[7-hydroxy-8-[(2-methoxyphenyl)azo]-2-naphthyl]trimethylammonium chloride

Endpoint summary

Description of key information

Skin irritation:
The substance was determined to ne non-irritant in various in vivo and in vitro tests for skin irritation.
Eye irritation:
Based on the various in vitro studies reported for the taregt as well as read across substances, the substance can be considered to be non irritant to eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

calculation (if not (Q)SAR)

Remarks:

Migrated phrase: estimated by calculation

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Principles of method if other than guideline:

In Vitro Skin Irritation: Reconstructed Human Epidermis Test MethodThe Test Guideline addresses the human health endpoint skin irritation. It is based on the in vitro test system of reconstructed human epidermis (RhE), which closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis. The RhE test system uses human derived non-transformed keratinocytes as cell source to reconstruct an epidermal model with representative histology and cytoarchitecture.Evaluation of test items in the EpiSkin™ skin irritation test in which both standard photometry (OD) and HPLC/UPLC-spectrophotometry are used as endpoint detection systems.

GLP compliance:

no

Species:

other: Reconstructed Human Epidermis

Strain:

not specified

Details on test animals or test system and environmental conditions:

Test TissueSource: The epidermal cells are taken from healthy volunteer donors negative to anti-HIV 1 and 2, and to hepatitis C antibodies, and to hepatitis B antigens.Type: Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen.Acclimation : No dataHousing conditions: No data

Type of coverage:

not specified

Preparation of test site:

not specified

Vehicle:

water

Remarks:

Distilled

Controls:

not specified

Amount / concentration applied:

10 ± 2mg (26.3 mg/cm2)

Duration of treatment / exposure:

15 minutes

Observation period:

42 hours post-exposure incubation time

Number of animals:

3 tissues/run

Details on study design:

TEST SITEArea of exposure: 0.38cm2% coverage: No dataType of wrap if used: No dataREMOVAL OF TEST SUBSTANCEWashing (if done): 25 ml sterile PBSTime after start of exposure: After 15 minutes exposureSCORING SYSTEM: The results are interpreted on the basis of a fixed 15 min exposure-time and 42 hours post-exposure incubation time. If cell viability is ≤ 50 %: the chemical is classified as Irritant (R38)If cell viability > 50%: the chemical is classified as Non Irritant (N C)

Irritation / corrosion parameter:

other: other: Cell viability index

Value:

108.5

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 42 hours post-exposure incubation time. Reversibility: no data. Remarks: determined by spectrophotometry: Non irritant. (migrated information)

Irritation / corrosion parameter:

other: other: Cell viability index

Value:

106.6

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 42 hours post-exposure incubation time. Reversibility: no data. Remarks: determined by HPLC: Non irritant. (migrated information)

Table 1: Test chemical identity and characteristics

Test chemical Name	Physical State and Color	Draize assay - In vivo Classification	EpiSkin™ tissue viability – In vitro Classification
Basic Red 76	Solid (Powder); Red	Not Classified	Non Irritant

 

Table 2: Reproducibility assessment for both OD and HPLC/UPLC-spectrophotometry EpiSkin™ tissue viability quantification from formazan tissue extracts

Part 1: Tissue viability OD (%)

Test chemical^b	Corrected relative viability			Corrected Final Mean viability (Uncorrected)	SD/*Diff
	Tissue 1	Tissue 2	Tissue 3
Basic Red 76	105.4	108.5	111.5	108.5 (110.4)	3.08

Part 2: Tissue viability HPLC/UPLC-Spectrophotometry

Test chemical^b	Corrected relative viability			Corrected Final Mean viability (Uncorrected)	SD/*Diff
	Tissue 1	Tissue 2	Tissue 3
Basic Red 76	106.3	102.5	111.0	106.6	4.25

Where

b – Color interference

SD – Standard deviation for 3 tissue replicates/mean values

*Diff – Difference between 2 tissue replicates

Interpretation of results:

not classified

Remarks:

Migrated informationCriteria used for interpretation of results: EU

Conclusions:

In an OECD TG 439: “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method", based on the cell viability values of 108.5 and 106.6 obtained in the standard photometry (OD) and HPLC/UPLC-spectrophotometry assays respectively, the test chemical was assessed to be a non irritant to re-constructed human skin.

Executive summary:

OECD TG 439: “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method” was used to assess the skin irritation potential of the Basic Red 76.

The Test Guideline addresses the human health endpoint skin irritation. It is based on thein vitrotest system of reconstructed humanepidermis(RhE), which closely mimics the biochemical and physiological properties of the upper parts of the human skin,i.e.theepidermis.The RhE test system uses human derived non-transformed keratinocytes as cell source to reconstruct an epidermal model with representative histology and cytoarchitecture.

