Registration Dossier

Administrative data

Description of key information

Experimental study indicates that Solvent Blue 4 with < 0.1 % Michler's Ketone is not irritant to the skin but it is an eye irritant.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Principles of method if other than guideline:
The objective of the study was to assess the irritant and/or corrosive effects of Solvent Blue 4 (CAS No. - 6786-83-0) after dermal application on the intact skin in Rabbits
GLP compliance:
yes
Test material information:
Composition 1
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Procured from LIVEON BIOLABS PVT LTD, Karnataka, India.
- Age at study initiation:4.5 to 5.0 Months (Approximately)
- Weight (Prior to Treatment):Minimum: 2.038 kg & Maximum: 2.548 kg (Prior to Treatment)
- Health Status : Healthy young adult rabbits were used for the study
-Diet:All animals were provided conventional laboratory rabbit diet (Nutrivet Life Sciences, Pune) ad libitum. Batch No.: 200004.
-Water:Aqua guard filtered tap water was provided ad libitum.
-Husbandry: The animals were housed individually in stainless steel cages.
-Room Sanitation:The experimental room floor and work tops were swept and mopped with disinfectant solution every day.
-Cages and water bottle:All the cages and water bottles were changed minimum twice a week
-Acclimatisation:Rabbits were acclimatised to the test conditions for a period of 6 days (Animal No.-1) and 8 days (Animal No.-2 and 3) prior to the application of the test item

ENVIRONMENTAL CONDITIONS
Temperature: Minimum: 20.30 °C Maximum: 22.70 °C
Relative humidity: Minimum: 48.30 % Maximum: 68.30 %
Light-dark-rhythm: 12:12
Air Changes:More than 12 changes per hour


Type of coverage:
other: porous gauze dressing and non-irritating tape
Preparation of test site:
clipped
Vehicle:
other: distilled water
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit):0.5 ml
- Concentration (if solution):N/A

VEHICLE
- Amount(s) applied (volume or weight with unit):500 mg of test item pulverised form (moistened with 0.5 ml distilled water
- Concentration (if solution):N/A
- Lot/batch no. (if required):N/A
- Purity:N/A
Duration of treatment / exposure:
4 hours
Observation period:
72 hours
Number of animals:
3 male rabbits
Details on study design:
TEST SITE
- Area of exposure:The dorsal lumbar region at contralateral sites.
- % coverage:Approximately 6 X 6 cm.
- Type of wrap if used:A porous gauze dressing and non-irritating tape (Micropore 3”).

REMOVAL OF TEST SUBSTANCE
- Washing (if done):The residual test item was removed by using cotton soaked in distilled water.
- Time after start of exposure:4 hour

SCORING SYSTEM:Draize Method
Irritation parameter:
erythema score
Basis:
animal #1
Time point:
other: At 24 hours
Score:
0.33
Max. score:
3
Reversibility:
no data
Remarks on result:
other: not irritating
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
other: 48hr
Score:
0.67
Max. score:
3
Reversibility:
no data
Remarks on result:
other: not irritating
Irritation parameter:
erythema score
Basis:
animal #3
Time point:
other: 72hr
Score:
0.67
Max. score:
3
Reversibility:
no data
Remarks on result:
other: not irritating
Irritation parameter:
edema score
Basis:
animal #1
Time point:
other: At 24, hours
Score:
0
Max. score:
3
Reversibility:
no data
Remarks on result:
other: not irritating
Irritation parameter:
edema score
Basis:
animal #2
Time point:
other: 48hrs
Score:
0
Max. score:
3
Reversibility:
no data
Remarks on result:
other: not irritating
Irritation parameter:
edema score
Basis:
animal #3
Time point:
other: 72hrs
Score:
0.33
Max. score:
3
Reversibility:
no data
Remarks on result:
other: not irritating
Irritant / corrosive response data:
The following were observed in treated rabbits.
The patch was removed after 4 hours and rabbits were observed for erythema and oedema at 1, 24, 48 and 72 hours after patch removal, evaluated and graded as per draize method.
Animal No. 1, showed very slight erythema (barely perceptible) and very slight oedema (barely perceptible) at 1 hour of observation. At 24 hour very slight erythema (barely perceptible) and no oedema was observed. At 48 and 72 hour observation no erythema and oedema was observed in animal no 1.
In Animals No. 2 and 3 at 1 hour observation post patch removal, revealed very slight erythema (barely perceptible) and very slight oedema (barely perceptible). At 24 hour observation post patch removal, revealed very slight erythema (barely perceptible) and no oedema in animal no. 2 whereas very slight erythema (barely perceptible) and very slight oedema (barely perceptible) was observed in animal no. 3. At 48 hour post patch removal very slight erythema (barely perceptible) and no oedema was observed. At 72 hour post patch removal, both the animals recovered to normal.
The individual mean score at 24, 48 and 72 hours for Animal Nos. 1, 2 and 3 were 0.33, 0.67, 0.67 and 0.00, 0.00, 0.33, for erythema and oedema formation, respectively..
Other effects:
Clinical Observation
No systemic toxicity was observed at treated rabbits during the experimental period.

