Disodium 4,4'-bis(5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-ylazo)-1,1'-biphenyl-2,6'-disulphonate

Endpoint summary

Description of key information

Sensitising

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From May 30 to December 09, 1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Magnusson B. - Kligman A. - The identification of contact allergens by animal assay. "The guinea pig maximization test". J. Invest. Dermatol 1969, 52, 3, 268-276.

Version / remarks:

1969

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.

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Iffa-Credo (69210 Saint Germain sur l'Arbresle, France)
- Weight at study initiation: mean bodyweight of animals in the control group was 617 ± 29 g for males and 526 ± 35 g for females. The mean bodyweight of animals in the treated group was 622 ± 28 g for males and 544 ± 50 g for females.
- Housing: animals housed individually in polycarbonate preautoclaved cages (0.48 x 0.27 x 0.20 m) on sawdust (Sociêtê Parisienne des Sciures, 93500 Pantin, France).
- Diet (e.g. ad libitum): animals were fed ad libitum during the study, with a certified pelleted diet :Ref. 106 (U.A.R., Villemoisson sur Orge, France).
- Water (e.g. ad libitum): free access to bottles containing tap water filtered with Millipore filters (0.22 micron). Routine bacteriological and chemical analysis of water were made,
in order to detect major contaminants, by the Laboratoire Municipal (76000 Rouen, France)
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 ± 3°C
- Humidity (%): 50 ± 20%.
- Air changes: the incoming non-recycled air was filtered with an absolute filter
- Photoperiod (hrs dark / hrs light): the light/dark cycle was 12 hours per day.

Route:

intradermal and epicutaneous

Vehicle:

other: Distilled water, NaCl 0.9 %, Freund's complete adjuvant

Concentration / amount:

- for the intradermal route at a concentration of 1% in distilled water; - for the epicutaneous route and the challenge patch test, undiluted

Adequacy of induction:

not specified

Route:

epicutaneous, occlusive

Vehicle:

other: Distilled water, NaCl 0.9 %, Freund's complete adjuvant

Concentration / amount:

0.5 g of the undiluted test sample and 0.5 ml of the vehicle

Adequacy of challenge:

not specified

No. of animals per dose:

control group: 10 animals (5 males - 5 females).
treated group : 20 animals (10 males - 10 females).

Details on study design:

MAIN STUDY
A. INDUCTION EXPOSURE
The animals were clipped and shaved on the scapula before each treatment.
a. Intradermal route
On Day 1, 3 x 2 intradermal injections of 0.1 ml each were performed on the scapula on a surface area of 4 x 2 cm (one injection of each solution per flank).
(1). Treated group
• Freund's complete adjuvant at 50 % in saline (NaCl0.9 %).
• Test sample at the concentration of 1% in distilled water.
• A 50/50 (v/v) mixture of Freund's complete adjuvant diluted at 50 % in saline (NaCl0.9 %) and of test sample at the concentration previously determined.
(2). Control group
• Freund's complete adjuvant at 50 % in saline (NaCl0.9 %)
• Vehicle : distilled water
• A 50/50 (v/v) mixture of Freund's complete adjuvant diluted at 50 % (v/v) in saline (NaCl0.9 %) and vehicle.
b. Epicutaneous route
On Day 8 after shaving the animals, 0.5 ml of sodium lauryl sulfate diluted at 10% in codex vaseline was applied to the scapular region
to provoke a local irritation.
On Day 9 : application to the scapular region, under an occlusive patch, during 48 h, on the 6 injections sites (4 x 2 cm):
(1) In the treated group, 0.5 g of the undiluted test sample per animal : the test sample was first applied to a hydrophilic gauze moistened with 0.5 ml of distilled water.
(2) In the control group, 0.5 ml of the vehicle : distilled water

B. CHALLENGE EXPOSURE
At the end of the rest period (on Day 25), the flanks of the animals of all groups were clipped and shaved. On Day 26, occlusive patches were applied on a 4 cm2 square for 24 hours with 0.5 g of the undiluted test sample per animal on the right flank, and 0.5 ml of the vehicle, distilled water on the left flank. The patches were kept in place by a hypo-allergenic tape.

Positive control substance(s):

not specified

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

0.5 g of the undiluted test sample and 0.5 ml of the vehicle

No. with + reactions:

8

Total no. in group:

20

CLINICAL EXAMINATION

1. Behaviour and clinical signs

Neither change in behaviour nor clinical signs because of treatment were observed during the study period.

2. Mortality

No mortality was recorded during the study.

MACROSCOPIC CUTANEOUS REACTIONS AFTER THE CHALLENGE PATCH TEST

In the control group:

No cutaneous reactions were observed at each scoring.