Evaluation of test items in the EpiSkin™ skin irritation test in which both standard photometry (OD) and HPLC/UPLC-spectrophotometry are used as endpoint detection systems.

 

10 ± 2mg (26.3 mg/cm²) of the test material was applied topically to the epidermal model (three epidermis units are used per test material, positive and negative controls) for 15 minutes at room temperature. Exposure to the test chemical was terminated by rinsing with phosphate buffered saline (PBS). Epidermis was then incubated at 37°C for 42 additional hours. For each test material, three independent tests with three different batches of EpiSkin^TMare used. Aliquots of culture media were kept frozen (-20°C) for cytokine (IL-1α) further measurements. The viability is assessed by incubating the tissues for 3 hours with MTT solution in a 12 well plate (0.3 mg/ml; 2 ml per well). The formazan precipitated is then extracted using acidified isopropanol (0.5 ml) and quantified spectrophotometrically at 570 nm using 96 well plates (200μl/well). Protein levels of IL-1α in the supernatant were expressed as pg/ml. SDS 5% and PBS-treated epidermis are used as positive and negative controls respectively. For each treated tissue the viability is expressed as a % relative to negative control tissues (mean).

 

The results are interpreted on the basis of a fixed 15 min exposure-time and 42 hours post-exposure incubation time.    

If cell viability is≤50 %: the chemical is classified as Irritant (R38)

If cell viability> 50%:the chemical is classified as Non Irritant (N C)

 

Based on the cell viability values of 108.5 and 106.6 obtained in the standard photometry (OD) and HPLC/UPLC-spectrophotometry assays respectively, the test chemical was assessed to be a non irritant to re-constructed human skin.

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:

4 (not assignable)

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

GLP compliance:

not specified

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALSAge at study initiation: Adult New Zealand White albino male rabbits

Vehicle:

not specified

Controls:

yes

Amount / concentration applied:

100 mg of test substance

Duration of treatment / exposure:

24 hours

Observation period (in vivo):

72 hours to 7 days

Number of animals or in vitro replicates:

3 rabbits – 1 male and 2 females

Details on study design:

TEST SITEArea of exposure: conjuctival sac of the left eye% coverage:Type of wrap if used:REMOVAL OF TEST SUBSTANCEWashing (if done): The substance remained in permanent contact with the eyes until rinsing with warm tap waterTime after start of exposure: 24 hours after instillation.SCORING SYSTEM: The eye irritation reactions were scored approximately 1, 24, 48 and 72 hours and 7 days after instillation of the test solution.

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

other: upto 72 hrs

Reversibility:

fully reversible within: 72 hrs

Remarks on result:

other: mild, early-onset and transient ocular changes, such as reddening of the conjunctivae and sclerae, discharge and chemosis.

Irritant / corrosive response data:

Slight red staining of the treated eyes by the test item was observed in all animals at the 1- and 24- hour reading and persisted in two animals up to 48 hours and in one animal up to 72 hours after treatment.

Interpretation of results:

not classified

Remarks:

Migrated informationCriteria used for interpretation of results: EU

Conclusions:

Eye irritation study was carried out on New Zealand White rabbits according to OECD 405 Guidelines to determine the irritation potency of Basic Red 76.The instillation of the undiluted Basic Red 76 into the eyes resulted in mild, early-onset and transient ocular changes, such as reddening of the conjunctivae and sclerae, discharge and chemosis.These effects were reversible and were no longer evident 72 hours after treatment. No abnormal findings were observed in the cornea or iris of any animal at any of the examinations. No corrosion was observed at any of the measuring intervals. Slight red staining of the treated eyes by the test item was observed in all animals at the 1- and 24- hour reading and persisted in two animals up to 48 hours and in one animal up to 72 hours after treatment. The study was terminated seven days after instillation.Under the conditions of the study, Basic Red 76 was considered to be not irritating to rabbit eyes.

Executive summary:

Eye irritation study was carried out on New Zealand White rabbits according to OECD 405 Guidelines to determine the irritation potency of Basic Red 76.

 

100 mg of Basic Red 76 was instilled into the conjunctival sac of the left eye of the test animals. The substance remained in permanent contact with the eyes until rinsing with warm tap water, 24 hours after instillation. The right eyes served as controls. The eye irritation reactions were scored approximately 1, 24, 48 and 72 hours and 7 days after instillation of the test solution.

 

The instillation of the undiluted Basic Red 76 into the eyes resulted in mild, early-onset and transient ocular changes, such as reddening of the conjunctivae and sclerae, discharge and chemosis.

 

These effects were reversible and were no longer evident 72 hours after treatment. No abnormal findings were observed in the cornea or iris of any animal at any of the examinations. No corrosion was observed at any of the measuring intervals. Slight red staining of the treated eyes by the test item was observed in all animals at the 1- and 24- hour reading and persisted in two animals up to 48 hours and in one animal up to 72 hours after treatment. The study was terminated seven days after instillation.

