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Diss Factsheets

Administrative data

Description of key information

Skin Irritation:

Administration of the test item at 2000 mg/kg did not result in any skin reaction at the site of application during the study period of 14 days. Also, the erythema and edema score of rats was calculated as 0. Administration of the test item did not result in any signs of toxicity and mortality during the study period of 14 days. Animals exhibited normal body weight gain through the study period of 14 days. Gross pathological examination did not reveal any abnormalities attributable to the treatment.

Hence, it was concluded that the test chemical was Non-Irritating to the skin of Sprague Dawley rats under the experimental conditions tested and Classified as “Category- Not Classified” as per CLP Classification.

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.

The mean of OD for test chemical was determined to be 2.240. The standard deviation of viabilities for test chemical were calculated to be 6.93.The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 107.4%. Hence, under the current experimental test conditions it was concluded that test chemical was considered to be non-irritating to human skin.

Eye Irritation:

The MAS score for the test chemical was calculated to be 8.7 and score measured at day 1 was found to be 7.7.

The test chemical was classified as Non irritant according to EEC(1984) directives and Mild irritant according toKay and Calandra (1962).

A multinational interlaboratory study was conducted to investigate the bovine corneal opacity and permeability (BCOP) assay.The Mean In vitro score for the test chemical was determined to be 12.9. Based on the classification scheme for BCOP assay, the test chemical was a non- irritant.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

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:
other: OECD Guideline 402 (Acute Dermal Toxicity)
Principles of method if other than guideline:
The study was designed and conducted to determine the dermal Irritation/corrosion potential of the test chemical in Sprague Dawley rats.
GLP compliance:
yes
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: National Institute of Biosciences, Pune.
- Females (if applicable) nulliparous and non-pregnant: No data available
- Age at study initiation: Young adult male and female rats aged between 8 – 12 weeks were used.
- Weight at study initiation: The weight range of approximately 211.3 to 235.7 grams at initiation of dosing.
Body weights at the start :
Male
Mean : 233.06 g (= 100 %)
Minimum : 228.8 g (- 1.83 %)
Maximum : 235.7 g (+ 1.13 %)
Total No. of animals : 5
Female
Mean : 216.42 g (= 100 %)
Minimum : 211.3 g (- 2.37 %)
Maximum : 220.0 g (+ 1.65 %)
Total No. of animals : 5
- Identification: Each rat was individually identified by the cage number.
- Fasting period before study: No data
- Housing: The rats were individually housed in polycarbonate cages with paddy husk as bedding.
- Diet (e.g. ad libitum): Rodent feed supplied by the Nutrivet Life Sciences, Pune, was provided ad libitum from individual feeders.
- Water (e.g. ad libitum): Water was provided ad libitum from individual bottles attached to the cages. All water was from a local source and passed through the reverse osmosis membrane before use.
- Acclimation period: 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6 to 22.6 degree centigrade.
- Humidity (%): 55.1% to 59.7%
- Air changes (per hr): Ten to fifteen air changes per hour.
- Photoperiod (hrs dark / hrs light): An artificial light and dark cycle of 12 hours each was provided to the room.

IN-LIFE DATES: 09-08-2017 to 24-08-2017
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
water
Remarks:
(Distilled water)
Controls:
not specified
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2000 mg/kg bw
- Concentration (if solution): No data available

VEHICLE
- Amount(s) applied (volume or weight with unit): No data available
- Concentration (if solution): No data available
- Lot/batch no. (if required): No data available
- Purity: No data

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): No data available
- Concentration (if solution): No data available

POSITIVE CONTROL
- Amount(s) applied (volume or weight): No data available
- Concentration (if solution): No data available
Duration of treatment / exposure:
24 hours
Observation period:
14 days
Number of animals:
10 (5/sex).
Details on study design:
TEST SITE
- Area of exposure: Trunk (dorsal surface and sides from scapular to pelvic area)
- % coverage: Approximately 10% of the body surface area.
- Type of wrap if used: Porous gauze dressing and non-irritating tape.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Distilled water was used to remove residual test item.

