Dodecan-1-ol, ethoxylated

Endpoint summary

Description of key information

Skin irritation

The dermal irritation potential of test chemical was assessed in various experimental studies conducted on rabbits. Based on the available data for the key and supporting study, it can be concluded that the test chemical is unable to cause skin irritation and thus considered as not irritating. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Not Classified”.

 

Eye irritation

The ocular irritation potential of target chemical was assessedin various in- vitro and in-vivo experimental studies.Based on the available key data and supporting studies,it can be concluded thatchemical is able to cause eye irritation and considered as irritating. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Category 2 (irritating to eyes) ''.

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 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 irritation/corrosive potential of the test chemical after dermal application on the intact skin of New Zealand White rabbits

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: LIVEON BIOLABS PVT. LTD
- Sex: Male
- Age at study initiation: 3-3.5 MONTHS
- Weight at study initiation: Minimum: 1.854 kg and Maximum: 2.568 kg (prior to treatment)
- Housing: The animals were housed individually in stainless steel cages.
- 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
- 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.
- Identification: During acclimatization marking was done with non toxic marker pen in the inside of left ear of rabbits and after acclimatization, animals were marked with permanent number in the inner side of right ear of rabbits. Permanent marker and cage card was used for identification. The individual cage cards were labelled with at least study no., study type, test system, sex, dose, experiment start date and experiment completion date.
- 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.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): Minimum: 20.50 °C Maximum: 22.30 °C
- Humidity (%): Minimum: 52.80 % Maximum: 67.40 %
- Air changes (per hr): More than 12 changes per hour
- Photoperiod (hrs dark / hrs light): 12:12

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

Preparation of Application Site
Approximately 24 h prior to treatment, the fur coat of each rabbit was removed from dorsal lumbar region approximately 6 X 6 cm at contralateral sites on each rabbit using clipper, one as control and other site as treatment. Rabbits with healthy intact skin were selected for the study.

Test Item Application Procedure
The 0.5 ml of test item (as such) was applied uniformly over clipped area (approximately 6 X 6 cm) of the trunk of each rabbit skin (treated site) and 0.5 ml distilled water was applied at control site. Test item was held in contact with the skin with a porous gauze dressing and non-irritating tape (Micropore 3”) throughout a 4-hour exposure period, to prevent access by the rabbits to the patch and resultant ingestion of the test item. At the end of the exposure period, residual test item was removed by using cotton soaked in distilled water.
A single rabbit (Animal No. 1) was used for initial testing. The patch was removed after 4 h and the responses graded, no erythema and oedema was observed at 1 hour, 24 hour, 48 hour and 72 hour in animal no. 1. Hence the confirmatory test was conducted on additional two rabbits (No. 2 and 3) after 24 hour to confirm the non irritant nature of the test item.

Duration of treatment / exposure:

4 hours

Observation period:

72 hours

Number of animals:

3

Details on study design:

Details on study design
TEST SITE
- Area of exposure: approximately 6 X 6 cm at contralateral sites
- Type of wrap if used: porous gauze dressing and non-irritating tape (Micropore 3”)
REMOVAL OF TEST SUBSTANCE
- Washing (if done): residual test item was removed by using cotton soaked in distilled water
- Time after start of exposure: 4hr
Control site: 0.5 ml distilled water was applied at control site.
SCORING SYSTEM: Draize Method

Irritation parameter:

erythema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

not specified

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

not specified

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

Evaluation of Skin Irritation:
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, revealed no erythema and no oedema at 1, 24, 48 and 72 hour observation post patch removal.
Animal No. 2 and 3, revealed no erythema and no oedema at 1, 24, 48 and 72 hour observation post patch removal.
The individual mean score at 24, 48 and 72 hours for animal nos. 1, 2 and 3 were 0.00, 0.00, 0.00 and 0.00, 0.00, 0.00, 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.

Skin Reaction

 

In Treated area Dose:0.5 ml of test item                                                                          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	Left	0	0	0	0	0	0	0	0
3		Left	0	0	0	0	0	0	0	0

 

 

 

 

 

In Control area  Dose: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	Left	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

Mean Individual Animal Score at 24, 48 and 72 hours

 

Animal Number                   Observations	1	2	3
Erythema	0.00	0.00	0.00
Oedema	0.00	0.00	0.00

 

Table 2

Individual Animal BodyWeight

Sex:Male

Animal No.	Body Weight (kg)
	Prior to Dosing	At termination
1	2.568	2.610
2	1.854	1.922
3	1.926	1.980

Key: kg = kilogram

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 0.00, 0.00, 0.00 and 0.00, 0.00, 0.00, 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 tested and Classified as “Category- Not Classified” as per GHS Classification.

Executive summary:

Acute Dermal Irritation/corrosion Study of the test chemical in Rabbits, 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, no erythema and no oedema observed at 1 hour of observation. At 24, 48 and 72 hours observation no erythema and oedema was observed in animal no 1.Hence the confirmatory test was conducted after 24 hours on additional two rabbits (No. 2 and 3)to confirm the non irritant nature of the test item. Animal No. 2 and 3, revealed no erythema and no oedema at 1, 24, 48 and 72 hour observation post patch removal. The patch was removed after 4 hours and rabbits wereobservedfor erythema and oedemaat 1, 24, 48 and 72 hours after patch removal, evaluated and graded as per Draize method. Animal No.1, 2 and 3 at 1, 24, 48 and 72 hour observation post patch removal, revealed no erythema and no oedema. The individual mean score at 24, 48 and 72 hours for animal nos. 1, 2 and 3 were 0.00, 0.00, 0.00 and 0.00, 0.00, 0.00, 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 tested and Classified as “Category- Not Classified” as per GHS Classification.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

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 the experimental reports

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:

