Registration Dossier

Administrative data

Description of key information

Skin irritation

The dermal irritation potential of test chemical was assessed in various experimental studies which were conducted in rabbits. Based on the available key data and supporting studies, it can be concluded that the test chemical is unable to cause skin irritation and 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 assessed in various in- vitro and in-vivo experimental studies for test chemical. Based on the available key data and supporting studies, it can be concluded that chemical 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 from experimental report.
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Principles of method if other than guideline:
The objective of the study was to assess the irritant and/or corrosive effects of test chemical after topical application on the intact skin in rabbits.
GLP compliance:
yes
Specific details on test material used for the study:
- Name of test material (as cited in study report): Sodium salicylate
- Molecular Formula: C7H6O3.Na
- Molecular Weight: 160.105 g/mol
- Smiles Notation: c1(c(cccc1)O)C(=O)[O-].[Na+]
- InChI: 1S/C7H6O3.Na/c8-6-4-2-1-3-5(6)7(9)10;/h1-4,8H,(H,9,10);/q;+1/p-1
- Substance Type: Organic
- Physical State: Solid
- AI Content (assay): 99.8%
- Manufactured data: March, 2013
- Expiry Date : February, 2015
- Storage conditions: Room temperature (20 - 30 °C)
Handling and Disposal
Safety precautions : Aprons, masks, caps, gloves and goggles were used to ensure the health and safety of the personnel.
Disposal : The remaining unused test item was disposed as per internal SOPs
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Sex: Male
- Source: LIVEON BIOLABS PVT LTD
- Age at study initiation: 3 to 4 Months (Approximately)
- Weight at study initiation: Minimum: Minimum: 1.764 kg & Maximum: 2.384 kg (Prior to Treatment)
- 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 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 8 days (Animal No.-2 and 3) prior to the application of the test item.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Minimum: 20.30 °C Maximum: 22.70 °C
- Humidity (%):Minimum: 48.30 % Maximum: 68.30 %
- Air changes (per hr): More than 12 changes per hour
- Photoperiod (hrs dark / hrs light):12:12 hours
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
water
Controls:
yes, concurrent vehicle
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 g moistened with 0.5 ml distilled water


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

REMOVAL OF TEST SUBSTANCE
- Washing (if done): test substance was removed by using cotton soaked in distilled water
- Time after start of exposure: 4 hrs

SCORING SYSTEM: Grading of irritation lesions was carried out as per Draize Method
Irritation parameter:
erythema score
Basis:
mean
Remarks:
Animal 1,2,3
Time point:
24/48/72 h
Score:
0
Max. score:
3
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritation parameter:
edema score
Basis:
mean
Remarks:
Animal 1,2 and 3
Time point:
24/48/72 h
Score:
0
Max. score:
3
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
Animal No. 1, showed very slight erythema (barely perceptible) and no oedema at 1 hour of observation. At 24, 48 and 72 hour observation no erythema and oedema was observed in animal No 1.
In Animals No. 2 and 3 at 1 hour observation post patch removal, revealed very Slight erythema (barely perceptible) and no oedema. At 24, 48 and 72 hour post patch removal, animals showed 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
Other effects:
Clinical Observation - No systemic toxicity was observed at treated rabbits during the experimental period.
Mortality - No mortality was observed during the observation period
Body Weights - Body weights were increased as compared to day 0 in all the three animals

Table 1: Skin Reaction

 

In Treated area 

Dose:500 mg of test item (moistened with 0.5 ml distilled water)            

Sex:Male 

Animal

No.

Test

Treated

 area*

Erythema score

Oedema score

1h

24h

48h

72h

1h

24h

48h

72h

1

Initial

Left

1

0

0

0

0

0

0

0

2

Confirmatory

Right

1

0

0

0

0

0

0

0

3

Right

1

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

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

Key: h = Hour.

Erythema                                                                                                       Oedema

0 =No erythema                                                                                           0 =No oedema

1 = Very slight erythema(barely perceptible)

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

2.314

2.360

2

2.384

2.420

3

1.764

1.836

Individual Animal Clinical Signs

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:
No erythema and oedema (skin irritation) were observed at the end of 72 hour observation period after patch removal. The individual mean score at 24, 48 and 72 hoursfor 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 given test chemical was Non-Irritating to the skin of Male New Zealand White rabbits under the experimental conditions tested
Executive summary:

