Reaction mass of disodium 4-amino-3-[[4-[(2,4-diaminophenyl)azo]phenyl]azo]-5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate and disodium 4-amino-3-[[4-[(2-amino-4-hydroxyphenyl)azo]phenyl]azo]-5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Test performed prior to the implementation of the current acknowledged OECD testing and GLP guidelines.The study was conducted according to the method of Magnusson and Kligman.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1973

Report date:

1973

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Method of Magnusson and Kligman

Version / remarks:

The Identification of Contact Allergens by Animal Assay. The Guinea Pig Maximization Test, The Journal of Investigative Dermatology, Vol 52, Issue 3, pages 268 - 276, 1969

GLP compliance:

no

Remarks:

pre-guideline study

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The study was conducted in 1973

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 4-amino-3-[[4-[(2-amino-4-hydroxyphenyl)azo]phenyl]azo]-5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate

EC Number:

270-629-5

EC Name:

Disodium 4-amino-3-[[4-[(2-amino-4-hydroxyphenyl)azo]phenyl]azo]-5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate

Cas Number:

68460-07-1

Molecular formula:

C28H22N8O8S2.2Na

IUPAC Name:

disodium 4-amino-3-({4-[(2-amino-4-hydroxyphenyl)diazenyl]phenyl}diazenyl)-5-hydroxy-6-(phenyldiazenyl)naphthalene-2,7-disulfonate

Constituent 2

Reference substance name:

Disodium 4-amino-3-[[4-[(2,4-diaminophenyl)azo]phenyl]azo]-5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate

EC Number:

272-559-0

EC Name:

Disodium 4-amino-3-[[4-[(2,4-diaminophenyl)azo]phenyl]azo]-5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate

Cas Number:

68877-33-8

Molecular formula:

C28H23N9O7S2.2Na

IUPAC Name:

disodium 4-amino-3-({4-[(2,4-diaminophenyl)diazenyl]phenyl}diazenyl)-5-hydroxy-6-(phenyldiazenyl)naphthalene-2,7-disulfonate

In vivo test system

Test animals

Species:

guinea pig

Strain:

not specified

Sex:

not specified

Details on test animals and environmental conditions:

- Weight at study initiation: 230 - 385 g (mean 282 g)

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

water

Concentration / amount:

5 % in deionized water
0.1 mL injected respectively

Day(s)/duration:

administered on days 1 to 7 of the study

Adequacy of induction:

not specified

Induction 2

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

5 % in deionized water
3 mL applied (patch) respectively on day 8 of the study

Day(s)/duration:

patch was removed after 48 hours followed by a period of 22 days without treatment

Adequacy of induction:

not specified

Challenge

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

5 % in deionized water
3 mL applied (patch) respectively on day 23 of the study

Day(s)/duration:

1 day

Adequacy of challenge:

not specified

No. of animals per dose:

30

Details on study design:

The GPMT is a highly sensitive method using Freund´s complete adjuvant as an immune enhancer. It includes both intradermal and topical induction treatment and closed challenge.

No details on study design remarked in the test report, but according to the method of Magnusson and Kligman, the following procedure is applied:

Three pairs of intradermal injections are given in the shoulder region which is cleared of hair: injection 1 is a 1:1 mixture of Freund's complete adjuvant and physiological saline, injection 2 is the test substance at the selected concentration in an appropriate vehicle and injection 3 is the test substance at the selected concentration formulated in a 1:1 mixture of Freund's complete adjuvant and physiological saline. On days 6-8, a filter paper loaded with test substance in a suitable vehicle is applied to the test area covered by occlusion for 48 hours.
Approximately one day before the application, if the substance is not a skin irritant, the test area is treated with 10 % sodium lauryl sulphate in vaseline, in order to create a local irritation. Control animals receive the same treatment using vehicle without test substance.
Challenge is carried out on days 20-22 in treated and control animals. A patch loaded with test substance is applied to one flank of the animals, a patch with the vehicle may also be applied
to the other flank. The patches are covered occlusively for 24 hours. The skin reaction is evaluated 24 and 48 hours after patch removal.

The following information was noted in the test report:

30 animals were used for the study, all treated with the test material

Induction phase: prior to application of the test substance the animals were weighed and shaved at the test sites.
Intradermal induction started on day 1 by injection of 0.1 mL of an aqueous 5 % solution of the test material respectively. After the last injection on day 7, animals were washed and again cleared of hair at the test sites. On day 8 a patch loaded with 3 mL of an 5 % aqueous solution of the test material was applied to the test area respectively and covered by occlusion for 48 hours. Then, the patch was removed.
After a rest period of 12 days without treatment, animals were washed and clipped free of hair. The challenge phase started on day 23 by application of a patch loaded with 3 mL of an 5 % aqueous solution of the test material and covered occlusively for 24 hours respectively.
The skin reaction was evaluated 24 and 48 hours after patch removal.

Challenge controls:

not specified

Positive control substance(s):

not specified

Results and discussion

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

3 mL of a 5 % aqueous solution

No. with + reactions:

0

Total no. in group:

24

Clinical observations:

no signs of skin sensitization observed

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Reading:

2nd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

3 mL of a 5 % aqueous solution

No. with + reactions:

0

Total no. in group:

24

Clinical observations:

no signs of skin sensitization observed

Remarks on result:

no indication of skin sensitisation

Any other information on results incl. tables

After dermal application the test areas were discolored by the test material.

One animal died on day 7, two animals during the rest period on days 12 and 16. Three further animals died during challenge period on days 25 and 26. Therefore only 24 animals were left for skin reaction evaluation.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Regulation (EC) no 1272/2008

Conclusions:

no indication of skin sensitization. Therfore, the test substance has not to be classified for skin sensitization according to Regulation (EC) no 1272/2008.

Executive summary:

In a guinea pig maximisation test according to the method of Magnusson and Kligman, an aqueous 5 % solution of the test item was applied to 30 animals. In the intradermal induction phase 0.1 mL was injected respectively. For the dermal application, induction and challenge, a patch loaded with 3 mL of the 5 % solution was used. After removals of the patches the skin of the test sites was discolored by the test material in all animals, but skin reaction evaluation was still possible. Overall 6 animals died at different phases of the test period.

No signs of skin sensitization were observed in any animal at any reading after challenge phase. Thus, the test substance has not to be classified for skin sensitization according to Regulation (EC) no 1272/2008.