Registration Dossier

Skin Irritation:

Exact evaluation for erythema was not possible due to staining of the skin by the substance. The skin findings on the sacrificed animal was confirmed by gross pathological examination after incision of the skin. The edema formation exceeded beyond the area of exposure with mean score after 48 hours was 2.6 and was not reversible even after 8 days.

Based on the observations and scores,N-(4-((4-(dimethylamino)phenyl)(4-(phenylamino)naphthalen-1-yl)methylene)cyclohexa-2,5-dienylidene)-N-methylmethanaminium acetate[Victoria Blue B liquid] was strongly irritating to the skin.

Eye Irritation:

Conjunctivae score was not assessed due to staining of the test chemcal. The effects observed in the rabbit eyes were not reversible even after 72 hours. The mean scores for corneal opacity, iris and chemosis after 24,72 hours of observation were 3.3, 1.9, 3.2 respectively.

Based on the scores, N-(4-((4-(dimethylamino)phenyl)(4-(phenylamino)naphthalen-1-yl)methylene)cyclohexa-2,5-dienylidene)-N-methylmethanaminium acetate can be considered to be irritating to rabbit eyes.

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:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

data is from experimental reports

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Principles of method if other than guideline:

To assess the dermal irritation potential of Victoria Blue B liquid in Vienna White rabbits

GLP compliance:

no

Specific details on test material used for the study:

- Name of test material: N-(4-((4-(dimethylamino)phenyl)(4-(phenylamino)naphthalen-1-yl)methylene)cyclohexa-2,5-dienylidene)-N-methylmethanaminium acetate- Molecular formula: C33H32N3C2H3O2- Molecular weight: 529.68 g/mol- Smiles notation : [N+](=C1/C=CC(\C=C1)=C(\c1ccc(c2ccccc12)Nc1ccccc1)c1ccc(cc1)N(C)C)(\C)C.C(C)(=O)[O-]- InChl : 1S/C33H31N3.C2H4O2/c1-35(2)27-18-14-24(15-19-27)33(25-16-20-28(21-17-25)36(3)4)31-22-23-32(30-13-9-8-12-29(30)31)34-26-10-6-5-7-11-26;1-2(3)4/h5-23H,1-4H3;1H3,(H,3,4)- Substance type: Organic- Physical state: Liquid- Common Name: Victoria Blue B liquid- Analytical purity: 44% Victoria Blue B 45 % acetic acid 11 % propyleneglycol

Species:

rabbit

Strain:

Vienna White

Details on test animals or test system and environmental conditions:

no data available

Type of coverage:

occlusive

Preparation of test site:

shaved

Vehicle:

unchanged (no vehicle)

Controls:

not specified

Amount / concentration applied:

2.5 x 2.5 cm

Duration of treatment / exposure:

4 h

Observation period:

1, 2 and 8 days

Number of animals:

2

Details on study design:

TEST SITE - Area of exposure: to the dorsal skin- % coverage: 2.5 x 2.5 cm

Irritation parameter:

erythema score

Basis:

other: not specified

Time point:

other: not specified

Reversibility:

not specified

Remarks on result:

other: Exact evaluation not possible due to staining of the skin by the substance

Irritation parameter:

edema score

Basis:

mean

Time point:

24/48 h

Score:

2.6

Max. score:

3

Reversibility:

not fully reversible within: 8 days

Remarks on result:

positive indication of irritation

Irritant / corrosive response data:

Exact evaluation for erythema was not possible due to staining of the skin by the substance.The skin findings on the sacrificed animal was confirmed by gross pathological examination after incision of the skin.

Observation Table:

Animal No.	4 hours		1 day		2 days		8 days
Animal No.	Reddening	Edema	Reddening	Edema	Reddening	Edema	Reddening	Edema
1	*	2^e	*	3^e	*	3^e	*sS^e	2^e
2	*	2	*	3^e	*	2^e	*S^e	1

S: scaling

s.S.: severe scaling

e: extending beyond the area of exposure

*Exact evaluation not possible due to staining of the skin by the substance.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Exact evaluation for erythema was not possible due to staining of the skin by the substance. The skin findings on the sacrificed animal was confirmed by gross pathological examination after incision of the skin.The edema formation exceeded beyond the area of exposure with mean score after 48 hours was 2.6 and was not reversible even after 8 days.Based on the observations and scores,N-(4-((4-(dimethylamino)phenyl)(4-(phenylamino)naphthalen-1-yl)methylene)cyclohexa-2,5-dienylidene)-N-methylmethanaminium acetate[Victoria Blue B liquid] was strongly irritating to the skin.

