Disodium 3-hydroxy-4-[(4-methyl-2-sulphonatophenyl)azo]-2-naphthoate

Endpoint summary

Description of key information

Skin irritation:

Skin irritation study was conducted to indicate the Comedogenicity and irritancy of D&C Red 6 mixed in propylene glycol at a 9 to 1 dilution for testing unless otherwise indicated (10% concentration). A colony of New Zealand albino rabbits that have genetically good ears and free from mites were used. Three rabbits, weighing two to three kilograms, were used for each assay. A dose of 1 ml of the test material was applied and spread once daily to the entire inner surface of once for five days per week for two weeks. The irritancy produced by repeated application of the chemical on the surface epidermis in the rabbit ear is evaluated on a scale of 0 to 5.D&C Red 6(CAS no: 5858-81-1)falls under Grade 0 (no irritation observed).Hence it can be concluded that D&C Red 6 was not irritating to rabbit ears.

 

Eye irritation:

The local eye irritative potential of test chemical D & C Red No. 6 (CAS no: 5858-81-1) was investigated in rabbits’ eye to assess the degree of eye irritancy caused by the chemical. Each rabbit received 2 installation of 0.2 ml of a 10% D & C Red No. 6 aqueous suspension into the conjunctival sac of rabbits 5 times per week for 4 weeks. The chemical produced only minimal irritative effects after 2 hours which could not confirmed the irritation potential. Hence, on the basis of findings the chemical D & C Red No. 6 (CAS no: 5858-81-1) can be considered as not irritating to the rabbits’ eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

data is from peer reviewed journals

Qualifier:

according to guideline

Guideline:

other: as mentioned below

Principles of method if other than guideline:

The test uses a single rabbit ear to indicate the Comedogenicity and irritancy of D & C Red No. 6 (CAS no: 5858-81-1).

GLP compliance:

not specified

Specific details on test material used for the study:

Name of the test chemical: disodium 4-[(E)-2-(4-methyl-2-sulfophenyl)diazen-1-yl]-3-oxidonaphthalene-2-carboxylateCommon Name: D&C Red 6Molecular Formula: C18H12N2Na2O6SMolecular Weight: 430.3468 g/molInChI: 1S/C18H14N2O6S.2Na/c1-10-6-7-14(15(8-10)27(24,25)26)19-20-16-12-5-3-2-4-11(12)9-13(17(16)21)18(22)23;;/h2-9,21H,1H3,(H,22,23)(H,24,25,26);;/q;2*+1/p-2/b20-19+;;Substance Type: OrganicPhysical State: Solid

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALSSource: No dataAge at study initiation: No dataWeight at study initiation: 3 kg Fasting period before study: No dataHousing: singly housed, in suspended cagesDiet (e.g. ad libitum): Purina Rabbit Chow, ad libitumWater (e.g. ad libitum): water, ad libitumAcclimation period: No dataENVIRONMENTAL CONDITIONS Temperature (°C): No data Humidity (%):No data Air changes (per hr): No data Photoperiod (hrs dark / hrs light): 12 hour light and 12 hr dark cycleIN-LIFE DATES: From: No data To: No data

Type of coverage:

other: Entire inner surface of one ear

Preparation of test site:

other: A colony of rabbits that had genetically good ears and which were free from mites were used for the assay

Vehicle:

other: The test chemical was mixed in propylene glycol at a 9 to 1 dilution for testing.

Controls:

yes

Amount / concentration applied:

Dose of 1ml of the test material was applied once daily for five days per week for 2 weeks.

Duration of treatment / exposure:

once daily for five days per week for 2 weeks.

