Bis(4-(1,1,3,3-tetramethylbutyl)phenyl)amine

Endpoint summary

Description of key information

Skin irritation

The dermal irritation potential of target chemical was assessedin various in- vitro and in-vivo experimental studies which were conducted for test chemical. Based on the available key data and supporting study,it can be concluded that the testchemical 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 assessedin various in- vitro and in-vivo experimental studies which were conducted for test chemical.Based on the available key data and supporting study,it can be concluded that the testchemical is unable to cause eye 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 ".

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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 study report

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Principles of method if other than guideline:

The objective of the study was to assess the irritant and/or corrosive effects of test chemical after dermal application on the intact skin in rabbits.

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Species:Rabbit (Oryctolagus cuniculus)
Strain :New Zealand White
Age :4.0 to 5.5 Months (Approximately)
Sex :Female
Number of Animals: Three
Supplier/Source:Procured from GENTOX BIOSERVICES PVT. LTD., HYDERABAD, India.
CPCSEA Registration No.: 1242/bc/08/CPCSEA.
Health Status :Healthy young adult rabbits were used for the study.
Females were nulliparous and non-pregnant.
Body weight of animals:Minimum: 2.042 kg & Maximum: 2.146 kg (Prior to Treatment)
Acclimatisation :Rabbits were acclimatised to the test conditions for a period of 10 days (Animal No. 1) and 13 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 study no., study type, test system, sex, dose, animal number, experimental start and completion date.

Diet:All animals were provided conventional laboratory rabbit diet (Nutrivet Life Sciences, Pune) ad libitum. Batch No.: 200009.
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.

Temperature : Minimum: 19.00 °C Maximum: 21.40 °C
Relative humidity: Minimum: 41.90% Maximum: 65.80%
Light-dark-rhythm: 12:12
Air Changes : More than 12 changes per hour

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):0.5 gm
VEHICLE (distilled water)
- Amount(s) applied (volume or weight with unit):0.5 ml of distilled water
- Lot/batch no. (if required):N/A
- Purity:N/A

Duration of treatment / exposure:

4 hours

Observation period:

72 hours

Number of animals:

3 female rabbits

Details on study design:

TEST SITE
- Area of exposure:The dorsal lumbar region at contralateral sites.
- % coverage:Approximately 6 X 6 cm.
- Type of wrap if used:A porous gauze dressing and non-irritating tape (Micropore 3”).
REMOVAL OF TEST SUBSTANCE
- Washing (if done):The residual test item was removed by using cotton soaked in distilled water.
- Time after start of exposure:4 hour
SCORING SYSTEM:Draize Method

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: 24 hrs,48 hrs,72 hrs

Score:

0

Max. score:

4

Reversibility:

no data

Remarks on result:

other: not irritating

Irritation parameter:

edema score

Basis:

mean

Time point:

other: 24 hrs,48 hrs,72 hrs

Score:

0

Max. score:

4

Reversibility:

no data

Remarks on result:

other: not irritating

Irritant / corrosive response data:

The following were observed in treated rabbits.
The patch was removed after 4 hours and rabbits were observed for erythema and oedema at 1, 24, 48 and 72 hours after patch removal, evaluated and graded as per draize method.
After 4 hours of exposure in Animal No. 1,very slight erythema (barely perceptible) and no oedema was observed at 1 hour of observation.At 24 , 48 and
72 hour no erythema and no oedema was observed.
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.
At 1,24,48 and 72 hours,no erythema and oedema was observed in Animal No. 2.
Animal no. 3 at 1 hour observation revealed very slight erythema (barely perceptible) and no oedema.At 24 , 48 and 72 hour,Animal No. 3 revealed no erythema and no oedema and was found to be normal throughout the experimental period.
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:

Body weight:
Increase in body weight at terminal sacrifice as compared to day 0 in all the three animals.
Clinical signs:
No systemic toxicity was observed at treated rabbits during the experimental period.
Mortality:
No mortality was observed during the observation period.

