A mixture of: disodium 6-[3-carboxy-4,5-dihydro-5-oxo-4-sulfonatophenyl)pyrazolin-4-yl-azo]-3-[2-oxido-4-(ethensulfonyl)-5-methoxyphenylazo]-4-oxidonaphthalene-2-sulfonate copper (II) complex; disodium 6-[3-carboxy-4,5-dihydro-5-oxo-4-sulfonatophenyl)pyrazolin-4-yl-azo]-3-[2-oxido-4-(2-hydroxyethylsulfonyl)-5-methoxyphenylazo]-4-oxidonaphthalene-2-sulfonate copper (II) complex

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 November 1995 to 08 December 1995

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Study was conducted prior to validation of the LLNA method

Test material

Test material information

Constituent 1

Reference substance name:

A mixture of: disodium 6-[3-carboxy-4,5-dihydro-5-oxo-4-sulfonatophenyl)pyrazolin-4-yl-azo]-3-[2-oxido-4-(ethensulfonyl)-5-methoxyphenylazo]-4-oxidonaphthalene-2-sulfonate copper (II) complex; disodium 6-[3-carboxy-4,5-dihydro-5-oxo-4-sulfonatophenyl)pyrazolin-4-yl-azo]-3-[2-oxido-4-(2-hydroxyethylsulfonyl)-5-methoxyphenylazo]-4-oxidonaphthalene-2-sulfonate copper (II) complex;

EC Number:

423-940-7

EC Name:

A mixture of: disodium 6-[3-carboxy-4,5-dihydro-5-oxo-4-sulfonatophenyl)pyrazolin-4-yl-azo]-3-[2-oxido-4-(ethensulfonyl)-5-methoxyphenylazo]-4-oxidonaphthalene-2-sulfonate copper (II) complex; disodium 6-[3-carboxy-4,5-dihydro-5-oxo-4-sulfonatophenyl)pyrazolin-4-yl-azo]-3-[2-oxido-4-(2-hydroxyethylsulfonyl)-5-methoxyphenylazo]-4-oxidonaphthalene-2-sulfonate copper (II) complex;

Cas Number:

85585-91-7

IUPAC Name:

4-[2-(7-{2-[4-(ethenesulfonyl)-2-hydroxy-5-methoxyphenyl]diazen-1-yl}-8-hydroxy-6-sulfonaphthalen-2-yl)diazen-1-yl]-5-oxo-1-(4-sulfophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid 4-[2-(8-hydroxy-7-{2-[2-hydroxy-4-(2-hydroxyethanesulfonyl)-5-methoxyphenyl]diazen-1-yl}-6-sulfonaphthalen-2-yl)diazen-1-yl]-5-oxo-1-(4-sulfophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid dicopper tetrasodium hydride

Test material form:

solid

Specific details on test material used for the study:

Identification: Pacified Reactive Black 31
Description: Dark blue crystals
Batch: 9T-55
Purity: 89%
Storage: At room temperature in the dark
Stability in vehicle: Stable in water for at least 96 hours

In vivo test system

Test animals

Species:

guinea pig

Strain:

other: Dunkin Hartley Crl

Sex:

female

Details on test animals and environmental conditions:

Number of animals
Experimental group: 10 females
control group: 5 females

Age at start of study
Approximately 5 weeks

Body weight prior to start
364 - 418 g

Identification
Ear tag

Conditions
Air-conditioned room with approximately 15 air changes per hour and the environment controlled with optimal conditions considered as being a temperature of 21°C and a relative humidity of 50%. Fluctuations from these optimal conditions were noted, but were considered not to have affected study integrity. Lighting was 12 hours artifical fluorescent light and 12 hours dark per day.

Accomodation
Housing of 1 or 2 animals per labelled metal cage with wire-mesh floors and equipped with an automatic drinking system. The acclimatisation period was at least 5 days before start of treatment under laboratory conditions.

Diet
Free access to standard guinea pig diet, including ascorbic acid. Certificates of analysis were examined and retained in the NOTOX archives. In addition, hay was provided once a week.

Water
Free access to tap water, diluted with decalcified water. Certificates of analysis were examined and t=retained in the NOTOX archives.

Study design: in vivo (non-LLNA)

Induction

Route:

intradermal

Vehicle:

water

Concentration / amount:

25% of 0.1 ml

Day(s)/duration:

10

Adequacy of induction:

not specified

Challenge

No.:

#1

Route:

other: epdermal

Concentration / amount:

0.05 ml of each of 25%, 10% and 5%

Day(s)/duration:

1 day

Adequacy of challenge:

not specified

No. of animals per dose:

Experimental group: 10 females
control group: 5 females

Details on study design:

MAIN STUDY

Induction - Experimental animals

Day 1
Three pairs of intradermal injections (0.1 ml/site) were made in the clipped scapular region as follows:
a) Freunds Complete Adjuvant, 50:50 with water for injection
b) The test substance at a 25% concentration.
c) The test substance, at twice the concentration used in b), emulsified in a 50:50 mixture of Freunds' complete Adjuvant.