OBSERVATION TIME POINTS
(indicate if minutes, hours or days) : Dermal reaction was observed daily for study period of 14 days.

SCORING SYSTEM: Draize Method.

OTHER OBSERVATIONS
Type and Frequency of Tests, Analyses and Measurements

Viability: Twice daily.

Clinical Observations and General Appearance:
Animals were observed for clinical signs, mortality, until sacrifice.
Onset, duration and severity of any sign were recorded. The clinical signs and mortality observations were conducted at 10, 30, 60 minutes, 2, 4 and 6 hours on the day of dosing and once daily thereafter for 14 day. Daily observation was done as far as possible at the same time.
The observations were included general clinical signs, observations of eyes, mucous membranes, respiratory, circulatory system and behavior pattern.

Body weights:
Individual animal body weights were recorded pre-test (prior to administration of the test item), day 7 and at termination on day 14.

Gross Pathology:
Necropsy was performed on animals surviving at the end of the study. Macroscopic examination of all the orifices, cavities and tissues were made and the findings were recorded. All animals surviving the study period were sacrificed by the carbon dioxide asphyxiation technique (day 15).

Histopathology:
No gross abnormalities were observed in animals sacrificed terminally hence, no histopathology was performed.
Irritation parameter:
erythema score
Basis:
mean
Time point:
14 d
Score:
0
Max. score:
4
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritation parameter:
edema score
Basis:
mean
Time point:
14 d
Score:
0
Max. score:
4
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
Overall result:
Sex : Male
Group I -
Animal treated at the dose level of 2000 mg/kg body weight did not result in any skin reaction during the study period of 14 days.

Sex : Female
Group I -
Animal treated at the dose level of 2000 mg/kg body weight did not result in any skin reaction during the study period of 14 days.
Other effects:
Other effects:
Clinical Signs of Toxicity and Mortality
Sex : Male
Group I -
Animal treated at the dose level of 2000 mg/kg body weight did not result in any signs of toxicity during the study period of 14 days. All animals survived through the study period of 14 days.

Sex : Female
Group I -
Animal treated at the dose level of 2000 mg/kg body weight did not result in any signs of toxicity during the study period of 14 days. All animals survived through the study period of 14 days.

Body Weight
Sex : Male
Group I (2000 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 10.37% and 21.31% respectively.

Sex : Female
Group I (2000 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 4.21% and 9.81% respectively.

Gross Pathological Findings
Gross pathological examination did not reveal any abnormalities in animals from 2000 mg/kg dose group.

Table No. I

Summary of Clinical Signs of Toxicity and Mortality

Test System : Sprague Dawley Rat

Sex : Male

Group

 No.

Dose mg/kg

                            Observed Signs

Total Number of

Animals

 

Animal Nos.

Period of signs in days

 From - to

 

Mortality

I

2000

No clinical signs observed

5

1 - 5

Day 0 - Day 14

0/5

 

 

Sex : Female

Group

 No.

Dose mg/kg

                            Observed Signs

Total Number of

Animals

 

Animal Nos.

Period of signs in days

 From - to

 

Mortality

I

2000

No clinical signs observed

5

6 - 10

Day 0 - Day 14

0/5

 

Table No. II

Summary of Evaluation of Dermal Reaction

Test System : Sprague Dawley Rat

Sex : Male 

Group

 No.

Dose mg/kg

                          

Dermal Reaction

Total Number of

Animals

 

Animal Nos.

Period of signs

in days

 From - to

 

Mortality

I

2000

No dermal reaction observed

5

1 - 5

Day 0 - Day 14

0/5

 

Sex : Female

Group

 No.

Dose mg/kg

                          

Dermal Reaction

Total Number of

Animals

 

Animal Nos.

Period of signs

in days

 From - to

 

Mortality

I

2000

No dermal reaction observed

5

6 - 10

Day 0 - Day 14

0/5

 

Appendix No.II

Individual Animal - Evaluation of Dermal Reaction

Test System : Sprague Dawley Rat

Sex : Male  

Group : I

Dose  : 2000 mg/kg body weight

Animal

Dermal

D A Y S

No.