Species : Rabbit (Oryctolagus cuniculus)
Strain : New Zealand White
Age : 2.0 to 4.0 Months (Approximately)
Sex : Female
Number of Animals :Three
Supplier/Source :Procured from SAINATH AGENCIES
Health Status :Healthy young adult rabbits were used. Females were nulliparous and non pregnant.
Body weight of animals: Minimum: 1.648 kg and Maximum: 1.752 kg (Prior to Treatment)
Acclimatisation Rabbits were acclimatised to the test conditions for a period of 7 days (Animal No.-1) and 10 days (Animal No. 2 and 3) prior to the application of the test item.
Identification:During acclimatization marking was done with non toxic marker pen in the inside of left ear of rabbits and after acclimatization, animals were marked with permanent number in the inner side of right ear of rabbits. Permanent marker and cage card was used for identification. The individual cage card was labelled with at least study no., study type, test system, sex, dose, experiment start date and experiment completion date.
Diet:All animals were provided conventional laboratory rabbit diet (Nutrivet Life Sciences, Pune) ad libitum. Batch No.: 200005 and 200006.
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.
Experimental Room Condition
Temperature : Minimum: 19.60 °C Maximum: 22.20 °C
Relative humidity : Minimum: 56.50 % Maximum: 69.20 %
Light-dark-rhythm : 12:12
Air Changes: More than 12 changes per hour

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0.1 ml test item (as such) was placed in the conjunctival sac of three rabbits

Duration of treatment / exposure:

24 hours

Observation period (in vivo):

All the animals were observed at 1, 24, 48, 72 hours,on day 7 and day 14 after instillation of test item.

Duration of post- treatment incubation (in vitro):

no data available

Number of animals or in vitro replicates:

three 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: hand-slit lamp / biomicroscope / fluorescein: Treated and control eyes of rabbits were examined with the help of ophthalmoscope at one hour after instillation of test item

Irritation parameter:

cornea opacity score

Basis:

other: animal: #1,#2 and #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

other: animal: #1,#2 and #3

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

other: animal: #1and #2

Time point:

24/48/72 h

Score:

1.67

Max. score:

3

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

other: animal: #3

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

other: animal: #1 and #3

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

positive indication of irritation

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

1.33

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

positive indication of irritation

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 - Some blood vessels definitely hyperaemic (injected) in all the animals; Chemosis: Some swelling above normal (includes nictitating membranes) in all the animals.
Observation at 24 hours 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 - Some blood vessels definitely hyperaemic (injected) was seen in animal no. 1 and 2 whereas diffuse, crimson color; individual vessels not easily discernible was seen in animal no. 3. Chemosis: Some swelling above normal (includes nictitating membranes) 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 30%, 20% and 30% damage in animal no. 1, 2 and 3 respectively.
Observation at 48 hours 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 - Diffuse, crimson color; individual vessels not easily discernible was observed in all the animals; Chemosis: Some swelling above normal (includes nictitating membranes) was observed in all the animals.
Observation at 72 hours 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 - Diffuse, crimson color; individual vessels not easily discernible was observed in all the 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 was seen in all the animals; Area of Opacity- Zero in all the animals; Iris: Normal in all the animals; Conjunctivae - Diffuse, crimson color; individual vessels not easily discernible was seen in animal no. 1 and 2 whereas some blood vessels definitely hyperaemic (injected) was seen animal no. 3; Chemosis: Some swelling above normal (includes nictitating membranes) was seen in all the animals.
Observation on day 14 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 all the animals; Chemosis: No swelling (Normal) 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 0.00, 0.00, 1.67, 1.00; 0.00, 0.00, 1.67, 1.33, and 0.00, 0.00, 2.00, 1.00, respectively.

Other effects:

Clinical Observation
No systemic toxicity was observed in treated rabbits during the experimental period (Refer Table 2).
Mortality
No mortality was observed during the observation period.
Body weight
Increase in body weights of all the animals weighed on test day 0 (prior to application) and at termination

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

Animal No.  Eye Reaction	1	2	3
Corneal Opacity	0.00	0.00	0.00
Iris	0.00	0.00	0.00
Conjunctiva	1.67	1.67	2.00
Chemosis	1.00	1.33	1.00

 

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
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
3	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1

Key:1 = Normal

Table 1 : Individual Animal Eye Irritation Scores

 

Treated Dose:0.1 ml of test item (as such, undiluted)                                           Sex:Female

 

Animal Number	1
Application Side	Right
Eye Reactions	*	Hour(s)				Day
		1	24	48	72	7	14
Cornea	0	0	0	0	0	0	0
Area of Opacity	0	0	0	0	0	0	0
Iris	0	0	0	0	0	0	0
Conjunctiva	0	1	1	2	2	2	0
Chemosis	0	1	1	1	1	1	0
Corneal Damage (%)	30

 

Animal Number	2
Application Side	Right
Eye Reactions	*	Hour(s)				Day
		1	24	48	72	7	14
Cornea	0	0	0	0	0	0	0
Area of Opacity	0	0	0	0	0	0	0
Iris	0	0	0	0	0	0	0
Conjunctiva	0	1	1	2	2	2	0
Chemosis	0	1	1	1	2	1	0
Corneal Damage (%)	20

 

Animal Number	3
Application Side	Right
Eye Reactions	*	Hour(s)				Day
		1	24	48	72	7	14
Cornea	0	0	0	0	0	0	0
Area of Opacity	0	0	0	0	0	0	0
Iris	0	0	0	0	0	0	0
Conjunctiva	0	1	2	2	2	1	0
Chemosis	0	1	1	1	1	1	0
Corneal Damage (%)	30

Key:*= Pre-treatment eye examination.

Interpretation of results:

Category 2 (irritating to eyes) 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 0.00, 0.00, 1.67, 1.00; 0.00, 0.00, 1.67, 1.33, and 0.00, 0.00, 2.00, 1.00, respectively.Under the experimental conditions tested, all the three animals were fully reversible within an observation period of 14 days. 
Hence under the experimental test conditions,the test chemical was “An Eye Irritant (Irritating to Eyes)" to New Zealand White Female rabbit eyes. it was classified under the category "Category 2" as per Globally Harmonized System of Classification and Labelling of Chemicals (GHS) classification.

Executive summary:

Acute eye irritation/corrosion study was conducted in rabbits to evaluate the eye irrritant nature of the test chemical. The study was performed as per OECD 405 Guidelines using 3 female New Zealand White rabbits.

