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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014

Materials and methods

Test guideline
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

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report):Solvent Blue 4 with < 0.1 % Michler's Ketone
- Molecular formula :C33H33N3O
- Substance type:Organic


Test animals / tissue source

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

Test system

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.

Results and discussion

In vivo

Resultsopen allclose all
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.


Any other information on results incl. tables

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

Applicant's summary and conclusion

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