Reaction

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

1

Erythema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

Oedema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

2

Erythema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

Oedema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

3

Erythema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

Oedema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

4

Erythema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

Oedema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

5

Erythema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

Oedema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

Sex : Female  

Group : I

Dose  : 2000 mg/kg body weight

Animal

Dermal

D A Y S

No.

Reaction

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

6

Erythema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

Oedema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

7

Erythema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

Oedema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

8

Erythema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

Oedema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

9

Erythema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

Oedema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

10

Erythema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

Oedema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

 

 

Table No.III

Mean Body Weight and Percent Body Weight Gain (g)

Test System : Sprague Dawley Rat

Sex : Male

Group No.

Dose

(mg/kg body weight)

 

Body weight Day 0

Body weight Day 7

% body weight gain

day 0-7

Body weight Day 14

% body weight gain

day 7- 14

% body weight gain

day 0- 14

I

2000

Mean

233.06

257.18

10.37

282.68

9.93

21.31

± SD

2.83

4.98

3.00

4.06

1.14

2.69

 

 

 

Sex : Female

Group No.

Dose

(mg/kg body weight)

 

Body weight Day 0

Body weight Day 7

% body weight gain

day 0-7

Body weight Day 14

% body weight gain

day 7- 14

% body weight gain

day 0- 14

I

2000

Mean

216.42

225.56

4.21

237.70

5.38

9.81

± SD

3.60

7.69

2.26

8.55

1.19

2.32

 

Table No.IV

Summary of Gross Pathological Findings

Test System : Sprague Dawley Rat

Sex : Male

Group No.

Dose

mg/kg

Animal Numbers

Animal Fate

Gross Pathological Findings

I

2000

1 - 5

TS

No abnormality detected

Sex : Female 

Group No.

Dose

mg/kg

Animal Numbers

Animal Fate

Gross Pathological Findings

I

2000

6 - 10

TS

No abnormality detected

  TS = Terminal Sacrifice

Interpretation of results:
other: Not irritating
Conclusions:
Administration of the test item at 2000 mg/kg did not result in any skin reaction at the site of application during the study period of 14 days. Also, the erythema and edema score of rats was calculated as 0. Administration of the test item did not result in any signs of toxicity and mortality during the study period of 14 days. Animals exhibited normal body weight gain through the study period of 14 days. Gross pathological examination did not reveal any abnormalities attributable to the treatment.
Hence, it was concluded that the test chemical was Non-Irritating to the skin of Sprague Dawley rats under the experimental conditions tested and Classified as “Category- Not Classified” as per CLP Classification.
Executive summary:

The study was designed and conducted to determine the dermal Irritation/corrosion potential of the test chemical in Sprague Dawley rats. This study was performed as per OECD guideline No. 402. Ten rats (5 male and 5 female) were used for conducting dermal irritation/ corrosion study.

 

The animals were kept in their cages for at least 5 days prior to administration for acclimatization to the laboratory condition and after acclimatization period, animals were randomly selected. Approximately 24 hours before application, the hair of each rat was closely clipped from the trunk (dorsal surface and sides from scapular to pelvic area) with an electric clipper, so as to expose at least 10% of the body surface area. The test item was moistened with distilled water. The test item was applied onto the exposed skin of the animal, taking care to spread the test item evenly over the entire area of approximately 10% of the total body surface area or as much of the area as can reasonably be covered. The test item was held in contact with the skin using a porous gauze dressing and non irritating tape around the animal to cover the exposure site for first 24 hours exposure period. Elizabethan collar was placed on each animal for first 24 hours after application of the test item. These collars prevent ingestion of test item. Following 24 hours of exposure, the wrapping was removed and the test site wiped free of excess test item. Distilled water was used to remove residual test item.

 

The test chemical was applied to shorn skin of 5 male and 5 female animals at 2000 mg/kg body weight. Administration of the test item at 2000 mg/kg did not result in any skin reaction at the site of application during the study period of 14 days. Also, the erythema and edema score of rats was calculated as 0. Administration of the test item did not result in any signs of toxicity and mortality during the study period of 14 days. Animals exhibited normal body weight gain through the study period of 14 days. Gross pathological examination did not reveal any abnormalities attributable to the treatment.