Acute Dermal Irritation/corrosion study was performed as per OECD guideline No. 404 in Rabbits for the given test chemical. Three healthy young adult male rabbits were used for conducting acute dermal irritation/corrosion study. The hairs of all the rabbits were clipped at contralateral sites, approximately 24 hours prior to treatment. A dose of 0.5 g of test item (moistened with 0.5 ml distilled water) 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 and0.5 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, very slight erythema (barely perceptible) 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. No severe skin lesions were observed hence a confirmatory test was conducted on additional two rabbits (No. 2 and 3) after 24 hours to confirm the non irritant nature of the test item. The patch was removed after 4 hours and rabbits were observed for erythema and oedema at 1, 24, 48 and 72 hours post patch removal, evaluated and graded as per Draize method. In animal no. 2 and 3 at 1 hour observation post patch removal, revealed very slight erythema (barely perceptible) and no oedema. At 24, 48 and 72 hour post patch removal, animals showed 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 given test chemical was Non-Irritating to the skin of male New Zealand White rabbits under the experimental conditions tested and classified as "Not Classified" as per CLP criteria.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

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:
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 in the (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 Lab. (Bratislava, Slovakia).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, Inc. 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 mg of solid test chemical
- 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 6 hrs ± 15 min for solid test articles, 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 and post soak, the tissues were rinsed and incubated at approximately 37°C, 5% CO2 in a humidified incubator for a post-exposure recovery time of 18 hours for solid test chemicals 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 6 hrs ± 15 min for solid 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 ~25 minutes for solid test articles and controls.Following the washing and post soak, the tissues were rinsed and incubated at approximately 37°C, 5% CO2 in a humidified incubator for a post-exposure recovery time of 18 hours for solid test chemicals 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:
Inserts are removed from the 24-well plate after 3 hrs of incubation and the bottom of the insert is blotted on absorbent material, and then transferred to a pre-labeled 6-well plate containing 1 ml isopropanol in each well so that no isopropanol is flowing into the insert. At the end of the non-submerged extraction inserts and tissues are discarded without piercing and 1 ml of isopropanol is added into each well. The extract solution is mixed and the optical density of the extracted formazan (200 μL/well of a 96-well plate) was determined using 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.2 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: When a solid was tested, 50 mg of the solid were added to the tissues. The tissues were placed into the ~37°C humidified incubator with 5% CO2 for the approximately 6 hrs ± 15 min.
3. Post exposure treatment:
After the exposure, the tissues were rinsed 20 times with sterile of 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:
When a solid was tested, 6 hours of the solid were added to the tissues. The tissues were placed into the ~37°C humidified incubator with 5% CO2 for the approximately 6 hrs ± 15 min.
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 6 hours for solid test articles and controls, at approximately37°C, 5% CO2 in a humidified incubator.
Following the post soak, the tissues were rinsed and incubated at approximately 37°C, 5% CO2 in a humidified incubator for a post-exposure recovery time totaling 18 hours for solid 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 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 meanthe 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 2 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:
2.4
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Remarks:
Mean O.D.=0.049; Irritant
Other effects / acceptance of results:
The MTT data show the assay quality controls were met.

Table - % viability

Code N°

Tissue No.

Raw data

Blank corrected data

mean of OD

% of viability

Aliq. 1

Aliq. 2

Aliq. 1

Aliq. 2

NC

 

1

2.0043

2.0745

1.970

2.040

2.005

99.4

2

2.0651

2.0633

2.030

2.029

2.029

100.6

PC

1

0.5856

0.6029

0.551

0.568

0.559

27.7

2

0.6292

0.6432

0.594

0.608

0.601

29.8

Test chemical

1

0.0809

0.0812

0.046

0.046

0.046

2.3

2

0.0864

0.0857

0.052

0.051

0.051

2.5

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 2.4%. Thus, the test chemical 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 solid test articles and control for approx.6 hours, followed by a 25 minute post-soak and approximately 18 hours 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 2.4 %. Hence, under the experimental test conditions it was concluded that test substance was considered to be severely irritating to the human eyes.

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:
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 given test chemical on eye, when exposed by the ocular route in rabbits.
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: LIVEON BIOLABS PVT. LTD
- Age at study initiation: 3 to 4 Months (Approximately)
- Weight at study initiation: Minimum: 2.120 kg and Maximum: 2.448 kg (Prior to Treatment)
- 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
- Water (e.g. ad libitum): Aqua guard filtered tap water was provided ad libitum
- Acclimation period: Rabbits were acclimatised to the test conditions for a period of 7 days (Animal No.-1) and 9 days (Animal No. 2 and 3) prior to the application of the test item

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Minimum: 20.30 °C Maximum: 22.70 °C
- Humidity (%): Minimum: 48.30 % Maximum: 68.30 %
- Air changes (per hr): More than 12 changes per hour
- Photoperiod (hrs dark / hrs light): 12:12