Mortality
No mortality was observed during the observation period.

Body weight
All rabbits showed increased in body weight at termination when compared to day 0

Table 1

Skin Reaction

In Treated area Dose:500 mg of test item (moistened with 0.5 ml distilled water)           Sex:Male

 

Animal

No.

Test

Treated

 area*

Erythema score

Oedema score

1h

24h

48h

72h

1h

24h

48h

72h

1

Initial

Left

1

1

0

0

1

0

0

0

2

Confirmatory

Left

1

1

1

0

1

0

0

0

3

Left

1

1

1

0

1

1

0

0

 

 

 

 

 

In Control areaDose:0.5 ml of distilled water                                                          Sex:Male

 

Animal

No.

Test

Treated area*

Erythema score

Oedema score

1h

24h

48h

72h

1h

24h

48h

72h

1

Initial

Right

0

0

0

0

0

0

0

0

2

Confirmatory

Right

0

0

0

0

0

0

0

0

3

Right

0

0

0

0

0

0

0

0

Key: h = Hour.

Erythema                                                                               Oedema

0 =No erythema                                                                   0 =No oedema

1 = Very slight erythema(barely perceptible)                       1 =very slight oedema (barely perceptible)

Table 1 Continued…

Mean Individual Animal Score at 24, 48 and 72 hours

 

                     Animal Number                  

Observations                      

1

2

3

Erythema

0.33

0.67

0.67

Oedema

0.00

0.00

0.33

 

  

Table 2

Individual Animal BodyWeight

Sex:Male

Animal

No.

Body Weight (kg)

Prior to Dosing

At termination

1

2.412

2.466

2

2.548

2.614

3

2.038

2.104

 

Table 3

Individual AnimalClinicalSigns

Sex:Male

Animal

No.

Days (Post dosing Observation)

0

1

2

3

1

1

1

1

1

2

1

1

1

1

3

1

1

1

1

Key: ./. = Not Applicable. 1 = Normal
Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The individual mean score at 24, 48 and 72 hours for animal nos. 1, 2 and 3 were 0.33, 0.67, 0.67 and 0.00, 0.00, 0.33, for erythema and oedema formation, respectively. Hence, it was concluded that Solvent Blue 4 (CAS No. 6786-83-0) was Non-Irritating to the skin of male New Zealand White rabbits under the experimental conditions as per CLP Regulation
.

Executive summary:

Acute Dermal Irritation/corrosion Study of Solvent Blue 4 (CAS No:6786-83-0)” was carried out in Rabbits, This study was performed as per OECD guideline No. 404.

Three healthy young adult male rabbits were used for conducting acute dermal irritation study.Body weights were recorded on day 0 (prior to application) and at termination.