 

Under the conditions of the study, Basic Red 76 was considered to be not irritating to rabbit eyes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin irritation:

An in vitro skin irritation test was conducted by Alépée et al. (2016) as per the OECD TG 439: “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method” to assess the skin irritation potential of the Basic Red 76. It is based on the in vitro test system of reconstructed human epidermis (RhE), which closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.

10 ± 2mg (26.3 mg/cm²) of the test material was applied topically to the epidermal model (three epidermis units are used per test material, positive and negative controls) for 15 minutes at room temperature. Exposure to the test chemical was terminated by rinsing with phosphate buffered saline (PBS). Epidermis was then incubated at 37°C for 42 additional hours. For each test material, three independent tests with three different batches of EpiSkin^TM are used. Aliquots of culture media were kept frozen (-20°C) for cytokine (IL-1α) further measurements. The viability is assessed by incubating the tissues for 3 hours with MTT solution in a 12 well plate (0.3 mg/ml; 2 ml per well). The formazan precipitated is then extracted using acidified isopropanol (0.5 ml) and quantified spectrophotometrically at 570 nm using 96 well plates (200μl/well). Protein levels of IL-1α in the supernatant were expressed as pg/ml. SDS 5% and PBS-treated epidermis are used as positive and negative controls respectively. For each treated tissue the viability is expressed as a % relative to negative control tissues (mean).

Based on the cell viability values of 108.5 and 106.6 obtained in the standard photometry (OD) and HPLC/UPLC-spectrophotometry assays respectively, the test chemical was assessed to be a non-irritant to re-constructed human skin.

Various in vivo skin irritation studieson New Zealand White rabbitsare reported in Scientific Committee on Cosmetic Products Opinion (2003).

In the first study, Basic Red 76 was applied without any vehicle at the level of 0.5 g/in2 to the backs of 3 rabbits of each sex with either shorn intact or scarified skin. The sample was covered and left in place for 24 hours. Readings were made according to Draize upon removal of the test material and daily for 14 days post administration.

There were no observable reactions to the dye. The test material was considered “not irritant” to rabbit skin.

 

In another study, 0.5 g of the test material was dampened with 0.5 ml distilled water and applied to an area of 1 square inchon the backs of 3 rabbits each with either shorn intact or scarified skin. The sample was covered by an impervious material and left in place for 24 hours. Skin reactions were recorded after 24 and 72 hours.

 

There were no observable reactions to the dye. The test material was considered “not irritant” to rabbit skin.

 

In another study, approximately 24 hours prior to the treatment, the dorsal fur was shaved to expose an area of about 150 cm². The intact shaved back skin of each animal was exposed to an aliquot of 0.5 g of the moistened test substance. The patch was removed 4 hours after semi-occlusive contact.

Animals were examined for signs of erythema, eschar and oedema formation. The skin reactions were assessed approximately 1, 24, 48 and 72 hours after termination of the exposure.

There were no visible signs of irritation. However, slight red staining was observed in all animals from the 1-hour reading up to 72 hours after treatment. Staining persisted in one animal up to a 14-day examination, the end of the observation period for all animals.

 

In an in vitro study reported by Scientific Committee on Consumer Safety SCCS (2010), The Episkin™ skin irritation assay was performed according to the validated and published ECVAM Standard Operating Procedure. The results are interpreted on the basis of a fixed 15 min exposure-time and 42 hours post-exposure incubation time. Based on the mean cell viability values of 72 -84 obtained in the Episkin™ assay, the test chemical was assessed to be a non-irritant.

Based on the various in vitro and in vivo studies, the substance can be considered to be non irritant to skin.

Eye irritation:

Scientific Committee on Consumer Safety SCCS (2011) reported an eye irritation study carried out on New Zealand White rabbits according to OECD 405 Guidelines to determine the irritation potency of Basic Red 76.

100 mg of Basic Red 76 was instilled into the conjunctival sac of the left eye of the test animals. The substance remained in permanent contact with the eyes until rinsing with warm tap water, 24 hours after instillation. The right eyes served as controls. The eye irritation reactions were scored approximately 1, 24, 48 and 72 hours and 7 days after instillation of the test solution.

The instillation of the undiluted Basic Red 76 into the eyes resulted in mild, early-onset and transient ocular changes, such as reddening of the conjunctivae and sclerae, discharge and chemosis.

These effects were reversible and were no longer evident 72 hours after treatment. No abnormal findings were observed in the cornea or iris of any animal at any of the examinations. No corrosion was observed at any of the measuring intervals. Slight red staining of the treated eyes by the test item was observed in all animals at the 1- and 24- hour reading and persisted in two animals up to 48 hours and in one animal up to 72 hours after treatment. The study was terminated seven days after instillation.