Table 1

Skin Reaction

 

In Treated area Dose:0.5 g of test item (Pulverized form)                       Sex:Female

 

Animal No.	Test	Treated  area	Erythema score				Oedema score
			1h	24h	48h	72h	1h	24h	48h	72h
1	Initial	Right	1	0	0	0	0	0	0	0
2	Confirmatory	Right	0	0	0	0	0	0	0	0
3		Left	1	0	0	0	0	0	0	0

 

 

 

 

In Control areaDose:0.5 ml of distilled water                                                        Sex:Female

 

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	Left	0	0	0	0	0	0	0	0
3		Right	0	0	0	0	0	0	0	0

Key: h = Hour.

Erythema                                                                                                       Oedema

0 =No erythema                                                                                           0 =No oedema

1 = Very slight erythema(barely perceptible)

Mean Individual Animal Score at 24, 48 and 72 hours (Treated Site)

 

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

 

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, the test chemical was re
garded as non-Irritating to the skin of Female New Zealand White rabbits under the experimental conditions tested and is thus not classified as a skin irritant.

Executive summary:

Acute Dermal Irritation/corrosion Study of test chemical was conducted in Rabbits.This study was performed as per OECD guideline No. 404.Three healthy young adult Female rabbits were used for conducting acute dermal irritation/corrosion study.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 g (pulverized form)test item moistened with 0.5 ml distilled water wasappliedto the skin,over an area of approximately 6 x 6 cm clippedof haironone 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,very slight erythema (barely perceptible) and no oedema was observed at 1 hour of observation. At 24, 48 and 72 hours observation no erythema and no oedema was observed.Hence the confirmatory test was conducted on additional two rabbits (No. 2 and 3)to confirm the non irritant nature of the test item. The patch was removed after 4 hours and rabbits wereobserved for erythema and oedemaat 1, 24, 48 and 72 hours after patch removal, evaluatedand graded as per draize method.At 1, 24, 48 and 72 hours, no erythema and oedema was observed in Animal No. 2. Animal No. 3 at 1 hour observation post patch removal revealed very slight erythema (barely perceptible) and no oedema. At 24, 48 and 72 hours, Animal No. 3 revealed no erythema and no oedema.The other untreated side revealed no erythema and no oedema and was found to be normal throughout the experimental period.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.  No erythema and no oedema (skin irritation) were found at the end of 72 hour observation period after patch removal.Hence, it was concluded that test chemical was Non-Irritating to the skin of Female New Zealand White rabbits under the experimental conditions.

Reference 2

Endpoint:

skin 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 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Principles of method if other than guideline:

The purpose of this study was to assess potential for the test article to be dermal irritants. The dermal irritation potential of test article may be predicted by measurement of their cytotoxic effect, as reflected in the assay, in the MatTek EpiDerm™ model.

GLP compliance:

no

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (Not applicable)-
Radiochemical purity: N/A-
Specific activity: N/A-
Locations of the label: N/A-
Expiration date of radiochemical substance: N/A
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL-
Storage condition of test material: Room temperature or Fridge storage-
Stability under test conditions: No data available
- Solubility and stability of the test substance in the solvent/vehicle: No data available
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No data available

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Prior to the main test, the test articles are tested for their ability to reduce/interact with MTT and their ability to stain the tissues itself. All tests are performed according to the by MatTek provided test protocol.
- Preliminary purification step (if any): No data available
- Final dilution of a dissolved solid, stock liquid or gel: No data available
- Final preparation of a solid: No data available
FORM AS APPLIED IN THE TEST:
SolidOTHER SPECIFICS: No data available

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: as provided by MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

Source strain:

other: Not applicable

Details on animal used as source of test system:

- 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 epidermis. 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 IdentificationAll of the EpiDerm™ 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 are included in this report. Tissue plates were appropriately labeled with study information.

Justification for test system used:

The 3-Dimensional Human Dermal Epithelial Model (EpiDerm™, MatTek, In Vitro Life Science Laboratories, Bratislava, Slovakia) is made up of normal human keratinocytes in serum free medium. The cells form an epithelial tissue that consists of organized basal, spinous, granular, and cornified layers analogous to those found in vivo. The EpiDerm™ model also contains epidermis-specific differentiation markers such as pro-filaggrin, the K1/K10 cytokeratin pair, involucrin, and type I epidermal transglutaminase, as well as keratohyalin granules, tonofilament bundles, desmosomes, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns characteristic of in vivo epidermis. 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). Tissue plates were appropriately labeled with study information. Bias was not a factor in this test system.

