Registration Dossier

Administrative data

Description of key information

Skin irritation:

The dermal irritation potential of target chemical was assessed in various in- vitro and in-vivo experimental studies.Based on the available studies,it can be concluded that the test chemical is unable to cause skin irritation and considered as not irritating. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Not Classified''.

Eye irritation:

The ocular irritation potential of target chemical was assessedin various in- vitro and in-vivo experimental studies.Based on the available studies,it can be concluded that the test chemical is unable to cause eye irritation and considered as not irritating. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Not Classified''.

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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from study report
Qualifier:
according to
Guideline:
other: OECD Guideline 402 (Acute Dermal Toxicity)
Principles of method if other than guideline:
The study was designed and conducted to determine the dermal reaction profile of [8-[(4-amino-3-nitrophenyl)azo]-7-hydroxy-2-naphthyl] trimethylammonium chloride in Sprague Dawley rats.
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Sustainability Support Services (Europe) AB, Sweden
- Lot/batch No.of test material: A171
- Expiration date of the lot/batch: 14/9/2021
- Purity test date: No data

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: No data
- Specific activity: No data
- Locations of the label: No data
- Expiration date of radiochemical substance: No data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient Temperature
- Stability under test conditions: No data
- Solubility and stability of the test substance in the solvent/vehicle: No data
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test item was moistened with distilled water before application.
- Preliminary purification step (if any):No data
- Final dilution of a dissolved solid, stock liquid or gel: No data
- Final preparation of a solid: No data

FORM AS APPLIED IN THE TEST (if different from that of starting material) : No data

OTHER SPECIFICS:
Safety Precautions: Safety precautions included use of protective clothing, gloves, masks and eye protection (glasses).
Test system:
other: No data
Source species:
other: No data
Cell type:
other: No data
Cell source:
other: No data
Source strain:
other: No data
Details on animal used as source of test system:
No data
Justification for test system used:
No data
Vehicle:
other: No data
Details on test system:
No data
Control samples:
other: No data
Amount/concentration applied:
No data
Duration of treatment / exposure:
No data
Duration of post-treatment incubation (if applicable):
No data
Number of replicates:
No data
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: National Institute of Biosciences, Pune.
- Age at study initiation: Young adult male and female rats aged between 6 – 9 weeks were used.
- Weight at study initiation: The weight ranges of approximately 219.6 to 262.5 grams at initiation of dosing were used.
Body weights at the start :
Male
Mean : 257.02 g (= 100 %)
Minimum : 251.5 g (- 2.15 %)
Maximum : 262.5 g (+ 2.13 %)
Total No. of animals : 5
Female
Mean : 225.00 g (= 100 %)
Minimum : 219.6 g (- 2.40 %)
Maximum : 232.7 g (+ 3.42 %)
Total No. of animals : 5
- Housing: The rats were individually housed in polycarbonate cages with paddy husk as bedding.
- Diet (e.g. ad libitum): Rodent feed supplied by the Nutrivet Life Sciences, Pune, was provided ad libitum from individual feeders.
- Water (e.g. ad libitum): Water was provided ad libitum from individual bottles attached to the cages. All water was from a local source and passed through the reverse osmosis membrane before use.
- Acclimation period: 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Room temperature was maintained at 20.0 to 21.9 degree centigrade.
- Humidity (%): Room humidity was maintained at 56.1% to 58.7%.
- Air changes (per hr): The animal room was independently provided with at least ten to fifteen air changes per hour of 100% fresh air that had been passed through the HEPA filters.
- Photoperiod (hrs dark / hrs light): An artificial light and dark cycle of 12 hours each was provided to the room.

IN-LIFE DATES: From: To: No data
Type of coverage:
occlusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2000 mg/kg bw
- Concentration (if solution): no

VEHICLE
- Amount(s) applied (volume or weight with unit):No data
- Concentration (if solution): No data
- Lot/batch no. (if required): No data
- Purity: No data

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): No data
- Concentration (if solution): No data

POSITIVE CONTROL
- Amount(s) applied (volume or weight): No data
- Concentration (if solution): No data
Duration of treatment / exposure:
24 hrs.
Observation period:
14 days
Number of animals:
10 (5/sex).
Details on study design:
TEST SITE
- Area of exposure: Dorsal surface and sides from scapular to pelvic area.
- % coverage: Approximately 10% of the total body surface area.
- Type of wrap if used: Porous gauze dressing and non-irritating tape.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Distilled water was used to remove residual test item.