Hence, it was concluded that the test chemical was Non-Irritating to the skin of Sprague Dawley rats under the experimental conditions tested and Classified as “Category- Not Classified” as per CLP Classification.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 05, 2017 to July 17, 2017
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 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, in the MatTek EpiDerm™ model (MatTek Corp., Ashland, MA).
GLP compliance:
no
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: EpiDerm™ 3-dimensional human tissues used in this study
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, Ashland, MA) 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 June 28, 2017 (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 for MTT auto-reduction and coloring was not performed for this study but was based on the results obtained from another study (CYP1690_R1b).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 2 hours, 57 minute and 25 second 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 2 hours 04 minutes and 11 seconds with gentle shaking. The optical density of the extracted formazan (200 µL/well of a 96-well plate) was determined using a Synergy H4 spectrophotometer 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 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. Tissues were exposed to controls and the test articles 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, 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 ArticleFor solid test article, the tissues were moistened with 25 μL of ultrapure water to improve contact of the tissue surface with the test article. Approximately 25 mg of each test article was evenly applied to the apical surface of each tissue (n=3). All the tissues were placed into the ~37°C incubator with 5% CO2. The exposure times were approximately 1 hour, with ~35 minutes exposure in the incubator and ~25 minutes at room temperature. 3.Post-exposure treatmentAfter the 1 hour exposure, the tissues were rinsed 20 to 25 times with 1 mL of 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 either 25 hours, 38 minutes and 23 seconds or for 24 hours, 10 minutes and 09 seconds (as there were numerous tissues, they had to be broken down into 2 sets to complete dosing in a timely manner). 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 17 hours, 03 minutes and 34 seconds prior to performing the MTT assay, 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: TwiceMTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE- MTT concentration: 300 µL MTT medium (1.0 mg/mL).- Incubation time: After 2 hours, 57 minute and 25 second MTT incubation- 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 (undiluted)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- 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 will be used per test compound and control.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Run 1
Value:
107.4
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The MTT data show the assay quality controls were met.
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 of OD for test chemical was determined to be 2.240.The standard deviation of viabilities for test chemical were calculated to be 6.93.The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 107.4%. 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 of OD for test chemical was determined to be 2.240. The standard deviation of viabilities for test chemical were calculated to be 6.93.The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 107.4%. Hence, under the current experimental test conditions it was concluded that test chemical was considered to be non-irritating to human skin.

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

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data from peer review publication
Qualifier:
according to guideline
Guideline:
other: EEC (1984 and 1991) and French (1984 and 1991) directives with few modifications
Principles of method if other than guideline:
The experiments were carried out according to the EEC (1984 and 1991) and French (1984 and 1991) directives, with few modifications: ocular lesions (cornea, iris and conjunctiva) were scored
GLP compliance:
not specified
Species:
rabbit
Strain:
not specified
Details on test animals or tissues and environmental conditions:
no data
Vehicle:
not specified
Controls:
not specified
Amount / concentration applied:
No data
Duration of treatment / exposure:
No data
Observation period (in vivo):
1 hr, then at 1, 2, 3, 4, 7 and 14 days.
Number of animals or in vitro replicates:
3
Details on study design:
REMOVAL OF TEST SUBSTANCE
Washing (if done): Yes , for 3 rabbits
Time after start of exposure: No data

SCORING SYSTEM: Only ocular lesions (cornea, iris and conjunctiva) were scored (Draize et al., 1944) at 1 hr, then at 1, 2, 3, 4, 7 and 14 days. In the case of positive score, eyes were also examined at 21 days.
Raw data were also used to classify chemicals according to the rating system described by Kay and Calandra (1962), and the EC criteria.
Irritation parameter:
other: Day 1 score
Basis:
mean
Time point:
other: 1 hr
Score:
7.7
Reversibility:
fully reversible within: 4 days
Remarks on result:
no indication of irritation
Irritation parameter:
other: Maximal Average Score (MAS)
Basis:
mean
Time point:
other: 1 hr
Score:
8.7
Reversibility:
fully reversible within: 4 days
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
Non irritant according to EEC(1984) directives and Mild irritant according to Kay and Calandra (1962)

CAS

In vivo Scores

Reversibility

(DAYS)

Class

MAS

DAY 1

K-C*

EEC#

92 -43 -3

8.7

7.7

4

Mild

NI

 

MAS – Maximal Average Score

* Kay and Calandra (1962):

PN=practically non-irritant; Min=minimally irritant; Mod=moderately irritant, Sev = severely irritant; Extr = extremely irritant; Max = maximally irritant.