Rabbits with good intact skin were selected for the study. The hairs of all the rabbits were clipped at contralateral sites, approximately 24 hours prior to treatment.A dose of 0.5 ml of test item (as such) was applied to the skin,over an area of approximately 6 x 6 cm clippedof hair on one side of rabbits.The other untreated side was kept as control area and 0.5 ml of distilled water was applied at this site. At the end of 4 hours, the gauze patch was removed and test item application site was wiped with water without altering the integrity of the epidermis. 

Initially, the test item was applied to the clipped area of skin of one rabbit. The test site was covered with gauze patch.After 4 hours of exposure in animal no. 1, there was very slight erythema (barely perceptible) and very slight oedema (barely perceptible) observed at 1 hour of observation. At 24 hour very slight erythema (barely perceptible) and no oedema was observed.Hence the confirmatory test was conducted on additional two animal (No. 2 and 3)to confirm the non irritant nature of the test item. At 48 and 72 hours observation no erythema and no oedema was observed in animal no 1.

The patch was removed after 4 hours and rabbits wereobservedfor erythema and oedemaat 1, 24, 48 and 72 hours after patch removal, evaluatedand graded as per draize method.In animals no. 2 and 3 at 1 hour observation post patch removal, revealedvery slight erythema (barely perceptible) and very slight oedema (barely perceptible). At 24 hour observation post patch removal, revealed very slight erythema (barely perceptible) and no oedema in aniaml no. 2 whereasvery slight erythema (barely perceptible) and very slight oedema (barely perceptible), in animal no. 3. At 48 hour post patch removal very slight erythema (barely perceptible) and no oedema was observed in animal no. 2 and 3. At 72 hour post patch removal, both the animals recovered to normal.The individual mean score at24, 48 and 72 hoursfor animal nos. 1, 2 and 3 were 0.33, 0.67, 0.67 and 0.00, 0.00, 0.33, for erythema and oedema formation, respectively.  

Hence, it was concluded that Solvent Blue 4 (CAS No. - 6786-83-0) was Non-Irritating to the skin of female New Zealand White rabbits under the experimental conditions tested and not Classified as a "skin irritant" as per CLP Regulation.

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
Reliability:
1 (reliable without restriction)
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Principles of method if other than guideline:
The objective of the study was to assess the irritant and/or corrosive effects of “Solvent Blue 4 (CAS No. - 6786-83-0)” on eye, when exposed by the ocular route in rabbits.
GLP compliance:
yes
Test material information:
Composition 1
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Procured from LIVEON BIOLABS PVT. LTD. Karnataka, India
- Age at study initiation:4.5 to 5.0 Months (Approximately)
- Weight at study initiation:Minimum: Minimum: 1.934 kg and Maximum: 2.256 kg (Prior to Treatment).
- Health Status :Healthy young adult rabbits were used. Females were nulliparous and non pregnant.
- Housing:The animals were housed individually in stainless steel cages.
- Room Sanitation:The experimental room floor and work tops were swept and mopped with disinfectant solution every day.
- Cages and water bottle:All the cages and water bottles were changed minimum twice a week.
- Diet (e.g. ad libitum):All animals were provided conventional laboratory rabbit diet (Nutrivet Life Sciences, Pune) ad libitum.
- Water (e.g. ad libitum):Aqua guard filtered tap water was provided ad libitum.
- Acclimation period: Rabbits were acclimatised to the test conditions for a period of 7 days (Animal No.-1) and 9 days (Animal No. 2 and 3) prior to the application of the test item

ENVIRONMENTAL CONDITIONS
- Temperature (°C):Minimum: 18.90 °C and Maximum: 22.70 °C
- Humidity (%):Minimum: 48.30 % and Maximum: 68.30 %
- Air changes (per hr): More than 12 changes per hour
- Photoperiod (hrs dark / hrs light): 12:12
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit):100 mg (0.1 gm)
- Concentration (if solution):N/A