Observation period:

2 weeks

Number of animals:

3 rabbits

Details on study design:

TEST SITEArea of exposure: Ear% coverage: A dose of 1ml of the test chemical is applied and spread once daily to the entire inner surface of one ear five days per week for 2 weeks.Type of wrap if used: No dataREMOVAL OF TEST SUBSTANCEWashing (if done): No dataTime after start of exposure: No dataSCORING SYSTEM: The irritancy produced by repeated application of a chemical or skin care product on the surface epidermis in the rabbit ear is evaluated on a scale of 0 to 5. The grades are summarized as follows: 0 = No irritation1 = few scales, no Erythema2 = diffuse scaling, no Erythema3 = Generalized scaling with Erythema4 = Scaling, Erythema and Edema5 = Epidermal necrosis and slough

Other effects / acceptance of results:

no data available

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

other: 2 weeks

Score:

0

Max. score:

5

Reversibility:

not specified

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

No erythema observed

Table 1: Ingredients and their Comedogenicity and Irritancy

Test chemical	Comedogenicity* [Grade 0-5]	Irritancy** [Grade 0-5]
D & C RED 6	1	0

 

*Comedogenicity or the ability of test substance to produce follicular hyperkeratosis

** Irritancy or ability of test substance to produce surface epithelial irritation

 

Interpretation of results:

other: not irritating

Conclusions:

D&C Red 6 falls under Grade 0 (no irritation observed).Hence it can be concluded that D & C Red No. 6 (CAS no: 5858-81-1)was not irritating to rabbit ears.

Executive summary:

The test uses a single rabbit ear to indicate the Comedogenicity and irritancy of D&C Red 6. D&C Red 6 was mixed in propylene glycol at a 9 to 1 dilution for testing unless otherwise indicated (10% concentration). A colony of New Zealand albino rabbits that have genetically good ears and free from mites were used. Three rabbits, weighing two to three kilograms, were used for each assay. Animals were housed singly in suspended cages and fed Purina Rabbit Chow and water ad libitum. Animals were maintained on a 12-hour light and 12-hour dark cycle. A dose of 1 ml of the test material was applied and spread once daily to the entire inner surface of once for five days per week for two weeks. The opposite untreated ear of each animal served as an untreated control.

The irritancy produced by repeated application of the chemical on the surface epidermis in the rabbit ear is evaluated on a scale of 0 to 5. The grades are summarized as follows:

 0 = No irritation; 1 = few scales, no Erythema; 2 = diffuse scaling, no Erythema; 3 = Generalized scaling with Erythema; 4 = Scaling, Erythema and Edema; 5 = Epidermal necrosis and slough.

D&C Red 6 falls under Grade 0 (no irritation observed).

Hence it can be concluded that D&C Red 6 was not irritating to rabbit ears.

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:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

data is from handbook or collection of data

Qualifier:

according to guideline

Guideline:

other: as mentioned below

Principles of method if other than guideline:

The local eye irritative potential of test chemical D & C Red No. 6 (CAS no: 5858-81-1) was investigated in rabbits’ eye to assess the degree of eye irritancy caused by the chemical.

GLP compliance:

not specified

Specific details on test material used for the study:

Name of the test chemical: disodium 4-[(E)-2-(4-methyl-2-sulfophenyl)diazen-1-yl]-3-oxidonaphthalene-2-carboxylateCommon Name: D & C Red No. 6/Lithol Rubine B (LRB)Molecular Formula: C18H12N2Na2O6SMolecular Weight: 430.3468 g/molInChI: 1S/C18H14N2O6S.2Na/c1-10-6-7-14(15(8-10)27(24,25)26)19-20-16-12-5-3-2-4-11(12)9-13(17(16)21)18(22)23;;/h2-9,21H,1H3,(H,22,23)(H,24,25,26);;/q;2*+1/p-2/b20-19+;;Substance Type: OrganicPhysical State: SolidSMILES:Cc1ccc(N=Nc2c3ccccc3cc(C(=O)O{-}.[Na]{+})c2O)c(S(=O)(=O)O{-}.[Na]{+})c1

Species:

rabbit

Strain:

not specified

Details on test animals or tissues and environmental conditions:

not specified

Vehicle:

other: aqueous suspension

Controls:

not specified

Amount / concentration applied:

2 instillations of 10% (0.2ml)

Duration of treatment / exposure:

24 hours

Observation period (in vivo):

4 weeks

Number of animals or in vitro replicates:

no data available

Details on study design:

- Area of exposure: conjunctival sac of rabbits

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

other: 4 weeks

Reversibility:

fully reversible

Remarks:

24 hours

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

Only minimal irritative effects after 2 hours.

