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

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:

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.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

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

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.

Route:

intradermal

Vehicle:

water

Remarks:

distilled

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.

Route:

epicutaneous, occlusive

Vehicle:

water

Remarks:

distilled

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

Vehicle:

other: Not applicable

Positive control results:

Historical results performed within the expected statistical spread.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

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.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

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.

Sensitisation was assessed using the Magnusson and Kligman sensitisation assay. During the induction phase of the study, no irritation reactions were noted in both control and test groups. After the challenge exposure none of the test animals exhibited skin reactions due to sensitising effects.

Skin Reactions Observed After Topical Challenge

75% w/w in Distilled Water

No skin reactions were noted at the challenge sites of test and control group animals at the 24 and 48-hour observations.

 

50% w/w in Distilled Water

Orange-coloured staining was noted at the challenge sites of three control group animals at the 24 and 48-hour observations. The staining prevented accurate evaluation of erythema at the challenge site of one control group animal at the 24 and 48-hour observations. Very slight erythema was noted at the challenge sites of four test group animals at the 24-hour observation. Small, superficial scattered scabs were noted at the challenge sites of two test group animals at the 24-hour observation and one test group animal at the 48-hour observation. Desquamation was noted at the challenge sites of two test and two control group animals at the 48-hour observation.

  

Skin Reactions Observed After Topical Rechallenge

Orange-coloured staining was commonly noted at the 50% and 25% challenge sites of the test and control group animals at the 24 and 48-hour observations. The staining did not prevent evaluation of the skin responses.

 

Very slight erythema (grade 1) was noted at the 50% and 25% challenge sites of one test group (number 3) animal at the 24 and 48-hour observations. Desquamation was also noted at the 48-hour observation. These reactions were attributed to non-specific skin irritation rather than skin sensitisation, since no skin reactions were elicited in this animal by 75% and 50% formulations of test material at the initial topical challenge.

 

Very slight erythema (grade 1) was also noted at the 25% challenge sites of two other test group animals (numbers 8 and 9) at the 24 and 48-hour observations. Desquamation was also noted at the 48-hour observation. These skin reactions were attributed to non-specific skin irritation since no skin reactions were noted at the 50% challenge sites of these animals, and a skin reaction was elicited in only one of these animals at the initial topical challenge.

 

No skin reactions were noted at the challenge sites of any control group animals at the 24 and 48-hour observations.

 

On the basis of the results, the substance cannot be considered to be a skin sensitiser.

Migrated from Short description of key information:
The test item cannot be considered to be a skin sensitiser.

Respiratory sensitisation

Endpoint conclusion

Additional information:

The registered chemical is a reactive dye. For this class of dyes it was generally agreed between the members of the Ecological and Toxicological Association of Dyes and Organic Pigments Manufacturers (ETAD)that a possible risk for respiratory sensitisation for workers exists at high exposure. However the following should be noted:

 

1) For the substance no history of respiratory problems, such as occupational asthma, is associated with the manufacture and use of the specific substance.

 

2)Due to the granular or well dedusted form of the substance (spay dried in closed system from aqueous solution directly after synthesis) no risk for inhalative exposure arises.

 

The potential to cause respiratory sensitisation is therefore not considered to be applicable for this substance.

No evidence of respiratory sensitisation was noted in any of the studies conducted, and it is proposed that the substance is not a respiratory sensitiser.

Justification for classification or non-classification

The above study has been ranked reliability 1 according to the Klimish et al system. This ranking was deemed appropriate because the studies were conducted to GLP an in compliance with agreed protocols. Sufficient dose ranges and numbers are detailed; hence it is appropriate for use based on reliability and animal welfare grounds.

The above results triggered no classification under the Dangerous Substance Directive (67/548/EEC) and the CLP Regulation (EC No 1272/2008). No classification for sensitisation effects is therefore required.