VEHICLE
- Amount(s) applied (volume or weight with unit):N/A
- Concentration (if solution):N/A
- Lot/batch no. (if required):N/A
- Purity:N/A
Duration of treatment / exposure:
24 hrs
Observation period (in vivo):
All the animals were observed at 1, 24, 48, 72 hours and at day7 after instillation of test item.
Number of animals or in vitro replicates:
3 female rabbits
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done):The treated eye of rabbit was washed with normal saline.
- Time after start of exposure:24 hours

SCORING SYSTEM:Draize Method

TOOL USED TO ASSESS SCORE: Ophthalmoscope and fluorescent strips.
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
other: 24,
Score:
0.67
Max. score:
3
Reversibility:
not specified
Remarks on result:
other: treated Eye
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
other: 48hr
Score:
1.33
Max. score:
3
Reversibility:
not specified
Remarks on result:
other: Treated eye
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
other: 72hr
Score:
1.33
Max. score:
3
Reversibility:
not specified
Remarks on result:
other: treated eye
Irritation parameter:
iris score
Basis:
animal: #1,#2 and #3
Time point:
other: 24,48,72 hrs
Score:
0
Max. score:
3
Reversibility:
not specified
Remarks on result:
other: treated Eye
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
other: 24hr
Score:
2.67
Max. score:
3
Reversibility:
not specified
Remarks on result:
other: treated Eye
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
other: 48hr
Score:
2.67
Max. score:
3
Reversibility:
not specified
Remarks on result:
other: treated Eye
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
other: 72hr
Score:
2.33
Max. score:
3
Reversibility:
not specified
Remarks on result:
other: treated Eye
Irritation parameter:
chemosis score
Basis:
animal: #1,#2 and #3
Time point:
other: 24,48,72 hrs
Score:
2
Max. score:
3
Reversibility:
not specified
Remarks on result:
other: treated Eye
Irritant / corrosive response data:
The following were observed in treated rabbits.
Observation at 1 hour after instillation of test item revealed: Cornea- No ulceration or opacity was seen in all the animals; Area of Opacity- Zero in all the animals; Iris: Normal in all the animals; Conjunctivae - Blood vessels normal in animal no. 1 whereas some blood vessels definitely hyperaemic (injected) in animal no. 2 and 3; Chemosis: No swelling (Normal) in animal no. 1 whereas some swelling above normal (includes nictitating membranes) in animal no. 2 and 3.
Observation at 24 hours after instillation of test item revealed: Cornea- No ulceration or opacity was seen animal no. 1 and scattered or diffuse areas of opacity (other than slight dulling of normal lustre); details of iris clearly visible in animal no. 2 and 3; Area of Opacity- Zero in animal no.1 and one quarter (or less) but not zero in animal no. 2 and 3; Iris: Normal in all the animals; Conjunctivae - Diffuse, crimson color; individual vessels not easily discernible was seen in all the animals; Chemosis: Obvious swelling with partial eversion of lids was seen in all the animals.
At 24 hours observation the rabbits were examined for corneal epithelium cell damage using sodium fluorescein strips and noticed 60%, 70% and 70% damage in animal no. 1, 2 and 3 respectively.
Observation at 48 hours after instillation of test item revealed: Cornea- Scattered or diffuse areas of opacity (other than slight dulling of normal lustre); details of iris clearly visible was seen in all the animals; Area of Opacity- One quarter (or less) but not zero in all the animals; Iris: Normal in all the animals; Conjunctivae - Diffuse beefy red was observed in animals no. 1 and 2 and diffuse, crimson color; individual vessels not easily discernible in animal no. 3; Chemosis: Obvious swelling with partial eversion of lids was observed in all the animals.
Observation at 72 hours after instillation of test item revealed: Cornea- Scattered or diffuse areas of opacity (other than slight dulling of normal lustre); details of iris clearly visible was observed in animal no. 1 and easily discernible translucent area; details of iris slightly obscured in animal no. 2 and 3; Area of Opacity- Greater than one quarter, but less than half was observed in all the animals; Iris: Normal in all the animals; Conjunctivae - Diffuse beefy red was observed in all the animals; Chemosis: Obvious swelling with partial eversion of lids was observed in all the animals.
Observation on day 7 after instillation of test item revealed: Cornea- Easily discernible translucent area; details of iris slightly obscured was seen in all the animals; Area of Opacity- Greater than one quarter, but less than half was observed in all the animals; Iris: Normal in all the animals; Conjunctivae- Diffuse beefy red was observed in all the animals; Chemosis: Swelling, with lids about half closed was observed in animal no. 1 and obvious swelling with partial eversion of lids was observed in animal no. 2 and 3.
Observation on day 14 after instillation of test item revealed: Cornea- Nacrous area; no details of iris visible; size of pupil barely discernible was observed in all the animals; Area of Opacity- Greater than half, but less than three quarters was observed in all the animals; Iris: Normal in all the animals; Conjunctivae - Diffuse beefy red was observed in all the animals; Chemosis: Swelling, with lids about half closed was observed in animal no. 1 and 2 and obvious swelling with partial eversion of lids was observed in animal no. 3.
Observation on day 21 after instillation of test item revealed: Cornea- Nacrous area; no details of iris visible; size of pupil barely discernible was observed in animal no. 1 and 3 and opaque cornea; iris not discernible through the opacity was observed in animal no. 2; Area of Opacity- Greater than half, but less than three quarters was observed in animal no. 1 and 3 whereas grreater than three quaters, upto whole area was observed in animal no. 2; Iris: Normal in all the animals; Conjunctivae - Diffuse beefy red was observed in all the animals; Chemosis: Swelling, with lids about half closed was observed in all the animals.
The individual mean score for animal nos. 1, 2 and 3 at 24, 48, 72 hours for corneal opacity, iris, conjunctiva and chemosis were found 0.67, 0.00, 2.67, 2.00; 1.33, 0.00, 2.67, 2.00, and 1.33, 0.00, 2.33, 2.00, respectively.
Other effects:
Clinical Observation
No systemic toxicity was observed in treated rabbits during the experimental period.
Mortality
No mortality was observed during the observation period.
Body weight
Increase in body weights of all the animals weighed at termination as compared to day 0 body weights.