Interpretation of results:

other: Not irritating

Conclusions:

The test chemical D & C Red No. 6 (CAS no: 5858-81-1) can be considered as not irritating to the rabbits’ eye.

Executive summary:

The local eye irritative potential of test chemicalD & C Red No. 6 (CAS no: 5858-81-1)was investigated in rabbits’ eye to assess the degree of eye irritancy caused by the chemical.

 

Each rabbit received 2 installation of 0.2 ml of a 10% D & C Red No. 6 aqueous suspension into the conjunctival sac of rabbits 5 times per week for 4 weeks.

 

The chemical producedonly minimal irritative effects after 2 hours which could not confirmed the irritation potential. Hence, on the basis of findingsthe chemical D & C Red No. 6 (CAS no: 5858-81-1)can be considered as not irritating to the rabbits’ eye.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Irritation:

Various studieshas been investigated for the test chemicalD & C Red No. 6 (CAS no: 5858-81-1)to observe the potential for dermal irritation to a greater or lesser extent. The studies are based on in vivo experiments in rabbits and humans for target chemicalD & C Red No. 6 (CAS no: 5858-81-1)and its structurally similar read across substancesDisodium 4-hydroxy-3-[(4-sulphonatonaphthyl)azo] naphthalenesulphonate [CAS: 3567-69-9] and Disodium 3-[(2,4-dimethyl-5-sulphonatophenyl)azo]-4-hydroxynaphthalene-1-sulphonate [CAS: 4548-53-2].The predicted data using theDanish QSAR databasehas also been compared with the experimental data and summarized as below;

 

The JAMES E. FULTON, JR (1989) conducted a test by using a single rabbit ear to indicate the Comedogenicity and irritancy of target chemical D&C Red 6 was mixed in propylene glycol at a 9 to 1 dilution for testing unless otherwise indicated (10% concentration). A colony of New Zealand albino rabbits that have genetically good ears and free from mites were used. Three rabbits, weighing two to three kilograms, were used for each assay. Animals were housed singly in suspended cages and fed Purina Rabbit Chow and water ad libitum. Animals were maintained on a 12-hour light and 12-hour dark cycle. A dose of 1 ml of the test material was applied and spread once daily to the entire inner surface of once for five days per week for two weeks. The opposite untreated ear of each animal served as an untreated control. The irritancy produced by repeated application of the chemical on the surface epidermis in the rabbit ear is evaluated on a scale of 0 to 5. The grades are summarized as follows: 0 = No irritation; 1 = few scales, no Erythema; 2 = diffuse scaling, no Erythema; 3 = Generalized scaling with Erythema; 4 = Scaling, Erythema and Edema; 5 = Epidermal necrosis and slough.D&C Red 6 falls under Grade 0 (no irritation observed).Hence it can be concluded that D&C Red 6 was not irritating to rabbit ears.

 

According toDanish QSAR database, skin irritation effects were estimated by using four different models i.e, Battery, Leadscope, SciQSAR and CASE Ultra for D & C Red No. 6 (CAS no: 5858-81-1). Based on estimation, no severe skin irritation effects were known when D & C Red No. 6 (CAS no: 5858-81-1) was exposed to rabbit skin.