OBSERVATION TIME POINTS
(indicate if minutes, hours or days) : Observations were conducted at 10, 30, 60 minutes, 2, 4 and 6 hours on the day of dosing and once daily thereafter for 14 day.

SCORING SYSTEM: Draize Method.
Irritation parameter:
overall irritation score
Basis:
animal: 1 - 10
Time point:
other: 0 - 14 d
Score:
0
Reversibility:
other: no data
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
Overall result:
Sex : Male
Group I -
Animal treated at the dose level of 2000 mg/kg body weight did not result in any skin reaction during the study period of 14 days.

Sex : Female
Group I -
Animal treated at the dose level of 2000 mg/kg body weight did not result in any skin reaction during the study period of 14 days.
Other effects:
Clinical Signs of Toxicity and Mortality
Sex : Male
Group I -
Animal treated at the dose level of 2000 mg/kg body weight did not result in any signs of toxicity during the study period of 14 days. All animals survived through the study period of 14 days.

Sex : Female
Group I -
Animal treated at the dose level of 2000 mg/kg body weight did not result in any signs of toxicity during the study period of 14 days. All animals survived through the study period of 14 days.

Body Weight
Sex : Male
Group I (2000 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 9.60% and 18.68% respectively.

Sex : Female
Group I (2000 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 5.03% and 10.54% respectively.

Gross Pathological Findings
Gross pathological examination did not reveal any abnormalities in animals from 2000 mg/kg dose group.

Table No. I

 

Summary of Evaluation of Dermal Reaction

Test System : Sprague Dawley Rat

Sex : Male 

Group

No.

Dose mg/kg

                          

Dermal Reaction

Total Number of

Animals

 

Animal Nos.

Period of signs in days

 From - to

 

Mortality

I

2000

No dermal reaction observed

5

1 - 5

Day 0 - Day 14

0/5

 

 

Sex : Female

 

Group

No.

Dose mg/kg

                          

Dermal Reaction

Total Number of

Animals

 

Animal Nos.

Period of signs in days

 From - to

 

Mortality

I

2000

No dermal reaction observed

5

6 - 10

Day 0 - Day 14

0/5

 

Table No. II

 

Individual Animal - Evaluation of Dermal Reaction

Test System : Sprague Dawley Rat

Sex : Male  

Group : I

Dose  : 2000 mg/kg body weight

Animal

Dermal

D A Y S

 

No.

Reaction

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

1

Erythema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

Oedema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

2

Erythema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

Oedema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

3

Erythema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

Oedema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

4

Erythema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

Oedema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

5

Erythema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

Oedema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

Sex : Female  

Group : I

Dose  : 2000 mg/kg body weight

Animal

Dermal

D A Y S

 

No.

Reaction

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

6

Erythema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

Oedema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

7

Erythema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

Oedema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

8

Erythema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

Oedema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

9

Erythema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

Oedema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

10

Erythema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

Oedema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Interpretation of results:
other: Not irritating
Remarks:
on the basis of EU Criteria
Conclusions:
From the study, it can be concluded that the test substance ‘[8-[(4-amino-3-nitrophenyl)azo]-7-hydroxy-2-naphthyl] trimethylammonium chloride’ 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 [8-[(4-amino-3-nitrophenyl)azo]-7-hydroxy-2-naphthyl] trimethylammonium chloride can be classified under nonirritant category.
Executive summary:

The study was designed and conducted to determine the dermal reaction profile of [8-[(4-amino-3-nitrophenyl)azo]-7-hydroxy-2-naphthyl] trimethylammonium chloride in Sprague Dawley rats. The test item was applied to shorn skin of 5 male and 5 female animals 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 ‘[8-[(4-amino-3-nitrophenyl)azo]-7-hydroxy-2-naphthyl] trimethylammonium chloride’ 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 [8-[(4-amino-3-nitrophenyl)azo]-7-hydroxy-2-naphthyl] trimethylammonium chloride can be classified under nonirritant category.