Table 1 : Individual Animal Eye Irritation Scores

 

In Treated area Dose:100 mg of test item                                                            Sex:Female

 

Animal Number

1

Application Side

Right

Eye Reactions

*

Hour(s)

Day

1

24

48

72

7

14

21

Cornea

0

0

0

1

1

2

3

3

Area of Opacity

0

0

0

1

1

2

3

3

Iris

0

0

0

0

0

0

0

0

Conjunctiva

0

0

2

3

3

3

3

3

Chemosis

0

0

2

2

2

3

3

3

Corneal Damage (%)

60

Animal Number

2

Application Side

Right

Eye Reactions

*

Hour(s)

Day

1

24

48

72

7

14

21

Cornea

0

0

1

1

2

2

3

4

Area of Opacity

0

0

1

1

2

2

3

4

Iris

0

0

0

0

0

0

0

0

Conjunctiva

0

1

2

3

3

3

3

3

Chemosis

0

1

2

2

2

2

3

3

Corneal Damage (%)

70

Animal Numbers

3

Application Side

Right

Eye Reactions

*

Hour(s)

Day

1

24

48

72

7

14

21

Cornea

0

0

1

1

2

2

3

3

Area of Opacity

0

0

1

1

2

2

3

3

Iris

0

0

0

0

0

0

0

0

Conjunctiva

0

1

2

2

3

3

3

3

Chemosis

0

1

2

2

2

2

2

3

Corneal Damage (%)

70

Key:*= Pre-treatment eye examination.

