Reaction mass of Cuprate(4-), [4,5-dihydro-4-[[8-hydroxy-7-[[2-hydroxy-5-methoxy-4-[[2-(sulfooxy)Vinyl]phenyl]azo]-6-sulfo-2-naphthalenyl]azo]-5-oxo-1-(4-sulfophenyl)-1H-pyrazole-3-carboxylato(6-)], trisodium salt and Cuprate(4-), [4,5-dihydro-4-[[8-hydroxy-7-[[2-hydroxy-5-methoxy-4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]azo]-6-sulfo-2-naphthalenyl]azo]-5-oxo-1-(4-sulfophenyl)-1H-pyrazole-3-carboxylato(6-)]-, sodium and Cuprate(4-), [4,5-dihydro-4-[[8-hydroxy-7-[[2-hydroxy-5-methoxy-4-[2-(sulfooxy)Ethanol]phenyl]azo]-6-sulfo-2-naphthalenyl]azo]-5-oxo-1-(4-sulfophenyl)-1H-pyrazole-3-carboxylato(6-)], trisodium salt

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From August 24 to September 25, 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

1984

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

1981

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Maximisation test was available.

Test material

Test material information

Constituent 1

Reference substance name:

Reactive Black 031

IUPAC Name:

Reactive Black 031

Test material form:

solid: particulate/powder

In vivo test system

Test animals

Species:

guinea pig

Strain:

Pirbright-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: HOECHST AG, Kastengrund, SPF breeding colony
- Weight at study initiation: 309-348 g
- Housing: in fully air-conditioned rooms in Makrolon cages (Type 4) on softwood granulate, in groups of 5 animals
- Diet: Altromin 3112 far guinea pigs and rabbits, ad libitum
- Water: tap water in plastic bottles, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 55 ± 20 %
- Photoperiod: 12 hours cycle dark/light

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

physiological saline

Remarks:

50 % Freund's adiuvant

Concentration / amount:

1 %, 0.1 ml

Day(s)/duration:

injection on day 1

Adequacy of induction:

other: concentration causing very slight to slight oedema and indurations

Induction 2

Route:

epicutaneous, occlusive

Vehicle:

physiological saline

Concentration / amount:

25 %, 0.5 ml

Day(s)/duration:

from day 8 to day 10

Adequacy of induction:

other: non irritant

Challenge

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

physiological saline

Concentration / amount:

25 %, 0.5 ml

Day(s)/duration:

1 day, i.e. day 22

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Determination of the primary non-irritant concentration: 6
Determination of the tolerance of intradermal injections: 3
Escort group: 5
Control group: 5
Treatment group: 10

Details on study design:

DETERMINATION OF THE PRIMARY NON-IRRITANT CONCENTRATION:
In a dermal occlusive test for primary skin irritation each of the following test concentrations was applied to the left flank of two Guinea pigs:
25 % in isotonic saline
5 % in isotonic saline
1 % in isotonic saline
The hair on the left flank of the animals was removed mechanically. 0.5 ml of the test substance preparation was applied to a 2 cm × 2 cm cellulose patch, which was then fixed to the left flank and covered occlusively for 24 hours with a bandage and film. 24 hours after removal of the patches, the treated skin areas were examined for erythema and oedema .

DETERMINING OF THE TOLERANCE OF INTRADERMAL INJECTIONS
To determine the tolerance of intradermal injections, each of the following preparations was administered twice by intradermal injectlon to 3 Guinea pigs. The injection sites were all within a dorsal area measuring 2 cm × 4 cm in the vicinity of the shoulder.
5 % in isotonic saline
1 % in isotonic saline
0.2 % in isotonic saline