 

 

These results are supported by the experimental study conducted in an OECD GLP laboratory (Sustainability Support Services (Europe) AB has the letter of access) for the structurally similar read across substance, Disodium 4-hydroxy-3-[(4-sulphonatonaphthyl)azo] naphthalenesulphonate [CAS: 3567-69-9]. The study was performed as per OECD Guidelines 402 and complying to the GLP procedures. The test item was applied to shorn skin of 5 male and 5 female Sprague Dawley rats at 2000 mg/kg body weight. Administration of the test item at 2000 mg/kg did not result in any skin reaction at the site of application during the study period of 14 days. Administration of the test item did not result in any signs of toxicity and mortality during the study period of 14 days. Animals exhibited normal body weight gain through the study period of 14 days. Gross pathological examination did not reveal any abnormalities attributable to the treatment. From the study, it can be concluded that the test substance ‘Disodium 4-hydroxy-3-[(4-sulphonatonaphthyl)azo] naphthalenesulphonate’ is nonirritant to the skin of Sprague Dawley rats when applied to the shorn skin of 5 male and 5 female animals at the tested dose level of 2000 mg/kg body weight. Also the erythema and edema score of rats was calculated as 0. Thus it can be concluded that the substance Disodium 4-hydroxy-3-[(4-sulphonatonaphthyl)azo] naphthalenesulphonate can be considered as not irritating .

 

The above results are further supported by the experimental study conducted in an OECD GLP laboratory (Sustainability Support Services (Europe) AB has the letter of access) for the structurally similar read across substance, Disodium 3-[(2,4-dimethyl-5-sulphonatophenyl)azo]-4-hydroxynaphthalene-1-sulphonate [CAS: 4548-53-2]. The study was performed as per OECD Guidelines 402 and complying to the GLP procedures. The test item was applied to shorn skin of 5 male and 5 Sprague Dawley rats at 2000 mg/kg body weight. Administration of the test item at 2000 mg/kg did not result in any skin reaction at the site of application during the study period of 14 days. Administration of the test item did not result in any signs of toxicity and mortality during the study period of 14 days. Animals exhibited normal body weight gain through the study period of 14 days. Gross pathological examination did not reveal any abnormalities attributable to the treatment. From the study, it can be concluded that the test substance ‘Disodium 3-[(2,4-dimethyl-5-sulphonatophenyl)azo]-4 -hydroxynaphthalene-1-sulphonate’ is nonirritant to the skin of Sprague Dawley rats when applied to the shorn skin of 5 male and 5 female animals at the tested dose level of 2000 mg/kg body weight. Also the erythema and edema score of rats was calculated as 0. Thus it can be concluded that the substance, Disodium 3-[(2,4-dimethyl-5-sulphonatophenyl)azo]-4-hydroxy naphthalene-1-sulphonate can be considered as not irritating .

 

Based on the available data for the target and read across substances and applying the weight of evidence approach, D & C Red No. 6 (CAS no: 5858-81-1)can be considered to be not irritating to skin. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Not Classified”.

 

 

Eye irritation:

In different studies, D & C Red No. 6 (CAS no: 5858-81-1) has been investigated for potential for ocular irritation to a greater or lesser extent. The studies are based on in vivo and in vitro experiments along with predicted data for target chemical D & C Red No. 6 and its structurally similar read across substances, Disodium 3-[(2,4 -dimethyl-5-sulphonatophenyl)azo]-4-hydroxynaphthalene-1-sulphonate [CAS: 4548-53-2] and 1,3-Naphthalenedisulfonic acid, 7-hydroxy-8-((4-sulfo-1-naphthalenyl)azo)-, trisodium salt (Acid Red 18) [2611-82-7]. The predicted data using the OECD QSAR toolbox has also been compared with the experimental data.

 

The local eye irritative potential of test chemical D & C Red No. 6 (CAS no: 5858-81-1) was investigated by IFA GESTIS (2017) in rabbits’ eye to assess the degree of eye irritancy caused by the chemical. Each rabbit received 2 installation of 0.2 ml of a 10% D & C Red No. 6 aqueous suspension into the conjunctival sac of rabbits 5 times per week for 4 weeks. The chemical produced only minimal irritative effects after 2 hours which could not confirmed the irritation potential. Hence, on the basis of findings the chemical D & C Red No. 6 (CAS no: 5858-81-1) can be considered as not irritating to the rabbits’ eye.