Executive summary:

The dermal irritation potential of N-(4-((4-(dimethylamino)phenyl)(4-(phenylamino)naphthalen-1-yl)methylene)cyclohexa-2,5-dienylidene)-N-methylmethanaminium acetate[Victoria Blue B liquid] was assessed in rabbits. The study was performed as per OECD 404 Guidelines.

Undiluted Victoria Blue B liquid was placed on a surgical gauze patch (2.5 cm x 2.5 cm) and applied to shaved dorsal skin of 2 Vienna White rabbits.The patch was covered with an occlusive dressing. The animals were exposed to the chemical for 4 hours. The animals were observed for signs of irritation at 1,2 and 8 days post exposure.

Exact evaluation for erythema was not possible due to staining of the skin by the substance. The skin findings on the sacrificed animal was confirmed by gross pathological examination after incision of the skin.

The edema formation exceeded beyond the area of exposure with mean score after 48 hours was 2.6 and was not reversible even after 8 days.

Based on the observations and scores,N-(4-((4-(dimethylamino)phenyl)(4-(phenylamino)naphthalen-1-yl)methylene)cyclohexa-2,5-dienylidene)-N-methylmethanaminium acetate[Victoria Blue B liquid] was strongly irritating to the skin.

Endpoint conclusion:

adverse effect observed (irritating)

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

data is from experimental reports

Qualifier:

according to guideline

Guideline:

other: Fed. Reg. 38, No. 187, § 1500.42, 1973 (Draize test)

Principles of method if other than guideline:

To assess the ocular irritation potential of N-(4-((4-(dimethylamino)phenyl)(4-(phenylamino)naphthalen-1-yl)methylene)cyclohexa-2,5-dienylidene)-N-methylmethanaminium acetate in rabbits

GLP compliance:

no

Specific details on test material used for the study:

- Name of test material: N-(4-((4-(dimethylamino)phenyl)(4-(phenylamino)naphthalen-1-yl)methylene)cyclohexa-2,5-dienylidene)-N-methylmethanaminium acetate- Molecular formula: C33H32N3C2H3O2- Molecular weight: 529.68 g/mol- Smiles notation : [N+](=C1/C=CC(\C=C1)=C(\c1ccc(c2ccccc12)Nc1ccccc1)c1ccc(cc1)N(C)C)(\C)C.C(C)(=O)[O-]- InChl : 1S/C33H31N3.C2H4O2/c1-35(2)27-18-14-24(15-19-27)33(25-16-20-28(21-17-25)36(3)4)31-22-23-32(30-13-9-8-12-29(30)31)34-26-10-6-5-7-11-26;1-2(3)4/h5-23H,1-4H3;1H3,(H,3,4)- Substance type: Organic- Physical state: Liquid

Species:

rabbit

Strain:

other: albino rabbit

Details on test animals or tissues and environmental conditions:

TEST ANIMALS- Weight at study initiation:ca. 3 - 4 kg- Housing:One animal per cage- Diet (e.g. ad libitum): a standardized animal laboratory diet, ad libitum- Water (e.g. ad libitum): tap water ad libitum.- Acclimation period:at least 3 daysENVIRONMENTAL CONDITIONS- Temperature (°C):22 +/- 1°C- Humidity (%):55 +/- 10%- Photoperiod (hrs dark / hrs light):12 h light, 12 h darkness

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

100 mg

Duration of treatment / exposure:

24 hours

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

6

Details on study design:

The test substance was not washed out.Readings: 1 h, 24 h, 48 h and 72 h after instillation

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

other: 24 - 72 hours

Score:

3.3

Max. score:

4

Reversibility:

not reversible

Remarks on result:

positive indication of irritation

Irritation parameter:

iris score

Basis:

mean

Time point:

other: 24 - 72 hours

Score:

1.9

Max. score:

2

Reversibility:

not reversible

Remarks on result:

positive indication of irritation

Irritation parameter:

chemosis score

Basis:

mean

Time point:

other: 24 - 72 hours

Score:

3.2

Max. score:

4

Reversibility:

not reversible

Remarks on result:

positive indication of irritation

Irritant / corrosive response data:

Due to substance colouring, conjunctivae score was not assessed.

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Conjunctivae score was not assessed due to staining of the test chemcal. The effects observed in the rabbit eyes were not reversible even after 72 hours. The mean scores for corneal opacity, iris and chemosis after 24,72 hours of observation were 3.3, 1.9, 3.2 respectively.Based on the scores, N-(4-((4-(dimethylamino)phenyl)(4-(phenylamino)naphthalen-1-yl)methylene)cyclohexa-2,5-dienylidene)-N-methylmethanaminium acetate can be considered to be irritating to rabbit eyes.

