Tetrabutylammonium bromide

Endpoint summary

Description of key information

Skin irritation:

The skin irritation study of test chemical was performed as per OECD Guidelines 404 in three female New Zealand White rabbits and complying with the GLP procedures. The animals were prepared 24 hours prior to application of test product. The furs from the dorsal area of trunk of animals were removed with electric clippers exposing an area measuring approximately 6 cm2 of body surface area of animal. The care was taken such that abrasion penetrated the Stratum corneum only and not dermis. 500gm (0.5g) of test compound was applied on a small area (approximately 6 cm2) of intact skin site. Each site of application was covered with impervious dressing which was secured in position with adhesive tape. The intact skin site of application of each animal was observed for signs of erythema and oedema at 60 min., 24, 48 and 72 hours after application and the responses were scored according to Draize method. Very slight erythema was observed after 4th to 24 hours of application of test compound. The skin irritation index of test compound was calculated as 0.50. Hence, under the test conditions,the test substancecan be concluded to be slightly irritating to New Zealand White rabbit skin.

 

Eye irritation:

Acute Eye Irritation/Corrosion Study of test chemical was performed in rabbits 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 (0.1 gm) of test item (pulverized) was applied into the conjunctival sac of the left eye of animal no.1 whereas the right eye of the rabbit served as the control. Ocular lesions were seen in animal no.1 at till 72 hours observation which recovered on day 7 hence a confirmatory test was conducted on additional two rabbits (animal no. 2 and 3); 100 mg of test item(pulverised)was instilled into the conjunctival sac of right eye of both the rabbits and left eye served as the control. Ocular lesions were seen in animal no. 2 and 3 till 72 hours observation which was recovered on day 7 observations. Untreated eye of all the three rabbits was normal throughout the experimental period of 72 hours. The following grading scores were observed in treated eye of tested rabbits The individual mean score for animal nos. 1, 2 and 3at 24, 48, 72 hoursfor corneal opacity, iris, conjunctiva and chemosis were found 1.33, 0.00, 1.67, 1.33; 1.67, 0.00, 2.00, 2.00; 1.00, and 1.00 0.00, 2.00, 1.67, respectively. Observation on day 7 after instillation of test item revealed: Cornea-No ulceration or opacity in all 3 animals; Area of Opacity- Zero percent was seen in all 3 animals;Iris:Normal in all the animals.Conjunctivae -Blood vessels normal was seen in all 3 animals;Chemosis:No swelling (Normal) was seen in all the 3 animals. Under the experimental conditions tested, eye irritation and reversibility of effects on eyes of rabbit no. 1, 2 and 3 was observed till 72 hours which were recovered on day 7. Hence under the experimental test conditions, the test chemical is “Mildly Irritating to Eyes” to New Zealand White female rabbit eyes and is being classified as 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)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from experimental report

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Principles of method if other than guideline:

To dertermine the skin irritation potential of test chemical.

GLP compliance:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Source : Institute for Industrial Research & Toxicology
Age : 10 to 12 weeks
Sex : Female
Body weight range : 1.80kg±200g
Identification : By cage tag and corresponding colour body marking
Environmental conditions : Air conditioned rooms with 10-15 air changes per hour, temperature between 22-25 deg C, relative humidity 40-60% and illumination cycle set to 12 hours artificial fluorescent light and 12 hours dark.
Accommodation : Animals were housed individually in stainless steel cages provided with stainless steel mesh bottom and facilities for food and water bottle.
Diet : Pelleted feed supplied by Pranav agro Industries Ltd., B7/6 Ramesh Nagar, Delhi
Water : Community tap water passed through ‘Aqua Guard on line water filter’, was kept in glass bottles, ad libitum

Type of coverage:

occlusive

Preparation of test site:

shaved

Vehicle:

unchanged (no vehicle)

Controls:

not specified

Amount / concentration applied:

0.5 gm of test compound

Duration of treatment / exposure:

no data

Observation period:

upto 14 days after application

Number of animals:

Three

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

other: 3 min - 14 days

Score:

2

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

other: The skin irritation index of test compound Tetrabutylammonium bromide was calculated as: 2.00/4 = 0.50

Irritant / corrosive response data:

Very slight erythema after 4th to 24 hrs of application of test compound. There were no other signs of edema and other irritation. These responses were graded according to Draize’s Scoring method.

