Reaction mass of tetrasodium 7,7'-(carbonyldiimino)bis[4-hydroxy-3-[(2-methyl-4-sulphonatophenyl)azo]naphthalene-2-sulphonate] and tetrasodium 4-[[1-hydroxy-6-[[[[5-hydroxy-6-[(2-methyl-4-sulphonatophenyl)azo]-7-sulphonato-2-naphthyl]amino]carbonyl]amino]-3-sulphonato-2-naphthyl]azo]benzoate and tetrasodium 4,4'-[carbonylbis[imino(1-hydroxy-3-sulphonatonaphthalene-6,2-diyl)azo]]dibenzoate

Endpoint summary

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Kleintierfarm Madoerin AG, CH-4414 Füllinsdorf, Switzerland
- Age at study initiation: males: 7 weeks, females: 8 weeks
- Weight at study initiation: males: 353 - 586 g, females: 333 - 390 g
- Housing: Individually in Makrolon type-3 cages with standard softwood bedding (Lignocel, Schill AG, 4132 Muttenz, Switzerland)
- Diet: Pelleted standard Kliba 342, Batch 50/89 and 51/89 guinea pig breeding/maintenance diet ("Kliba", Klingentalmühle AG, CH-4303 Kaiseraugst), ad libitum
- Water: tap water ad libitum
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 40-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Route:

intradermal and epicutaneous

Vehicle:

other: distilled water for intradermal applications; petrolatum oil for epicutaneous applications

Concentration / amount:

- Intradermal induction: 3%
- Epicutaneous induction: 5%
- Challenge: 3%

Route:

epicutaneous, occlusive

Vehicle:

other: distilled water for intradermal applications; petrolatum oil for epicutaneous applications

Concentration / amount:

- Intradermal induction: 3%
- Epicutaneous induction: 5%
- Challenge: 3%

No. of animals per dose:

- Control group: 5 males + 5 females
- Test group: 10 males + 10 females

Details on study design:

RANGE FINDING TESTS:
- Intradermal injections: Intradermal injections (0.1 mL/site) were made into the clipped flank of two guinea-pigs at concentrations of 1, 3 and 5% of the test article in distilled water. The resulting dermal reactions were assessed 24 hours later.
- Epidermal applications: Patches of filter paper (2 cm x 2 cm) were saturated with concentrations of 3, 5, 10 and 25% of the test article in petrolatum oil and applied to the clipped and shaved flanks of each of four guinea-pigs. The patches were covered by a strip of aluminum foil and firmly secured by elastic plaster wound round the trunk and covered with impervious adhesive tape. This procedure ensured the intensive contact of the test article with the guinea pig skin. The dressings were removed after an exposure period of 24 hours and the reaction sites were assessed for erythema and edema on a numerical basis according to Draize. Further examination of the sites was performed 24 and 48 hours after removal of the dressings. The allocation of the different test sites on the animals were alternated in order to minimize site to site variation in responsiveness.

MAIN STUDY
A. INDUCTION EXPOSURE
- Intradermal injections: An area of dorsal skin from the scapular region (approximately 6 cm x 8 cm) was clipped free of hair. Three pairs of intradermal injections (0.1 mL/site) were made at the border of a 4 cm x 6 cm area in the clipped region as follows:
1) Freund's complete adjuvant 50:50 with distilled water.
2) The test article, diluted to 3% with distilled water.
3) The test article at the concentration used in (2), emulsified in a 50:50 mixture of Freund's complete adjuvant, and the vehicle used in (2).
- Control group: The control group was treated accordingly with the omission of the test article.

- Epidermal applications: One week after the injections, the scapular area (approximately 6 cm x 8 cm) was again clipped and shaved free of hair. A 2 cm x 4 cm patch of filter paper was saturated with the test article (5% in petrolatum oil) and placed over the injection sites of the test animals. The patch was covered by aluminum foil and firmly secured by an elastic plaster wound round the trunk of the animal and secured with impervious adhesive tape. The dressings were left in place for approximately 48 hours. The epidermal application procedure described ensured intensive contact of the test article with the guinea pig skin. The guinea-pigs of the control group were treated as described above with the omission of test article. The reaction sites were assessed for erythema and edema immediately, 24 and 48 hours after removal of the dressing, using the numerical grading system according to Draize.

B. CHALLENGE EXPOSURE
The test and control guinea-pigs were challenged two weeks after the epidermal induction application. Hair was clipped and shaved from a 5 cm x 5 cm area on the left and right flank of each guinea-pig. Two patches (2 cm x 2 cm) of filter paper were saturated with a) non-irritant concentration (3% in petrolatum oil) of the test article and b) with the vehicle only and applied to the (a) left flank and (b) right flank respectively using the same method as for the epidermal application. The dressings were removed approximately 24 hours later. The sites were assessed for erythema and edema immediately, 24 and 48 hours after removal of the dressing, using the numerical scoring system according to Draize. The control animals were treated in the same way as described above.