Vehicle:

unchanged (no vehicle)

Details on test system:

The tissues were exposed to the test article neat (undiluted) on April 25, 2018 (Run 1 of 1). EpiDerm™ 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 1 hour, with 35 minutes in an approximately 37°C, 5% CO2 humidified incubator and the remaining 25 minutes at room temperature. Following the exposure time, the tissues were rinsed and placed in fresh media for approximately 24 hours. The media was then changed again and the tissues were incubated in fresh media for another ~18 hours for a total of approximately 42 hour post-exposure recovery period. The tissue viability was then assessed by MTT assay. The tissue CoA was used instead of verification of barrier properties of the tissue.MTT and Color Pre-testsPretesting has actually been conducted for all chemicals, although the first intitial 8 test chemicals a pretesting was not conducted (for skin).MTT AssayFollowing the rinsing period, the MTT assay was performed by transferring the tissues to 24-well plates containing 300 µL MTT medium (1.0 mg/mL). After 3 hours 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 time was approximately 3 hours with gentle shaking. 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 is 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 EpiDerm™ tissue cultures were placed in 0.9 mL of fresh Maintenance medium (in a 6-well plate). The culture inserts are incubated for ~one hour. The tissues were then transferred to 6-well plates containing 0.9 mL fresh Maintenance medium and they were incubated overnight (18 ± 3 hrs) at ~37°C, 5% CO2 in a humidified incubator.2. Control and Test Article Exposures: On the day of dosing, the tissues are then removed from the incubator and the controls and the test article are applied topically to tissues by pipette(liquid) Tissues were exposed to controls and the test article for one hour, with ~35 minutes in a 37°C, 5% CO2 humidified incubator and the remaining 25 minutes at room temperature.a) Controls30 µL of negative control DPBS and 30 μL of the positive control 5% SDS was applied topically to the tissue and gently spread by placing a nylon mesh on the apical surface of each tissue, if necessary.b) Test Articles 25 mg of the test article was applied topically to the tissue 3. Post-exposure treatmentAfter the 1 hour exposure, the tissues were rinsed 15 times with sterile DPBS. After the 15th rinse from washing bottle, each insert wasw completely submerge 3 times in 150 ml DPBS. The apical surface was gently blotted with a cotton swab. The tissues were placed in 0.9 mL of fresh Maintenance medium (6-well plate) for 24 ± 2 hours. After this initial ~24 hour incubation, the tissues were placed in 6-well plates containing 0.9 mL fresh Maintenance medium and incubated for another 18 ± 3 hours, for a total of an approximately 42 hour post-exposure incubation.RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE- Model used: The EpiDerm™ 3 dimensional human tissue model- Tissue Lot number(s): 26459- Date of initiation of testing: 6/08/2017TEMPERATURE USED FOR TEST SYSTEM- Temperature used during treatment / exposure: 37°C- Temperature of post-treatment incubation (if applicable): 37°CREMOVAL OF TEST MATERIAL AND CONTROLS-Volume and number of washing steps: The test substance was rinsed from the tissues with sterile DPBS by filling and emptying the tissue insert 15 times to remove any residual test material. This was followed by completely submerge the insert 3 times in 150 ml DPBS.MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE- MTT concentration: 300 µL MTT medium (1.0 mg/mL).- Incubation time: After 3 hours- Spectrophotometer: Synergy H4 spectrophotometer - Wavelength: 570 nm- Filter: No data- Filter bandwidth: No data- Linear OD range of spectrophotometer: No dataNUMBER OF REPLICATE TISSUES: 3CALCULATIONS and STATISTICAL METHODSAll data were background subtracted before analysis. MTT data are presented as % viable compared to negative control. Data were generated as follows: MTT AssayBlanks:·        The optical density (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. 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. Tested compound :·        Calculate the blank corrected value ODTT= ODTTraw– ODblank. ·        The OD mean per tissue was calculated. ·        The viability per tissue was calculated: %TT = [ODTT/ mean ODNC] x 100. ·        The mean viability for all tissues was calculated: Mean TT = Σ %TT / number of tissues. ·        The standard deviation (SD), standard error of the mean (SEM) and the percent coefficient of variation (% CV) was calculated. Data Correction Procedure for MTT Interfering Compounds (if applicable)True viability = Viability of treated tissue – Interference from test article = ODtvt– ODktwhere ODkt= (mean ODtkt– mean ODukt).ODtvt= optical density of treated viable tissueODkt= optical density of killed tissuesODtkt= optical density of treated killed tissueODukt= optical density of untreated killed tissue (NC treated tissue) Data Correction Procedure for Colored Compounds (if applicable)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 mediaODvt= optical density of viable tissues incubated in media alone - Evaluation of data The results of the assay was evaluated and compared to negative control. Table: Criteria for in vitro Interpretation: In VitroResults In VivoPredictionMean tissue viability ≤50% Irritant (I), R38Mean tissue viability >50% Non-irritant (NI)- Assay quality controls- Negative Controls (NC)The Dulbecco’s phosphate buffered saline (DPBS) was used as a NC. The assay passed all acceptance criteria if the ODs of the negative control exposed tissues were between ≥0.8 and ≤2.8.  - Positive Controls (PC)5% solution of sodium dodecyl sulfate was used as a PC. The assay is meeting the acceptance criteria if the viability of the PC is ≤20% of the negative control.   - Standard Deviation (SD)The standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates was ≤18.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL- Amount(s) applied (volume or weight with unit):25 mg- Concentration (if solution): neat VEHICLE (Not used)- Amount(s) applied (volume or weight with unit): none- Concentration (if solution): none- Lot/batch no. (if required): none- Purity: noneNEGATIVE CONTROL- Amount(s) applied (volume or weight): 30 µL sterile DPBS- Concentration (if solution): neatPOSITIVE CONTROL- Amount(s) applied (volume or weight): 30 µL- Concentration (if solution): 5% solution of sodium dodecyl sulfate