Other effects:

The test compound Tetrabutylammonium bromide applied on the shaven back skin of rabbit did not produce any clinical signs of toxicity throughout the examination period of 14 days.

TABLE – 1

INDIVIDUAL ANIMAL DERMAL IRRITATION SCORES

Rabbit No.	Sex	INTACT SKIN
		3 Min.		4 Hours		24 Hours		48 Hours		72 Hours		14 days
		Erythema	Oedema	Erythema	Oedema	Erythema	Oedema	Erythema	Oedema	Erythema	Oedema	Erythema	Oedema
01	F	0	0	1	0	1	0	0	0	0	0	0	0
02	F	-	-	1	0	1	0	0	0	0	0	0	0
03	F	-	-	1	0	1	0	0	0	0	0	0	0
Total		0	0	3	0	3	0	0	0	0	0	0	0
Mean		0	0	1.00	0	1.00	0	0	0	0	0	0	0
Grand Total		2.00

Dermal Irritation Index: 2.00/4 = 0.50

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

The result obtained from present study concludes that the test chemical is slightly irritatant on intact skin of New Zealand white rabbit under the test condition and has therefore been classified in the category of Skin irritant 2

Executive summary:

The skin irritation study of test chemical was performed as per OECD Guidelines 404 in three female New Zealand White rabbits and complying with the GLP procedures. The animals were prepared 24 hours prior to application of test product. The furs from the dorsal area of trunk of animals were removed with electric clippers exposing an area measuring approximately 6 cm2 of body surface area of animal. The care was taken such that abrasion penetrated the Stratum corneum only and not dermis. 500gm (0.5g) of test compound was applied on a small area (approximately 6 cm2) of intact skin site. Each site of application was covered with impervious dressing which was secured in position with adhesive tape. The intact skin site of application of each animal was observed for signs of erythema and oedema at 60 min., 24, 48 and 72 hours after application and the responses were scored according to Draize method. Very slight erythema was observed after 4th to 24 hours of application of test compound. The skin irritation index of test compound was calculated as 0.50. Hence, under the test conditions,the test substancecan be concluded to be slightly irritating to New Zealand White rabbit skin.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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

Justification for type of information:

Data is from experimental report

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 “Tetrabutylammonium bromide (CAS No. - 1643-19-2)” on eye, when exposed by the ocular route in rabbits.

GLP compliance:

yes

Specific details on test material used for the study:

- Name of test material (as cited in study report):Tetrabutylammonium bromide
- Molecular formula :C16H36N.Br
- Molecular weight :322.37 g/mol
- Substance type:Organic
- Physical state:White Solid (Crystals)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source:Procured from Rabbiroof, Hyderabad, India.
- Age at study initiation:4.5 to 5.5 Months (Approximately)
- Weight at study initiation:Minimum: 1.932 kg and Maximum: 2.438 kg (Prior to Treatment).
- Health Status :Healthy young adult, 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 6 days (Animal No.-1) and 11 days (Animal No. 2 and 3) prior to the application of the test item.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):Minimum: 19.50 °C and Maximum: 22.70 °C
- Humidity (%):Minimum: 41.20 % and Maximum: 67.30 %
- Air changes (per hr): More than 12 changes per hour
- Photoperiod (hrs dark / hrs light): 12:12

Vehicle:

unchanged (no vehicle)

Controls:

yes

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,#2 and #3

Time point:

other: 24,48,72 hrs and 7 days

Score:

0

Max. score:

3

Reversibility:

not specified

Remarks on result:

other: Untreated (Control Eye)

