A mixture of: trisodium (2,4(or 2,6 or 4,6)-bis(3,5-dinitro-2-oxidophenylazo)-5-hydroxyphenolato)(2(or 4 or 6)-(3,5-dinitro-2-oxidophenylazo)-5-hydroxy-4(or 2 or 6)-(4-(4-nitro-2-sulfonatoanilino)phenylazo)phenolato)ferrate(1-); trisodium bis(2,4(or 2,6 or 4,6)-bis(3,5-dinitro-2-oxidophenylazo)-5-hydroxyphenolato)ferrate(1-); trisodium (2,4(or 2,6 or 4,6)-bis(3,5-dinitro-2-oxidophenylazo)-5-hydroxyphenolato)(2(or 4 or 6)-(3,5-dinitro-2-oxidophenylazo)-5-hydroxy-4(or 2 or 6)-(4-nitro-2-sulfonatophenylazo)phenolato)ferrate(1-); trisodium (2,4(or 2,6 or 4,6)-bis(3,5-dinitro-2-oxidophenylazo)-5-hydroxyphenolato)(2(or 4 or 6)-(3,5-dinitro-2-oxidophenylazo)-5-hydroxy-4(or 2 or 6)-(3-sulfonatophenylazo)phenolato)ferrate(1-); disodium 3,3'-(2,4-dihydroxy-1,3(or 1,5 or 3,5)-phenylenediazo)dibenzenesulfonate

Endpoint summary

Description of key information

skin sensitizer

Key value for chemical safety assessment

Skin sensitisation

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

In order to assess the cutaneous allergenic potential of test substance, the GPMT and the Buhler assay were performed in accordance with OECD Guideline 406 (1981) and the method B.6 of EEC-Directive 96/54 EEC.

GUINEA PIG MAXIMISATION TEST (GPMT, OECD TG 406)

A GPMT was performed in order to obtain information on the potential of the test substance to induce delayed contact hypersensitivity (skin sensitisation) in the guinea pig, according to OECD guideline 406 (1981).

The intracutaneous route of administration (first induction at 5 %) was selected in order to obtain optimal contact between the test substance and elements of the immunosystem. Freunds Complete Adjuvant (FCA) was added to attract circulating immunocompetent cells to the injected area. The dermal route of administration (second induction and challenge) was performed. The test and control guinea pigs were challenged two weeks after the epidermal induction application. The dressings and residual test substance were removed after approximately 24 hours. The sites were assessed for redness and sweiling 24 and 48 hours after removal of the dressings. A positive control was tested in a concurrent study in order to verify the test validity. The following results were obtained:

POSITIVE SKIN REACTIONS TO THE CHALLENGE

	Test Substance Concentration
	25%	10%	5%	0%
EXPERIMENTAL GROUP Number of animals with positive reaction	4	0	0	0
Sensitisation rate	20	0	0	0
CONTROL GROUP Number of animals with positive reaction	0	0	0	0

BUEHLER ASSAY (OECD TG 406)

The substance was tested for skin sensitisation according to the OECO Guideline 406 (1981) in a Buehler test. For this study 30 Himalayan females albino guinea pigs were distributed as follows: ten females for the control  group and twenty  females for the experimental group. The five  remaining  animals were used for the primary  irritation  test, one week before the main  study.

The induction phase consisted of nine repeated occluded topical applications to the same area of the scapula region (left side). The treated area was clipped prior to each application.

Experimental animals were exposed to 0.5 ml of the test substance suspended to 25% (w/w) in milli-RO water. The applications were made 9 times during a period of 3 weeks (days 1, 3, 5, 8, 10, 12, 15, 17 and 19). After each application the remaining test substance was removed from the skin with a tissue moistened with tap-water. The control animals were treated as described above with the omission of the test substance.

The treated skin area of experimental and control animals was scored immediately after removal of the dressings on day 19.

Ten days after the last induction exposure (day 29), the experimental and control animals were challenged to 0.5 mi of the test substance : 25% (w/w) in milli-RO water and to 0.5 ml of the vehicle alone.

Hair was clipped and shaved from a 5 x 5 cm area on the right flank of each guinea-pig. A volume of 0.5 ml of the test substance or the vehicle was placed onto a similar patch as used in the induction phase. The patches were placed to the shaved area and kept with an elastic bandage (Coban). After 6 hours, the dressings and residual test article were removed using a tissue moistened with tap-water.

The challenge sites of experimental and control animals were assessed for redness and swelling 40, 48 and 72 hours after removal of the dressings, using the numerical grading system described below.

The test sites were shaved with an electric razor immediately after the skin reading, 40 hours after bandage removal.

A second challenge was performed one week after the first challenge, due to inconclusive results being obtained from the first challenge, In this case, all animals were re-challenged with a series of three test

substance concentrations: 25 %, 10 %, 5 % (w/w) and the vehicle (milli-RO water). From each concentration or the vehicle, 0.05 ml was placed into the Square chambers. The patches were placed to the contralateral shaved flank. The dressing and the residual test substance were removed after approximately 6 hours. The sites were assessed for redness and swelling 24 and 48 hours after the removal of the dressing, using the numerical grading system described previously. Immediately after the 24 hours skin reading the treated sites were shaved. All animals were killed at the end of the test period by carbon dioxide asphyxiation.

No animals showed a positive reaction in response to the 25%, 10% or 5% concentrations . Four animals showed red spots in response to the 25% concentration.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In the CLP Regulation (EC) no. 1272/2008 a skin sensitizer is defined as “a substance that will lead to an allergic response following skin contact”. A substance classified as skin sensitiser (Category 1) may be allocated to one of the two sub-categories 1A or 1B in accordance with the criteria given in Annex I, Part 3, Table 3.4.2.. If classification is based on results obtained from studies conducted in experimental animals, the test results from the LLNA, GPMT and the Buehler assay can be used directly for classification. Guidance values are presented in the CLP Regulation (EC) no. 1272/2008 in Annex I, Part 3, Table 3.4.3 for classification in sub-category 1A and in Annex I, Part 3, Table 3.4.4 for classification in sub-category 1B.

Based on the results from the GPMT performed, the substance induces a sensitisation rate of 80 -100 % after the challenge epidermal application of the test item at 15 % in PEG. This result meets the classification criterion for Skin sens. Cat. 1B “≥ 30 % responding at > 1 % intradermal induction dose”. On the contrary, results from the Buhler assay shown no skin reactions after the challenge treatment with the test item at 25 % in PEG 300. Even if the two different tests were both performed according to the OECD Guideline 406, clearly the two results are opposing.

Considering that:

- no significant deviations from the standardised followed methods that could lead to a lower reliability of the studies can be found;

- it should be recognised that there is often a degree of uncertainty associated with the derivation of allergenic potencies from both the GPMT and the Buhler, as also the “Guidance on the Application of the CLP Criteria – Version 5.0, July 2017” reported it should be considered a worst case approach;

- the substance is listed in the Annex VI of the CLP Regulation (EC) 1272/2008

the substance should be classified as Skin sens. Cat. 1B.