Rabbitsfree 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; 0.1 mlof test itemwas instilled in the other (treated) eye of each rabbit.The eye was observed at 1, 24, 48, 72 hour, day 7 and day 14 for all the animals after test item instillation.Ophthalmoscope was used for scoring of eye lesions.

In the initial test,0.1 ml of test item (as such, undiluted)was applied 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 a confirmatory test was conducted on additional two rabbits (animal no. 2 and 3); 0.1 mlof 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 -Some blood vessels definitely hyperaemic (injected) in all the animals;Chemosis:Some swelling above normal (includes nictitating membranes) in all the animals.

Observation at 24 hours 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 -Some blood vessels definitely hyperaemic (injected) was seen in animal no. 1 and 2 whereas diffuse, crimson color; individual vessels not easily discernible was seen in animal no. 3.Chemosis:Some swelling above normal (includes nictitating membranes) 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 30%, 20% and 30% damage in animal no. 1, 2 and 3 respectively.

Observation at 48 hours 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 - Diffuse, crimson color; individual vessels not easily discernible was observed in all the animals;Chemosis:Some swelling above normal (includes nictitating membranes) was observed in all the animals.

Observation at 72 hours 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 - Diffuse, crimson color; individual vessels not easily discernible was observed in all the 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 was seen in all the animals;Area of Opacity-Zero inall the animals;Iris:Normal in all the animals;Conjunctivae - Diffuse, crimson color; individual vessels not easily discernible was seen in animal no. 1 and 2 whereas some blood vessels definitely hyperaemic (injected) was seenanimal no. 3;Chemosis:Some swelling above normal (includes nictitating membranes) was seen in all the animals.

Observation on day 14 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 all the animals;Chemosis:No swelling (Normal) in all the animals.

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

Under the experimental conditions tested, all the three animals were fully reversiblewithin an observation period of 14 days.  Hence under the experimental test conditions,the test chemical was “An Eye Irritant (Irritating to Eyes)"  to New Zealand White Female rabbit eyes. it was classified under the category "Category 2" as per  Globally Harmonized System of Classification and Labelling of Chemicals (GHS) classification.

Reference 2

Endpoint:

eye irritation: in vitro / ex 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 study report.

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Principles of method if other than guideline:

The purpose of this study was to assess potential for the test article to be ocular irritants. The ocular irritation potential of a test article may be predicted by measurement of its cytotoxic effect, as reflected inthe 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, in the MatTek EpiOcular™ model

GLP compliance:

no

Species:

human

Strain:

other: Not applicable

Details on test animals or tissues and environmental conditions:

- Description of the cell system used:
The normal human-derived keratinocytes were cultured at the air-liquid interface in a chemically defined medium on a permeable polycarbonate insert (surface 0.5 cm2). They were cultured in chemically defined serum free medium to form a multi-layered epithelium similar to that found in native corneal mucosa. Each lot of tissues was Quality Assured by MatTek according to specific QC standards including: histology, tissue viability (MTT mean optical density), reproducibility (SD) and tissue thickness.

- Test System Identification
All of the EpiOcular™ 3-dimensional human tissues used in this study were identified by the date of arrival and the lot number. Certificate of Analysis for the tissues is included in this report. Tissue plates were appropriately labeled with study information. Bias was not a factor in this test system.

- Justification of the test method and considerations regarding applicability
EpiOcularTM Eye Irritation (OCL) by MatTek In Vitro Life Science Laboratories, Bratislava, Slovakien.
The test articles and controls were evaluated for potential ocular irritancy using the EpiOcular™ 3 dimensional human tissue model purchased from MatTek In Vitro Life Science Laboratories, Bratislava, Slovakien.
The EpiOcular tissue construct is a nonkeratinized epithelium prepared from normal human keratinocytes (MatTek). It models the cornea epithelium with progressively stratified, but not cornified cells. These cells are not transformed or transfected with genes to induce an extended life span in culture. The “tissue” is prepared in inserts with a porous membrane through which the nutrients pass to the cells. A cell suspension is seeded into the insert in specialized medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test material to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo. Each lot of tissues was Quality Assured by MatTek In Vitro Life Science Laboratories according to specific QC standards including: histology (cell layers), tissue viability (MTT mean optical density) and reproducibility (SD)

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 μL of liquid test article
- Concentration (if solution): neat (undiluted)

VEHICLE (no vehicle)
- Amount(s) applied (volume or weight with unit): none
- Concentration (if solution): none
- Lot/batch no. (if required): none
- Purity: none

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Concentration (if solution): neat

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Concentration (if solution): neat

Duration of treatment / exposure:

Tissues were exposed for approximately 30 minutes for liquid test article and controls, at approximately 37°C, 5% CO2 in a humidified incubator.

Observation period (in vivo):

Not applicable

Duration of post- treatment incubation (in vitro):

Following the washing step and the post-soak, the tissues were incubated at approximately 37°C, 5% CO2 in a humidified incubator for a post-exposure recovery time of ~2 hours for liquid test articles , or 18 hrs for solid test articles, and controls.

Number of animals or in vitro replicates:

2 tissues were used for test compound and control.

Details on study design:

- Details of the test procedure used
The tissues were exposed to the test article neat (undiluted). EpiOcular™ tissues were purchased from MatTek. Quality control of the tissues was performed by MatTek and the Certificate of Analysis (CoA) for the tissues is provided and is kept in the study binder. Tissues were exposed for approximately 30 minutes for liquid test articles and controls, at approximately 37°C, 5% CO2 in a humidified incubator. After the exposure, the test article was rinsed off the tissues and the tissues were soaked in media for ~12 minutes for liquid test articles and controls. Following the washing step and the post-soak, the tissues were incubated at approximately 37°C, 5% CO2 in a humidified incubator for a post-exposure recovery time of ~2 hours for liquid test articles and controls. Tissue viability was assessed by 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

- MTT Auto reduction and colouring assessment
MTT Pre-test
The test article was assessed for the potential to interfere with the assay. Approximately 50 µL of liquid test article was added to 1 mL of MTT media (~1 mg/mL) and incubated in a humidified incubator at approximately 37°C and approximately 5% CO2 for 3 hours. 50 µL of ultrapure water was used as a negative control.