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:
key study
Study period:
06 July 2004 - 19 July 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from study report
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Qualifier:
according to
Guideline:
other: EC Commission Directive 92/69/EEC, B.5, "Acute Eye Irritation/Corrosion" (1992)
Qualifier:
according to
Guideline:
EPA OPPTS 870.2400 (Acute Eye Irritation)
Qualifier:
according to
Guideline:
other: JMAFF guidelines (2000).
Principles of method if other than guideline:
The purpose of this acute eye irritation/corrosion study was to assess the possible irritation or corrosion potential when a single dose of the test substance was placed in the conjunctival sac of the rabbit eye.
The absence of eye pigmentation in the albino rabbit facilitates the evaluation of induced eye reactions. The ocular route was selected because the test substance may accidentally come into contact with the eyes during manufacture, handling and/or use.
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Test Item Identification: B 007 ([8-[(4-amino-3-nitrophenyl)azo]-7-hydroxy-2-naphthyl]trimethylammonium chloride)
- Batch No.of test material: NDKS 1944
- Expiration date of the lot/batch: 31 December 2013
- Purity: 96.3% (HPLC)

RADIOLABELLING INFORMATION (Not applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In refrigerator in the dark.
- Stability under test conditions: Stable
- Solubility and stability of the test substance in the solvent/vehicle: No data
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The powdery test substance was instilled as delivered by the sponsor.
- Preliminary purification step (if any): No data
- Final dilution of a dissolved solid, stock liquid or gel: No data
- Final preparation of a solid: No data


Species:
rabbit
Strain:
New Zealand White
Remarks:
(SPF-Quality)
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Kisslegg, Germany
- Age at study initiation: Animals used within the study were at least 6 weeks old
- Weight at study initiation: Body weights were at least 1.0 kg.
- Identification: Earmark.
- Housing: Individually in labelled cages with perforated floors (Scanbur, Denmark, dimensions 56x44x37.5 cm).
- Diet (e.g. ad libitum):Standard laboratory rabbit diet (Charles River Breeding and Maintenance Diet for Rabbits,Altromin, Lage, Germany) approx. 100 g. per day. In addition, hay was provided at least three times a week.
- Water (e.g. ad libitum): Free access to tap-water.
- Acclimation period: 5 days before start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0°C (actual range: 19.0-20.7°C)
- Humidity (%): 30-70% (actual range: 38 - 80%)
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours darkne ss per day.

IN-LIFE DATES: From:06 July 2004 To:19 July 2004
Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 45.4 (43.8 - 47.1) mg of the test substance (a volume of appraximately 0.1 ml).

VEHICLE (No vehicle)
- Amount(s) applied (volume or weight with unit): None
Observation period (in vivo):
The eyes of each animal were examined approximately 1, 24, 48 and 72 hours and 7 days after instillation of the test substance.
Duration of post- treatment incubation (in vitro):
No data
Number of animals or in vitro replicates:
3 Males.
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): No data
- Time after start of exposure: No data

SCORING SYSTEM:The irritation was assessed according to the numerical scoring system.

TOOL USED TO ASSESS SCORE: No data
Other effects / acceptance of results:
All available data relevant to the potential eye irritation/corrosivity of the substance indicated that no severe effects were to be expected. No severe reactions were noted in the skin irritation study. An in-vitro test was considered, but a negative test result was anticipated that still would have to be confirmed in an in-vivo study.
Irritation parameter:
cornea opacity score
Basis:
animal: 39,64,65
Time point:
7 d
Score:
0
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal: 39,64,65
Time point:
7 d
Score:
0
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal: 39,64,65
Time point:
7 d
Score:
0
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal: 39,64,65
Time point:
7 d
Score:
0
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal: 39
Time point:
24/48/72 h
Score:
0
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal: 39
Time point:
24/48/72 h
Score:
0
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal: 39
Time point:
24/48/72 h
Score:
1.7
Reversibility:
not specified
Irritation parameter:
chemosis score
Basis:
animal: 39
Time point:
24/48/72 h
Score:
0.3
Reversibility:
not specified
Irritation parameter:
cornea opacity score
Basis:
animal: 64
Time point:
24/48/72 h
Score:
0
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal: 64
Time point:
24/48/72 h
Score:
0.3
Reversibility:
not specified
Irritation parameter:
conjunctivae score
Basis:
animal: 64
Time point:
24/48/72 h
Score:
2
Reversibility:
not specified
Irritation parameter:
chemosis score
Basis:
animal: 64
Time point:
24/48/72 h
Score:
1
Reversibility:
not specified
Irritation parameter:
cornea opacity score
Basis:
animal: 65
Time point:
24/48/72 h
Score:
0
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal: 65
Time point:
24/48/72 h
Score:
0
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal: 65
Time point:
24/48/72 h
Score:
1.7
Reversibility:
not specified
Irritation parameter:
chemosis score
Basis:
animal: 65
Time point:
24/48/72 h
Score:
0.3
Reversibility:
not specified
Irritant / corrosive response data:
Irritation: Instillation of 45 mg of B 007 (a volume of approximately 0.1 ml) into one eye of each of three rabbits resulted in effects on the iris in two animals and on the conjunc tivae in all animals. Iridial 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 corneaI opacity was observed, and treatment of the eyes with 2% fluorescein, 24 hours after test substance instillation revealed no corneaI epithelial damage.