Executive summary:

The ocular irritation potential of N-(4-((4-(dimethylamino)phenyl)(4-(phenylamino)naphthalen-1-yl)methylene)cyclohexa-2,5-dienylidene)-N-methylmethanaminium acetate was assessed in rabbits according to Fed. Reg. 38, No. 187, § 1500.42, 1973 (Draize test) Guidelines. 100 mg of the undiluted test chemical was instilled into one eye of the 6 albino rabbits.The untreated eye served as control. The animals were exposed to the test chemical for 24 hours, and the treated eyes remained unwashed throughout the study. Rabbits were observed for signs of irritation 1 h, 24 h, 48 h and 72 h after instillation of the test chemical. The ocular reactions at 24 and 72 hours were scored according to method of Draize.

Conjunctivae score was not assessed due to staining of the test chemical. The effects observed in the rabbit eyes were not reversible even after 72 hours. The mean scores for corneal opacity, iris and chemosis after 24,72 hours of observation were 3.3, 1.9, 3.2 respectively.

Based on the scores, N-(4-((4-(dimethylamino)phenyl)(4-(phenylamino)naphthalen-1-yl)methylene)cyclohexa-2,5-dienylidene)-N-methylmethanaminium acetate can be considered to be irritating to rabbit eyes.

Endpoint conclusion:

adverse effect observed (irritating)

Endpoint conclusion:

no study available

Skin Irritation:

The dermal irritation potential of N-(4-((4-(dimethylamino)phenyl)(4-(phenylamino)naphthalen-1-yl)methylene)cyclohexa-2,5-dienylidene)-N-methylmethanaminium acetate[Victoria Blue B liquid] was assessed[Sustainability Support Services (Europe) AB has letter of access from BASF SE,1983] in rabbits. The study was performed as per OECD 404 Guidelines.

Undiluted Victoria Blue B liquid was placed on a surgical gauze patch (2.5 cm x 2.5 cm) and applied to shaved dorsal skin of 2 Vienna White rabbits. The patch was covered with an occlusive dressing. The animals were exposed to the chemical for 4 hours. The animals were observed for signs of irritation at 1,2 and 8 days post exposure.

Exact evaluation for erythema was not possible due to staining of the skin by the substance. The skin findings on the sacrificed animal were confirmed by gross pathological examination after incision of the skin. The edema formation exceeded beyond the area of exposure with mean score after 48 hours was 2.6 and was not reversible even after 8 days.

Based on the observations and scores, N-(4-((4-(dimethylamino)phenyl)(4-(phenylamino)naphthalen-1-yl)methylene)cyclohexa-2,5-dienylidene)-N-methylmethanaminium acetate[Victoria Blue B liquid] was strongly irritating to the skin.

N-(4-((4-(dimethylamino)phenyl)(4-(phenylamino)naphthalen-1-yl)methylene)cyclohexa-2,5-dienylidene)-N-methylmethanaminium acetate can considered to be Severe Skin Irritant and can be classified under the category “Category 2” as per CLP regulation.

The dermal irritation potential of test article was determined according to the OECD 439 In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method”. The MatTek EpiDerm™ model was used to assess the potential dermal irritation of the test article by determining the viability of the tissues following exposure to the test article via MTT. The objective of this study was to assess the dermal irritation potential of test article Tissues were exposed to test article and controls for ~one hour, followed by a 42 hour post-exposure recovery period. The viability of each tissue was determined by MTT assay.

The MTT data shows that the assay quality controls were met, as the OD of the negative control tissues was between 1.195 and 1.430. Also, the positive control, 5% sodium dodecyl sulfate (SDS), reduced tissue viability to 4.5% of negative control.

The Mean % tissue viability compared to negative control (n=3) of the [4-[[4-anilino-1-naphthyl][4-(dimethylamino)phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium acetate, CAS No. 83803-79-6 was determined to be 88.7%.

Hence, under the experimental test conditions it was concluded that test substance [4-[[4-anilino-1-naphthyl][4-(dimethylamino)phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium acetate, CAS No. 83803-79-6 was considered to be not irritating to the human skin and being classified as “Not Classified'' as per CLP Regulation.

Even though the results of in–vitro study substantiate the claim that the chemical can indeed be a non skin irritating , but the results from the in vivo study present a stronger evidence of its skin-irritating properties.

Eye Irritation:

In different studies, N-(4-((4-(dimethylamino)phenyl)(4-(phenylamino)naphthalen-1-yl)methylene)cyclohexa-2,5-dienylidene)-N-methylmethanaminium acetate has been investigated for potential for ocular irritation to a greater or lesser extent. The studies are based on in vivo experiments in rabbits for target chemical.