In the treated group:

- 24 hours after patch removal, erythema could not be detected because of the staining of the skin by the test sample. No oedema was observed.

- 48 hours after patch removal , a skin dryness was observed on the right flank of one male (184). A very slight erythema was observed on the right flank of three males (187, 189, 190) and two females (196, 197). A well-defined erythema was observed on the right flank of one female (200). No cutaneous reactions were observed on the left flank (vehicle).

MICROSCOPIC EXAMINATIONS

- several animals showed, on either flank, some cutaneous reactions attributed to irritation

- 8 animals out of 20 in Group II showed cutaneous reactions on their right side of a type and severity that were attributed to a sensitization reaction (184, 187, 189, 190,194, 196, 197, 299)

- 2 animals out of 20 in group II showed equivocal cutaneous reactions on both sides (185 and 200).

There was no trace of the dye staining the surface of the treated skin after processing techniques.

Interpretation of results:

other: Category 1B (indication of skin sensitising potential) based oon Regulation 1272/2008

Conclusions:

Sensitising

Executive summary:

Method

The sensitizing potential of the test substance was tested after intradermal injections and cutaneous applications in male and female albino guinea pigs, according to Magnusson B. - Kligman A. ( J. Invest. Dermatol 1969).

Results

The intradermal induction was performed at the concentration of 1 % in distilled water. The epicutaneous induction and the challenge patch test were performed with the undiluted test sample. The behaviour of the animals was not influenced by the treatment. 24 hours after patch removal, cutaneous reactions could not be detected because of the staining of the skin by the test sample. 48 hours after patch removal, a slight erythema was observed on the right flank of five animals, and a well-defined erythema in one female. Skin samples were taken and subjected to microscopic examination. Histological evidence of a sensitization reaction due to the test sample was observed in eight animals.

Conclusion

Sensitising.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available (further information necessary)

Additional information:

The sensitizing potential of the test substance was tested after intradermal injections and cutaneous applications in male and female albino guinea pigs, according to Magnusson B. - Kligman A. ( J. Invest. Dermatol 1969).

The intradermal induction was performed at the concentration of 1 % in distilled water. The epicutaneous induction and the challenge patch test were performed with the undiluted test sample. The behaviour of the animals was not influenced by the treatment. 24 hours after patch removal, cutaneous reactions could not be detected because of the staining of the skin by the test sample. 48 hours after patch removal, a slight erythema was observed on the right flank of five animals, and a well-defined erythema in one female. Skin samples were taken and subjected to microscopic examination. Histological evidence of a sensitization reaction due to the test sample was observed in eight animals.

Justification for classification or non-classification

SKIN SENSITIZATION

Category 1

Substances shall be classified as skin sensitizers in Category 1 where data are not sufficient for sub-categorisation in accordance with the following criteria:

(a) if there is evidence in humans that the substance can lead to sensitisation by skin contact in a substantial number of persons; or

(b) if there are positive results from an appropriate animal test.

Specific criteria of animal test:

when an adjuvant type test method for skin sensitisation is used, a response of at least 30 % of the animals is considered as positive.

For a non-adjuvant Guinea pig test method a response of at least 15 % of the animals is considered positive.

Furthermore, stimulation index of three or more is considered a positive response in the local lymph node assay.

Sub-category 1A

Substances showing a high frequency of occurrence in humans and/or a high potency in animals can be presumed to have the potential to produce significant sensitisation in humans. Severity of reaction may also be considered.

Specific criteria:

Local lymph node assay-EC3 value ≤ 2 %

Guinea pig maximisation test-≥ 30 % responding at ≤ 0,1 % intradermal induction dose or ≥ 60 % responding at > 0,1 % to ≤ 1 % intradermal induction dose

Buehler assay - ≥ 15 % responding at ≤ 0,2 % topical induction dose or ≥ 60 % responding at > 0,2 % to ≤ 20 % topical induction dose

Sub-category 1B

Substances showing a low to moderate frequency of occurrence in humans and/or a low to moderate potency in animals can be presumed to have the potential to produce sensitisation in humans. Severity of reaction may also be considered.

Local lymph node assay - EC3 value > 2 %

Guinea pig maximisation test- ≥ 30 % to < 60 % responding at > 0,1 % to ≤ 1 % intradermal induction dose or ≥ 30 % responding at > 1 % intradermal induction dose.

Buehler assay - ≥ 15 % to < 60 % responding at > 0,2 % to ≤ 20 % topical induction dose or ≥ 15 % responding at > 20 % topical induction dose.

Based on the results of the animal test (Guinea pig maximisation test) performed, according to the paragraph 3.4. of the CLP Regulation n. 1272/2008, the test substance is classified in SubCategory 1B, as 40 % of the animals were positive after treatment (intradermal dose = 1 % of test substance).