 

Table 1 Continued…

Untreated Control                                                                                                                 Sex:Female

Animal Number

1

Application Side

Left

Eye Reactions

*

Hour(s)

Day

1

24

48

72

7

14

21

Cornea

0

0

0

0

0

0

0

0

Area of Opacity

0

0

0

0

0

0

0

0

Iris

0

0

0

0

0

0

0

0

Conjunctiva

0

0

0

0

0

0

0

0

Chemosis

0

0

0

0

0

0

0

0

Corneal Damage (%)

0

 

Animal Number

2

Application Side

Left

Eye Reactions

*

Hour(s)

Day

1

24

48

72

7

14

21

Cornea

0

0

0

0

0

0

0

0

Area of Opacity

0

0

0

0

0

0

0

0

Iris

0

0

0

0

0

0

0

0

Conjunctiva

0

0

0

0

0

0

0

0

Chemosis

0

0

0

0

0

0

0

0

Corneal Damage (%)

0

 

Animal Number

3

Application Side

Left

Eye Reactions

*

Hour(s)

Day

1

24

48

72

7

14

21

Cornea

0

0

0

0

0

0

0

0

Area of Opacity

0

0

0

0

0

0

0

0

Iris

0

0

0

0

0

0

0

0

Conjunctiva

0

0

0

0

0

0

0

0

Chemosis

0

0

0

0

0

0

0

0

Corneal Damage (%)

0

Key:*= Pre-treatment eye examination

Table 1 Continued…

 

Eye Irritation Scores - Mean Values after 24, 48, 72 Hours (Treated eye)

            Animal No.

 Eye Reaction

1

2

3

Corneal Opacity

0.67

1.33

1.33

Iris

0.00

0.00

0.00

Conjunctiva

2.67

2.67

2.33

Chemosis

2.00

2.00

2.00

 Formula :

 

Mean Eye Irritation Score =

                          

Sum of the Individual Animal Score for eye reaction at 24, 48 and 72 hours

Number of the Observations (3)

Table 2 : Individual AnimalClinicalSigns

 

Sex:Female

Animal No.

Days (Post application observation)

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

2

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

3

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

Key:1 = Normal

Table 3: Individual Animal Body Weight

Sex :Female

Animal No.

Animal Body Weight (kg)

Prior to application

At termination

1

2.012

2.460

2

1.934

2.190

3

2.256

2.750

Key:kg = Kilogram
Interpretation of results:
Category I
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the experimental conditions tested, all the three animals were fully irreversible within an observation period of 21 days.
Hence under the experimental test conditions, “Solvent Blue 4 (CAS No. - 6786-83-0)” solid is “ “An Eye Irritant ” to New Zealand White female rabbit eyes and is being classified as an eye irritant in 'category I' as per the CLP regulation.
Executive summary:

Acute Eye Irritation/Corrosion Study of “Solvent Blue 4 (CAS No. - 6786-83-0)" was carried out in Rabbits, This study was performed as per OECD guideline no. 405.

Rabbits free from injury of eye were selected for the study. The eyes of all the rabbits were examined 24 hours prior to treatment. One eye of each rabbit served as control and other as treated. Control eye was left untreated whereas; 100 mgof test item (pulverised)was instilled in the other (treated) eye of each rabbit.The eye was observed at 1, 24, 48, 72 hour and at day 7 after test item instillation.Ophthalmoscope was used for scoring of eye lesions.

In the initial test,100 mg of test itemwas instilled into the conjunctival sac of the right eye of animal no.1 whereas the left eye of the rabbit served as the control. As animal no. 1 showed no severe ocular lesions; hence a confirmatory test was conducted on additional two rabbits (animal no. 2 and 3); 100 mgof test itemwas instilled into the conjunctival sac of right eye of both the rabbits and left eye served as the control.

Untreated eye of all the three rabbits were normal throughout the experimental period

The following grading scores were observed in treated eye of tested rabbits.