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 100 mg
Duration of treatment / exposure:
24 hours
Observation period (in vivo):
All the animals were observed at 1, 24, 48 and 72 hours and on day 7 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:Grading of irritation lesions was carried out as per Draize Method
TOOL USED TO ASSESS SCORE: Ophthalmoscope and fluorescent strips.
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
Animal 1,2 and 3
Time point:
24/48/72 h
Score:
> 0.67 - <= 1
Max. score:
3
Reversibility:
fully reversible within: 7 days observation period
Remarks on result:
probability of mild irritation
Irritation parameter:
iris score
Basis:
mean
Remarks:
Animal 1,2 and3
Time point:
24/48/72 h
Score:
0
Max. score:
3
Reversibility:
not specified
Remarks on result:
probability of mild irritation
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
animal 1,2 and 3
Time point:
24/48/72 h
Score:
2
Max. score:
3
Reversibility:
fully reversible within: 7 days observation period
Remarks on result:
probability of mild irritation
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
Animal 1, 2 and 3
Time point:
24/48/72 h
Score:
1
Max. score:
3
Reversibility:
fully reversible within: 7 days observation period
Remarks on result:
probability of mild 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 in all 3 animals; Area of Opacity- Zero in all the animals; Iris: Normal in all the animals. Conjunctivae - Some blood vessels definitely hyperaemic (injected) was observed in all the animals; Chemosis: Some swelling above normal (includes nictitating membranes) was observed in all the animals.
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 animal no. 1 whereas animal no. 2 and 3 showed no ulceration or opacity; Area of Opacity- One quarter (or less) but not zero in animal no. 1 and Zero in animal no. 2 and 3; Iris: Normal in all the animals. Conjunctivae – Diffuse, crimson color; individual vessels not easily discernible was observed in all animals; Chemosis: Some swelling above normal (includes nictitating membranes) was observed in all animals.
At 24 hours observation the rabbits were examined for corneal epithelium cell damage using sodium fluorescein strips and noticed 40%, 20%, 20% damage in animal no. 1, 2 and 3 respectively.
Observation at 48 hour 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 animals; Area of Opacity- One quarter (or less) but not zero in all 3 animals; Iris: Normal in all the animals. Conjunctivae – Diffuse, crimson color; individual vessels not easily discernible was observed in all animals; Chemosis: Some swelling above normal (includes nictitating membranes) was observed in all animals.
Observation at 72 hour 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 animals; Area of Opacity- One quarter (or less) but not zero in all 3 animals; Iris: Normal in all the animals. Conjunctivae – Diffuse, crimson color; individual vessels not easily discernible was observed in all animals; Chemosis: Some swelling above normal (includes nictitating membranes) was observed in all animals.
Observation on day 7 after instillation of test item revealed: Cornea- No ulceration or opacity in all the animals; Area of Opacity- Zero in all 3 animals; Iris: Normal in all the animals. Conjunctivae – Blood vessels were normal in all 3 animals; Chemosis: No swelling (Normal) was observed in all 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, 0.00, 2.00, 1.00; 0.67, 0.00, 2.00, 1.00 and 0.67, 0.00, 2.00, 1.00 respectively.
Other effects:
Clinical Observation - No systemic toxicity was observed in treated rabbits during the experimental period
Mortality - No mortality was observed during the observation period
Body weight - 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 (0.1gm) of test item                                              

 Sex:Female

Animal Numbers

1

2

3

Application Side

Left

Left

Left

Eye Reactions

At hour

At hour

At hour

*

1

24

48

72

Day 7

*

1

 24

48

72

Day 7

*

1

24

48

72

Day 7

Corneal Opacity

0

0

1

1

1

0

0

0

0

1

1

0

0

0

0

1

1

0

Area of Opacity

0

0

1

1

1

0

0

0

0

1

1

0

0

0

0

1

1

0

Iris

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Conjunctiva

0

1

2

2

2

0

0

1

2

2

2

0

0

1

2

2

2

0

Chemosis

0

1

1

1

1

0

0

1

1

1

1

0

0

1

1

1

1

0

Corneal Damage%

40

20

20

 

Dose:Untreated (Control Eye)                                                                       Sex:Female

 

Animal Numbers

1

2

3

Application Side

Left

Left

Left

Eye Reactions

At hour

At hour

At hour

*

1

24

48

72

Day 7

*

1

 24

48

72

Day 7

*

1

24

48

72

Day 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.

 

 

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

            Animal No.

 Eye Reaction

1

2

3

Cornea

1.00

0.67

0.67

Iris

0.00

0.00

0.00

Conjunctiva

2.00

2.00

2.00

Chemosis

1.00

1.00

1.00

 

 Formula :

 Mean Irritation Score= Sum of the Individual Animal Score for eye reactions at 24, 48 and 72 hours

Number of the Observations (3)

Table 2 : Individual Animal Clinical Signs

 

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 = Norma

Individual Animal Body Weight

Sex :Female

Animal No.

