Disodium 7-[[2-(acetylamino)-4-[(5-chloro-2,6-difluoro-4-pyrimidinyl)amino]phenyl]azo]naphthalene-1,3-disulphonate

Endpoint summary

Description of key information

Skin Sens 1B (H317), according to the CLP Regulation (EC) No 1272/2008

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 September 13th to October 21st, 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

1981

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:

When the test was conducted, the GPMT system was chosen because the guinea pig had been shown to be a suitable species for this type of study and it was recommended in the test method.

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: main study animals were supplied by David Hall Limited, Burton-on-Trent, Staffordshire, U.K.
- Age at study initiation: approximately eight to twelve weeks old.
- Weight at study initiation: 307 - 431 g
- Housing: animals were housed in groups of up to three in solid-floor polypropylene cages furnished with softwood shavings.
- Diet: Guinea Pig FDI Diet, Special Diet Services Limited, Witham, Essex, U.K. was allowed throughout the study, ad libitum.
- Water: tap water, ad libitum.
- Acclimation period: a minimum acclimatisation period of five days.

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 23 °C
- Relative humidity: 47 - 69 %
- Air changes: approximately 15 changes per hour.
- Photoperiod: lighting was controlled by a time switch to give 12 hours ljght and 12 hours darkness.

Route:

intradermal

Vehicle:

water

Concentration / amount:

10 %

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

50 %

Day(s)/duration:

2

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

50 %

Day(s)/duration:

1

No. of animals per dose:

20 tested group and 10 control group

Details on study design:

RANGE FINDING TESTS
The concentrations of test material to be used at each stage of the main study were determined by 'sighting tests' 'in which groups of guinea pigs were treated with various concentrations of test material.

- Selection of Concentration for Intradermal Induction
Individual animals were intradermally injected with a 1, 5, 10 or 25 % (w/v) preparation of test material in distilled water. The highest concentration that did not cause local necrosis, ulceration or systemic toxicity, was selected for the intradermal induction stage of the main study.
- Selection of Concentration for Topical Induction
Two guinea pigs (intradermally injected with Freund's complete Adjuvant sixteen days earlier) were treated with 50, 25, 10 and 5 % (w/w) preparations of the test material in distilled water. The highest concentration producing only mild to moderate dermal irritation after a 48-hour occlusive exposure, was selected for the topical induction stage of the main study.
- Selection of Concentrations for Topical Challenge
On day fourteen of the main study 50, 25, 10 and 5 % (w/w) preparations of the test material in distilled water were applied occlusively to the flanks of two guinea pigs for a period of 24 hours. These guinea pigs had been treated identically to the control animals of the main study, up to day fourteen. The maximum non-irritant concentration of the test material was selected for the topical challenge stage of the main study.

MAIN STUDY
A. INDUCTION EXPOSURE
Intradermal induction of the Test Animals
- Exposure area: the hair was removed from an area approximately 40 mm x 60 mm on the shoulder region of each animal with veterinary clippers.
- No of exposures: a row of three injections (0.1 ml each) was made on each side of the mid-line. The injections were: Freund's Complete Adjuvant plus distilled water in the ratio 1:1; a 10 % (w/v) dilution of test material in distilled water; a 10 % (w/v) dilution of test material in a 1:1 preparation of Freund's Complete Adjuvant plus distilled water.
- Control: identical procedure to that used for the test animals, except that the injections were: Freund's Complete Adjuvant plus distilled water in the ratio 1:1; distilled water; Freund's Complete Adjuvant plus distilled water in the ratio 1:1.

Epicutaneous induction of the Test Animals
- Application after intradermal induction: one week later.
- Exposure area: the same area on the shoulder region used previously for intradermal injections was clipped again.
- Application: the test material formulation (0.2 - 0.3 ml) was applied on filter paper (approximate size 40 mm x 20 mm) which was held in place by a strip of surgical adhesive tape (approximate size 60 mm x 25 mm) and covered with an overlapping length of aluminium foil. The patch and foil were further secured by a strip of elastic adhesive bandage (approximate size 250 mm x 35 mm) wound in a double layer around the torso of each animal.
- Exposure duration: the occlusive dressing was kept in place for 48 hours.
- Observation: erythematous reactions were quantified one and twenty-four hours following removal of the patches.
- Control: the topical applications followed the same procedure as for the test animals except that the vehicle alone was applied to the filter paper.

B. CHALLENGE EXPOSURE
- Days after induction: day 21
- Exposure area: an area, approximately 50 mm x 70 mm on both flanks of each animal, was clipped free of hair with veterinary clippers.
- Application: a quantity of 0.1 - 0.2 ml of the test material formulation was applied to the shorn right flank of each animal on a 20 mm x 20 mm square of filter paper which was held in place by a strip of surgical adhesive tape (approximate size 40 mm x 50 mm). The vehicle alone was similarly applied to the left shorn flank. The patches were occluded with an overlapping length of aluminium foil and secured by a strip of elastic adhesive bandage (approximate size 250 mm x 75 mm) wound in a double layer around the torso of each animal.
- Exposure duration: after 24 hours, the dressing was carefully cut using blunt-tipped scissors, removed and discarded.