 

# EEC (1984) risk phrases for ocular irritancy;

NI = non-irritant; R36 = irritant; R41 = severely irritant.

Interpretation of results:
other: not irritating
Conclusions:

The MAS score for the test chemical was calculated to be 8.7 and score measured at day 1 was found to be 7.7.
The test chemical was classified as Non irritant according to EEC(1984) directives and Mild irritant according toKay and Calandra (1962).
Executive summary:

The test chemical was evaluated for in vivo eye irritation in rabbits.The tests were performed according to EEC (1984 and 1991) and French (1984 and 1991) directives with few modifications: ocular lesions (cornea, iris and conjunctiva) were scored.

Three rabbits were used per test compound, and maximal average score (MAS) as well as score at day 1 were calculated. At the request of the participants, any mass of material present in the conjunctival sac was removed after the 1-hr observation. Also, a fluorescein solution was used for observation of corneal lesions. Only ocular lesions (cornea, iris and conjunctiva) were scored (Draize et al.,1944) at 1 hr, then at 1, 2, 3, 4, 7 and 14 days. In the case of positive score, eyes were also examined at 21 days. Raw data were also used to classify chemicals according to the rating system described by Kay and Calandra (1962), and the EC criteria (1984). The MAS score for the test chemical was calculated to be 8.7 and score measured at day 1 was found to be 7.7.

The test chemical was classified as Non irritant according to EEC(1984) directives and Mild irritant according toKay and Calandra (1962).

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:
10.3
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
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 OD % of viability
  n Aliq. 1 Aliq. 2 Aliq. 1 Aliq. 2
NC 1 2.205 2.1744 2.170 2.139 2.155 102.3
  2 2.0851 2.097 2.050 2.062 2.056 97.7
PC 1 0.6042 0.6195 0.569 0.584 0.577 27.4
  2 0.6827 0.699 0.648 0.664 0.656 31.1

92-43-3 1 0.2434 0.2507 0.208 0.216 0.212 10.1
  2 0.2562 0.2553 0.221 0.220 0.221 10.5

  mean Dif. mean of Dif. Dif./2 Classification
  of OD of OD viabilities [%] of viabilities      
NC 2.105 0.099 100.0 4.69 2.34 NI qualified
PC 0.616 0.079 29.3 3.75 1.88 I qualified

92-43-3 0.216 0.009 10.3 0.41 0.21 I qualified
Interpretation of results:
other: 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 be10.3.%. 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 be10.3%. Hence, under the experimental test conditions it was concluded that test substance was considered to be irritating to the human eyes and can thus be classified as‘’Irritating to eyes in Category 2” as per CLP Regulation

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin Irritation

Various studies have been summarized to determine the degree of irritation caused by the test chemical in living organisms. These results include in vivo as well as in vitro experimental data for the test chemical.

The study was designed and conducted to determine the dermal Irritation/corrosion potential of the test chemical in Sprague Dawley rats. This study was performed as per OECD guideline No. 402. Ten rats (5 male and 5 female) were used for conducting dermal irritation/ corrosion study.