Duration of treatment / exposure:

The exposure times were approximately 1 hour, with ~35 minutes exposure in the incubator and ~25 minutes at room temperature.

Duration of post-treatment incubation (if applicable):

For a total of an approximately 42 hour post-exposure incubation.

Number of replicates:

3 tissues were used for test compound and control.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Run 1

Value:

103.5

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Non irritant

Other effects / acceptance of results:

The MTT data show the assay quality controls were met.

Code N°	Tissue	Raw data		Blank corrected data		mean	% of viability
	n	Aliq. 1	Aliq. 2	Aliq. 1	Aliq. 2	of aliquotes
NC	1	2.1514	2.211	2.116	2.164	103.7	2.1514
	2	2.2157	2.121	2.181	2.151	103.0	2.2157
	3	1.9602	1.969	1.925	1.947	93.3	1.9602
PC	1	0.0819	0.048	0.047	0.047	2.3	0.0819
	2	0.0786	0.044	0.044	0.044	2.1	0.0786
	3	0.0858	0.053	0.051	0.052	2.5	0.0858
C4	1	1.8047	1.8249	1.770	1.790	1.780	85.3
	2	2.2468	2.3414	2.212	2.306	2.259	108.2
	3	2.5173	2.4352	2.482	2.400	2.441	117.0

Interpretation of results:

other: not irritating

Conclusions:

The dermal irritation potential of test article was determined according to the OECD 439 test guideline followed for this study. The Mean % tissue viability compared to negative control (n=3) of the test substance was determined to be 103.5%. Thus, test chemical was considered to be not irritating to the human skin.

Executive summary:

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 103.5%.

Hence, under the current experimental test conditions it was concluded that test substance was considered to be not irritating to human skin and can thus be classified as “Not Classified'' as per CLP Regulation.

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 experimental study report

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Principles of method if other than guideline:

The objective of the study was to assess the irritant and/or corrosive effects of 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 : 4.0 to 5.5 Months (Approximately)
Sex : Male
Number of Animals: Three
Supplier/Source: Procured from LIVEON BIOLABS PVT. LTD., INDIA
(CPCSEA Reg. No. 1610/PO/RcBiBt/S/2012/CPCSEA).
Health Status : Healthy young adult animals were used for the study.
Body weight of animals: Minimum: 1.832 kg and Maximum: 2.530 kg
(Prior to Treatment)
Acclimatisation : Rabbits were acclimatised to the test conditions for a period of 5 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 card was labelled with at least study no., study type, test system, sex, dose, animal no. experiment start date and experiment completion date.