Animal Body Weight (kg)

Prior to application

At termination

1

2.448

2.612

2

2.120

2.156

3

2.236

2.402

Key:kg = Kilogram

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 1.00, 0.00, 2.00, 1.00; 0.67, 0.00, 2.00, 1.00 and 0.67, 0.00, 2.00, 1.00 respectively. Under the experimental conditions tested, eye irritation and reversibility of effects on eyes of rabbits was observed within day 7. Hence, under the experimental test conditions, the given test chemical is “Mildly Irritating to eyes” of New Zealand White female rabbit eyes and is being classified "Category 2"as per the CLP regulation.
Executive summary:

Acute Eye Irritation/Corrosion study was performed according to OECD 405 Guideline by using the given test chemical in Rabbits. 3 female young adult rabbits were used for the study. 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 itemwas instilled in the other (treated) eye of each rabbit.The eye was observed at 1, 24, 48, 72 hour and on day 7 after test item instillation.Ophthalmoscope was used for scoring of eye lesions. In the initial test,100 mg of test item (pulverized form)was instilled into the conjunctival sac of the right eye of animal no.1 whereas the left eye of the rabbit served as the control. As animal no. 1 showed no severe ocular lesions a confirmatory test was conducted on additional two rabbits (animal no. 2 and 3); 100 mg of test item 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 at 1, 24, 48 and 72 hour observation which recovered at day 7. Untreated eye of all the three rabbits was normal throughout the experiment.

The following grading scores were observed in treated eye of treated rabbits. Observation at 1 hour after instillation of test item revealed: Cornea-No ulceration or opacity in all 3 animals; Area of Opacity-Zero in all the animals;Iris:Normal in all the animals.Conjunctivae -Some blood vessels definitely hyperaemic (injected) was observed in all the animals;Chemosis:Some swelling above normal (includes nictitating membranes) was observed in all the animals.

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 animal no. 1 whereas animal no. 2 and 3 showed no ulceration or opacity; Area of Opacity-One quarter (or less) but not zero in animal no. 1 and Zero in animal no. 2 and 3;Iris:Normal in all the animals.Conjunctivae –Diffuse, crimson color; individual vessels not easily discernible was observed in all animals;Chemosis:Some swelling above normal (includes nictitating membranes) was observed in all animals.

At 24 hours observation the rabbits were examined for corneal epithelium cell damage using sodium fluorescein strips and noticed 40%, 20%, 20% damage in animal no. 1, 2 and 3 respectively.

Observation at 48 hour 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 animals; Area of Opacity-One quarter (or less) but not zero in all 3 animals;Iris:Normal in all the animals.Conjunctivae –Diffuse, crimson color; individual vessels not easily discernible was observed in all animals;Chemosis:Some swelling above normal (includes nictitating membranes) was observed in all animals.

Observation at 72 hour 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 animals; Area of Opacity-One quarter (or less) but not zero in all 3 animals;Iris:Normal in all the animals.Conjunctivae –Diffuse, crimson color; individual vessels not easily discernible was observed in all animals;Chemosis:Some swelling above normal (includes nictitating membranes) was observed in all animals.

Observation on day 7 after instillation of test item revealed: Cornea- No ulceration or opacity in all the animals; Area of Opacity- Zero in all 3 animals; Iris: Normal in all the animals. Conjunctivae – Blood vessels were normal in all 3 animals; Chemosis: No swelling (Normal) was observed in all 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, 0.00, 2.00, 1.00; 0.67, 0.00, 2.00, 1.00 and 0.67, 0.00, 2.00, 1.00 respectively. Under the experimental conditions tested, eye irritation and reversibility of effects on eyes of rabbits was observed within day 7. Hence, under the experimental test conditions, the given test chemical is “Mildly Irritating to eyes” of New Zealand White female rabbit eyes and is being classified "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:

Various studies have 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 and in vitro experiments which are summarized as follows -

 

Acute Dermal Irritation/corrosion study was performed as per OECD guideline No. 404 in Rabbits for the given test chemical. Three healthy young adult male rabbits were used for conducting acute dermal irritation/corrosion study. The hairs of all the rabbits were clipped at contralateral sites, approximately 24 hours prior to treatment. A dose of 0.5 g of test item (moistened with 0.5 ml distilled water) 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 and0.5 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, very slight erythema (barely perceptible) 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. No severe skin lesions were observed hence a confirmatory test was conducted on additional two rabbits (No. 2 and 3) after 24 hours to confirm the non irritant nature of the test item. The patch was removed after 4 hours and rabbits were observed for erythema and oedema at 1, 24, 48 and 72 hours post patch removal, evaluated and graded as per Draize method. In animal no. 2 and 3 at 1 hour observation post patch removal, revealed very slight erythema (barely perceptible) and no oedema. At 24, 48 and 72 hour post patch removal, animals showed 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 given test chemical was Non-Irritating to the skin of male New Zealand White rabbits under the experimental conditions tested and classified as "Not Classified" as per CLP criteria.

