Reaction mass of 1,5-Naphthalenedisulfonic acid, 3,3'-[(4-methyl-1,2-phenylene)bis[imino(6-chloro-1,3,5-triazine-4,2-diyl)imino[2-(acetylamino)-5-methoxy-4,1-phenylene]-2,1-diazenediyl]]bis-, sodium salt (1:4) and 1,5-Naphthalenedisulfonic acid, 3,3'-[(3-methyl-1,2-phenylene)bis[imino(6-chloro-1,3,5-triazine-4,2-diyl)imino[2-(acetylamino)-5-methoxy-4,1-phenylene]-2,1-diazenediyl]]bis-, sodium salt (1:4)

Skin sensitisation

Some information in this page has been claimed confidential.

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From November 1988 to December 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

Magnusson and Kligman method

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The study was run before the requirement for LLNA testing was implemented.

Test material

Test material information

Constituent 1

Test material form:

solid

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Barriered animal breeding unit, alderley park, macclesfield, cheshire, UK.
- Age at study initiation: Young adults
- Weight at study initiation: 250-307 g
- Housing: Guinea pigs were housed indiviually in suspended cages (37 cm lenght × 32 cm widht × 20 cm height). The floor adn the back of the each cage were made of approximately 1.2 cm square stainless steel mesh, the sides were maded of solid stainless steel and the front was made of polycarbonate.
- Diet (e.g. ad libitum): Free access
- Water (e.g. ad libitum): Free acess
- Acclimation period: 6 d prior to experimentation
- Indication of any skin lesions:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19±2°C
- Humidity (%): 50±10%
- Air changes (per hr): 20-30
- Photoperiod (hrs dark / hrs light): 12

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

other: deionised water

Concentration / amount:

10% 0.05-0.1 mL

Induction 2

Route:

epicutaneous, occlusive

Vehicle:

other: deionised water

Concentration / amount:

50% (w/v) preparation in deionised water

Day(s)/duration:

48 h

Challenge

Route:

epicutaneous, occlusive

Vehicle:

other: deionised water

Concentration / amount:

50% (w/v) and 30% (w/v) preparation of test substance in deionised water at 0.05-0.1 mL was applied on left and right flank respectively.

Day(s)/duration:

24 h

No. of animals per dose:

Test group: 20
Control group: 10

Details on study design:

Sighting study:

The dose levels for each of the three stages of the main study were determined by a sighting study in which groups of two or more guinea pigs were used and up to four dose levels were tested on each group of animals. The procedure was as follows:

1) Intradermal injection (induction): Preparations of the test substance in deionised water were tested to determine the highest concentration that could be well tolerated locally and systemically;
2) Topical application (induction): Preparations of the test substance in deionised water were tested to determine the highest concentration which did not cause greater than a mild to moderate irritation response, in animals that had been injected with Freund's Complete Adjuvant at least fourteen days previously;
3) Topical application (challenge): Preparations of the test substance in deionised water were tested to determine the highest concentration which did not produce irritation in animals that had been injected with Freund's Complete Adjuvant at least fourteen days previously.

Main study:

Induction:

Intradermal injection:
-Three intradermal injections of (0.05 to 0.1 m/L site) on clipped scapular region of guinea pig as follows:
- Top: 1:1 ratio of FCA plus deionised water.
- Middle: 10% preparation of test substance in deionised water.
- Bottom: 10% preparation of test substance in a 1:1 preparation of FCA plus deionised water.

The injections checked for 48 h for any adverse effects.

One week later, the scapular area was clipped again and treated with a topical application of the test sample as a 50% (w/v) preparation in deionised water. This preparation (0.27-0.34g) was applied on filter paper (approximate size 4cm x 2cm) which was held in place by a piece of surgical tape. The tape was covered by a strip of adhesive bandage (approximate size 20-30cm x 5cm) and secured by a piece of self-adhesive PVC tape. This occlusive dressing was kept in place for 48 hours.

The application sites were checked approximately 24 hours after removal of the dressings.

Induction of the control animals:

Intradermal injections were administered using an identical procedure to that used for the test animals, except that.
- Top: 1:1 ratio of FCA plus deionised water.
- Middle: in deionised water.
- Bottom: 1:1 preparation of FCA plus deionised water.