Under the conditions of the study, Basic Red 76 was considered to be not irritating to rabbit eyes.

In another study reported by Scientific Committee on Cosmetic Products (2003),eye irritation study was carried out on New Zealand White rabbits to determine the irritation potency of Basic Red 76.

0.1 ml of 0.5% solution Basic Red 76 was instilled into the conjunctival sac of the left eye of three rabbits. The right eye was treated with 0.1 ml of the vehicle and served as a control.

Eye reactions were recorded at 30 and 60 minutes and 1 and 2 days following and evaluated by the Draize method.

The treatment provoked no effects on the cornea or iris in any of the test animals, however, there was a discoloration of the conjunctivae

Under the conditions of the study, Basic Red 76 was not irritating to rabbit eyes.

In studies conducted on similar substance (CAS: 68391-32-2) reported in Scientific Committee on Consumer Products SCCP Opinion on Basic Brown 17 (2003 and 2008), eye irritation study was performed on rabbits (male/female).

The test chemical solution (0.1 ml of a 0.5 % (w/v) in 0.9% saline solution) was instilled into the conjunctival sac of the left eye of each of three rabbits. No washing of test material solution was carried out. Physiological saline was used as a vehicle. The right eye was treated with 0.1 ml of the vehicle and served as a control.  

The eye irritation reactions were read 30 and 60 minutes and after one and two days following instillation of the test material. Irritation effects observed in the eye of the test animal were scored according to the Draize method.  

Apart from a discoloration of the conjunctivae, no effects were noted on the cornea and iris in any of the test animal. Thus, the test substance (8-((4-Amino-3-nitrophenyl)azo)-7-hydroxy-2-naphthyl)trimethylammonium chloride was considered to be not irritant to rabbit eyes.

Another Eye irritation study was performed on male albino rabbits. Single samples of approximately 45 mg of test substance (a volume of approximately 0.1 ml) were instilled into one eye of each of three rabbits. Observations were made 1, 24, 48 and 72 hours and 7 days after instillation.

After the 7 days of observation period, irritation effect was noted on the iris in two test animals and on the conjunctivae in all animals. lridial irritation grade 1 was observed and had resolved within 24 or 72 hours. The irritation of the conjunctivae consisted of redness, chemosis and discharge and had completely resolved within 7 days in all animals.  

No corneal opacity was observed, and treatment of the eyes with 2% fluorescein, 24 hours after test substance instillation revealed no corneal epithelial damage.

Thus, the test substance (8-((4-Amino-3-nitrophenyl)azo)-7-hydroxy-2-naphthyl)trimethylammonium chloride was considered to be not irritant to rabbit eyes.

In a study for similar substance (CAS: 26381-41-9), reported in Scientific Committee on Consumer Safety (2012), The test substance was applied by instillation of 0.1 g into the left eye of each of three young adult New Zealand White rabbits. The treated and untreated eyes were rinsed with lukewarm tap water 48 hours after instillation. Scoring of irritation effects was performed

approximately 1, 24, 48 and 72 hours, as well as 7, 10 and 14 after test item instillation.The mean score was calculated across 3 scoring times (24, 48 and 72 hours after instillation) for each animal for corneal opacity, iris, redness and chemosis of the conjunctivae, separately.

The individual mean scores for corneal opacity were 0.33, 0.67 and 0.33, respectively. The individual mean scores for the iris were 0.00 for all three animals. The individual mean scores for the conjunctivae were 1.67, 2.00 and 2.00 for reddening and 1.67, 1.33 and 1.67 for chemosis, respectively.

Thus from overall observation of experiment the test substance Basic Brown 16 was observed to be not irritating to rabbit eye.

Based on the various in vitro studies reported for the taregt as well as read across substances, the substance can be considered to be non irritant to eyes.

Justification for selection of skin irritation / corrosion endpoint:
OECD TG 439: “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method” was used to assess the skin irritation potential of the Basic Red 76.
Based on the cell viability values of 108.5 and 106.6 obtained in the standard photometry (OD) and HPLC/UPLC-spectrophotometry assays respectively, the test chemical was assessed to be a non-irritant to re-constructed human skin.

Justification for selection of eye irritation endpoint:
Eye irritation study was carried out on New Zealand White rabbits according to OECD 405 Guidelines to determine the irritation potency of Basic Red 76.
The instillation of the undiluted Basic Red 76 into the eyes resulted in mild, early-onset and transient ocular changes, such as reddening of the conjunctivae and sclerae, discharge and chemosis.
These effects were reversible and were no longer evident 72 hours after treatment.
Under the conditions of the study, Basic Red 76 was considered to be not irritating to rabbit eyes.

Justification for classification or non-classification

On the basis of available data, the substance Basic red 76 is not likely to exhibit skin and eye irritation and can be classified as 'non-hazardous' as per the CLP classification criteria.