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

Endpoint summary

Description of key information

The test item is not a skin or eye irritant.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

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

Adequacy of study:

key study

Study period:

21 June 1996 to 05 July 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. e.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Three New Zealand White rabbits supplied by David Percival Ltd, Moston, Sandbach, Cheshire, UK were used. At the start of the study the animals weighed 2.44 to 2.81 kg and were twelve to sixteen weeks old. After a minimum acclimatisation period of five days each animal was given a number unique within the study which was written with a black indelible marker-pen on the inner surface of the ear and on the cage label.

The animals were individually housed in suspended metal cages. Free access to mains drinking water and food (STANRAB SQC Rabbit Diet, Special Diets Services Ltd, Witham, Essex, UK) was allowed throughout the study.

The animal room was maintained at a temperature of 17 to 21⁰C and relative humidity of 58 to 70%. 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.

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

water

Remarks:

distilled

Controls:

no

Amount / concentration applied:

500 mg, moistened with 0.5ml water.

Duration of treatment / exposure:

4 h

Observation period:

72 hours. Additional observations were made on Days 7 and 14 to assess the reversibility of skin reactions.

Number of animals:

3

Details on study design:

On the day before the test each rabbit was clipped free of fur from the dorsal flank area using veterinary clippers. Only animals with a healthy intact epidermis by gross observation were selected for the study.

On the day of the test a suitable test site was selected on the back of each rabbit. A quantity of 0.5 g of the test material, moistened with 0.5 ml of distilled water, was introduced under a 2.5 cm x 2.5 cm cotton gauze patch and placed in position on the shorn skin. The patch was secured in position with a strip of surgical adhesive tape (BLENDERM: approximate size 2.5 cm x 4.0 cm). To prevent the animals interfering with the patches, the trunk of each rabbit was wrapped in an elasticated corset (TUBIGRIP) and the animals were returned to their cages for the duration of the exposure period.

Four hours after application the corset and patches were removed from each animal and any residual test material removed by gentle swabbing with cotton wool soaked in 74% Industrial Methylated Spirits.

Approximately one hour following the removal of the patches, and 24, 48 and 72 hours later, the test sites were examined for evidence of primary irritation and scored according to the following scale from Draize J H, (1977) "Dermal and Eye Toxicity Tests" In: Principles and Procedures for Evaluating the Toxicity of Household Substances, National Academy of Sciences, Washington DC.

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal #1

Time point:

other: overall 24, 48, 72h

Score:

0.7

Max. score:

4

Reversibility:

fully reversible

Remarks:

within 72 hours

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal #2

Time point:

other: overall 24, 48, 72h

Score:

2

Max. score:

4

Reversibility:

fully reversible

Remarks:

within 7 days

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal #3

Time point:

other: overal 24, 48, 72h

Score:

0.7

Max. score:

4

Reversibility:

fully reversible

Remarks:

within 72 hours

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal #1

Time point:

other: overall 24, 48, 72h

Score:

0.3

Max. score:

4

Reversibility:

fully reversible

Remarks:

within 48 hours

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal #2

Time point:

other: overall 24, 48, 72h

Score:

1.7

Max. score:

4

Reversibility:

fully reversible

Remarks:

within 7 days

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal #3

Time point:

other: overal 24, 48, 72h

Score:

0.3

Max. score:

4

Reversibility:

fully reversible

Remarks:

within 48 hours

Irritant / corrosive response data:

Very slight to well-defined erythema was noted at all treated skin sites at the 24 and 48-hour observations. Well-defined erythema persisted at one treated skin site at the 72-hour observation. No other evidence of erythema was noted.

Very slight oedema was noted at all treated skin sites one hour after patch removal with very slight to slight oedema at the 24-hour observation. Slight oedema persisted at one treated skin site at the 48-hour observation with very slight oedema at the 72-hour observation.
Moderate desquamation was noted at one treated skin site at the 7-day observation. No skin reactions were noted at ail treated skin sites at the 14 day observation

Other effects:

Orange/yellow-coloured staining was noted at all treated skin sites throughout the study. The staining prevented accurate evaluation of erythema at all treated skin sites one hour after patch removal. Erythematous reactions were evaluated as less than or equal to grade 1 redness. The staining did not affect evaluation of erythema at all treated skin sites at the 24-hour observation and at subsequent observations.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Mild erythema and oedema formation was observed up to 72 hours after test item application.. All effects besides staining of the skin were reversible within the 14-day observation period. The test item is not a skin irritant.

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.

The study was performed to assess the irritancy potential of the test material following a single, 4-hour, semi-occluded application to the intact rabbit skin. The method used followed the recommendations of the OECD Guidelines for Testing of Chemicals No. 404 "Acute Dermal Irritation/Corrosion" (adopted 17 July 1992) and Method B4 of Commission Directive 92/69/EEC. Study was conducted in compliance with GLP and reported with a GLP certificate.

 

Orange/yellow-coloured staining was noted at all treated skin sites throughout the study. The staining prevented accurate evaluation of erythema at all treated skin sites one hour after patch removal. Erythematous reactions were evaluated as less than or equal to grade 1 redness. The staining did not affect evaluation of erythema at all treated skin sites at the 24-hour observation and at subsequent observations.