The animals were kept in their cages for at least 5 days prior to administration for acclimatization to the laboratory condition and after acclimatization period, animals were randomly selected. Approximately 24 hours before application, the hair of each rat was closely clipped from the trunk (dorsal surface and sides from scapular to pelvic area) with an electric clipper, so as to expose at least 10% of the body surface area. The test item was moistened with distilled water. The test item was applied onto the exposed skin of the animal, taking care to spread the test item evenly over the entire area of approximately 10% of the total body surface area or as much of the area as can reasonably be covered. The test item was held in contact with the skin using a porous gauze dressing and non irritating tape around the animal to cover the exposure site for first 24 hours exposure period. Elizabethan collar was placed on each animal for first 24 hours after application of the test item. These collars prevent ingestion of test item. Following 24 hours of exposure, the wrapping was removed and the test site wiped free of excess test item. Distilled water was used to remove residual test item.

The test chemical was applied to shorn skin of 5 male and 5 female animals at 2000 mg/kg body weight. Administration of the test item at 2000 mg/kg did not result in any skin reaction at the site of application during the study period of 14 days. Also, the erythema and edema score of rats was calculated as 0. Administration of the test item did not result in any signs of toxicity and mortality during the study period of 14 days. Animals exhibited normal body weight gain through the study period of 14 days. Gross pathological examination did not reveal any abnormalities attributable to the treatment.

Hence, it was concluded that the test chemical was Non-Irritating to the skin of Sprague Dawley rats under the experimental conditions tested and Classified as “Category- Not Classified” as per CLP Classification.

The in vivo result is supported by an in vitro study performed according to the OECD 439 test guideline for the test chemical. 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 of OD for test chemical was determined to be 2.240. The standard deviation of viabilities for test chemical were calculated to be 6.93.The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 107.4%. Hence, under the current experimental test conditions it was concluded that test chemical was considered to be non-irritating to human skin.

A single dose skin irritation study was performed on guinea pigs to assess the dermal irritation potential of the test chemical.

Single dose of the test chemical was applied to depilated abdomen skin of 3 guinea pigs under occlusion and observed for effects. (duration, observation period not mentioned). The dermal reactions were scored according to the scale- slight, moderate, severe. Slight irritation was observed in 3 of the 3 guinea pigs tested with the test chemical. Hence, the test chemical can be considered to be slightly irritating to skin.

The above single application is further supported by a repeated application skin irritation study performed on guinea pigs to assess the dermal irritation potential of the test chemical. Undiluted test chemical was applied daily to the open dorsal skin of 5 guinea pigs for 10 days. The treated skin was observed throughout the 10 days for signs of irritation.

Repeated application of the test chemical caused mild exacerbation of the initial response obtained in the single exposure test. Hence, the test chemical can be considered to be slightly irritating to skin when subjected to repeated exposure.

In another Skin irritation study was conducted in White Vienna rabbits to observe irritation potential of the test chemical. The study was performed according to OECD TG 404.About 500mg of the unchanged test substance (moistened with water) was applied onto a shaven area of 2.5 x 2.5 cm of the upper third of the back or flanks of 6 rabbits under semi occlusion. The untreated skin served as negative control. The test site was exposed for 4 hours and skin reactions were evaluated at 24, 48 and 72 hours after test substance application.

No oedema was noted in any rabbit at any time. Slight erythema (grade 1) was observed in all animals 1 day after the exposure period. This finding persisted until day 2 after exposure in two out of six rabbits. The total mean values were 0.1 for redness and 0.0 for oedema. Since there were no findings the study was terminated after 72 hours after exposure. Hence the test chemical was considered to be not irritating to the skin of White Vienna rabbits.

Eventhough the results of the single exposure and repeated exposure to guinea pigs gave indication of slight irritation to skin, but the in vivo and in vitro results claim otherwise. Also , when tested at the maximum applicable dose of 2000mg/kg in rats, no signs of erythema or edema were observed till 14 days of observation. Keeping all these factors in to consideration, the test chemical can be considered to be not irritating to skin. Comparing the above annotations with the criteria of CLP Regulation, the test chemical can be classified under the category “Not Classified”.

Eye irritation:

Various studies have been summarized to determine the degree of irritation caused by the test chemical in living organisms. These results include in vivo as well as in vitro experimental data for the test chemical.

The test chemical was evaluated for in vivo eye irritation in rabbits. The tests were performed according to EEC (1984 and 1991) and French (1984 and 1991) directives with few modifications: ocular lesions (cornea, iris and conjunctiva) were scored.