Day 3
The dermal reactions caused by the intradermal injections wre assessed for erythema or, in case of necrosis, the diameter of necrosis.

Day 7
The clipped scapular area was rubbed with 10% sodium dodecyl sulphate in vaseline using a spatula. This concentration provokes a mildly inflammatory reaction.

Day 8
The clipped area between the injection sites was treated with 0.5 ml of a 25% test substance concentration using a patch mounted on tape and secured with an elastic bandage.

Day 10
After 48 hours, the dressings were removed and the skin cleaned of residual test substance. Subsequently, all dermal reactions caused by the epidermal exposure were assessed.


Challenge

Day 22
All animals were treated epidermally with 0.05 ml of each of the following test substance concentrations: 25%, 10% and 5% and the vehicle on a clipped flank, using chambers attached to tape and secued with an elastic banadage

Day 23
After approximately 24 hours, the dressings were removed. To remove the stainign caused by the test substance and to facilitate scoring, an approved depillatory cream was placed on the patch sites and surrounding areas for approximately 5 minutes. The cream was then gently removed using a plastic spatula. the skin was further cleaned by washing with lukewarm, running tap-water.Subsequently, the animals wer dried with a disposable towl and returned to their cages. The traeted sites were assessed for erythema and swelling 24 and 48 hours after removal of the dressings.

Challenge controls:

The control animals were treated as described for the experimental animals, except that the vehicle only was administered.

Positive control substance(s):

no

Results and discussion

Positive control results:

Not applicable

In vivo (non-LLNA)

Results

Key result

Reading:

other: Challenge phase

Hours after challenge:

24

Group:

test chemical

Dose level:

25%, 10% and 5%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

Please see "Info on other results" section

Any other information on results incl. tables

Preliminary study

Based on the results, the test substance concentrations for the main study were selected to be a 25% concentration for the intradermal induction and a 25% concentration forthe apidermal induction exposure.

Since no signs of irritation were observed to the concentrations selected for the epidermal induction, it was decided to treat all animals with 10% SDS approximately 24 hours before the epidermal induction.

A 25%, 10% and 5% concentrations were selected for the challenge phase.

Main study

Induction phase

The reactions noted in the control animals after the epidermal induction exposure, were considered to be enhanced by the SDS treatment.

Challenge phase

No skin reactions were evident after the challenege expopsure in the experimental and control animals.

Toxicity symptoms / mortality

One control animal showed deep respiration, dark coloured eyes and emaciation on Day 24 and was removed from the study on the same day. Another control animal was found dead on Day 25. Both animals showed dark red areas in the lungs at necropsy. These findings indicate acute pneumonia.

It was considered that the study outcome, based on the survivig animals, was not adversely affected.

Body weights

Body weight gain of experimental animals remained in the same range as controls over the study period.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

No evidence was obtained that Pacified Reactive Black 31 had caused skin hypersensitivity in the gunea pig, since no responses were observed in the experimental animals in the challenge phase. This result led to as sensitisation rate of 0 %.

Applying the rating of allergenicity described by Kligman on the results obtained in this test, Pacified Reactive Black 31 is considered to have weak sensitising properties.

Executive summary:

The study was carried out in accordance with the OECD Guideline No. 406, "Skin sensitisation", the EEc Directive 92/6/EEC, Part B.6 "Skin snsitisation" and based on the method described by Magnussen and Kligman.

Test substance concentrations (in distilled water) selected for the main study were v=based on the results of the preliminary study.

In the main study, the experimental animals were intradermally injected with a 25% concentration and epidermally exposed to a 25% concentration. The control animals were similarly treated, but with omission of the test substance.

Approximately 24 hours after the epidermal induction exposure all animals were treated with 10% SDS. Two weeks after the epidermal application all animals were challenged with a 25%, 10% and 5% test substance concentraion and the vehicle. All animals were treated with a depiliatory cream after removal of the dressings, to remove the staining caused by the test substance and to facilitate scoring the animals.

In the challenge phase, no skin reactions were evident in the control and experimental animals.

No evidence was obtained that Pacified Reactive Black 31 had caused skin hypersensitivity in the gunea pig, since no responses were observed in the experimental animals in the challenge phase. This result led to as sensitisation rate of 0 %.

Applying the rating of allergenicity described by Kligman on the results obtained in this test, Pacified Reactive Black 31 is considered to have weak sensitising properties.