals.
- Test group: adjuvant/saline mixture 1:1 (v/v); 5 % of test item in physiological saline (w/v); 5 % of test item in the adjuvant/saline mixture (w/v).
- Control group: adjuvant/saline mixture 1:1 (v/v); adjuvant/saline mixture 1:1 (v/v); physiological saline.

A2. INDUCTION EXPOSURE - epidermal applications
- Exposure period: performed on test day 8th.
- No. of exposures: one application.
- Application: substance was incorporated in vaseline (w/w) and applied on a filterpaper patch to the neck of the animals (patch 2x4 cm)
- Substance amout: approx. 0.4 g per patch.
- Type of wrap: occluded administration.
- Exposure period: 48 hours.
- Test group: 50 % of test item in vaseline
- Control group: treated with the vehicle only.

B. CHALLENGE EXPOSURE
- Exposure period: performed on test day 21th.
- Application: the test and control group animals were tested on one flank with test item in vaseline (w/w) and on the other flank with the vehicle alone (patch 2x2 cm)
- Substance amout: approx. 0.2 g per patch.
- Type of wrap: occluded administration.
- Exposure period: 24 hours.
- Test group: 10 % of test item in vaseline
- Control group: treated with the vehicle only.

POSITIVE CONTROL
The sensitivity of the strainis checked once or twice a year with a known mild to moderate sensitiser, such as mercaptobenzothiazole, hexyl cinnamic aldehyde or potassiumdichromate.

OBSERVATIONS
After the intradermal and the epidermal induction application irritant reactions are normally induced by the adjuvant and the high test article concentration. Because most of the reactions are treatment related and not compound related, the reactions are only described in special cases in the section of results.
Twenty four and forty eight hours after removing the dressings, the challenge reactions were graded according to the Draize scoring scale.

The body weight was recorded at start and end of the test.

Results and discussion

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

5 % of test item, intradermal induction

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

no indication of skin sensitisation

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

5 % of test item, intradermal induction

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

no indication of skin sensitisation

Any other information on results incl. tables

Number of positive animals per group after occlusive epidermal application

	after 24 hours	after 48 hours
Control group
Vehicle control	0/10	0/10
Test article	0/10	0/10
Test group
Vehicle control	0/20	0/20
Test article	0/20	0/20

Applicant's summary and conclusion

Interpretation of results:

other: not classified, according to the CLP Regulation (EC 1272/2008)

Conclusions:

Non skin sensitizer.

Executive summary:

Method

To assess the allergenic potential of test item in albino guinea pigs the Maximization-Test of B. Magnusson and A.M. Kligman was used. Five male and five female were used as control group and 10 male and 10 female were used as test group. The intradermal injections was performed with 5 % w/v test item in physiological saline and in the adjuvant/saline mixture. After 8 days, epidermal induction was performed with 50 % test item in vaseline. Challenge has been administered after 21 days at 10 % test item in vaseline by epidermal oacclusive patch system.

Under the experimental conditions employed, none of the animals of the test group showed skin reactions 24 and 48 hours after removing the dressings. Therefore, the substance resulted to be non skin sensitiser.

Results

Under the experimental conditions employed, none of the animals of the test group showed skin reactions 24 and 48 hours after removing

the dressings. Therefore, the test article is considered to be a non sensitizer.