Diet: All animals were provided conventional laboratory rabbit diet (Nutrivet Life Sciences, Pune) ad libitum. Batch No. 200010 and 200011.
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.

Temperature : Minimum: 19.30 °C Maximum: 21.20 °C
Relative humidity: Minimum: 49.10 % Maximum: 61.50 %
Light-dark-rhythm: 12:12
Air Changes: More than 12 changes per hour

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):100 mg
- Concentration (if solution):N/A
VEHICLE
- Amount(s) applied (volume or weight with unit):N/A
- Concentration (if solution):N/A
- Lot/batch no. (if required):N/A
- Purity:N/A

Duration of treatment / exposure:

24 hr

Observation period (in vivo):

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

Number of animals or in vitro replicates:

3 female rabbits

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The treated eye of rabbit was washed with normal saline
- Time after start of exposure: 24 hrs

SCORING SYSTEM: Grading of irritation lesions was carried out as per Draize Method

TOOL USED TO ASSESS SCORE: ophthalmoscope

Irritation parameter:

cornea opacity score

Basis:

animal: #1,#2 and #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

not specified

Remarks on result:

other: no indication of irritation for treated eye and untreated eye

Irritation parameter:

iris score

Basis:

animal: #1,#2 and #3

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

not specified

Remarks on result:

other: no indication of irritation For treated eye and untreated eye

Irritation parameter:

conjunctivae score

Basis:

animal: #1,#2 and #3

Time point:

24/48/72 h

Score:

0

Max. score:

3

Reversibility:

not specified

Remarks on result:

other: no indication of irritation For treated eye and untreated eye

Irritation parameter:

chemosis score

Basis:

animal: #1,#2 and #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

not specified

Remarks on result:

other: no indication of irritation For treated eye and untreated eye

Irritant / corrosive response data:

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 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 observed in all the animals; Chemosis: No swelling (Normal) was observed 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: Blood vessels normal in all the animals; Chemosis: No swelling (Normal) was observed in all the animals.
At 24 hours observation the rabbits were examined for corneal epithelium cell damage using sodium fluorescein strips and noticed 0%, 0% and 0% damage in animal no. 1, 2 and 3 respectively.
Observation at 48 and 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: Blood vessels normal in all the animals; Chemosis: No swelling (Normal) was observed in all the animals.The individual mean score (treated site) 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, 0.00, 0.00; 0.00, 0.00, 0.00, 0.00 and 0.00, 0.00, 0.00, 0.00 respectively

Other effects:

Clinical Observation:
No systemic toxicity was observed in treated rabbits during the experimental period.
Mortality:
No mortality was observed during the observation period.
Body weight:
Increase in body weights of all the animals at terminal sacrifice as compared to day 0 in all the three animals was observed

Table 1 : Individual Animal Eye Irritation Scores

 

Treated Dose:0.1 g of test item (in pulverized form)                                              Sex:Male

Animal Number	1
Side of Instillation	Right
Eye Reactions	*	Hour(s)
		1	24		48		72
Cornea	0	0	0		0		0
Area of Opacity	0	0	0		0		0
Iris	0	0	0		0		0
Conjunctiva	0	1	0		0		0
Chemosis	0	0	0		0		0
Corneal Damage (%)	0%	./.	0%	./.		./.

 

Animal Number	2
Side of Instillation	Right
Eye Reactions	*	Hour(s)
		1	24	48	72
Cornea	0	0	0	0	0
Area of Opacity	0	0	0	0	0
Iris	0	0	0	0	0
Conjunctiva	0	1	0	0	0
Chemosis	0	0	0	0	0
Corneal Damage (%)	0%	./.	0%	./.	./.