The ocular irritation potential of N-(4-((4-(dimethylamino)phenyl)(4-(phenylamino)naphthalen-1-yl)methylene)cyclohexa-2,5-dienylidene)-N-methylmethanaminium acetate was assessed[Sustainability Support Services (Europe) AB has letter of access from BASF SE, 1978] in rabbits according to Fed. Reg. 38, No. 187, § 1500.42, 1973 (Draize test) Guidelines. 100 mg of the undiluted test chemical was instilled into one eye of the 6 albino rabbits. The untreated eye served as control. The animals were exposed to the test chemical for 24 hours, and the treated eyes remained unwashed throughout the study. Rabbits were observed for signs of irritation 1 h, 24 h, 48 h and 72 h after instillation of the test chemical. The ocular reactions at 24 and 72 hours were scored according to method of Draize.

Conjunctivae score was not assessed due to staining of the test chemical. The effects observed in the rabbit eyes were not reversible even after 72 hours. The mean scores for corneal opacity, iris and chemosis after 24, 72 hours of observation were 3.3, 1.9, 3.2 respectively.

Based on the scores, N-(4-((4-(dimethylamino)phenyl)(4-(phenylamino)naphthalen-1-yl)methylene)cyclohexa-2,5-dienylidene)-N-methylmethanaminium acetate can be considered to be irritating to rabbit eyes.

This result is supported by the experimental study performed according to BASF-test protocol to assess[Sustainability Support Services (Europe) AB has letter of access from BASF SE, 1976]the irritation potential of N-(4-((4-(dimethylamino)phenyl)(4-(phenylamino)naphthalen-1-yl)methylene)cyclohexa-2,5-dienylidene)-N-methylmethanaminium acetate[Victoria Blue B Liquid]. 50 µl of the test substance was applied to the conjunctival sac of one eye in 2 Vienna White rabbits. The adjacent eye served as saline control. The animals were observed after 10 min, 1 and 3 hours on the day of treatment and up to 8 days afterwards. The eyes were not washed out after 24 hours.

Severe suppuration and secretion was observed even after 8 days of observation in all the test animals. The mean corneal opacity and mean chemosis score after 24 hours were 3.0 and 2.0 respectively. The observations were not reversible even after 8 days.

Based on these observations and scores, N-(4-((4-(dimethylamino)phenyl)(4-(phenylamino)naphthalen-1-yl)methylene)cyclohexa-2,5-dienylidene)-N-methylmethanaminium acetate can be considered to be severely irritating to rabbit eyes.

The above studies are further supported by the experimental result reported in the study performed according to Fed. Reg. 28, No. 187, § 1500.42, 1973 (Draize test) Guidelines [Sustainability Support Services (Europe) AB has letter of access from BASF SE, 1983]. 3 Vienna White rabbits were used for the study.0.1 ml undiluted test chemical was instilled in the conjunctival sac of one eye of 3 Vienna White rabbits, and the untreated eye served as control. The eyes were exposed to the test chemical for 24 hours and the treated eyes remained unwashed throughout the study duration. The treated eyes were observed and scored for signs of irritation at 24, 48, 72 hours and 8 days.

Reddening and clouding cannot be assessed due to staining of the eye by the test substance. White mucosa was discharged from the nictitating membranes after 8 days of observation. The Primary Irritation Index was 12.9 and the effects observed were not reversible by 8 days.

Based on the scores and observations, N-(4-((4-(dimethylamino)phenyl)(4-(phenylamino)naphthalen-1-yl)methylene)cyclohexa-2,5-dienylidene)-N-methylmethanaminium acetate[Victoria Blue B liquid] can be considered to be severely irritating to rabbit eyes.

Available studies for N-(4-((4-(dimethylamino)phenyl)(4-(phenylamino)naphthalen-1-yl)methylene)cyclohexa-2,5-dienylidene)-N-methylmethanaminium acetate indicate a very strong possibility of it being severely irritating to rabbit eyes.

Hence, N-(4-((4-(dimethylamino)phenyl)(4-(phenylamino)naphthalen-1-yl)methylene)cyclohexa-2,5-dienylidene)-N-methylmethanaminium acetate can considered to be Severe Eye Irritant and can be classified under the category “Category 2” as per CLP regulation.

Available studies for N-(4-((4-(dimethylamino)phenyl)(4-(phenylamino)naphthalen-1-yl)methylene)cyclohexa-2,5-dienylidene)-N-methylmethanaminium acetate suggests that it is very likely to cause severe irritation to skin and eyes. Hence,N-(4-((4-(dimethylamino)phenyl)(4-(phenylamino)naphthalen-1-yl)methylene)cyclohexa-2,5-dienylidene)-N-methylmethanaminium acetate can be classified under the category “Category 2” as per CLP regulation.