 

The above result are supported by another dermal irritation study conducted on New Zealand white rabbits in accordance with OECD 404 to assess the irritation parameter of the given test chemical. 3 female New Zealand White rabbits were used for the study. The animals were prepared 24 hrs 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. 0.5 gm of the test compound was applied on a small area 6 square cms of the intact test site. Each site of application was covered with an impervious dressing which was secured with an adhesive tape. The animals were housed individually and restrained by the use of plastic collars. After patch removal [4 hours later] the unabsorbed test chemical was removed and the test site was washed with lukewarm water. The intact skin site of application was observed for erythema and edema at 1, 24,48 and 72 hours till 14 days after application and scored according to Draize method. Test chemical produced slight redness to skin at 4 hours of observation. Furthermore, no clinical signs of skin irritation were observed at 24 hours of observation. It did not produce any clinical signs of toxicity throughout the examination period of 14 days. The dermal irritation index was calculated as 0.00 and test compound can be classified under the category "Not Classified".              

 

Above in-vivo studies are contradicted with the in-vitro study. The dermal irritation potential of test chemical 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 3.9%. 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 ''Irritating to skin in Category 2” as per CLP Regulation.

 

Based on the available data for key (in-vivo) and supporting studies, it can be concluded that test chemical is unable to cause skin irritation and 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 ocular irritation to a greater or lesser extent. The studies are based on in vivo and in-vitro experiments in rabbits which are summarized as below –

 

An in-vitro study was conducted to determine 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 solid test articles and control for approx.6 hours, followed by a 25 minute post-soak and approximately 18 hours 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 2.4 %. Hence, under the experimental test conditions it was concluded that test substance was considered to be severely irritating to the human eyes.

The above in-vitro study result is supported with another study conducted on test chemical. 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. 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 μgml–1 recombinant human insulin, 10 ngml–1 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. The test chemical was selected according to the globally harmonized system of classification and labeling (GHS) classification for eye irritation (United Nations, 2013). 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 chemical was 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 Rsample 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 sensitivity, specificity and accuracy of the Vitrigel-eye irritancy test method were 82.3%, 89.5% and 83.7%, respectively. The score plateau level for the given test chemical in the Vitrigel-eye irritancy test method was 80. According to classification criteria of Vitrigel-eye irritancy test method, it can be regarded as an Eye irritant.

 

Further, the above studies are supported with in-vitro study performed for predicting eye irritation potential of the given test chemical by Short time exposure (STE) test. SIRC (ATCC CCL-60) cells were obtained from American Type Culture Collection (Manassas, VA, USA). SIRC cells were cultured in Eagle’s MEM containing 10% (v/v) fetal bovine serum, 2 mM L-glutamine, 50 units/ml penicillin, and 50 μg/ml streptomycin. When the cells proliferated in the culture flask to confluence, the cells were dispersed with trypsin-EDTA solution. The dispersed cells were spread into 96-well flat-bottomed plates at 3.0 × 103cells/well. After incubation (37°C, 5% CO2) for 5 days or (6.0 × 103cells/well for 4 days), the cells reached confluence. Briefly, physiological saline was used as first vehicle for test chemicals. The cells cultured in 96-well plates were exposed to 200 microliters of 5% and 0.05% test chemical solution. After exposure, the cells were washed with phosphate buffered saline (-) (PBS (-))twice and 200 μl of methylthiazolydiphenyl-tetrazolium bromide (MTT, Sigma Aldrich) solution (0.5 mg MTT/ml of medium) was added. After a 2-hr reaction time, MTT formazan was extracted with 0.04 N HCl-isopropanol (Kanto Chemical Co., Inc.) for 30 min, and the absorbance of the extract was measured at 570 nm with a plate reader. The ratio of absorbance (%) on each test sample to that of control was represented as relative viability (triplicate determinations). The control group cells were exposed to physiological saline, saline with 5% DMSO, or mineral oil. The mean of three wells for each test concentration was calculated. This was the mean relative viability for one independent test. A total of three independent tests were conducted for each concentration of a test material, and the calculated overall mean of three independent tests was used for estimation of eye irritation. Irritation results from the STE test were converted to a point system in order to establish a potency ranking. A lower rank point score was given to the experimental condition that had a lower cytotoxicity. For the lower test concentration results, a higher rank point score was awarded due to the potential cytotoxicity observed with the lower concentration. Once a score was given for each test condition (i.e., with 5% or 0.05% test concentrations), the total points for each test material were added to get a rank category of a 1, 2, or 3. A Rank of 1 meant that the test material was a ‘‘minimal irritant” while a Rank of 2 meant that the material was a ‘‘moderate irritant”, and a Rank of 3 meant that the test material was a ‘‘severe irritant”. The GHS eye irritation ranking were classified as NI(Not classified; not an eye irritant),Category 2(Cat.2, irritating to eyes) or category 1 (Cat. 1, irreversible effects on the eye) based on the Draize eye irritation test . To evaluate the predictive capacity of the STE test, the correspondence between STE Rank 1 and GHS NI; STE Rank 2 and GHS Cat. 2; and STE Rank 3 and Cat. 1 was calculated. The mean viability for 5% test chemical was 89.7, 88.7, 84.5 and that of 0.05% test chemical was 100.7, 95.4, 101.3 for the three labs. The CVs of the cells treated with test chemical were 2.7, 3.2 for 5% and 0.05% respectively. Based on the STE scores and ranking, it was considered to be an irritant and the GHS Classification was correlated to be Category 1.