Corrosion
There was no evidence of ocular corrosion.
Other effects:
- clinical observations:Colouration / Remnants
Remnants of the test substance were present in the eye 1 hour after instillation. And remnants were present on the outside of the eyelids 24 and 48 hours after instillation in one animal.Yellow-brown staining of the fur on the head and paws, caused by the test substance, was noted during the observation period.

- Other observations:Toxicity / Mortality
No symptoms of systemic toxicity were observed in the animals during the test period and no
mortality occurred.
Interpretation of results:
other: Not irritating
Remarks:
based on CLP criteria
Conclusions:
Based on the above study the test item B 007 ([8-[(4-amino-3-nitrophenyl)azo]-7-hydroxy-2-naphthyl]trimethylammonium chloride) does not have to be classified for eye irritation.
Executive summary:

Acute eye irritation/corrosion study with B 007 ([8-[(4-amino-3-nitrophenyl)azo]-7-hydroxy-2-naphthyl]trimethylammonium chloride) in the rabbit. The study was carried out based on the guidelines described in: "Acute Toxicity - Eye irritation",OECD NoA05 (2002); EC Commission Directive 92169/EEC, B.5, "Acute Eye Irritation/Corrosion" (1992); US EPA, OPPTS 870.2400, Acute Eye Irritation, (1998) and JMAFF guidelines (2000). Single samples of approximately 45 mg of B 007 (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. Instillation of the test substance resulted in effects on the iris in two animals and on the conjunctivae in all animals. Iridial 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. Remnants of the test substance were present in the eye 1 hour after instillation. And remnants were present on the outside of the eyelids 24 and 48 hours after instillation in one animal.Yellow-brown staining of the fur on the head and paws, caused by the test substance, was noted during the observation period. Based on these results and according to the CLP classification criteria: B 007 ([8-[(4-amino-3-nitrophenyl)azo]-7-hydroxy-2-naphthyl]trimethylammonium chloride) does not have to be classified for eye irritation.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation:

Various data available for the target chemical including lab reports, study reports and publications were reviewed to determine the skin irritation potential for the test chemical of [8-[(4-amino-3-nitrophenyl)azo]-7-hydroxy-2-naphthyl] trimethylammonium chloride. The studies are as mentioned below:

 

The study was designed and conducted (Sustainability Support Services, 2016) to determine the dermal reaction profile of [8-[(4-amino-3-nitrophenyl)azo]-7-hydroxy-2-naphthyl] trimethylammonium chloride in Sprague Dawley rats. The test item was applied to shorn skin of 5 male and 5 female animals 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 ‘[8-[(4-amino-3-nitrophenyl)azo]-7-hydroxy-2-naphthyl] trimethylammonium chloride’ 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 [8-[(4-amino-3-nitrophenyl)azo]-7-hydroxy-2-naphthyl] trimethylammonium chloride can be classified under nonirritant category.