- Test Article Color Test
Approximately 50 µL of liquid test article was added to 1.0 mL of ultrapure water and 2.0 mL isopropanol and incubated in a humidified incubator at approximately 37°C and approximately 5% CO2 for 2 hours, 04 minutes and 35 seconds. Samples were then added to the wells of a clear 96-well plate and the plate was read on a Thermo Scientific Multiskan FC Microplate Photometer to 570 nm. Test articles that tested positive for excessive coloration (OD >0.08) were assessed on living-tissue controls that were incubated in both culture media and MTT media as well (n=3 for both conditions).

- MTT Assay:
After the recovery period, the MTT assay was performed on run 1 tissues by transferring the tissues to 24-well plates containing 300 µL MTT medium (1.0 mg/mL). After 3 hours of MTT incubation at approximately 37°C, approximately 5% CO2 in a humidified incubator.The blue formazan salt was extracted by submerging tissues in 2 mL isopropanol in a 24-well plate. The extraction for liquid exposed tissues was overnight incubation. The optical density of the extracted formazan (200 µL/well of a 96-well plate) was determined using a Thermo Scientific Multiskan FC Microplate Photometer at 570 nm. Relative cell viability was calculated for each tissue as % of the mean negative control tissues

- Evaluation of Test Article in the cell Models
1. Cell System:
Upon receipt, the MatTek EpiOcular™ tissue cultures were placed in 1.0 mL of fresh Maintenance medium (in a 6-well plate) for 60 minutes. After the 60 minutes incubation, the Maintenance medium was exchanged with fresh medium and the tissues were incubated overnight (16-24 hrs) at approximately 37°C, approximately 5% CO2 in a humidified incubator.
2. Control and Test Article Exposures:
20 µL of calcium and magnesium free DPBS was added to each tissue and the tissues placed back into the incubator for 30 minutes. The controls and the test article will be applied topically to tissues by pipette. Three tissues will be used per test compound and control.
a)Controls: 50 µL of negative control sterile ultrapure water and positive control methyl acetate were added to the tissues. The tissues were placed into the ~37°C humidified incubator with 5% CO2 for the approximately 30 minute exposure time.
b)Test Article: 50 µL of liquid test article were added to the tissues. The tissues were placed into the ~37°C humidified incubator with 5% CO2 for the approximately 30 minute exposure time.
3. Post exposure treatment:
After the exposure, the tissues were rinsed 20 times with sterile DPBS to remove test material. The apical surface was gently blotted with a cotton swab and cultures were immediately transferred to a 12-well plate containing 5 mL of media per well. Tissues exposed to liquid test articles (and the respective control) were incubated, submerged in the media for ~12 minutes at room temperature.For liquid test articles, tissues, Tissuses were then transferred to 6-well plates containing 1.0 mL fresh Maintenance medium per well and incubated for a post-exposure recovery period for 2 hours at approximately 37 degC, 5% CO2 in a humidified incubator.
- Doses of test chemical and control substances used
Test Article:
50 µL of liquid test article were added to the tissues. The tissues were placed into the ~37°C humidified incubator with 5% CO2 for the approximately 30 minute exposure time.
Controls: 50 µL of negative control sterile ultrapure water, positive control methyl acetate were added to the tissues. The tissues were placed into the ~37°C humidified incubator with 5% CO2 for the approximately 30 minute exposure time.
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: Tissues were exposed for approximately 30 minutes for liquid test articles and controls, at approximately 37°C, 5% CO2 in a humidified incubator. Following the washing step and the, the tissues were rinsed and incubated at approximately 37°C, 5% CO2 in a humidified incubator for a post-exposure recovery time totaling ~2 hours for liquid test articles and controls.
- Justification for the use of a different negative control than ultrapure H2O (Not applicable
- Justification for the use of a different positive control than neat methyl acetate (Not applicable)
- Number of tissue replicates used per test chemical and controls: 2 tissues were used for test compound and control.
- Description of the method used to quantify MTT formazan
The blue formazan salt was extracted by submerging tissues in 2 mL isopropanol in a 24-well plate. The extraction for liquid exposed tissues was overnight incubation with a 20 minute 24 second shake the following morning. The optical density of the extracted formazan (200 µL/well of a 96-well plate) was determined using a Thermo Scientific Multiskan FC Microplate Photometer at 570 nm. The blue formazan salt was extracted by placing the tissue insterts in 1 mL isopropanol in a 6-well plate. The extraction for solid exposed tissues was 3 hrs incubation. After an addition of 1 ml isopropanol and mixing, the optical density of the extracted formazan (200μL/well of a 96-well plate) was determined using a Thermo Scientific Multiskan FC Microplate Photometer at 570 nm.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
Calculations and Statistical Methods
MTT Assay
Blanks:
· The OD mean from all replicates for each plate (ODblank).
Negative Controls (NC):
· The blank corrected value was calculated: ODNC= ODNCraw– ODblank.
· The OD mean per NC tissue was calculated.
· The mean OD for all tissues corresponds to 100% viability.
· The mean, standard deviation (SD), standard error of the mean (SEM) and the percent coefficient of variation (% CV) was calculated.
ODblank= optical density of blank samples (isopropanol alone).
ODNCraw= optical density negative control samples.
ODNC= optical density of negative control samples after background subtraction.
Positive Control (PC):
· Calculate the blank corrected value: ODPC= ODPCraw– ODblank.
· The OD mean per PC tissue was calculated.
· The viability per tissue was calculated: %PC = [ODPC/ mean ODNC] x 100.
· The mean viability for all tissues was calculated: Mean PC = Σ %PC / number of tissues.
· The standard deviation (SD), standard error of the mean (SEM) and the percent coefficient of variation (% CV) was calculated.
ODPCraw= optical density positive control samples.
ODPC= optical density of positive control samples after background subtraction.
Tested Articles:
· Calculate the blank corrected value ODTT= ODTTraw– ODblank.
· The OD mean per tissue is calculated.
· The viability per tissue is calculated: %TT = [ODTT/ mean ODNC] x 100.
· The mean viability for all tissues is calculated: Mean TT = Σ %TT / number of tissues.
· The standard deviation (SD) and the percent coefficient of variation (% CV)for the controls and the test articles will be calculated.
ODTTraw= optical density test article samples.
ODPC= optical density of test article samples after background subtraction.
Data Correction Procedure for MTT Interfering Compounds
True viability = Viability of treated tissue – Interference from test article = ODtvt – ODkt where ODkt = (mean ODtkt – mean ODukt).
ODtvt = optical density of treated viable tissue
ODkt = optical density of killed tissues
ODtkt = optical density of treated killed tissue
ODukt = optical density of untreated killed tissue (NC treated tissue)