MAIN STUDY
A1. INDUCTION EXPOSURE
- No. of exposures: 2 intradermal injections
- Exposure period: day 1
- Test groups: 10
- Control group: 5
- Site: dorsal area measuring 2 × 4 cm in the vicinity of the animals shoulder. The injection sites were left uncovered.
- Concentrations:
Site 1: 2 × 0.1 ml 50 % Freund's Adjuvant
Site 2: 2 × 0.1 ml 1 % solution of test substance in isotonic saline
Site 3: 2 × 0.1 ml 1 % solution of test substance in 50 % Freund's Adjuvant

A2. DERMAL INDUCTION TREATMENT
- No. of exposures: 1 patch application
- Exposure period: day 8
- Test groups: 10
- Control group: 5
- Site: dorsal area measuring 2 cm × 4 cm in the vicinity of the animals shoulder. Intradermal induction area.
- Frequency of applications: 1 application
- Duration:48 hours
- Concentrations:
Treatment group: 25 % (0.5 ml) test substance in isotonic saline
Control group: isotonic saline

B. CHALLENGE EXPOSURE
- No. of exposures: one application
- Day of challenge: 22
- Exposure period: 24 hours
- Test groups: 10
- Control group: 5
- Site: one area of approx. 5 cm × 5 cm an the left flank
- Concentrations: 25 % (0.5 ml) test substance in isotonic saline (treated and control group)
- Evaluation: 48 and 72 hours after challenge

Positive control substance(s):

no

Results and discussion

In vivo (non-LLNA)

Results

Results 1

Group:

positive control

Remarks on result:

not measured/tested

Results 2

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25 % in isotonic saline

No. with + reactions:

9

Total no. in group:

10

Clinical observations:

blue discolored

Remarks on result:

positive indication of skin sensitisation

Results 3

Reading:

2nd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

25 % in isotonic saline

No. with + reactions:

9

Total no. in group:

10

Clinical observations:

dry, rough, skin scabbed, blue discolored

Remarks on result:

positive indication of skin sensitisation

Results 4

Reading:

1st reading

Hours after challenge:

48

Group:

negative control

Dose level:

25 % in isotonic saline

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

no signs of irritation

Results 5

Reading:

2nd reading

Hours after challenge:

72

Group:

negative control

Dose level:

25 % in isotonic saline

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

no signs of irritation

Any other information on results incl. tables

Determination of the primary non-irritant concentration

No signs of irritation occurred after application of the different test concentrations, i.e. 25 %, 5 % and 1 % in isotonic saline.

Tolerance of intradermal injections

The intradermal injections with the 0.2 % preparations caused no oedema. After injection of the 1.0 % preparation very slight to slight oedema and indurations occurred. The application areas treated with the 5.0 % preparation showed slight to moderate oedema with indurations and encrustations. Evaluation of erythema was not possible due to blue discoloured skin. Based on this preliminary test, a 1 % preparation was selected for the intradermal injections in the main test.

Signs and bodyweight

Treated animals showed no clinical signs of intoxication throughout the study. Body weight gains of treated animals were not impaired.

Challenge treatment

24 and 48 hours after removal of the occlusive bandage 9 of 10 animals of the treated group showed very slight to severe erythema as well as very slight to slight oedema. In some cases the skin of the treated area was dry, rough and scabbed. Additionally, the treated area was discoloured blue.

No signs of irritation were observed 24 and 48 hours after removal of the occlusive bandage in the control group.

Applicant's summary and conclusion

Interpretation of results:

Category 1A (indication of significant skin sensitising potential) based on GHS criteria

Conclusions:

Based on the results in Guinea pig maximisation test, the substance is a skin sensitiser.

Executive summary:

Method

Testing for sensitising properties of test material was performed in female Guinea pigs according to the Magnusson & Kligman method. Intradermal induction was performed using 1.0 % of test substance in isotonic saline as well as in 50 % Freund's adjuvant. Dermal induction and challenge treatment were carried out with 25 % of test item in isotonic saline.

Results

Positive response was seen in 9/10 animals upon observation at 48 and 72 hours after challenge in treated animas. Therefore, the substance is considered as skin sensitiser.