 

Very slight to well-defined erythema was noted at all treated skin sites at the 24 and 48-hour observations. Well-defined erythema persisted at one treated skin site at the 72-hour observation. No other evidence of erythema was noted.

 

Very slight oedema was noted at all treated skin sites one hour after patch removal with very slight to slight oedema at the 24-hour observation. Slight oedema persisted at one treated skin site at the 48-hour observation with very slight oedema at the 72-hour observation.

No skin reactions were noted at all treated skin sites at the 14-day observation.

The test material did not meet the criteria for classification as irritant or corrosive. No symbol and risk phrase are required.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

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

Adequacy of study:

key study

Study period:

25 June 1996 to 23 July 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.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Three New Zealand White rabbits, supplied by David Percival Ltd, Moston, Sandbach, Cheshire, UK, were used. At the start of the study the animals weighed 2.71 to 3.39 kg and were twelve to sixteen weeks old. After a minimum acclimatisation period of five days each animal was given a number unique within the study which was written with a black indelible marker-pen on the inner surface of the ear and on the cage label.

The animals were individually housed in suspended metal cages. Free access to mains drinking water and food (STANRAB SQC Rabbit Diet, Special Diets Services Ltd, Witham, Essex, UK) was allowed throughout the study.

The animal room was maintained at a temperature of 17 to 22⁰C and relative humidity of 51 to 70%. 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.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

A volume of 0.1 ml of the test material, which was found to weigh approximately 86 mg

Duration of treatment / exposure:

Single application of the test material

Observation period (in vivo):

Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment. Additional observations were made on Days 7, 14 and 21 to assess the reversibility of the ocular effects.

Number of animals or in vitro replicates:

3

Details on study design:

Immediately before the start of the test, both eyes of the provisionally selected test rabbits were examined for evidence of ocular irritation or defect with the aid of a light source from a standard ophthalmoscope. Animals showing evidence of ocular lesions were rejected and replaced.

One rabbit was initially treated. A volume of 0.1 ml of the test material, which was found to weigh approximately 86 mg (as measured by gently compacting the required volume into an adapted syringe) was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after application, to prevent loss of the test material, and then released. The left eye remained untreated and was used for control purposes. Immediately after administration of the test material, an assessment of the initial pain reaction was made.

After consideration of the ocular responses produced in the first treated animal, two additional animals were treated. In order to minimise pain on application of the test material, one drop of local anaesthetic ("Ophthaine", 0.5% proxymetacaine hydrochloride, E R Squibb & Sons Limited, Hounslow, Middlesex, UK) was instilled into both eyes of these animals 1 to 2 minutes before treatment.

Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment, according to the numerical evaluation, (from Draize J H (1977) "Dermal and Eye Toxicity Tests" In: Principles and Procedures for Evaluating the Toxicity of Household Substances, National Academy of Sciences, Washington DC).

Any other ocular effects were also noted. Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

other: overall 24, 48, 72h

Score:

0.44

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

iris score

Basis:

mean

Time point:

other: overall 24, 48, 72 h

Score:

0.11

Max. score:

2

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

other: overall 24, 48, 72h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Basis:

mean

Time point:

other: overall 24, 48, 72h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Irritant / corrosive response data:

Residual test material was noted around the treated eye of all animals throughout the study.

Orange-coloured staining was noted in all treated eyes throughout the study.

Dulling of the normal lustre of the cornea was noted in two treated eyes one hour after treatment. Diffuse corneal opacity was noted in one treated eye one hour after treatment, in all treated eyes at the 24-hour observation and in one treated eye at the 48-hour observation. No other corneal effects were noted.

Orange-coloured staining prevented accurate evaluation of the iris in all treated eyes one hour after treatment, in two treated eyes at the 24-hour observation and in one treated eye at the 48-hour observation. Iridial inflammation was noted in one treated eye at the 24-hour observation. No other iridial effects were noted.

Moderate conjunctival irritation was noted in all treated eyes one hour after treatment with minimal to moderate conjunctival irritation at the 24-hour observation and minimal conjunctival irritation at the 48-hour observation. Minimal conjunctival redness persisted in one treated eye at the 72-hour observation.


No ocular effects were noted at the 72-hour or 7-day observation.

One animal was found dead on Day 18. The cause of death was not determined but was considered not to be treatment related. The absence of this animal did not affect the purpose or integrity of the study.

Interpretation of results:

not classified

Remarks:

Migrated information See conclusion below 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.
A study was performed to assess the irritancy potential of the test material to the eye of the New Zealand White rabbit. The method used followed that described in the OECD Guidelines for Testing of Chemicals No. 405 "Acute Eye Irritation/Corrosion" (adopted 24 February 1987) and Method B5 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.
A single application of the test material to the non-irrigated eye of three rabbits produced diffuse corneal opacity, iridial inflammation and moderate conjunctival irritation. Dulling of the normal lustre of the cornea was noted in two treated eyes one hour after treatment. No ocular effects were noted at the 72-hour or 7-day observations.