Data Correction Procedure for Colored Compounds
True viability = Viability of treated tissue incubated in MTT media – Viability of treated tissue incubated in media without MTT = ODtvt – ODvt.
ODtvt = optical density of treated viable tissue incubated in MTT media
ODvt = optical density of viable tissues incubated in media alone.
Proposed Statistical methods
The mean, standard deviation (SD) and the percent coefficient of variation (% CV) for the controls and the test article will be calculated.
- Evaluation of data
The results of the assay was evaluated and compared to negative control.
Table: Irritancy Prediction
In VitroResults In VivoPrediction
Mean tissue viability ≤60% Irritant (I) – Category 1 or 2
Mean tissue viability >60% Non-irritant (NI) – No Category
- Assay quality controls
- Negative Controls (NC)
The assay is meeting the acceptance criterion if the mean viability of the NC in terms of Optical Density (OD570) of the NC tissues (treated with sterile ultrapure water) in the MTT assay are >0.8 to <2.5. This is an indicator of tissue viability following shipping and conditions under use.
- Positive Controls (PC)
Methyl acetate was used as a PC and tested concurrently with the test article. The assay is meeting the acceptance criteria if the viability of the PC is <50% of the negative control.
- Standard Deviation (SD)Each test of ocular irritancy potential is predicted from the mean viability determined on 3 single tissues. The assay meets the acceptance criteria if SD calculated from individual percent tissue viabilities of the replicates is <18% for three replicate tissues.

Irritation parameter:

other: mean % tissue viability

Run / experiment:

Run 1

Value:

34.4

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

Mean O.D.:0.752; Irritant

Other effects / acceptance of results:

The MTT data show the assay quality controls were met.

Code N°	Tissue No.	Raw data		Blank corrected data		mean of OD	% of viability
		Aliq. 1	Aliq. 2	Aliq. 1	Aliq. 2
NC	1	2.2839	2.3263	2.249	2.292	2.270	103.8
	2	2.1466	2.1316	2.112	2.097	2.104	96.2
PC	1	0.841	0.8408	0.806	0.806	0.806	36.9
	2	0.9048	0.9138	0.870	0.879	0.875	40.0
Test chemical	1	0.8565	0.8563	0.822	0.822	0.822	37.6
	2	0.7212	0.7117	0.687	0.677	0.682	31.2

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

The ocular irritation potential of test article was determined according to the OECD 492 test guideline followed for this study. The mean % tissue viability of test substance was determined to be 34.4%. Thus, substance was considered to be irritating to the human eyes.

Executive summary:

The ocular irritation potential of test article was determined according to the OECD 492 test guideline for this study. The MatTek EpiOcular™ model was used to assess the potential ocular irritation of the test articles by determining the viability of the tissues following exposure to the test article via MTT.

Tissues were exposed to liquid test article (50 μL) and controls for ~30 minutes, followed by a ~12 minute post-soak and approximately 2 hour recovery after the post-soak. The viability of each tissue was determined by MTT assay.

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

The mean % tissue viability of test substance was determined to be 34.4%. Thus, substance was considered to be irritating to the human eyes. and can thus be classified as ‘’Irritating to eyes in Category 2” as per CLP Regulation.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

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

 

Acute Dermal Irritation/corrosion Study of the test chemical in Rabbits, 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 clipped of 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, no erythema and no oedema observed at 1 hour of observation. At 24, 48 and 72 hours observation no erythema and oedema was observed in animal no 1.Hence the confirmatory test was conducted after 24 hours on additional two rabbits (No. 2 and 3)to confirm the non-irritant nature of the test item. Animal No. 2 and 3, revealed no erythema and no oedema at 1, 24, 48 and 72 hour observation post patch removal. 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, 2 and 3 at 1, 24, 48 and 72 hour observation post patch removal, revealed no erythema and no oedema. The individual mean score at 24, 48 and 72 hours for animal nos. 1, 2 and 3 were 0.00, 0.00, 0.00 and 0.00, 0.00, 0.00, 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 tested.

 

The above result was supported by another dermal irritation study conducted on New Zealand white rabbits in accordance with OECD 404 to assess the irritation parameter of the test chemical. 3 female New Zealand White rabbits were used for this study. In the initial test one healthy rabbit of body weight 2.33 kg was selected for study after acclimatization. The test compound in the amount of 0.5 ml 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. The test substance in the amount of 0.5 ml 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. Redness of skin was observed after four hours patch removal upto 24 hours. 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 ml 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, the intact skin site of application of each animal was observed for signs of erythema and oedema and the responses were scored following Draize’s method at 60 min., 24, 48 and 72 hours after application. The test chemical when applied dermally on New Zealand White rabbit in the amount of 0.5 ml produced redness of skin after four hours patch removal upto the 24 hours. Furthermore, no other corrosion or irritation and clinical signs of toxicity were observed during the entire observation period.   The dermal irritation index of the test chemical in New Zealand white rabbits was calculated as 0.92. Since the effects were fully reversible by 14 days, the test chemical can be considered as not irritating to New Zealand White rabbit skin.