EVALUATION OF SKIN REACTIONS
Approximately 24 and 48 hours after dressing removal, erythematous reactions were quantified using the four-point scale shown below:
0 - no reaction
1 - scattered mild redness
2 - moderate and diffuse redness
3 - intense redness and swelling

Positive control substance(s):

yes

Remarks:

formaldehyde solution (37 to 41 %)

Group:

test chemical

Dose level:

50 % (w/w) in distilled water

No. with + reactions:

10

Total no. in group:

19

Remarks on result:

positive indication of skin sensitisation

Group:

negative control

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Group:

positive control

Dose level:

10 % formaldehyde solution (37 to 41 %)

No. with + reactions:

20

Total no. in group:

23

Remarks on result:

positive indication of skin sensitisation

Skin Reactions Observed After Topical Induction

Orange-coloured staining caused by the test material was commonly noted following bandage removal. This prevented accurate evaluation of erythema.

Skin Reactions Observed After Topical Challenge

The test material produced a 53 % (10/19) sensitisation rate.

Pale yellow-orange-coloured staining caused by the test material was commonly noted following bandage removal. This did not prevent accurate evaluation of erythema.

One test group animal was not dosed due to ill-health.

Positive skin responses (redness grade 2) were noted at the test material sites of ten test animals at the 24-hour observation. The response persisted in three animals at the 48-hour observation.

Scattered mild redness was noted at the test material sites of seven test animals at the 24-hour observation and at the test material sites of ten test animals at the 48-hour observation. Desquamation was commonly noted at the 48-hour observation.

Scattered mild redness (grade 1) was noted at the test material sites of six control animals at the 24-hour observation. The response persisted in one control animal at the 48-hour observation.

The test material, therefore, produced a 53 % (10/19) sensitisation rate and was classified as a moderate sensitiser to guinea pig skin.

Bodyweight

Bodyweight gains of guinea pigs in the test group, between day 0 and day 24, were comparable to those observed in the control group animals over the same period.

Interpretation of results:

other: Skin Sens 1B (H317), according to the CLP Regulation (EC) No 1272/2008

Conclusions:

Skin sensitizer.

Executive summary:

A study was performed to assess the skin sensitisation potential of the test material in the albino guinea pig. The method used followed that described in the OECD Guidetines for Testing of Chemicals (1981) No. 406 referenced as Method B6 in Commission Directive B4/449/EEC.

Twenty test and ten control animals were used for the main study. Based on the results of sighting tests, the concentrations of test material for the induction and challenge phases were selected as follows: intradermal induction at 10 % (w/w) in distilled water; topical induction at 50 % (w/w) in distilled water; topical challenge at 50 % (w/w) in distilled water.

The test material produced a 53 % (10/19) sensitisation rate and was classified as a moderate sensitiser to guinea pig skin.

Conclusion

Skin sensitizer; more than 30 % responding at an intradermal induction dose higher than 1 %. Therefore, the susbtance meets the criteria to be classifies as skin sensitizer, sub-category 1B (H317), of the CLP Regulation (EC) No 1272/2008.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

A study was performed to assess the skin sensitisation potential of Reactive Yellow 125 in the albino guinea pig. The method used followed that described in the OECD Guidetines for Testing of Chemicals (1981) No. 406 referenced as Method B6 in Commission Directive B4/449/EEC.

Twenty test and ten control animals were used for the main study. Based on the results of sighting tests, the concentrations of test material for the induction and challenge phases were selected as follows: intradermal induction at 10 % (w/w) in distilled water; topical induction at 50 % (w/w) in distilled water; topical challenge at 50 % (w/w) in distilled water.

The test material produced a 53 % (10/19) sensitisation rate and was classified as a moderate sensitiser to guinea pig skin.

Respiratory sensitisation

Endpoint conclusion

Additional information:

There is no experimental literature data regarding the respiratory sensitisation potential of Reactive Yellow 125.

However, a letter between farm responsible and physician reporting an episode of sensitisation is available.

In October 1986, a case of respiratory hypersensitivity (rhinitis, cough and asthma) was found in the Medical Center in Lund (Schueden). After consultation, it was recognized by the physician as a case of sensitization. Unforunately, no additional documentation is available. Thus, it is not know if additional investigations were perfromed.

The episode is here nmentioned only for completeness sake.

Justification for classification or non-classification

According to the CLP Regulation (EC) No 1272/2008, 3.4.2.2 skin sensitisation section, skin sensitizer means a substance that will lead to an allergic response following skin contact. Effects seen in either humans or animals will normally justify classification in a weight of evidence approach for skin sensitizers.

In the experiments performed, more than 30 % of animals showed reactions responding to an intradermal induction dose higher than 1 %.

Therefore, the susbtance meets the criteria to be classifies as skin sensitizer, sub-category 1B (H317), of the CLP Regulation (EC) No 1272/2008.