Challenge controls:

Treatment of the control group with the test substance formulation

Positive control substance(s):

yes

Remarks:

1-chloro-2,4-dinitro-benzol

Positive control results:

A control group (Dinitro-chloro-benzene) is tested twice a year in the testing laboratory for sensitivity check of the guinea pig strain. The most recent test was run during September 1988. In 6 out of 9 animals tested in this study sensitisation was observed

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

3% in petrolatum oil

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 3% in petrolatum oil. No with. + reactions: 0.0. Total no. in groups: 10.0.

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

3% in petrolatum oil

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 3% in petrolatum oil. No with. + reactions: 0.0. Total no. in groups: 10.0.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

3% in petrolatum oil

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 3% in petrolatum oil. No with. + reactions: 0.0. Total no. in groups: 20.0.

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

3% in petrolatum oil

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 3% in petrolatum oil. No with. + reactions: 0.0. Total no. in groups: 20.0.

Pretest:

- Intradermal injection: According to Magnusson - Kligman and to the findings observed, the concentration selected for the main study was 3 %. The application area was red discolored and therefore a possible erythema could not be observed. Edema was observed at all concentrations.

- Epidermal application: No edema was observed, but erythema was observed in all four animals immediately after after removal of the bandage. After 24 hours in 3/4, 2/4, 1/4 and 0/4 animals exposed to 25, 10, 5, and 3% test substance, respectively, erythema was observed. After 48 hours only in 2 out 0f 4 animals of the 25% dose group erythema was observed. According to Magnusson - Kligman, and to the findings observed, the concentration selected for the induction period was 5 % and for the challenge procedure 3 %. Prior to observation the skin of all animals was depilated to facilitate the observation of a possible erythema.

Main test:

- The highest non-irritating concentration was 3%. No systemic toxic symptoms were evident in the guinea pigs of either the control or test group. No death occurred. The body weight gain of all animals was not affected during the test.

- Control group local effects: The application area around the injection site 1 was found to show erythema and edema from day 2 to 7. Necroses were observed at day 8 and day 10 to 18. Exfoliation was observed from day 19 to 25 (termination of test). The application area around the injection site 3 was found to show erythema and edema from day 2 to 7. Necroses were observed on day 8 and from day 10 to 16. Exfoliation was observed from day 17 to 25 (temination of test). Additionally discoloration was observed from day 23 to 25 at the first challenge application.

- Test group local effects: The application area around the injection site 1 was found to show erythema from day 2 to 7 and edema on day 2 and 3. Necroses were observed at day 8 and days 10 to 18. Exfoliation was observed from day 19 to 25 (termination of test). The application areas around the injection sites 2 and 3 were found to show edema from day 2 to 7 and discoloration was observed from day 2 to 8 and from day 10 to 16. Necroses were found on day 8 and from day 10 to 16. Exfoliation was observed from day 17 to 25 (termination of test). Additionally discoloration was observed from day 10 to 16 after the epidermal application and from day 22 to 25 at the first challenge application. On day 9 of the test no observation could be performed because the animals were treated semi occlusively.

Interpretation of results:

not sensitising

Remarks:

Migrated information

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Skin sensitisation

In a GLP compliant OECD 406 guideline study, Dunkin-Hartley guinea pigs were used to assess the sensitizing potential of the test substance. Ten animals per sex were used in the test group and five per sex in the control group. The purity of the tested substance was ca. 75%. Concentrations used in the test were derived from the results of a pretest. For intradermal injections of the test group, 0.1 mL of a 3% solution in water, Freund’s complete adjuvant (50:50), or 3% in Freud’s adjuvant was used. One week after the injections, 5% test substance in petrolatum oil was applied to the animals for 48 hours. For challenge one week later, the highest non-irritating dose of 3% in petrolatum oil was used. One and two days after challenge, neither animals of the control nor of the test group showed a positive reaction for erythema. No reactions were observed after treatment with vehicle only. The test substance was therefore not considered to be skin sensitizing.

Migrated from Short description of key information:
The test substance is not considered to be a skin sensitizer under the conditions of a GLP-compliant guinea pig study (OECD Guideline 406).

Justification for selection of skin sensitisation endpoint:
There is only one study available.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

No skin sensitizing effects were observed in animals exposed to the test substance. Based on this information classification for skin sensitisation is not warranted in accordance with EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.

As no study is available, classfication is not possible for respiratory sensitisation in accordance with EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008