Three rabbits were used per test compound, and maximal average score (MAS) as well as score at day 1 were calculated. At the request of the participants, any mass of material present in the conjunctival sac was removed after the 1-hr observation. Also, a fluorescein solution was used for observation of corneal lesions. Only ocular lesions (cornea, iris and conjunctiva) were scored (Draize et al.,1944) at 1 hr, then at 1, 2, 3, 4, 7 and 14 days. In the case of positive score, eyes were also examined at 21 days. Raw data were also used to classify chemicals according to the rating system described by Kay and Calandra (1962), and the EC criteria (1984). The MAS score for the test chemical was calculated to be 8.7 and score measured at day 1 was found to be 7.7.

The test chemical was classified as Non irritant according to EEC(1984) directives and Mild irritant according to Kay and Calandra (1962).

This is supported by the results of a Draize test performed in rabbits to assess the irritation potential of the test chemical. Undiluted test chemical was instilled in the eyes of rabbits 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 mean corneal opacity score of undiluted of the test chemical at 24, 48 and 72 was 0. Hence, the test chemical was considered to be not irritating to eyes, was classified under the category “Not Classified”.

The above in vivo results are supported by a multinational interlaboratory study conducted to investigate the bovine corneal opacity and permeability (BCOP) assay. The assays were carried out in 12 European laboratories with different types of activity. In each of these laboratories 52 substances, with a wide range of structure, physical form and irritant properties, were tested andin vitro scores were compared. The Bovine Corneal Opacity and Permeability (BCOP) assay, which is based on the method of Muir, is an in vitro test which has been developed recently. Originally, it was devoted to predicting the irritant potential of process intermediates compounds in development.

 

Bovine eyes were collected from a commercial abattoir in a plastic jar containing 1 litre HBSS for approximately 25 eyes. Buffer storage and eyes transportation to the laboratories were performed at ambient temperature, and the eyes were used within 2 hr of the killing of the animals. During dissection great care was taken to avoid damage of corneal surfaces (epithelial and endothelial). Each cornea was carefully examined, and those presenting defects, such as neovascularization, pigmentation, opacity or scratches were discarded. Globes were first dissected free of surrounding tissues and placed in a jar containing fresh HBSS. Selected corneas were dissected with a 2-3-mm rim of sclera for easier handling, and stored in a petri dish containing HBSS until use. Corneas were then mounted in holders filled with MEM, and incubated for 1 hr in a water-bath at 32°C. At the end of the incubation period, the medium was removed from both compartments of the holders, using a needle attached to a vacuumpump or a syringe. The posterior compartment wasthen refilled with MEM, whereas the anterior compartmentwas filled with the test compound or itsvehicle.

Solids were prepared as an approximate 20% solution or suspension (200mg + 1 ml) in MEM using a mortar and pestle for homogeneous preparations, and a volume of 0.75 ml was applied on the epithelium with an appropriate syringe and needle. Corneas were placed in a horizontal position for 4 hr at 32°C. The compound was then removed and the epithelium was washed at least three times (until the cornea was free of particles; gentle swirling movements of the holders were sometimes necessary) with approximately 4ml medium. Both compartments were refilled with medium, and opacity measurement was performed immediately without any further incubation. The number of corneas per experiment was 15, including a control group of three corneas treated with MEM, and two groups of six corneas treated with test compounds.

The opacitometer (Electro- Design, Riom, France) determines the difference in light transmission between a treated and a control cornea, and displays a numerical opacity value (arbitrary units).

The second step of the assay was performed immediately after the measurement of opacity. The medium was again removed from both chambers of holders, and replaced by fresh medium in the posterior compartment, and by 1 ml fluorescein solution (0.4% for liquids and surfactants, 0.5% for solids) in contact with the epithelium. Corneas were then incubated in a horizontal position for 90 min at 32°C. Medium from the posterior chamber was then removed, and its optical density (O.D.) determined with a spectrophotometer at 490 nm.

The Mean In vitro score for the test chemical was determined to be 12.9. Based on the classification scheme for BCOP assay, the test chemical was a non- irritant.