Irritation parameter:

iris score

Basis:

animal: #1,#2 and #3

Time point:

other: 24,48,72 hrs and 7 days

Score:

0

Max. score:

3

Reversibility:

not specified

Remarks on result:

other: Untreated (Control Eye)

Irritation parameter:

conjunctivae score

Basis:

animal: #1,#2 and #3

Time point:

other: 24,48,72 hrs and 7 days

Score:

0

Max. score:

3

Reversibility:

not specified

Remarks on result:

other: Untreated (Control Eye)

Irritation parameter:

chemosis score

Basis:

animal: #1,#2 and #3

Time point:

other: 24,48,72 hrs and 7 days

Score:

0

Max. score:

3

Reversibility:

not specified

Remarks on result:

other: Untreated (Control Eye)

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

other: After 24, 48, 72 Hours

Score:

1.33

Max. score:

3

Reversibility:

not specified

Remarks on result:

other: Treated eye

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

other: After 24, 48, 72 Hours

Score:

1.67

Max. score:

3

Reversibility:

not specified

Remarks on result:

other: Treated eye

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

other: After 24, 48, 72 Hours

Score:

1

Max. score:

3

Reversibility:

not specified

Remarks on result:

other: Treated eye

Irritation parameter:

cornea opacity score

Basis:

animal: #1,#2 and #3

Time point:

other: Day 7

Score:

0

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: after 24, 48, 72 Hours and day 7

Score:

0

Max. score:

3

Reversibility:

not specified

Remarks on result:

other: Treated eye

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

other: After 24, 48, 72 Hours

Score:

1.67

Max. score:

3

Reversibility:

not specified

Remarks on result:

other: Treated eye

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

other: After 24, 48, 72 Hours

Score:

2

Max. score:

3

Reversibility:

not specified

Remarks on result:

other: Treated eye

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

other: After 24, 48, 72 Hours

Score:

2

Max. score:

3

Reversibility:

not specified

Remarks on result:

other: Treated eye

Irritation parameter:

conjunctivae score

Basis:

animal: #1,#2 and #3

Time point:

other: Day 7

Score:

0

Max. score:

3

Reversibility:

not specified

Remarks on result:

other: Treated eye

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

other: After 24, 48, 72 Hours

Score:

1.33

Max. score:

3

Reversibility:

not specified

Remarks on result:

other: Treated eye

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

other: After 24, 48, 72 Hours

Score:

2

Max. score:

3

Reversibility:

not specified

Remarks on result:

other: Treated eye

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

other: After 24, 48, 72 Hours

Score:

1.67

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: Day 7

Score:

0

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 animal no. 1 and 3 whereas scattered or diffuse areas of opacity (other than slight dulling of normal lustre) details of iris clearly visible was seen in animal no. 2; Area of Opacity- Zero in animal no. 1 and 3 whereas one quarter (or less) but not zero was seen in animal no.2; Iris: Normal in all the animals. Conjunctivae - Diffuse, crimson color; individual vessels not easily discernible in animal no. 2 and some blood vessels definitely hyperaemic (injected) animal no. 1 and 3; Chemosis: Some swelling above normal (includes nictitating membranes) in animal no. 1 and obvious swelling with partial eversion of lids was observed in animal no. 2 and 3.

Observation at 24 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 in all 3 animals; Area of Opacity- Greater than one quarter, but less than half was seen in animal no. 1 and 2 whereas one quarter (or less) but not zero was seen in animal no.3; Iris: Normal in all the animals. Conjunctivae - Diffuse, crimson color; individual vessels not easily discernible was seen in all 3 animals; Chemosis: Some swelling above normal (includes nictitating membranes) was seen in animal no.1 whereas obvious swelling with partial eversion of lids was seen in animal no.2 and 3.