 

 

Animal Number	3
Side of Instillation	Right
Eye Reactions	*	Hour(s)
		1	24	48		72
Cornea	0	0	0	0		0
Area of Opacity	0	0	0	0		0
Iris	0	0	0	0		0
Conjunctiva	0	1	0	0		0
Chemosis	0	0	0	0		0
Corneal Damage (%)	0%	./.	0%		./.	./.

Key:*= Pre-treatment eye examination;  ./.= Not Applicable

 

 

 

 

 

 

Table 1 Continued…

Untreated Control                                                                                                                 Sex:Male

Animal Number	1
Side of Instillation	Left
Eye Reactions	*	Hour(s)
		1	24	48	72
Cornea	0	0	0	0	0
Area of Opacity	0	0	0	0	0
Iris	0	0	0	0	0
Conjunctiva	0	0	0	0	0
Chemosis	0	0	0	0	0
Corneal Damage (%)	0%	./.	0%	./.	./.

 

 

Animal Number	2
Side of Instillation	Left
Eye Reactions	*	Hour(s)
		1	24	48		72
Cornea	0	0	0	0		0
Area of Opacity	0	0	0	0		0
Iris	0	0	0	0		0
Conjunctiva	0	0	0	0		0
Chemosis	0	0	0	0		0
Corneal Damage (%)	0%	./.	0%		./.	./.

 

 

Animal Number	3
Side of Instillation	Left
Eye Reactions	*	Hour(s)
		1	24	48	72
Cornea	0	0	0	0	0
Area of Opacity	0	0	0	0	0
Iris	0	0	0	0	0
Conjunctiva	0	0	0	0	0
Chemosis	0	0	0	0	0
Corneal Damage (%)	0%	./.	0%	./.	./.

Key:*= Pre-treatment eye examination;  ./.= Not Applicable

Interpretation of results:

other: not irritating

Conclusions:

The individual mean score (treated site) 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, 0.00, 0.00; 0.00, 0.00, 0.00, 0.00 and 0.00, 0.00, 0.00, 0.00 respectively.Hence, test chemical is “Non Irritant” to New Zealand White Female rabbit eyes and is thus not classified as a eye irritant.

Executive summary:

Acute Eye Irritation/Corrosion Study of test chemical was conducted in Rabbits. This study was performed as per OECD guideline no. 405.Rabbits free from injury of eye were selected for the study. The eyes of all the rabbits were examined 24 hours prior to treatment. One eye of each rabbit served as control and other as treated. Control eye was left untreated whereas; 0.1 gof test item (pulverised form)was instilled in the other (treated) eye of each rabbit.The eye was observed at 1, 24, 48 and 72 hour after test item instillation.Ophthalmoscope was used for scoring of eye lesions. In the initial test,0.1 g 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 hence a confirmatory test was conducted on additional two rabbits (Animal No. 2 and 3); 0.1 gof test item (pulverized form)was 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 was 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) was observed in all the animals;Chemosis:No swelling (Normal) was observed 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:Blood vessels normal in all the animals;Chemosis:No swelling (Normal) was observed in all the animals. At 24 hours observation the rabbits were examined for corneal epithelium cell damage using sodium fluorescein strips and noticed 0%, 0% and 0% damage in animal no. 1, 2 and 3 respectively. Observation at 48 and 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:Blood vessels normal in all the animals;Chemosis:No swelling (Normal) was observed in all the animals.The individual mean score (treated site) 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, 0.00, 0.00; 0.00, 0.00, 0.00, 0.00 and 0.00, 0.00, 0.00, 0.00 respectively.Hence, test chemical is “Non Irritant” to New Zealand White Female rabbit eyes and is thus not classified as a eye irritant.

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 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:

88.2

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

Mean O.D.:1.883; Non-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	2.2532	2.2372	2.219	2.203	2.211	103.6
	2	2.098	2.0888	2.064	2.054	2.059	96.4
PC	1	0.5744	0.5927	0.540	0.558	0.549	25.7
	2	0.7763	0.7786	0.742	0.744	0.743	34.8
Test chemical	1	1.8047	1.824	1.770	1.790	1.780	83.4
	2	2.0148	2.0246	1.980	1.990	1.985	93.0

Interpretation of results:

other: not irritating

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 88.2%. Thus, the test chemical was considered to be not 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 (50mg) 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 88.2%.

