Sodium 2,4-diamino-5-(4-(2-sulfoxyethanesulfonyl)phenylazo)benzenesulfonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

10 June 1996 to 03 August 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted to recent EU test guidance in compliance with GLP and reported with a GLP certificate. Read across is applicable based on the content of sodium ions. The presence of these is determined by the pH of the isolation of the dyestuff itself. Therefore the substance to be registered is deemed to be a mixture of free acid, mono and di sodium salts. Upon comparison of the NMR-Spectra of the substance to be registered and the read across chemical it is evident that the chemical shifts as well as the integrations are the same, hence it is difficult to quantify free acid, mono and di variants. The CAS number proposed for the substance to be registered covers the sodium element. The associated free acid has a unique CAS number; this is referenced in the substance identity, as does the disodium variant, which is also referenced.

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Test material

Test material information

Constituent 1

Reference substance name:

27624-67-5

EC Number:

608-116-9

Cas Number:

27624-67-5

IUPAC Name:

27624-67-5

Details on test material:

See below

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male

Details on test animals and environmental conditions:

Twenty-seven male, albino Dunkin Hartley guinea pigs supplied by David Hall Limited, Burton-on-Trent, Staffordshire, UK were used. At the start of the main study the animals weighed 357 to 448g, and were approximately eight to twelve weeks old. After an acclimatisation period of at least five days, each animal was selected at random and given a number unique within the study which was written on a small area of clipped rump using a black indelible marker-pen.

The animals were housed singly or in pairs in solid-floor polypropylene cages furnished with woodflakes. Free access to mains tap water and food (Guinea Pig FD1 Diet, Special Diets Services Limited, Witham, Essex, UK) was allowed throughout the study.

The animal room was maintained at a temperature of 19 to 24⁰C and relative humidity of 52 to 70%. On two occasions the temperature was above the limit specified in the protocol (23⁰C). This deviation was considered not to affect the purpose or integrity of the study. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light and twelve hours darkness.

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

water

Remarks:

distilled

Induction 2

Concentration / amount:

Intradermal Induction : 10% w/v in distilled water
Topical Induction : 75% w/w in distilled water
Topical Challenge : 75% and 50% w/w in distilled water

A topical rechallenge was performed at concentrations of 50% and 25% w/w in distilled water.

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

water

Remarks:

distilled

Challenge 2

Concentration / amount:

Intradermal Induction : 10% w/v in distilled water
Topical Induction : 75% w/w in distilled water
Topical Challenge : 75% and 50% w/w in distilled water

A topical rechallenge was performed at concentrations of 50% and 25% w/w in distilled water.

No. of animals per dose:

Ten test and five control animals were used for the main study.

Details on study design:

Main Study
A group of fifteen guinea pigs was used for the main study, ten test and five control. The bodyweight of each animal was recorded on Day 0 (start of the study) and on Day 24. The bodyweight of each test group animal was also recorded on Day 37.

Two main phases were involved in the main study; (a) an induction of a response and (b) a challenge of that response.

Induction
Induction of the Test Animals: Shortly before treatment on Day 0 the hair was removed from an area approximately 20 mm x 40 mm on the shoulder region of each animal with veterinary clippers. 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 suspension of the test material in distilled water
a 10% w/v emulsion of the test material in a 1:1 preparation of Freund's Complete Adjuvant plus distilled water.

Approximately 24 and 48 hours after intradermal injection the degree of erythema at the test material injection sites (ie. injection site b) was evaluated. One week later (Day 7), the same area on the shoulder region used previously for intradermal injections was clipped again and treated with a topical application of the test material formulation. A filter paper patch (WHATMAN No.4: approximate size 40 mm x 20 mm), loaded with the test material formulation (75% w/w in distilled water) as a thick, even layer was applied to the prepared skin and held in place with a strip of surgical adhesive tape (BLENDERM: approximate size 50mm x 30mm) covered with an overlapping length of aluminium foil. The patch and foil were further secured with a strip of elastic adhesive bandage (ELASTOPLAST: approximate size 250mm x 35mm) wound in a double layer around the torso of each animal. This occlusive dressing was kept in place for 48 hours.