At 24 hours observation the rabbits were examined for corneal epithelium cell damage using sodium fluorescein strips and noticed 50%, 60% and 45% 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 in all 3 animals; Area of Opacity- One quarter (or less) but not zero was seen in animal no.1 and 3 whereas greater than one quarter, but less than half was seen in animal no. 2; Iris: Normal in all the animals. Conjunctivae - Some blood vessels definitely hyperaemic (injected) was seen in animal no. 1 whereas diffuse, crimson color; individual vessels not easily discernible was seen in animal no. 2 and 3; Chemosis: Obvious swelling with partial eversion of lids was seen in all 3 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 in all 3 animals; Area of Opacity- One quarter (or less) but not zero was seen in all 3 animals; Iris: Normal in all the animals. Conjunctivae - Diffuse, crimson color; individual vessels not easily discernible was seen in all 3 animals; Chemosis: Some swelling above normal (includes nictitating membranes) was seen in animal no. 1 and 3 whereas obvious swelling with partial eversion of lids was seen in animal no. 2.

Observation on day 7 after instillation of test item revealed: Cornea- No ulceration or opacity in all 3 animals; Area of Opacity- Zero percent was seen in all 3 animals; Iris: Normal in all the animals. Conjunctivae - Blood vessels normal was seen in all 3 animals; Chemosis: No swelling (Normal) was seen in all the 3 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 1.33, 0.00, 1.67, 1.33; 1.67, 0.00, 2.00, 2.00; 1.00, and 1.00 0.00, 2.00, 1.67, respectively.

Observation at 1 hour after instillation of test item revealed: Cornea- No ulceration or opacity was seen in animal no. 1 and 3 whereas scattered or diffuse areas of opacity (other than slight dulling of normal lustre) details of iris clearly visible was seen in animal no. 2; Area of Opacity- Zero in animal no. 1 and 3 whereas one quarter (or less) but not zero was seen in animal no.2; Iris: Normal in all the animals. Conjunctivae - Diffuse, crimson color; individual vessels not easily discernible in animal no. 2 and some blood vessels definitely hyperaemic (injected) animal no. 1 and 3; Chemosis: Some swelling above normal (includes nictitating membranes) in animal no. 1 and obvious swelling with partial eversion of lids was observed in animal no. 2 and 3.

Observation at 24 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 in all 3 animals; Area of Opacity- Greater than one quarter, but less than half was seen in animal no. 1 and 2 whereas one quarter (or less) but not zero was seen in animal no.3; Iris: Normal in all the animals. Conjunctivae - Diffuse, crimson color; individual vessels not easily discernible was seen in all 3 animals; Chemosis: Some swelling above normal (includes nictitating membranes) was seen in animal no.1 whereas obvious swelling with partial eversion of lids was seen in animal no.2 and 3.

At 24 hours observation the rabbits were examined for corneal epithelium cell damage using sodium fluorescein strips and noticed 50%, 60% and 45% 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 in all 3 animals; Area of Opacity- One quarter (or less) but not zero was seen in animal no.1 and 3 whereas greater than one quarter, but less than half was seen in animal no. 2; Iris: Normal in all the animals. Conjunctivae - Some blood vessels definitely hyperaemic (injected) was seen in animal no. 1 whereas diffuse, crimson color; individual vessels not easily discernible was seen in animal no. 2 and 3; Chemosis: Obvious swelling with partial eversion of lids was seen in all 3 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 in all 3 animals; Area of Opacity- One quarter (or less) but not zero was seen in all 3 animals; Iris: Normal in all the animals. Conjunctivae - Diffuse, crimson color; individual vessels not easily discernible was seen in all 3 animals; Chemosis: Some swelling above normal (includes nictitating membranes) was seen in animal no. 1 and 3 whereas obvious swelling with partial eversion of lids was seen in animal no. 2.
Observation on day 7 after instillation of test item revealed: Cornea- No ulceration or opacity in all 3 animals; Area of Opacity- Zero percent was seen in all 3 animals; Iris: Normal in all the animals. Conjunctivae - Blood vessels normal was seen in all 3 animals; Chemosis: No swelling (Normal) was seen in all the 3 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 1.00, 1.00, 1.00; 1.33, 1.67, 1.00; 1.67, 2.00, 2.00 and 1.33, 2.00, 1.67, 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
All rabbits were weighed on test day 0 (prior to application) and at termination.