One animal was found dead on Day 18. The cause of death was not determined but was considered not to be treatment related. The absence of this animal did not affect the purpose or integrity of the study.

The test material caused staining of the iris at the 21-day observation. However, review of REGULATION (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures, specifically section 3.3.2.6. Irreversible effects on the eye/serious damage to eyes (Category 1) states that:

3.3.2.6.1. Substances that have the potential to seriously damage the eyes are classified in Category 1 (irreversible effects on the eye). Substances are classified in this hazard category on the basis of the results of animal testing, in accordance with the criteria listed in Table 3.3.1. These observations include animals with grade 4 cornea lesions and other severe reactions (e.g., destruction of cornea) observed at any time during the test, as well as persistent corneal opacity, discoloration of the cornea by a dye substance, adhesion, pannus, and interference with the function of the iris or other effects that impair sight. In this context, persistent lesions are considered those which are not fully reversible within an observation period of normally 21 days. Substances are also classified in Category 1 if they fulfil the criteria of corneal opacity ≥ 3 or iritis > 1,5 detected in a Draize eye test with rabbits, recognising that such severe lesions usually do not reverse within a 21-day observation period.

As a dye substance, the test material was not considered to cause discoloration of the cornea at the 21-day observation point as specified in the regulation, and hence is not considered to fulfil the criteria for classification as a Category 1 eye irritant. No classification is therefore 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 irritancy potential of the test material to the eye of the New Zealand White rabbit. The method used followed that described in the OECD Guidelines for Testing of Chemicals No. 405 "Acute Eye Irritation/Corrosion" (adopted 24 February 1987) and Method B5 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.

A single application of the test material to the non-irrigated eye of three rabbits produced diffuse corneal opacity, iridial inflammation and moderate conjunctival irritation. Dulling of the normal lustre of the cornea was noted in two treated eyes one hour after treatment. No ocular effects were noted at the 72-hour or 7-day observations.

 

One animal was found dead on Day 18. The cause of death was not determined but was considered not to be treatment related. The absence of this animal did not affect the purpose or integrity of the study.

 

Irritation parameter	Basis	Time point	Score	Max. score	Reversibility
cornea score	mean	24, 48, 72h	0.44	4	fully reversible within: 72 hours
iris score	mean	24, 48, 72 h	0.11	2	fully reversible within: 72 hours
conjunctivae score	mean	24, 48, 72h	1	3	fully reversible within: 7 days
chemosis score	mean	24, 48, 72h	0.33	4	fully reversible within: 48 hours

 

The test material also caused staining of the iris at the 21-day observation. However, review of REGULATION (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures, specifically section 3.3.2.6.Irreversible effects on the eye/serious damage to eyes (Category 1) states that:

 

3.3.2.6.1. Substances that have the potential to seriously damage the eyes are classified in Category 1 (irreversible effects on the eye). Substances are classified in this hazard category on the basis of the results of animal testing, in accordance with the criteria listed in Table 3.3.1. These observations include animals with grade 4 cornea lesions and other severe reactions (e.g., destruction of cornea) observed at any time during the test, as well as persistent corneal opacity,discoloration of the cornea by a dye substance, adhesion, pannus, and interference with the function of the iris or other effects that impair sight. In this context, persistent lesions are considered those which are not fully reversible within an observation period of normally 21 days. Substances are also classified in Category 1 if they fulfil the criteria of corneal opacity≥3 or iritis > 1,5 detected in a Draize eye test with rabbits, recognising that such severe lesions usually do not reverse within a 21-day observation period.

 

As a dye substance, the test material was not considered to cause discoloration of the cornea at the 21-day observation point as specified in the regulation, and hence is not considered to fulfil the criteria for classification as a Category 1 eye irritant. No classification is therefore required.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

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.

Skin irritation / Corrosion.

A single study was evaluated on this endpoint. In these the substance was tested for primary dermal irritation and corrosiveness. Due to staining of the skin, redness could not be sufficiently evaluated 1 hour after test item removal. Mild erythema/eschar or oedema formation became apparent within the observation period of 7 days. Signs of toxicity were not observed. This evidence indicates a slight initial irritating effect to the skin; however is not classified as irritating to skink according to CLP. No risk phrase or classification is required.

 

Eye irritation.

A single study was evaluated on this endpoint. In these the substance was tested for acute irritation and was found not to be an irritant to the rabbit eye. Temporary effects were noted in all animals assessed; however as a dye substance, the test material did not result in persistent discoloration of the cornea, and hence does not have to be classified as Category 1 eye irritant according to Regulation (EC) No 1272/2008. No classification is therefore required.

 

Respiratory irritation

Respiratory irritation was not assessed; however no effects on the animals were noted in any associated studies.

Justification for classification or non-classification

The above studies have all been ranked reliability 1 according to the Klimisch 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 classification under the Dangerous Substance Directive (67/548/EEC) for the eyes, however, do not result in a classification according to the CLP Regulation (EC No 1272/2008). Classification for irritation effects is therefore R41 “Risk of serious damage to eyes” for the Dangerous Substance Directive (67/548/EEC) on the basis of the staining effects noted and “not classified” for Regulation (EC No 1272/2008).