The dermal irritation potential of test article was determined according to the OECD 439 test guideline for this study. The MatTek EpiDerm™ model was used to assess the potential dermal irritation of the test article by determining the viability of the tissues following exposure to the test article via MTT. Tissues were exposed to the test article and controls for ~one hour, followed by a 42 hour post-exposure recovery period. The viability of each tissue was determined by MTT assay. The MTT data show the assay quality controls were met and passed the acceptance of criteria.The Mean % tissue viability compared to negative control (n=3) of the test substance was determined to be 2.7%.Hence, under the current experimental test conditions it was concluded that test substance was considered to be irritating to human skin and can thus be classified as “Not Classified'' or ''Irritating to skin in Category 2” as per CLP Regulation.

Based on the available data for key and supporting study, it can be concluded that test chemical is unable to cause skin irritation and thus considered as not irritating. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Not Classified”.

 

 

Eye Irritation:

In different studies, the test chemical has been investigated for potential for dermal irritation to a greater or lesser extent. The studies are based onin- vitro and in-vivo experimental conductedfor test chemical which have been summarized as below;

An in-vitro study was conducted on test chemical to determine the ocular irritation potential of test article was determined according to the OECD 492 test guideline. The MatTek EpiOcular™ model was used to assess the potential ocular irritation of the test articles by determining the viability of the tissues following exposure to the test article via MTT. Tissues were exposed to liquid test article (50 μL) and controls for ~30 minutes, followed by a ~12 minute post-soak and approximately 2 hour recovery after the post-soak. The viability of each tissue was determined by MTT assay. The MTT data show the assay quality controls were met, passing the acceptance criteria. The mean % tissue viability of test substance was determined to be 34.4%. Thus, substance was considered to be irritating to the human eyes

 

Acute eye irritation/corrosion study was conducted in rabbits to evaluate the eye irritant nature of the test chemical. The study was performed as per OECD 405 Guidelines using 3 female New Zealand White rabbits. 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; 0.1 mlof test itemwas instilled in the other (treated) eye of each rabbit.The eye was observed at 1, 24, 48, 72 hour, day 7 and day 14 for all the animals after test item instillation. Ophthalmoscope was used for scoring of eye lesions. In the initial test,0.1 ml of test item (as such, undiluted)was applied 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 a confirmatory test was conducted on additional two rabbits (animal no. 2 and 3); 0.1 mlof 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 individual mean score for animal nos. 1, 2 and 3at 24, 48, 72 hours for corneal opacity, iris, conjunctiva and chemosis were found 0.00, 0.00, 1.67, 1.00; 0.00, 0.00, 1.67, 1.33, and 0.00, 0.00, 2.00, 1.00, respectively. Under the experimental conditions tested, all the three animals were fully reversible within an observation period of 14 days.  Hence under the experimental test conditions, the test chemical was considered as “An Eye Irritant (Irritating to Eyes)” to New Zealand White Female rabbit eyes.

 

Another ocular irritation study was conducted on New Zealand white rabbits in accordance with OECD 405 to assess the irritation parameter of the test chemical. 3 female New Zealand White rabbits were used for the study. In the initial test, one healthy rabbit of body weight 2.20 kg was selected for study after acclimatization. Both eyes of rabbits were examined for any abnormal discharge such as eye irritation, ocular defects or pre-existing corneal injury from eye 24 hours prior to application of test compound. The test compound was applied in the conjunctival sac of rabbit after gently pulling the lower lid away from the eyeball at the dose rate of 0.1ml. The lids were then gently held for about one second in order to prevent loss of the material. The other eye which remain untreated, served as a control. The acute irritation to eye conjunctiva, cornea and iris was evaluated at 1, 24, 48 and 72 hours after the treatment. 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. Any other lesions in the eye viz pannus, staining were observed and scored accordingly. Examination of reactions was facilitated by use of biomicroscope and hand slit lamp. Individual animal weight before and during the study was observed. The test chemical produced severe eye discharge and redness of the eye upto 72 hours after 1 hour application of the test chemical. No other clinical signs were observed. The result obtained from the initial test was confirmed in additional two animals of same sex and same dose level.In the confirmatory test the test compound was applied in the amount of 0.1 ml in the conjunctival sac of two more rabbits after gently pulling the lower lid away from the eyeball. The other eye which remain untreated, served as a control. The acute irritation to eye conjunctiva, cornea and iris was evaluated at 1, 24, 48 and 72 hours after the treatment. The grades of ocular reaction (conjunctiva, cornea and iris) were recorded at each observation. To determine the reversibility of the effect the animals were observed normally for 21 days. Any other lesions in the eye viz pannus, staining were observed and scored accordingly. Examination of reactions was facilitated by use of biomicroscope and hand slit lamp. Individual animal weight before and during the study was observed. In the confirmatory test, the test chemical when applied to the eyes of New Zealand white rabbit at the dose level of 0.1 ml produced severe eye discharge and redness of eye upto 72 hours after 1 hour of application of test compound. However, any lesions such as pannus, staining were not recorded throughout the observation period of 72 hours. The overall irritation index was calculated to be 24.6. Based on the scores and observations, the test chemical was considered to be mildly irritating to New Zealand White rabbit eyes.

 

Next eye irritation study of test chemical was assessed in rabbits according to Draize method. Twenty-five laboratories supplied data for a joint study of the variability of test results for the eye irritation caused by a series of test chemicals tested according to Draize method. 6 young adult male albino rabbits were used for the study. The eyes were free of defects and irritation, and 24 hr prior to application of test material the eyes were stained with an aqueous 5% solution of sodium fluorescein. The stain was allowed to contact the eyes for 20 set and was flushed thoroughly with distilled water at room temperature, The eyes were then examined for possible corneal lesions not otherwise visible.0.1mlUndiluted test chemical was instilled into three left and 3 right eyes of the test animals.The lower lid was pulled away from the eye to form a cup into which The lid was held open for a few seconds, then raised to close with the upper lid. The 2 lids were held gently together for a few seconds before the animal was released. The eyes were not washed following instillation. The eyes were examined and the degree of irritation recorded at 1, 24, and 72 hr and 7 days after application of the sample. Observations on any animal were discontinued after 24 or 72 hr if the eyes were free of irritation. Fluorescein staining was done at 24 and 72 hr and 7 days on the eyes of animals not previously negative. The grading of eye irritation was according to the scale of Draize et al. (1944). Injuries such as pannus, keratoconus, and sloughing of corneal epithelium were not included in the Draize system and were recorded separately. The Median of Laboratory Scores at 24, 72 hours and 7 days were 28.2, 29.5 and 16 respectively. Based on these scores, the test chemical can be considered to be irritating to rabbit eyes.