The degree of erythema and oedema was quantified one and twenty-four hours following removal of the patches.

Any other reactions were also recorded.

Challenge
Shortly before treatment on Day 21, an area of approximately 50 mm x 70 mm on both flanks of each animal, was clipped free of hair with veterinary clippers.
A square filter paper patch (WHATMAN No.4: approximate size 20 mm x 20 mm), loaded with a thick, even layer of test material at the maximum non-irritant concentration (75% w/w in distilled water) was applied to the shorn right flank of each animal and was held in place with a strip of surgical adhesive tape (BLENDERM: approximate size 40 mm x 50 mm). To ensure that the maximum non-irritant concentration was used at challenge, the test material at a concentration of 50% w/w in distilled water was similarly applied to a skin site on the left shorn flank. The patches were occluded with an overlapping length of aluminium foil and secured with a strip of elastic adhesive bandage (ELASTOPLAST: approximate size 250 mm x 75 mm) wound in a double layer around the torso of each animal.

After 24 hours, the dressing was carefully cut using blunt-tipped scissors, removed and discarded. The challenge sites were swabbed with cotton wool soaked in distilled water to remove residual material. The position of the treatment sites was identified by using a black indelible marker-pen.

Prior to the 24-hour observation the flanks were chemically depilated using Immac Cream Hair Remover (Anne French, London, UK).

Challenge controls:

Induction of the Control Animals: Intradermal injections were administered using an 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
a 50% w/v formulation of formulation in Freund's Complete Adjuvant/distilled water 1:1

The topical applications followed the same procedure as for the test animals except that the vehicle alone was applied to the filter paper. Skin reactions were quantified as for the test animals.

Positive control substance(s):

yes

Remarks:

Laboratory historical data utilised on the following substances: Ethyl 4-aminobenzoate, 2,4-dinitrochlorobenzene, neomycin sulphate, 2-mercaptobenzothiazole

Study design: in vivo (LLNA)

Vehicle:

other: Not applicable

Results and discussion

Positive control results:

Historical results performed within the expected statistical spread.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

75%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 75%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Results 2

Reading:

2nd reading

Hours after challenge:

24

Group:

test chemical

Dose level:

75%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 24.0. Group: test group. Dose level: 75%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50%

No. with + reactions:

4

Total no. in group:

10

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50%. No with. + reactions: 4.0. Total no. in groups: 10.0.

Results 4

Reading:

2nd reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Results 5

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

75%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 75%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Results 6

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

75%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 75%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Results 7

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

50%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Results 8

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

50%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Results 9

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

75%

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 75%. No with. + reactions: 0.0. Total no. in groups: 5.0.

Results 10

Reading:

2nd reading

Hours after challenge:

24

Group:

negative control

Dose level:

75%

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 75%. No with. + reactions: 0.0. Total no. in groups: 5.0.

Results 11

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

50%

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 5.0.

Results 12

Reading:

2nd reading

Hours after challenge:

24

Group:

negative control

Dose level:

50%

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 5.0.

Results 13

Reading:

1st reading

Hours after challenge:

48

Group:

negative control

Dose level:

75%

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 75%. No with. + reactions: 0.0. Total no. in groups: 5.0.

Results 14

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

75%

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 75%. No with. + reactions: 0.0. Total no. in groups: 5.0.

Results 15

Reading:

1st reading

Hours after challenge:

48

Group:

negative control

Dose level:

50%

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 5.0.

Results 16

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

50%

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 5.0.