The above results are supported by an eye irritation study performed on rabbits to assess the eye irritation potential of the test chemical.

Undiluted test chemical was instilled in to the eyes of 6 rabbits. The eyes of 3 rabbits were washed and the remaining eyes remained unwashed throughout the test. The eyes were stained with fluorescein to observed effects on the cornea and adnexa.

Application of the test chemical to the washed and unwashed rabbit eyes resulted in slight irritation. Prompt washing had a palliative effect as the unwashed eyes had fluorescein stained adnexa but the washed eyes did not.

Hence, the test chemical can be considered to be slightly irritating to eyes.

In another study, the EpiOcular-EIT tissue model was used to assess the irritation potential of the test chemical.

The EpiOcular tissue model (OCL-200] was obtained from MATTEK Corporation was used for this assay.EpiOcular tissues were transferred from the agarose into 6-well plates containing 1ml of assay medium (provided with the testing kit) and pre-incubated for 1 hour underStandard culture conditions (SCC), which are defined as an atmosphere with a relative humidity of 95 ± 3%, 5 ± 0.5% (v/v) Carbon dioxide, 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 Standard culture conditions (SCC). On day 1 of the test, the tissues were pre-treated for 30 minutes with 20μl of calcium and magnesium- free DPBS [Dulbecco’s Phosphate Buffered Saline]. If the DPBS did not spread across the tissue surface, the plate was tapped to ensure that the entire tissue surface was in contact with the liquid. Next, 50μl of the Negative Control (ultrapure H2O), the Positive Control (methyl acetate) or approximately 50 mg of the test chemical was topically applied to the EpiOcular tissues. Each chemical [test and controls] were tested in duplicates. The treated tissues were incubated under Standard culture conditions (SCC) for 90 minutes. For rinsing the tissues, three 150ml beakers were filled with 100ml DPBS for each test article. After a 90-minute exposure to the test articles or controls, each pair of duplicate tissues was successively rinsed by dipping, swirling, and decanting through its set of three beakers. 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 thecell culture inserts [CCIs] and thecell culture inserts [CCIs] containing the tissues were transferred to a 6-well plate containing 1ml of warm medium (37°C) and post-incubated for 2 hours under Standard culture conditions [SCC].The tissues exposed to test chemical were post- incubated for 18 hours.After the 18-hour post-incubation period, tissue viability was determined by using the MTT assay.

The test chemicals were classified as an irritant (GHS Category 1 or 2), if the tissue viability was ≤ 60%, and as a non-irritant (GHS unclassified), if the viability was > 60%. Since the Cell culture insert[CCI] insert 1 exhibited poor performance to some chemicals, EIT studies were conducted and results were reported for EpiOcular tissues grown on the other three alternative Cell culture inserts[CCI]. The mean viabilities (%) obtained from these Cell culture inserts[CCI] were compared with that of Cell culture inserts[CCI] insert 1.

Variability in tissue viability between trials was high for the test chemical. The differences between runs exceeded 30%.

The mean percent tissue viability for the test chemical in the EpiOcular-EIT assay was 69.57%.According to the GHS classification criteria, test chemical can be classified under the category “Not Classified”.

In an another in vitro assay performed according to the OECD 492 test guideline to determine the ocular irritation potential of the test chemical. 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 be10.3%. Hence, under the experimental test conditions it was concluded that test substance was considered to be irritating to the human eyes and can thus be classified as ‘’Irritating to eyes in Category 2” as per CLP Regulation

Even though the results of one in vivo test in rabbits and one in vitro assay claim that the test chemical can cause irritation to eyes, but the remaining in vivo and in vitro studies provide stronger evidence regarding the non-irritant nature of the test chemical. Taking into consideration the all these factors, the test chemical can be considered to be not irritating to eyes. Comparing the above annotations with the criteria of CLP Regulation, the test chemical can be classified under the category “Not Classified”.

Justification for classification or non-classification

Available studies for the test chemical indicate a strong possibility that the test chemical can be considered to be not irritating to skin and eyes. Hence, the test chemical can be classified under the category “Not Classified”.