 

This study supported with EpiOcularTMEye Irritation Test (EpiOcularTMEIT) performed to determine the degree of ocular damage caused by the given test chemical. The study was conducted according to procedures mentioned in OECD 492 Guidelines. The EpiOcular™ tissue model OCL-200 (herein referred to as EpiOcular™) was produced using good manufacturing practice (GMP) in the MatTek production facilities. EpiOcular™ tissues were packaged on a proprietary agarose formulation and stored overnight at 4 ± 2°C to mimic shipping conditions to customers. The next day, the tissues were transferred from the agarose into 6-well plates containing 1mL of medium (provided with the OCL-200 kit) and pre-incubated for one hour under standard culture conditions (SCC), which are defined as an atmosphere with 90 ± 10% relative humidity, 5± 0.5% v/v) CO2, and a temperature of 37 ± 1°C. After 1 hour, the medium was changed and the EpiOcular™ cultures were further pre-incubated overnight (16–18 hours) under SCC. On day 1 of the test, the tissues were pre-treated for 30 minutes with 20 μL of calcium and magnesium-free DPBS. If the DPBS did not spread across the tissue surface.Next, 50 μL of the control substances (H2O and methyl acetate), or approximately 50mg of solid test material, were applied topically to the EpiOcular™ tissues, the latter by using a calibrated tool. Sodium Salicylate and control was tested in duplicate tissues.After a 30-minute exposure to the test chemical or controls, each pair of tissues was successively rinsed by dipping, swirling, and decanting through a set of three 150 ml beakers prefilled with DPBS; separate beakers were used for each test chemical or control. After the final rinse and decanting, the tissues were immersed in 5mL of EpiOcular™ assay medium in a 12-well plate for 12 minutes (post-soak) at room temperature. After the post-soak period, the medium was decanted from the CCIs and the CCIs were transferred to a 6-well plate containing 1mL of warm medium (37°C) and post-incubated for 2 hours under SCC.tissues exposed to solid test samples were post-incubated for 18 hours. After the 18-hour post-incubation period, tissue viability was determined by using the MTT assay.Tissue viability was determined with the MatTek MTT viability assay kit (MTT-100) which contains MTT Concentrate (5mg/mL), MTT Diluent (Dulbecco’s Modified Eagle Medium - DMEM) and MTT Extractant (Isopropanol p.a.). Approximately 1 hour prior to use, the MTT concentrate was thawed and diluted with the MTT Diluent resulting in a final concentration of 1mg/mL. A 300 μL aliquot of the diluted MTT solution was pipetted into each well of a 24-well plate. EpiOcular™ tissue inserts were rinsed in DPBS and placed in the MTT plate, making sure that no air bubbles were trapped underneath the CCI. The tissues were then incubated under SCC for 3 hours. After incubation, each EpiOcular™ insert was removed from the plate, blotted on an absorbent paper, and transferred into a 24-well plate containing 2mL of the MTT Extractant. Alternatively, tissues were extracted for 2 hours at room temperature on a plate shaker covered with foil. After the formazan extraction period was completed, the inserts were discarded and the extract was homogenised by pipetting up and down 2–3 times. Duplicate samples of the extract (200 μL) were pipetted into separate wells of a 96-well microtitre plate, and the optical density of the samples measured at 570 nm on a plate reader. The mean viability of the positive control (Methyl acetate) was 37.0 % and the mean OD for the Negative control in the established range of 1.0 – 2.5. The mean tissue viability for Sodium Salicylate was 5.0% and 5.1% respectively.  The results of the EpiOcular EIT protocol were in concordance with the Draize test results, indicating a classification of Category 1 for the given test chemical. Hence, it was considered to be highly irritating to eyes.