 

THE SCIENTIFIC COMMITTEE ON COSMETIC PRODUCTS AND NON-FOOD PRODUCTS (SCCNFP, 2004) conductedan in-vitro study of test chemicalD & C Red No. 6 (CAS no: 5858-81-1)in the Hen’s egg test on the chorioallantoic membrane (HET-CAM) to determine its eye irritation potential.A HET-CAM assay was performed with a 1% aqueous dilution of Pigment Red 57. The diluted test item was applied onto the CAM of fertilised chicken eggs at day 9 of incubation and irritation parameters such as haemorrhage, lysis of blood vessels and protein coagulation were evaluated.The test item was rinsed off 30 sec after application onto the CAM and evaluation of the parameters was done.The 1% aqueous solution did not cause any damaging effect on the CAM as the obtained evaluation resulted in score 0. ence,the chemical D & C Red No. 6 (CAS no: 5858-81-1)can be considered as not irritating in the Hen’s egg test on the chorioallantoic membrane (HET-CAM).

 

In a prediction done by SSS (2017) using the OECD QSAR toolbox with log kow as the primary descriptor, the ocular irritation potential was estimated for aluminium(3+) ion (5E)-6-oxo-5-[2-(4-sulfonatonaphthalen-1-yl)hydrazin-1-ylidene]-5,6-dihydronaphthalene-1,3-disulfonate (CAS No:15876-47-8). It was estimated that D & C Red No. 6 (CAS no: 5858-81-1) was not irritating to eyes of New Zealand White rabbits.

 

These results are supported by the experimental study summarized in European Commission (EC) - Scientific Committee on Cosmetology (SCC), 1988; for the structurally similar read across substance, Disodium 3-[(2,4-dimethyl-5-sulphonatophenyl)azo]-4-hydroxynaphthalene-1-sulphonate [CAS: 4548-53-2].This studywas performed on rabbits to determine its eye irritation potential.Negative effects were known when the chemical FD and C red 4 installed into the eye of each rabbit at a dose of 5%. Hence the chemicalFD and C red 4(CAS No.4548-53-2)was considered to be not irritating to the eye of rabbits.

 

 

These results are further supported by the experimental study reported byTHE SCIENTIFIC COMMITTEE ON COSMETIC PRODUCTS AND NON-FOOD PRODUCTS (SCCNFP, 2004)for the structurally similar read across substance, 1,3-Naphthalenedisulfonic acid, 7-hydroxy-8-((4-sulfo-1-naphthalenyl)azo)-, trisodium salt (Acid Red 18) [2611-82-7]. The ocular irritation potential ofAcid Red 18 (CAS no: 2611-82-7)was assessed by determining theCytotoxicity in the two independent neutral red uptake assay (NRU) on human keratinocyteswith the modification that human keratinocytes of the HaCaT cell line were used. The treatment was performed in serum-free culture medium. Doses from 681 – 10000 μg/ml [1% in water] were applied together with appropriate controls. No NRU-50 value could be determined from the cytotoxicity curves as the viability was 61% in the first assay and 70% in the second, so the NRU-50 was reported to be > 10000 μg/ml.Hence, Acid red 18(CAS no: 2611-82-7)can be considered to be not irritating to Human Keratinocytes in the neutral red uptake assay.

 

 

Based on the available data for the target and read across substances and applying the weight of evidence approach, D & C Red No. 6 (CAS no: 5858-81-1)can be considered to be not irritating to eyes. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Not Classified”.

Justification for classification or non-classification

The skin and eye irritation potential of test chemicalD & C Red No. 6 (CAS no: 5858-81-1) and its structurally similar read across substanceswere observed in various studies. The results obtained from these studies indicates that the test chemical D & C Red No. 6 (CAS no: 5858-81-1)is not likely to cause skin and eye irritation. Hence D & C Red No. 6 (CAS no: 5858-81-1)can be classified under the category “Not Classified” for skin and eye as per CLP.