 

In a study by Sustainability Support Services, 2004, Primary skin irritation/corrosion study with B 007 ([8-[(4-amino-3-nitrophenyl)azo]-7-hydroxy-2-naphthyl]trimethylammonium chloride) in the rabbit (4-hour semi-occlusive application). The study was carried out based on the guidelines described in: OECD NoA04, "Acute Dermal Irritation/Corrosion" (2002); EC Commission Directive 92169/EEC, 804, "Acute Toxicity - Skin irritation" (1992); US EPA, OPPTS 870.2500, Acute Dermal Irritation (1998) and .IMAFF,Japanese Test Guidelines (2000). Three rabbits were exposed to 0.5 grams of B 007, applied onto clipped skin for 4 hours using a semi-occlusive dressing. Observations were made 1, 24, 48 and 72 hours after exposure. Exposure to B 007 resulted in very slight erythema and/or very slight oedema and/or slight oedema in the treated skin-areas of the rabbits, which had resolved within 48 hours. Yellow-brown staining of the treated skin by the test substance was observed throughout the observation period, which did not hamper the scoring of the skin reactions. No remnants of the test substance were present on the skin. Based on these results and according to the CLP classification criteria: B007 ([8-[(4-amino-3-nitrophenyl)azo]-7-hydroxy-2-naphthyl]trimethylammonium chloride) does not have to be classified for skin irritation.

 

Alépée et al (Toxicology in vitro, 2016) performed skin irritation study on Reconstructed human tissue (RhT). Standard Operating Procedures (SOP) in accordance with OECD guideline TG 439 was used for the study. Both positive and negative controls were run in parallel with the test items. The test chemical was tested in the EpiSkin™in vitro skin irritation test method in one run. Water was used as a vehicle for the study. The test chemical conc. used for the study was10 ± 2 mg (26.3 mg/cm2) alongwith exposure duration of 15 – 60 mins.Test chemical powder of conc.26.3 mg/cm2was applied to the epidermis surface.Exposure of test animal to the test substance was terminated by rinsing with phosphate buffered saline (PBS). The evaluation of direct MTT reduction involved incubation of the test item with MTT solution. If the solution turned blue, killed tissue adapted controls incubated with MTT to determine non-specific MTT reduction (% NSMTT) were performed. Killed tissues were prepared according to the SOP for each test method. The evaluation of colour interference involved incubation of the test chemical with solvent or water depending on thein vitroRhT test method. If the solution absorbed at 570 nm or became coloured following incubation with the chemical, living tissue adapted controls incubated with medium instead of MTT to define non-specific colour (%NSCliving) were performed. The run was considered as valid for chemicals and all adapted controls if the following criteria were met: difference of viability between the two replicates tissues was≤20% for skin corrosion or Standard Deviation (SD) of viability between 3 replicate tissues was≤18% for skin irritation in accordance with the respective test method SOPs. The resulting formazan tissue extracts were analysed by photometry (OD) in accordance with the SOP for each test method after testing and also in parallel by HPLC/UPLC-spectrophotometry. Result of the study was interpretated on the basis of cell viability- If cell viability is ≤ 50%: the chemical was classified as irritant and If cell viability is > 50% : the chemical was classified as non – irritant. Based on the cell viability of reconstructed human tissue (RhT) after exposure to the test substance, the test chemical was evaluated to be non-irritant.

 

Skin irritation study was performed (Scientific Committee on Consumer Safety, 2003) on rabbits (New Zealand White). The test material was applied undiluted at the level of 0.5 g per square inch either to the shorn intact or to scarified skin on the back of six albino rabbits of each sex. The sample was covered by a linen cloth and plastic foil which were fixed by an elastic bandage and left in place for 24 hours. Readings were made according to Draize upon removal of the test material and then daily for the following 14 days. During observation period of 14 days,noirritation effects were noted on intact or to scarified skin on the back of the test animal. Thus, the test substance(8-((4-Amino-3-nitrophenyl)azo)-7-hydroxy-2-naphthyl)trimethylammonium chloridewas considered to be not irritant to rabbit skin.

 

In Scientific Committee on Consumer Safety report (2008), Skin irritation study was performed on rabbits. Test rabbits were exposed to 0.5 grams of the test substance, applied onto clipped skin for 4 hours using a semi-occlusive dressing.Observations were made 1, 24, 48 and 72 hours after exposure. During the observation period,very slight erythema and/or very slight to slight oedema in the treated skin-areas of the rabbits was noted, which had resolved within 48 hours. Yellow-brown staining of the treated skin by the test substance was observed throughout the observation period, which did not hamper the scoring of the skin reactions. No remnants of the test substance were present on the skin. Thus, based on the above results, the test substance (8-((4-Amino-3-nitrophenyl)azo)-7-hydroxy-2-naphthyl) trimethylammonium chloride was considered to be not irritant to rabbit s