 

All the above in-vitro studies are supported with in-vivo study performed according to OECD 405 Guideline by using the given test chemical in Rabbits. 3 female young adult rabbits were used for the study. 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 was instilled in the other (treated) eye of each rabbit. The eye was observed at 1, 24, 48, 72 hour and on day 7 after test item instillation. Ophthalmoscope was used for scoring of eye lesions. In the initial test, 100 mg of test item (pulverized form)was instilled into the conjunctival sac of the right eye of animal no.1 whereas the left eye of the rabbit served as the control. As animal no. 1 showed no severe ocular lesions a confirmatory test was conducted on additional two rabbits (animal no. 2 and 3); 100 mg of test item 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 at 1, 24, 48 and 72 hour observation which recovered at day 7. Untreated eye of all the three rabbits was normal throughout the experiment. The following grading scores were observed in treated eye of treated rabbits. Observation at 1 hour after instillation of test item revealed: Cornea-No ulceration or opacity in all 3 animals; Area of Opacity-Zero in all the animals;Iris:Normal in all the animals.Conjunctivae -Some blood vessels definitely hyperaemic (injected) was observed in all the animals;Chemosis:Some swelling above normal (includes nictitating membranes) was observed in all the animals. 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 animal no. 1 whereas animal no. 2 and 3 showed no ulceration or opacity; Area of Opacity-One quarter (or less) but not zero in animal no. 1 and Zero in animal no. 2 and 3;Iris:Normal in all the animals.Conjunctivae –Diffuse, crimson color; individual vessels not easily discernible was observed in all animals; Chemosis: Some swelling above normal (includes nictitating membranes) was observed in all animals. At 24 hours observation the rabbits were examined for corneal epithelium cell damage using sodium fluorescein strips and noticed 40%, 20%, 20% damage in animal no. 1, 2 and 3 respectively. Observation at 48 hour 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 animals; Area of Opacity-One quarter (or less) but not zero in all 3 animals;Iris:Normal in all the animals. Conjunctivae –Diffuse, crimson color; individual vessels not easily discernible was observed in all animals; Chemosis: Some swelling above normal (includes nictitating membranes) was observed in all animals. Observation at 72 hour 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 animals; Area of Opacity-One quarter (or less) but not zero in all 3 animals;Iris:Normal in all the animals. Conjunctivae –Diffuse, crimson color; individual vessels not easily discernible was observed in all animals; Chemosis: Some swelling above normal (includes nictitating membranes) was observed in all animals. Observation on day 7 after instillation of test item revealed: Cornea- No ulceration or opacity in all the animals; Area of Opacity- Zero in all 3 animals; Iris: Normal in all the animals. Conjunctivae – Blood vessels were normal in all 3 animals; Chemosis: No swelling (Normal) was observed in all 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, 0.00, 2.00, 1.00; 0.67, 0.00, 2.00, 1.00 and 0.67, 0.00, 2.00, 1.00 respectively. Under the experimental conditions tested, eye irritation and reversibility of effects on eyes of rabbits was observed within day 7. Hence, under the experimental test conditions, the given test chemical is “Mildly Irritating to eyes” of New Zealand White female rabbit eyes and is being classified "Category 2"as per the CLP regulation.

 

The above results are further supported by the Draize test conducted according to the OECD guidelines for the given test chemical. In the first validation, 100 ml aqueous solution or a suspension of test chemical (10%) was applied to the right eyes of male New Zealand white rabbits (2.30±2.98 kg, 13 wk of age). Left eyes remained untreated as a control. Eyes were observed at 1 hr, 4 hr, and every 24 hr thereafter for 7 days. Three rabbits were used for test. MAS [Maximum average scores] and scores at 24 hr after application calculated for cornea, iris, conjunctivae and the sum of these scores (total average score) were used for comparison with in vitro data. Isotonic sodium chloride solution was also used as a negative control. According to the classification by Kay and Calandra (1962), chemicals can be classified as follows based on their MAS scores: non-irritants = (0≤MAS <0.5),  slight irritants = (0.5≤MAS< 15), mild irritants = (15≤MAS< 25), moderate irritants = (25≤MAS <50), severe irritants  = (50≤MAS). The Maximum Average Score[MAS] obtained for 100% test chemical instilled in rabbits eyes was 83.7; whereas for 10% solution the MAS score was 0.0. According to the classification based on the MAS scores it can be considered to be an eye irritant at higher concentrations.