Table 1 : Individual Animal Eye Irritation Scores

 

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

Animal Numbers		1							2								3
Application Side		Left							Right								Right
Eye Reactions		At hour						Day		At hour						Day			At hour						Day
		*		1		24		48		72		7		*		1		24		48		72			7		*		1		24		48		72			7
Corneal Opacity		0		0		1		1		1		0		0		1		1		1		1			0		0		0		1		1		1			0
Area of Opacity		0		0		2		1		1		0		0		1		2		2		1			0		0		0		1		1		1			0
Iris		0		0		0		0		0		0		0		0		0		0		0			0		0		0		0		0		0			0
Conjunctiva		0		2		2		1		2		0		0		1		2		2		2			0		0		1		2		2		2			0
Chemosis		0		1		1		2		1		0		0		2		2		2		2			0		0		2		2		2		1			0
Corneal Damage%		50%							60%								45%

 

Dose:Untreated (Control Eye)                                                                       Sex:Female

Animal Numbers	1							2							3
Application Side	Right							Left							Left
Eye Reactions	At hour					Day		At hour					Day		At hour					Day
	*	1	24	48	72		7	*	1	24	48	72		7	*	1	24	48	72		7
Corneal Opacity	0	0	0	0	0		0	0	0	0	0	0		0	0	0	0	0	0		0
Area of Opacity	0	0	0	0	0		0	0	0	0	0	0		0	0	0	0	0	0		0
Iris	0	0	0	0	0		0	0	0	0	0	0		0	0	0	0	0	0		0
Conjunctiva	0	0	0	0	0		0	0	0	0	0	0		0	0	0	0	0	0		0
Chemosis	0	0	0	0	0		0	0	0	0	0	0		0	0	0	0	0	0		0
Corneal Damage%	0							0							0

Key:*= Pre-exposure 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	1.33	1.67	1.00
Iris	0.00	0.00	0.00
Conjunctiva	1.67	2.00	2.00
Chemosis	1.33	2.00	1.67

 

 

Formula :

 

Mean Eye Irritation Score =

                          

Sum of the Individual Animal Score for eye reactionat24, 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
1	1	1	1	1	1	1	1	1
2	1	1	1	1	1	1	1	1
3	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	2.208	2.387
2	1.932	2.214
3	2.438	2.646

Key:kg = Kilogram

Interpretation of results:

Category II

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the experimental conditions tested, eye irritation and reversibility of effects on eyes of rabbit no. 1, 2 and 3 was observed till 72 hours which were recovered on day 7.
Hence under the experimental test conditions, “Tetrabutylammonium bromide (CAS No. - 1643-19-2) solid is “Mildly Irritating to Eyes” to New Zealand White female rabbit eyes and is being classified as an eye irritant in 'category 2' as per the CLP regulation.

Executive summary:

Acute Eye Irritation/Corrosion Study of test chemical in Rabbits, sponsored bySustainability Support Services (Europe) AB was conducted at sa-FORD (Sanctuary for Research and Development), Maharashtra, India. 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 (0.1 gm) of test item (pulverized)was applied into the conjunctival sac of the left eye of animal no.1 whereas the right eye of the rabbit served as the control. Ocular lesions were seen in animal no.1 at till 72 hours observation which recovered on day 7 hence a confirmatory test was conducted on additional two rabbits (animal no. 2 and 3); 100 mgof test item(pulverised)was instilled into the conjunctival sac of right eye of both the rabbits and left eye served as the control. Ocular lesions were seen in animal no. 2 and 3 till 72 hours observation which was recovered on day 7 observation.

 

Untreated eye of all the three rabbits was normal throughout the experimental period of 72 hours.

