α,α-bis[4-(dimethylamino)phenyl]-4-(phenylamino)naphthalene-1-methanol

Endpoint summary

Description of key information

Skin Irritation

Acute Dermal Irritation/corrosion Study of the test chemical was carried out in Rabbits, This study was performed as per OECD guideline No. 404.

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 the test chemical was Non-Irritating to the skin of male New Zealand White rabbits under the experimental conditions as per CLP Regulation.

Eye Irritation

Acute Eye Irritation/Corrosion Study of the test chemical was carried out in female New Zealand White Rabbits, This study was performed as per OECD guideline no. 405.

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. 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, the test chemical was an “An Eye Irritant (Causing Irreversible Effect on Eye)” of New Zealand White Female rabbit eyes and classified under the category "Category 1" as per GHS Classification.

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 experimental reports

Qualifier:

according to guideline

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 the test chemical after dermal application on the intact skin in Rabbits

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system 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:

not specified

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:

24 h

Score:

0.33

Max. score:

3

Reversibility:

not specified

Remarks on result:

no indication of irritation

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

48 h

Score:

0.67

Max. score:

3

Reversibility:

not specified

Remarks on result:

no indication of irritation

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

72 h

Score:

0.67

Max. score:

3

Reversibility:

not specified

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24 h

Score:

0

Max. score:

3

Reversibility:

not specified

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

animal #2

Time point:

48 h

Score:

0

Max. score:

3

Reversibility:

not specified

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

animal #3

Time point:

72 h

Score:

0.33

Max. score:

3

Reversibility:

not specified

Remarks on result:

no indication of irritation

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:

other: not irritating

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 the test chemical 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 the test chemical 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 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 the test chemical was Non-Irritating to the skin of male New Zealand White rabbits under the experimental conditions 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)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

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 the test chemical on eye, when exposed by the ocular route in rabbits.

GLP compliance:

yes

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:

yes, concurrent no treatment

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 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

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. 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, the test chemical was an “An Eye Irritant (Causing Irreversible Effect on Eye)” of New Zealand White Female rabbit eyes and classified under the category "Category 1" as per GHS Classification.

Executive summary:

Acute Eye Irritation/Corrosion Study of the test chemical was carried out in female New Zealand White 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 mg of 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 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. 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, the test chemical was an “An Eye Irritant (Causing Irreversible Effect on Eye)” of New Zealand White Female rabbit eyes and classified under the category "Category 1" as per GHS Classification.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Irritation

Various studies have been summarized to ascertain the degree of dermal irritation caused by test chemical in living organisms. These results include in vivo experimental studies performed on rabbits for the test chemical.

Acute Dermal Irritation/corrosion Study of the test chemical 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 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 the test chemical was Non-Irritating to the skin of male New Zealand White rabbits under the experimental conditions as per CLP Regulation.

This is supported by another OECD 404 Guideline study performed on female New Zealand white rabbits to assess the irritation parameter of the test chemical.

In the initial test one healthy rabbit of body weight 2.13 kg selected for study after acclimatization. The test compound in the amount of 0.5 gm was applied at the different sites on the shaven back skin of animal. The hairs of back sides were removed (approximately 6 cm2) one day earlier before the treatment. 0.5 gm of the test chemical was applied to a small area (approximately 6 cm2) of skin and covered with a gauze patch, which was held in place with non-irritating tape. The first patch was applied on the shaven back skin of rabbit and removed after three minutes. No serious reaction was observed at the site of application. The second patch was applied on the different shaven back side and removed after one hour. There were no signs of skin reaction observed at this site of application. Finally, a third patch was applied at a different site and was removed after four hour.No skin reactionwas observed after four hours patch removal.Finally, the animal was observed for 14 days, for any irritation and corrosion.

Because no corrosive effect observed in the initial test, a confirmatory test was done in order to confirm the irritant or negative response of the test substance by using two additional animals. In the confirmatory test the test compound in the amount of 0.5 gm was applied on the shaven back skin of two animals, each with one patch, for an exposure period of four hours. After four hours the patch was removed and the skin reactions were graded according to Draize’s method.

0.5 gm  of the test chemical when applied on shaven back skin (approximately 6 cm2) of rabbit did not produce any irritation to skin during period of observation. The dermal irritation index of the test chemical in New Zealand white rabbits was calculated as 0.00 and test compound can be classified under "Not Classified".

Available Guideline studies indicate a strong possibility that the test chemical is indeed not irritating to skin. Hence, the test chemical can be considered to be not irritating to skin.

Comparing the above annotations with the criteria of CLP Regulations, the test chemical can be classified under the category “Not Classified” .

Eye Irritation

Acute Eye Irritation/Corrosion Study of the test chemical was carried out in female New Zealand White 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 mg of 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 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. 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, the test chemical was an “An Eye Irritant (Causing Irreversible Effect on Eye)” of New Zealand White Female rabbit eyes and classified under the category "Category 1" as per GHS Classification.

 

Justification for classification or non-classification

Available Guideline studies indicate a strong possibility that the test chemical is indeed not irritating to skin. Hence, the test chemical can be considered to be not irritating to skin.

Comparing the above annotations with the criteria of CLP Regulations, the test chemical can be classified under the category “Not Classified” .

Available Guideline studies indicate a strong possibility that the test chemical is indeed severely irritating to eyes. Hence, the test chemical can be considered to be irritating to eyes.

Comparing the above annotations with the criteria of CLP Regulations, the test chemical can be classified under the category “Category 1” .