Observation at 1 hour after instillation of test item revealed: Cornea-No ulceration or opacity was seen in all the animals;Area of Opacity-Zero inall the animals;Iris:Normal in all the animals;Conjunctivae -Blood vessels normal in animal no. 1 whereas some blood vessels definitely hyperaemic (injected) in animal no. 2 and 3;Chemosis:No swelling (Normal) in animal no. 1 whereas some swelling above normal (includes nictitating membranes) in animal no. 2 and 3

Observation at 24 hours after instillation of test item revealed: Cornea-No ulceration or opacity was seen animal no. 1 and scattered or diffuse areas of opacity (other than slight dulling of normal lustre); details of iris clearly visible in animal no. 2 and 3;Area of Opacity-Zero in animal no.1 and one quarter (or less) but not zeroin animal no. 2 and 3;Iris:Normal in all the animals;Conjunctivae -Diffuse, crimson color; individual vessels not easily discernible was seen in all the animals;Chemosis:Obvious swelling with partial eversion of lids was seen in all the animals. At 24 hours observation the rabbits were examined for corneal epithelium cell damage using sodium fluorescein strips and noticed 60%, 70% and 70% damage in animal no. 1, 2 and 3 respectively

Observation at 48 hours after instillation of test item revealed: Cornea- Scattered or diffuse areas of opacity (other than slight dulling of normal lustre); details of iris clearly visible was seen in all the animals;Area of Opacity-One quarter (or less) but not zero inall the animals;Iris:Normal in all the animals;Conjunctivae -Diffuse beefy redwas observed in animals no. 1 and 2 and diffuse, crimson color; individual vessels not easily discernible in animal no. 3;Chemosis:Obvious swelling with partial eversion of lids was observed in all the animals Observation at 72 hours after instillation of test item revealed: Cornea- Scattered or diffuse areas of opacity (other than slight dulling of normal lustre); details of iris clearly visible was observed in animal no. 1 and easily discernible translucent area; details of iris slightly obscured in animal no. 2 and 3;Area of Opacity-Greater than one quarter, but less than half was observed inall the animals;Iris:Normal in all the animals;Conjunctivae -Diffuse beefy redwas observed in all the animals;Chemosis:Obvious swelling with partial eversion of lids was observed in all the animalsObservation on day 7 after instillation of test item revealed: Cornea-Easily discernible translucent area; details of iris slightly obscured was seen in all the animals;Area of Opacity-Greater than one quarter, but less than half was observed inall the animals;Iris:Normal in all the animals;Conjunctivae-Diffuse beefy red was observed in all the animals;Chemosis:Swelling, with lids about half closed was observed in animal no. 1 and obvious swelling with partial eversion of lids was observed in animal no. 2 and 3. Observation on day 14 after instillation of test item revealed: Cornea-Nacrous area; no details of iris visible; size of pupil barely discernible was observed in all the animals;Area of Opacity-Greater than half, but less than three quarters was observed in all the animals;Iris:Normal in all the animals;Conjunctivae -Diffuse beefy red was observed in all the animals;Chemosis:Swelling, with lids about half closed was observed in animal no. 1 and 2 and obvious swelling with partial eversion of lids was observed in animal no. 3. Observation on day 21 after instillation of test item revealed: Cornea-Nacrous area; no details of iris visible; size of pupil barely discernible was observed in animal no. 1 and 3 and opaque cornea; iris not discernible through the opacity was observed in animal no. 2;Area of Opacity-Greater than half, but less than three quarters was observed in animal no. 1 and 3 whereas grreater than three quaters, upto whole area was observed in animal no. 2;Iris:Normal in all the animals;Conjunctivae -Diffuse beefy red was observed in all the animals;Chemosis:Swelling, with lids about half closed was observed in all the animal.