 

This result is supported with an eye irritation study in rabbits conducted to determine the degree of eye damage and histopathological changes when exposed to the given test chemical for longer durations. 3 New Zealand White rabbits of both sexes were used for the study. Test chemical was dissolved in Physiological saline. 15mg/ml/day of chemical was instilled in the right conjunctival sac of the rabbits.The lower lid was pulled away from the eye ball and the chemical solution was added drop wise into the conjunctival sac. The eyelids were held together for a few seconds so as to ensure contact with the ocular tissue.The left eye was the control (for scoring and histopathology).The test chemical was dosed daily for 7 days.At the end of the 7th day, the animals were kept for observation for 72 hr. A score from 0-3 (3-being the most severe) was given to eyes of each animal as per Draize test. Then they were sacrificed by administering high dose of thiopentone through the marginal ear vein and eyes, spleen, liver and brain were collected for different experiments. The mean scores at the end of 7 days of repeated exposure to test chemical for corneal opacity, iris, conjunctival redness, chemosis and discharge were 1, 3, 2, 3, 2, 2 respectively. It can be seen that prolonged treatment caused swelling in the cornea. Redness, chemosis and discharge from the conjunctiva was evident.  It showed highest degree of irritation when compared to values of control animals.Histopathologically, Animals treated with test chemical, the cornea had increased in size in comparison to control, due to oedema. Mild acute inflammation of the ciliary body was also noticed. Hence, based on the scores and histopathological observations, Test chemical was regarded as a severe to highly irritating chemical to rabbit eyes.

 

Along with these results, an eye irritation study in rabbits was conducted to determine the degree of eye damage and histopathological changes when exposed to the given test chemical for shorter durations. 3 New Zealand White rabbits of both sexes were used for the study. Test chemical was dissolved in Physiological saline. 100mg/ml of chemical was instilled in the right conjunctival sac of the rabbits. The lower lid was pulled away from the eye ball and the chemical solution was added drop wise into the conjunctival sac. The eyelids were held together for a few seconds so as to ensure contact with the ocular tissue. The left eye was the control (for scoring and histopathology).After 24 hours, the eyes were thoroughly washed with physiological saline. The animals were then kept for further observation for 72 hours and scored periodically (0, 24, 48, and 72 h). At the end of 72 hours blood was collected from the animal for hematological and biochemical parameters and then sacrificed. The mean scores at the end of 72 hours of exposure to test chemical for corneal opacity, iris, conjunctival redness, chemosis and discharge were 1,2,2,3,2,2 respectively. It showed highest degree of irritation when compared to values of control animals. Histopathologically, animals treated with test chemical showed corneal morphology similar to control. However, chronic inflammation was visible in the ciliary body and mild inflammation was evident in the choroid and retina of chemical treated animals. Hence, based on the scores and histopathological observations, it was regarded as a severe to highly irritating chemical to rabbit eyes.

 

This study further supported with an eye irritation study in rabbits was conducted to assess the irritation potential of the given test chemical. The study was performed according to Draize method. Undiluted test chemical was instilled in the eyes of 3 rabbit and observed for signs of irritation till 21 days. The reactions observed were scored according to Draize method. Mean scores are calculated for each animal from gradings at 24, 48, and 72 h after instillation of the test chemical and these “severity scores” are then used to determine the classification of the test chemical. The study was terminated on Day 14 as corneal opacity was 4 in 2 of 3 rabbits tested which was not reversible by day 21. Iris and conjunctival chemosis, conjunctival redness scores on day 14 were 0 and 2, 2 respectively. Based on these observations and scores, it can be considered to highly irritating to eyes and was classified under the category “Category 1”.

 

All these in-vivo studies are supported with the rabbit Draize ocular irritation test performed to obtain Draize Score 0 (DS0) values for the given test chemical. 0.1 ml of each test solution was placed directly into the conjunctival cul-de-sac of one eye in each animal. The other eye remained untreated and served as control. Eyes were observed and scored, according to the scale of Draize et al. (1944), at 1, 3, 6, 24, 96, and 168 hr after dose. Three to six animals per dosage group were used, and at least three dosage groups were tested to obtain DS 0.Draize Score 0 (DS0) was defined as the maximum concentration of test compound at not showing any ocular irritation.The DS 0 values were calculated from the dose-response curves obtained. The Draize score 0(DS0) concentrations for test chemical was obtained at 5% w/v. It did not show any ocular irritation, even at the high concentrations which reached the limits of their solubilities and/or physiological osmotic pressure, and their DS0 values could not be obtained. Hence, it was considered that non-ocular irritation dose which can be used for ophthalmic preparations was 5% w/v.

 

Thus, based on the available data for the given test chemical, it can be concluded that test chemical is able to cause severe 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”.

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 severe eye damage. Hence the test chemical can be classified under the category “Not Classified” for skin and “Category 2” for eye as per CLP.