 

Further, an eye irritation study of test chemical was conducted on albino rabbits to observe the presence or absence of ocular lesions caused by the test chemical. Young adult albino rabbits of both sexes were restrained in stocks before treatment and kept in that position for the following 6 hours. One-tenth milliliter of the (10-100%) sample was placed in the conjunctival sac of one eye and the other eye served as the untreated control. In some instances the sample was kept in the conjunctival sac for 4 seconds and then the eye was flushed with physiological saline (post treatment). Observation of the eyes was made after 1 and 4 hours; and 1, 2, 3, and 7 days following treatment. Scoring was done according to the method of Draize and Kelley, which permits a maximum score of 110. The treated rabbits caused ocular lesion at tested concentrations of 10-100%.Under the test conditions, the test material was therefore considered to be irritating to the rabbit eye.

 

The above results were further supported by an ocular irritation study of test chemical conducted on three albino rabbits according to French legislation.About 100µl test compound was instilled into the conjunctival sac of one eye of each rabbit. To avoid unnecessary suffering, solid compounds were diluted at 10% (w/v) in aqueous solution. The ocular lesions (conjunctiva, iris and cornea) were collected 1 hr and 1, 2, 3, 4 and 7 days after treatment. The daily scores for each rabbit were summed and the maximum average score (MAS) was determined as the highest score obtained on a scale of 110. Four classes of irritants were distinguished relative to the MAS.The test chemical caused mild eye irritation in treated rabbits withMAS of 1.3. Therefore,the test material was considered to be irritating to the rabbit eye.

All these in vivo studies lead to conclusion that the test chemical is indeed not irritating to the eyes.

Further, various in-vitro studies were conducted for test chemical by using different in-vitro models which were described as below;

The test chemical was evaluated for eye irritation. The tests were performed according to EEC (1984 and 1991) and French (1984 and 1991) directives with few modifications: ocular lesions (cornea, iris and conjunctiva) were scored. Three rabbits were used per test compound, and maximal average score (MAS) as well as score at day 1 were calculated. At the request of the participants, any mass of material present in the conjunctival sac was removed after the 1-hr observation. Also, a fluorescein solution was used for observation of corneal lesions. Only ocular lesions (cornea, iris and conjunctiva) were scored (Draizeet al.,1944) at 1 hr, then at 1, 2, 3, 4, 7 and 14 days. In the case of positive score, eyes were also examined at 21 days. Raw data were also used to classify chemicals according to the rating system described by Kay and Calandra (1962), and the EC criteria. The MAS score for the test chemical was 1.3 and the score measured on day 1 was 0.0. The effects observed were fully reversible by day 1. The test chemical was considered as Non-irritant according to EEC(1984) directives and French (1984 and 1991) directives.

 

The STE test method was a cytotoxicity-basedin vitro assay that was performed on a confluent monolayer of Statens Seruminstitut Rabbit Cornea (SIRC) cells, cultured on a 96-well polycarbonate microplate .After five-minute exposure to a test chemical, the cytotoxicity was quantitatively measured as the relative viability of SIRC cells using the MTT assay .Decreased cell viability was used to predict potential adverse effects leading to ocular damage. The cells in the 96-well plates were exposed to 200 microliters of 5% (w/w) and 0.05% (w/w) test chemical solutions prepared using physiological saline for 5 minutes. Three wells were used for each concentration. After exposure, the, cells were washed with phosphate buffered saline (-) (twice, and 200µl of methylthiazolydiphenyl-tetrazolium bromide solution (0.5 mg MTT/ml of medium) was added. After a 2-h reaction time, MTT formazan was extracted with 0.04 N HCl-isopropanol) for 30 min, and the absorbance of the extract was measured at 570 nm with a plate reader. The ratio (%) of MTT formazan absorbance for each test chemical to the absorbance of MTT formazan for control represented cell viability (triplicate determinations). The control group cells were exposed to physiological saline. The mean of three wells for each test concentration was calculated. This was the mean cell viability for one independent test. A total of three independent tests were conducted for each concentration of a test chemical, and the calculated overall mean of three independent tests (thereafter termed the ‘‘relative viability’’) was used in the final analysis. After obtaining relative viability in the STE test, which used a 5 min exposure to test chemicals in physiological saline, category and rank classifications were determined for each test concentration. The mean viability of 5% and 0.05% test chemical in saline were 72.3, 105.5 respectively. Based on the relative viability index obtained in STE test , the test chemical was considered to be not irritating. Similarly from the Draize 100% data the test chemical was considered to be not irritating with irritation index of 0.0. Based on the scores of the Draize test and the mean viabilities of the test chemical obtained in the STE assay, the test chemical can be considered as not irritating.