 

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 animal no. 1 and 3 whereas scattered or diffuse areas of opacity (other than slight dulling of normal lustre) details of iris clearly visible was seen in animal no. 2;Area of Opacity-Zero in animal no. 1 and 3 whereas one quarter (or less) but not zero was seen in animal no.2;Iris:Normal in all the animals.Conjunctivae -Diffuse, crimson color; individual vessels not easily discerniblein animal no. 2 and some blood vessels definitely hyperaemic (injected) animal no. 1 and 3;Chemosis:Some swelling above normal (includes nictitating membranes) in animal no. 1 and obvious swelling with partial eversion of lids was observed in animal no. 2 and 3.

 

Observation at 24 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 in all 3 animals; Area of Opacity-Greater than one quarter, but less than half was seen in animal no. 1 and 2 whereas one quarter (or less) but not zero was seen in animal no.3;Iris:Normal in all the animals.Conjunctivae -Diffuse, crimson color; individual vessels not easily discernible was seen in all 3 animals;Chemosis:Some swelling above normal (includes nictitating membranes) was seen in animal no.1 whereas obvious swelling with partial eversion of lids was seen in animal no.2 and 3.

 

At 24 hours observation the rabbits were examined for corneal epithelium cell damage using sodium fluorescein strips and noticed 50%, 60% and 45% 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 in all 3 animals; Area of Opacity-One quarter (or less) but not zero was seen in animal no.1 and 3 whereas greater than one quarter, but less than half was seen in animal no. 2;Iris:Normal in all the animals.Conjunctivae -Some blood vessels definitely hyperaemic (injected) was seen in animal no. 1 whereas diffuse, crimson color; individual vessels not easily discernible was seen in animal no. 2 and 3;Chemosis:Obvious swelling with partial eversion of lids was seen in all 3 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 in all 3 animals; Area of Opacity-One quarter (or less) but not zero was seen in all 3 animals;Iris:Normal in all the animals.Conjunctivae - Diffuse, crimson color; individual vessels not easily discernible was seen in all 3 animals;Chemosis:Some swelling above normal (includes nictitating membranes) was seen in animal no. 1 and 3 whereas obvious swelling with partial eversion of lids was seen in animal no. 2.

 

Observation on day 7 after instillation of test item revealed: Cornea-No ulceration or opacity in all 3 animals; Area of Opacity- Zero percent was seen in all 3 animals;Iris:Normal in all the animals.Conjunctivae -Blood vessels normal was seen in all 3 animals;Chemosis: No swelling (Normal) was seen in all the 3 animals.

 

The individual mean score for animal nos. 1, 2 and 3at 24, 48, 72 hours for corneal opacity, iris, conjunctiva and chemosis were found 1.33, 0.00, 1.67, 1.33; 1.67, 0.00, 2.00, 2.00; 1.00, and 1.00 0.00, 2.00, 1.67, respectively.

 

Under the experimental conditions tested, eye irritation and reversibility of effects on eyes of rabbit no. 1, 2 and 3 was observed till 72 hours which were recovered on day 7.

Hence under the experimental test conditions, the test chemical solid is “Mildly Irritating to Eyes” to New Zealand White female rabbit eyes and is being classified as an eye irritant in 'category 2' 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:

The experimental studyhas been investigated for the test chemicalto observe the potential for dermal irritation to a greater or lesser extent. The study is based on in vivo experiments in rabbits and summarized as below;

 

The skin irritation study of test chemical was performed as per OECD Guidelines 404 in three female New Zealand White rabbits and complying with the GLP procedures. The animals were prepared 24 hours prior to application of test product. The furs from the dorsal area of trunk of animals were removed with electric clippers exposing an area measuring approximately 6 cm2 of body surface area of animal. The care was taken such that abrasion penetrated the Stratum corneum only and not dermis. 500gm (0.5g) of test compound was applied on a small area (approximately 6 cm2) of intact skin site. Each site of application was covered with impervious dressing which was secured in position with adhesive tape. The intact skin site of application of each animal was observed for signs of erythema and oedema at 60 min., 24, 48 and 72 hours after application and the responses were scored according to Draize method. Very slight erythema was observed after 4th to 24 hours of application of test compound. The skin irritation index of test compound was calculated as 0.50. Hence, under the test conditions,the test substancecan be concluded to be slightly irritating to New Zealand White rabbit skin.