Challenge:
Two weeks after the topical inductions, an area, approximately 15cm x 5cm, on one or both flanks of all the test and control animals, was clipped free of hair with a pair of veterinary clippers. An occlusive dressing was prepared which consisted of two pieces of filter paper (approximate size 1cm x 1.5-2.0cm) stitched to a piece of rubber sheeting (approximate size 12cm x 5cm). A 50% (w/v) preparation of the test sample (approximately 0.17g) in deionised water was applied to one piece of filter paper and a 30% (w/v) preparation in deionised water (0.05-0.1mL) was applied to the second piece of filter paper. The dressing was placed onto the guinea pig so that the 50% (w/v) preparation was on the left shorn flank and the 30% (w/v) preparation was on the right shorn flank. It was then covered with a strip of adhesive bandage (approximate size 25-40cm x 7.5cm) which was secured by self-adhesive PVC tape. After 24 hours, the dressing was carefully cut, using blunt-tipped scissors. Removed and discarded. The position of the pieces of filter paper on the skin were identified using a black waterproof marker-pen.

After a further 24 and 48 hours any erythematous reactions were quantified.

Positive control substance(s):

yes

Remarks:

Formaldehyde as a 40% solution in water

Results and discussion

Positive control results:

One test and one control animal died prior to challenge. These deaths were considered to be unrelated to treatment. Following challenge with a 30% (w/v) dilution of the formaldehyde solution, scattered mild redness to intense redness and swelling was seen in all the test animals. Control animals were unaffected. The net response was 100% and, therefore, the test sample elicited an extreme sensitisation response in previously-induced guinea pigs.

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

other: Overall

Hours after challenge:

48

Group:

test chemical

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Reading:

other: Overall

Hours after challenge:

48

Group:

positive control

Dose level:

40%

No. with + reactions:

20

Total no. in group:

20

Remarks on result:

positive indication of skin sensitisation

Results 3

Key result

Reading:

other: Overall

Hours after challenge:

48

Group:

negative control

Remarks on result:

not determinable

Any other information on results incl. tables

Results: 

The bandage slipped from one test animal following challenge. There was no erythematous response at either challenge site, therefore it could not be determined whether the test sample had been in contact with the skin for a period long enough to elicit a skin sensitisation response. This animal was excluded from the analysis of the results.

 

Challenge with the 50% (w/v) preparation of the test substance:

 

Stained the test site in all but one of the remaining test animals and in all but one of the control animals. However, this did not interfere with the assessment of erythema. The erythematous response seen in one test animal was doubtful and it was therefore excluded from the analysis of the challenge results. Five out of the remaining eighteen test animals showed scattered mild redness or moderate diffuse redness (one animal). One of the ten control animals showed scattered mild redness. The net percentage response was calculated to be 18%.

 

Therefore, challenge with a 50% (w/v) preparation of test substance elicited a mild skin sensitisation response in previously-induced guinea pigs.

 

Challenge with the 30% (w/v) preparation of the test substance:

 

Stained the test site in all but one test animal. No erythematous responses were seen in any of the animals, test or control. Therefore, challenge with a 30% (w/v) preparation of test substance did not elicit a skin sensitisation response in previously-induced guinea pigs.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the guinea-pig maximisation study, the test substance was not considered to be a skin sensitiser.

Executive summary:

A study was conducted to assess the sensitising potential of the test substance in a Guinea pig maximisation test according to OECD Guideline 406, in compliance with GLP. Intradermal induction was carried out with a 10% dilution of the test substance in deionised water and in FCA + deionized water. One week later, epicutaneous induction was conducted under occlusion with 50% test substance in deionised water. Two weeks after the epicutaneus induction application, the challenge was completed by epicutaneus application of the test substance at 50 and 30% in deionised water to the left and right shorn flank of the guinea pigs under occlusive dressing. The animals in the control group were induced with deionised water and FCA + deionised water and challenged similarly to those in the test groups. Cutaneous reactions (erythema, eschar and oedema formation) were evaluated at 24 and 48 h after removal of the dressing. Challenge with 50% test substance stained the test sites in all animals but did not interfere with the assessment of erythema. At 24 and 48 h after removal of the adhesive bandage, the reaction was scored. In this study, 18% of the animals of the test group were observed with mild skin reactions after treatment with the non irritating test substance concentration of 50% in deionised water, however the same was not observed in 30% challenge of test substance in deionised water. The author concluded that the substance was a mild skin sensitizer, however the CLP (EC 1272/2008) classification criteria were not met. Under the conditions of the Guinea pig maximisation study, the test substance was therefore not considered to be a sensitiser (Ishmael, 1989).