Hence, under the experimental test conditions it was concluded that test substance was considered to be not irritating to the human eyes and can thus be classified as "Not Classified" as per CLP Regulation.

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin irritation

Various studieshave 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-vitro and in-vivo experiments conducted for target chemicalwhich have beensummarized as below;

 

Acute Dermal Irritation/corrosion Study of test chemical was conducted in Rabbits. This study was performed as per OECD guideline No. 404.Three healthy young adult Female rabbits were used for conducting acute dermal irritation/corrosion study. 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 g (pulverized form)test item moistened with 0.5 ml distilled water was appliedto 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,very slight erythema (barely perceptible) and no oedema was observed at 1 hour of observation. At 24, 48- and 72-hours observation no erythema and no oedema was observed. Hence the confirmatory test was conducted on additional two rabbits (No. 2 and 3)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 after patch removal, evaluated and graded as per draize method. At 1, 24, 48 and 72 hours, no erythema and oedema was observed in Animal No. 2. Animal No. 3 at 1 hour observation post patch removal revealed very slight erythema (barely perceptible) and no oedema. At 24, 48 and 72 hours, Animal No. 3 revealed no erythema and no oedema.The other untreated side revealed no erythema and no oedema and was found to be normal throughout the experimental period. 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.  No erythema and no oedema (skin irritation) were found at the end of 72 hour observation period after patch removal. Hence, it was concluded that test chemical was Non-Irritating to the skin of Female New Zealand White rabbits under the experimental conditions.

The above in-vivo result was supported by an in-vitro test conducted for test chemical according to the OECD 439 test guideline. 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 103.5%. Hence, under the current experimental test conditions it was concluded that test substance was not irritating to human.

 

Based on the above summarized studies for target chemical,it can be concluded that the testchemical 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:

Various studieshas been investigated for the test chemical to observe the potential for ocular irritation to a greater or lesser extent. The studies are based on in-vitro and in-vivo experiments conducted for target chemicalwhich have beensummarized as below;

 

Acute Eye Irritation/Corrosion Study of test chemical was conducted in Rabbits. This study was performed as per OECD guideline no. 405.Rabbits free from injury of eye were selected for the study. The eyes of all the rabbits were examined 24 hours prior to treatment. One eye of each rabbit served as control and other as treated. Control eye was left untreated whereas; 0.1 g of test item (pulverised form)was instilled in the other (treated) eye of each rabbit. The eye was observed at 1, 24, 48 and 72 hour after test item instillation. Ophthalmoscope was used for scoring of eye lesions. In the initial test,0.1 g 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 hence a confirmatory test was conducted on additional two rabbits (Animal No. 2 and 3); 0.1 gof test item (pulverized form)was 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 was 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) was observed in all the animals;Chemosis:No swelling (Normal) was observed 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:Blood vessels normal in all the animals;Chemosis:No swelling (Normal) was observed in all the animals. At 24 hours observation the rabbits were examined for corneal epithelium cell damage using sodium fluorescein strips and noticed 0%, 0% and 0% damage in animal no. 1, 2 and 3 respectively. Observation at 48 and 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:Blood vessels normal in all the animals;Chemosis:No swelling (Normal) was observed in all the animals.The individual mean score (treated site) 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, 0.00, 0.00; 0.00, 0.00, 0.00, 0.00 and 0.00, 0.00, 0.00, 0.00 respectively. Hence, test chemical is “Non Irritant” to New Zealand White Female rabbit eyes and is thus not classified as an eye irritant.

 

The above in-vivo result was supported by an in-vitro test conducted for test chemical 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 solid test articles (50mg) 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 88.2%. Hence, under the experimental test conditions it was concluded that test substance was considered to be not irritating to the human eyes.

 

Based on the above summarized studies for target chemical,it can be concluded that the testchemical is unable to cause eye 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”.

Justification for classification or non-classification

 The skin and eye irritation potential of test chemical was observed in experimental studies. The results obtained from these studies indicates that the chemical is not likely to cause skin irritation and eye irritation. Hence the test chemical can be classified under the category “Not Classified” for skin irritation and eye irritation as per CLP.