Any other information on results incl. tables

INDIVIDUAL SKIN REACTIONS IN TEST ANIMALS AT CHALLANGE

CHALLANGE CONCENTRATIONS: 75% AND 50% w/w                                          VEHICLE: DISTILLED WATER

Animal Number	Skin Reactions (Hours After Removal of Dressing)
	24 Hours						48 Hours
	75%			50%			75%			50%
	Er	Oe	Other	Er	Oe	Other	Er	Oe	Other	Er	Oe	Other
1	0	0	-	0	0	-	0	0	-	0	0	-
2	0	0	-	0	0	-	0	0	-	0	0	-
3	0	0	-	0	0	-	0	0	-	0	0	-
4	0	0	-	1	0	Ss	0	0	-	0	0	D
5	0	0	-	1	0	-	0	0	-	0	0	-
6	0	0	-	0	0	Ss	0	0	-	0	0	Ss
7	0	0	-	0	0	-	0	0	-	0	0	-
8	0	0	-	0	0	-	0	0	-	0	0	-
9	0	0	-	1	0	-	0	0	-	0	0	D
10	0	0	-	1	0	-	0	0	-	0	0	-

Er = erythema                                   D = desquamation

Or = oedema                                     Ss = small superficial scattered scabs

- = no other reactions noted

 

INDIVIDUAL SKIN REACTIONS IN CONTROL ANIMALS AT CHALLANGE 

CHALLLENGE CONCENTRAIONS: 75 % AND 50% w/w                                         VEHICLE: DISTILLED WATER

Animal Number	Skin Reactions (Hours After Removal of Dressing)
	24 Hours						48 Hours
	75%			50%			75%			50%
	Er	Oe	Other	Er	Oe	Other	Er	Oe	Other	Er	Oe	Other
11	0	0	-	0	0	-	0	0	-	0	0	-
12	0	0	-	?s	0	-	0	0	-	?s	0	D
13	0	0	-	0	0	-	0	0	-	0	0	-
14	0	0	-	0	0	STA	0	0	-	0	0	STAD
15	0	0	-	0	0	STA	0	0	-	0	0	STA

Er = erythema                                    D = desquamation

Oe = oedema                                     STA = Orange-coloured staining

- = no other reactions noted            ?s = orange-coloured staining prevents accurate evaluation of erythema

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The results are read across from the free acid form. Read across is applicable based on the content of sodium ions. The presence of these is determined by the pH of the isolation of the dyestuff itself. Therefore the substance to be registered is deemed to be a mixture of free acid, mono and di sodium salts. Upon comparison of the NMR-Spectra of the substance to be registered and the read across chemical it is evident that the chemical shifts as well as the integrations are the same, hence it is difficult to quantify free acid, mono and di variants. The CAS number proposed for the substance to be registered covers the sodium element. The associated free acid has a unique CAS number; this is referenced in the substance identity, as does the disodium variant, which is also referenced.

The test material, EVERZOL YELLOW GSP, produced a 0% (0/10) sensitisation rate and was classified as a NON-SENSITISER to guinea pig skin. The test material did not meet the criteria for classification as a sensitiser according to EU labelling regulations. No risk phrase is required.

Executive summary:

The results are read across from the free acid form. Read across is applicable based on the content of sodium ions. The presence of these is determined by the pH of the isolation of the dyestuff itself. Therefore the substance to be registered is deemed to be a mixture of free acid, mono and di sodium salts. Upon comparison of the NMR-Spectra of the substance to be registered and the read across chemical it is evident that the chemical shifts as well as the integrations are the same, hence it is difficult to quantify free acid, mono and di variants. The CAS number proposed for the substance to be registered covers the sodium element. The associated free acid has a unique CAS number; this is referenced in the substance identity, as does the disodium variant, which is also referenced.

A study was performed to assess the contact sensitisation potential of the test material in the albino guinea pig. The study was performed in compliance with the OECD Guidelines for Testing of Chemicals No. 406 "Skin Sensitisation" (adopted 17 July 1992) and Method B6 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC). Study conducted in compliance with GLP and reported with a GLP certificate.

 

Based on the results of sighting tests, the concentrations of test material for the induction and challenge phases were selected as follows:

 

Intradermal Induction  :     10% w/v in distilled water

Topical Induction        :    75% w/w in distilled water

Topical Challenge        :     75% and 50% w/w in distilled water

 

A topical rechallenge was performed at concentrations of 50% and 25% w/w in distilled water.

The test material produced a 0% (0/10) sensitisation rate and was classified as a non-sensitiser to guinea pig skin. The test material did not meet the criteria for classification as a sensitiser according to EU labelling regulations. No risk phrase is required.