The individual mean score for animal nos. 1, 2 and 3at 24, 48, 72 hoursfor corneal opacity, iris, conjunctiva and chemosis were found 0.67, 0.00, 2.67, 2.00; 1.33, 0.00, 2.67, 2.00, and 1.33, 0.00, 2.33, 2.00, respectively

Under the experimental conditions tested, all the three animals were fully irreversiblewithin an observation period of 21 days. 

Hence under the experimental test conditions,“Solvent Blue 4 (CAS No. - 6786-83-0)”is“An Eye Irritant to New Zealand White Female rabbit eyes and is being classified as

an eye irritant in'category 1'as per the CLP regulation

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation:

Acute Dermal Irritation/corrosion Study of Solvent Blue 4 (CAS No. - 6786-83-0)” was carried out in Rabbits, This study was performed as per OECD guideline No. 404. Three healthy young adult male rabbits were used for conducting acute dermal irritation study.Body weights were re­corded on day 0 (prior to application) and at termination. Rabbits with good intact skin were selected for the study. The hairs of all the rabbits were clipped at contralateral sites, approximately 24 hours prior to treatment.A dose of 0.5 ml of test item (as such) was applied to the skin,over an area of approximately 6 x 6 cm clippedof hair on one side of rabbits.The other untreated side was kept as control area and 0.5 ml of distilled water was applied at this site. At the end of 4 hours, the gauze patch was removed and test item application site was wiped with water without altering the integrity of the epidermis.  The individual mean score at24, 48 and 72 hoursfor animal nos. 1, 2 and 3 were 0.33, 0.67, 0.67 and 0.00, 0.00, 0.33, for erythema and oedema formation, respectively. Hence, it was concluded that Solvent Blue 4 (CAS No. - 6786-83-0) was Non-Irritating to the skin of female New Zealand White rabbits under the experimental conditions tested and not Classified as per CLP Regulation

Eye Irritation:

Acute Eye Irritation/Corrosion Study of “Solvent Blue 4 (CAS No. - 6786-83-0)” was carried out in Rabbits, This study was performed as per OECD guideline no. 405.Rabbits free from injury of eye were selected for the study. The eyes of all the rabbits were examined 24 hours prior to treatment. One eye of each rabbit served as control and other as treated. Control eye was left untreated whereas; 100 mgof test item (pulverised)was instilled in the other (treated) eye of each rabbit.The eye was observed at 1, 24, 48, 72 hour and at day 7 after test item instillation.Ophthalmoscope was used for scoring of eye lesions. In the initial test,100 mg of test itemwas instilled into the conjunctival sac of the right eye of animal no.1 whereas the left eye of the rabbit served as the control. As animal no. 1 showed no severe ocular lesions; hence a confirmatory test was conducted on additional two rabbits (animal no. 2 and 3); 100 mgof test itemwas instilled into the conjunctival sac of right eye of both the rabbits and left eye served as the control.

The individual mean score for animal nos. 1, 2 and 3at 24, 48, 72 hoursfor corneal opacity, iris, conjunctiva and chemosis were found 0.67, 0.00, 2.67, 2.00; 1.33, 0.00, 2.67, 2.00, and 1.33, 0.00, 2.33, 2.00, respectively.Under the experimental conditions tested, all the three animals were fully irreversiblewithin an observation period of 21 days. Hence under the experimental test conditions,“Solvent Blue 4 (CAS No. - 6786-83-0)”is“An Eye Irritant ”of New Zealand White Female rabbit eyes and is being classified as Category 1 as per CLP criteria of classification.


Justification for selection of skin irritation / corrosion endpoint:
K1 level expermental data from OECD GLP certified laboratory

Justification for selection of eye irritation endpoint:
K1 level expermental data from OECD GLP certified laboratory. The chemical was found to be irritating to the eye of rabbit.

Effects on eye irritation: corrosive

Justification for classification or non-classification

Based upon the data for the target chemical (Solvent Blue 4) in the key study as well as using the supporting study data, it can be concluded that Solvent Blue 4 is unlikely to be a skin irritant though it is an eye irritant in category 1.