 

Based on the observed result obtained in above study, it can be concluded that the chemical is able to cause skin reactions and thus can be considered as irritating to the skin. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Category 2 (irritant)”.

 

Eye irritation:

Various studieshas been investigated for the test chemical to observe the potential for ocular irritation to a greater or lesser extent. The studies are based on in vivo experiments in rabbits and summarized as below;

 

Acute Eye Irritation/Corrosion Study of test chemical was performed in rabbits 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 (0.1 gm) of test item (pulverized) was applied into the conjunctival sac of the left eye of animal no.1 whereas the right eye of the rabbit served as the control. Ocular lesions were seen in animal no.1 at till 72 hours observation which recovered on day 7 hence a confirmatory test was conducted on additional two rabbits (animal no. 2 and 3); 100 mg of test item(pulverised)was instilled into the conjunctival sac of right eye of both the rabbits and left eye served as the control. Ocular lesions were seen in animal no. 2 and 3 till 72 hours observation which was recovered on day 7 observations. Untreated eye of all the three rabbits was normal throughout the experimental period of 72 hours. The following grading scores were observed in treated eye of tested rabbits The individual mean score for animal nos. 1, 2 and 3at 24, 48, 72 hoursfor corneal opacity, iris, conjunctiva and chemosis were found 1.33, 0.00, 1.67, 1.33; 1.67, 0.00, 2.00, 2.00; 1.00, and 1.00 0.00, 2.00, 1.67, respectively. Observation on day 7 after instillation of test item revealed: Cornea-No ulceration or opacity in all 3 animals; Area of Opacity- Zero percent was seen in all 3 animals;Iris:Normal in all the animals.Conjunctivae -Blood vessels normal was seen in all 3 animals;Chemosis:No swelling (Normal) was seen in all the 3 animals. Under the experimental conditions tested, eye irritation and reversibility of effects on eyes of rabbit no. 1, 2 and 3 was observed till 72 hours which were recovered on day 7. Hence under the experimental test conditions, the test chemical is “Mildly Irritating to Eyes” to New Zealand White female rabbit eyes and is being classified as an eye irritant.

 

Another eye irritation study of test chemical was performed in New Zealand white rabbit in accordance with OECD Guideline 405 (Acute Eye Irritation / Corrosion). The test chemical was placed in the conjunctival sac of one eye of each animal at the dose level of 0.1 gm after gentle pulling the lower eye lid away from the eye ball. The eye lids are the gently held together for about one second to prevent the loss of the material. The other eyes which remains untreated, served as control. The eyes of the test animal did not washed for at least 24 hours following instillation of the test compound. The eyes were examined at 1, 24, 48 and 72 hours after test substance application. The grades of ocular reaction (conjunctiva, cornea and iris) were recorded at each observation. To determine the reversibility of the effect the animal was observed normally for 21 days. The test compound produced some blood vessels of conjunctiva hyperemic to diffuse hyperemia and some swelling of eye lid at 24th hour after the application of test substance. Furthermore all the clinical signs of irritation were subsided after 48-72 hrs of test compound application. The test compound applied in conjunctival sac of rabbits did not show any observable clinical signs throughout the observation period of 21 days. Since the compound caused slight ocular irritation at a dose of 0.1gm, the chemical was considered as irritating to the eye of New Zealand white rabbit.

 

Based on the available data for target chemical, it can be concluded that the chemical is able to cause ocular lesion and thus can be considered as irritating to the eyes. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Category 2 (irritant)”.

Justification for classification or non-classification

The skin and eye irritation potential of test chemical was observed in various studies. The results obtained from these studies indicate that the chemical is likely to cause skin and eye irritation. Hence can be classified under the category “Category 2 (irritant)” for skin and eye as per CLP.