 

A multinational interlaboratory study was conducted to investigate the bovine corneal opacity and permeability (BCOP) assay. The assays were carried out in 12 European laboratories with different types of activity. The Bovine Corneal Opacity and Permeability (BCOP) assay, which was based on the method of Muir, was anin vitrotest which has been developed recently. Originally, it was devoted to predicting the irritant potential of process intermediates compounds in development. The assay uses corneas, one of the target tissues of ocular irritancy, from freshly collected bovine eyes, and two endpoints were investigated, namely corneal opacity and the disruption of the corneal barrier as assessed by the passage of a fluorescent dye. Bovine eyes were collected from a commercial abattoir in a plastic jar containing 1 litre HBSS for approximately 25 eyes. Buffer storage and eyes transportation to the laboratories were performed at ambient temperature, and the eyes were used within 2 hr of the killing of the animals. During dissection great care was taken to avoid damage of corneal surfaces (epithelial and endothelial). Each cornea was carefully examined, and those presenting defects, such as neovascularization, pigmentation, opacity or scratches were discarded. Globes were first dissected free of surrounding tissues and placed in a jar containing fresh HBSS. Selected corneas were dissected with a 2-3-mm rim of sclera for easier handling, and stored in a petri dish containing HBSS until use. Corneas were then mounted in holders filled with MEM, and incubated for 1 hr in a water-bath at 32°C. At the end of the incubation period, the medium was removed from both compartments of the holders, using a needle attached to a vacuumpump or a syringe. The posterior compartment wasthen refilled with MEM, whereas the anterior compartmentwas filled with the test compound or itsvehicle. Two treatment protocols were used depending on the physical state (liquid or solid) of the product evaluated. Liquids were applied pure, and surfactants were diluted at 10% in MEM, and a volume of 0.75 ml was put into the anterior compartment with an appropriate syringe and needle. Corneas were incubated in a horizontal position for 10minutes at 32°C. The test substance was then removed and the epithelium was washed at least three times (until the medium was clear) with approximately 4 ml MEM. The anterior compartment was refilled with medium, and a first measurement of opacity was performed. Corneas were again incubated at 32°C, for 2 hr, before a second measurement of opacity was made. The values obtained at this second measurement were the ones used for calculations, while the first reading was considered indicative of early effects. The opacitometer (Electro- Design, Riom, France) determines the difference in light transmission between a treated and a control cornea, and displays a numerical opacity value (arbitrary units). The apparatus was previously calibrated with standardized opaque sheets of polyester, and values obtained with test substances generally ranged between 0 and 150. This second step of the assay was performed immediately after the measurement.T his second step of the assay was performed immediately after the measurement of opacity. The medium was again removed from both chambers of holders, and replaced by fresh medium in the posterior compartment, and by 1 ml fluorescein solution (0.4% for liquids and surfactants, 0.5% for solids) in contact with the epithelium. Corneas were then incubated in a horizontal position for 90 min at 32°C. Medium from the posterior chamber was then removed, and its optical density (O.D.) determined with a spectrophotometer at 490 nm. The Mean In vitro score for the test chemical was 0.1. Based on the classification scheme for BCOP assay, the test chemical was a non- irritant.

 

 

The Vitrigel-EIT method is composed of two parts, i.e., the construction of a human corneal epithelium (HCE) model in a collagen vitrigel membrane chamber and the prediction of eye irritancy by analyzing the time-dependent profile of transepithelial electrical resistance values for 3 min after exposing a chemical to the HCE model. The aim of the study was to estimate the predictive performance of the Vitrigel-EIT method. A SV40-immortalized HCE cell strain (HCE-T cells, RCB no. 2280) was obtained from the RIKEN BioResource Center (Tsukuba, Japan). The cells were maintained in the following culture medium: 1 : 1 mixture of Dulbecco’s modified eagle medium and nutrient mixture F-12 supplemented with 5% heat-inactivated fetal bovine serum, 5 μg/ml recombinant human insulin, 10 ng/ml recombinant human epidermal growth factor, 0.5% dimethyl sulfoxide 100 units/ml penicillin and 100 μg/ml streptomycin. Cells were grown at 37 °C in a humidified atmosphere of 5% CO2 in air. A collagen xerogel membrane chamber (ad-MED VitrigelTM) was purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). The collagen xerogel membrane chamber was set in the well of a 12-well plate. Then, the collagen xerogel membrane was immersed in the above culture medium by pouring 1.5 ml outside and 0.5 ml inside the chamber in the well for 10 min to convert the xerogel into a vitrigel immediately before use. Every test chemical solution was prepared in a culture medium at a concentration of 2.5 (weight/volume) % appropriate for measuring TEER values without being influenced by the test chemical-dependent electrical resistance. Here, the chemicals were dissolved in the medium by using an appropriate technique(s) as follows: vortex mixing within 1 min, sonication within 20 min and/or heating in a water bath <70 °C. In case test chemicals are insoluble or immiscible by the above technique(s), the test chemical solution was prepared as a homogeneous suspension that the chemical was mixed well in the medium by vortex within 1 min immediately before use. The pH level of each 2.5 w/v % test chemical solution was measured using Universal pH test paper from ADVANTEC (Tokyo, Japan). The HCE models on day 6 were subjected to the exposure experiment of test chemicals. At first, 500 μl of culture medium was poured in the chamber and the value of the Rsample, before chemical exposures, was measured to obtain the initial TEER value of each model. Next, the medium inside the chamber was changed to 500 μl of test chemical solution and the periodical values of R sample were measured by the TEER recorder at intervals of 10 s for 3 min after exposure of each test solution. Three independent models were subjected to the exposure experiment for each test solution to plot the average time-dependent profile of TEER values on a chart. The chemical exposure experiment was conducted in the ambient temperature of 28 ± 2 °C. The average time-dependent profile of TEER values after exposing each test chemical solution in three-independent experiments was analyzed by three indexes for time lag, intensity and plateau level. The eye irritant potential of test chemicals was classified into two categories, irritant and non-irritant, according to the criteria for the scores of three indexes. The pH of the test chemical was measured to be 7.The scores for lag time, intensity and plateau regions recorded while measuring the TEER values were 180, -0.01, 0 respectively. Based on these values, the test chemical was considered to be not irritating to eyes in the Vitrigel-eye irritancy method.

 

Even though few references claims that there is a no possibility of test chemical to cause eye irritation, but it is strongly negated by the results of the other studies for the test chemical which indicate that the test chemical is likely to cause eye damage and hence it 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 (irritating to eyes) ''.

Justification for classification or non-classification

The skin and eye irritation potential of test chemical were observed in various studies. The results obtained from these studies indicates that the chemical is unlikely to cause skin irritation but can cause eye damage. Hence the test chemical can be classified under the category “Not Classified” for skin and “Category 2 (irritating to eyes) '' eye as per CLP.