Yet another study was also mentioned in Scientific Committee on Consumer Safety, 2010. Skin irritation study was performed according to ECVAM Standard Operating Procedures (SOP) on human tissue usingthe EpiSkin™in vitro skin irritation test method. Both positive and negative controls were used for the study. Test chemical was applied to the epidermis surface of the skin of human for 15 mins. Exposure of test animal to the test substance was terminated by rinsing with phosphate buffered saline (PBS) (using a cotton bud if necessary).After the termination of exposure, further incubation of the epidermis at 37°C for 42 ± 1 hrs (37°C, 5% CO2, 95% humidity) and assessment of cell viability was done by incubating the tissues for 3 hours with MTT solution, after whichthe precipitated formazan is extracted and quantified spectrophotometrically at 570 nm. For each treated tissue the viability is expressed as a percentage of the mean of the negative control tissues’ values. For each test material, three independent tests with three different batches of Episkin™ samples have been carried out. In case viability of the tissues is > 50% after treatment, a second endpoint, namely interleukin-1α(IL-1α) release into the culture medium, is measured. To that end, samples are taken after the 42 hours of incubation and frozen until subjected to an ELISA (enzyme linked immunosorbant assay) test. Result of the study was interpretated on the basis of cell viability and/or IL-1α release. Based on the cell viability of human tissue > 50% (i.e. 84 and 93% of sample 1 & 2) andIL - 1α release of < 50 pg/ml (i.e. 6 and -0.5 pg/ml of sample 1 & 2) after exposure to the test substance, the test chemical Basic brown 17 was evaluated to be non-irritant.

Eye irritation:

In different studies, the test chemical has been investigated for potential for ocular irritation to a greater or lesser extent. The studies are based on in- vitro and in-vivo experimental conducted in rabbits conducted which have been summarized as below:

 

Acute eye irritation/corrosion study (Sustaianability Support Services, 2004) with B 007 ([8-[(4-amino-3-nitrophenyl)azo]-7-hydroxy-2-naphthyl]trimethylammonium chloride) in the rabbit. The study was carried out based on the guidelines described in: "Acute Toxicity - Eye irritation",OECD NoA05 (2002); EC Commission Directive 92169/EEC, B.5, "Acute Eye Irritation/Corrosion" (1992); US EPA, OPPTS 870.2400, Acute Eye Irritation, (1998) and JMAFF guidelines (2000). Single samples of approximately 45 mg of B 007 (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. Instillation of the test substance resulted in effects on the iris in two animals and on the conjunctivae in all animals. Iridial 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. Remnants of the test substance were present in the eye 1 hour after instillation. And remnants were present on the outside of the eyelids 24 and 48 hours after instillation in one animal.Yellow-brown staining of the fur on the head and paws, caused by the test substance, was noted during the observation period. Based on these results and according to the CLP classification criteria: B 007 ([8-[(4-amino-3-nitrophenyl)azo]-7-hydroxy-2-naphthyl]trimethylammonium chloride) does not have to be classified for eye irritation.

The ocular irritation potential of test article was determined according to the OECD 492 test guideline for this study. The MatTek EpiOcular™ model was used to assess the potential ocular irritation of the test articles by determining the viability of the tissues following exposure to the test article via MTT. Tissues were exposed to liquid test articles and controls for ~30 minutes, followed by a ~12 minute post-soak and approximately 2 hour recovery after the post-soak. The viability of each tissue was determined by MTT assay.

The MTT data show the assay quality controls were met, passing the acceptance criteria.

The mean of OD for test chemical was determined to be 1.770.The mean % tissue viability of test chemical was determined to be 97.2%. Hence, under the experimental test conditions it was concluded that test chemical was considered to be not irritating to the human eyes.

 

Eye irritation study was performed (Scientific Committee on Consumer Safety, 2003) 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 chloridewas considered to be not irritant to rabbit eyes.

 

In another study reported in Scientific Committee on Consumer Safety, 2008, 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.

All these studies lead to a conclusion that Test chemical is indeed not irritating to eye. Hence, comparing the above annotations with the criteria of CLP regulation, Test chemical can be classified under the category “Not Classified”.

Justification for classification or non-classification

The skin and eye irritation potential of test chemical was observed in various studies. The results obtained from these studies indicate that the test chemical is not likely to cause skin and eye irritation. Hence the test chemical can be classified under the category “Not Classified” for skin and eye as per CLP.