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Administrative data

Description of key information

Skin Irritation

A study was designed and conducted to determine the dermal reaction profile of the test chemical in Sprague Dawley rats. The study was performed as per OECD Guidelines 402 and complying to the GLP procedures. 5 females (nulliparous and non-pregnant) were used for the study.

The overall irritation score of the substance was determined to be 0 and no erythema and edema (skin irritation) were found at the end of 14 days observation period after patch removal.

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.

An in vitro study was performed according to the OECD 439 test guideline to determine the irritation potential of the test chemical.

The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 108.2%.

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

Eye irritation

The ocular irritation potential of test article was determined according to the OECD 492 test guideline for this study.

The mean of OD for test chemical was determined to be 2.185.The mean % tissue viability of test chemical was determined to be 103.3%. Hence, under the experimental test conditions it was concluded that test chemical was considered to be not irritating to the human eyes.

An in vivo study was performed as per OECD 404 to determine the acute Eye Irritation/Corrosion potential of the test chemical in Rabbits. 3 young adult female New Zealand White rabbits were used for the study.

In the confirmatory test, the test compound when applied to the conjunctival sac of the rabbits in the amount of 0.1 gm did not produce any eye irritation or inflammation during the entire observation period.. However, some blood vessels observed hyperemic upto 24 hours after the application of test compound. There were no other signs observed throughout the observation period of 21 days.

 

Based on above findings, it can be concluded that the test compound can be considered as practically non-irritant when applied in the amount of 0.1 gm in the conjunctival sac of the rabbits under the test condition.

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:
comparable to guideline study
Justification for type of information:
data is from experimental reports
Qualifier:
according to
Guideline:
other: OECD Guideline 402 (Acute Dermal Toxicity)
Principles of method if other than guideline:
To determine the dermal reaction profile of the test chemical in Sprague Dawley rats.
GLP compliance:
yes
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: National Institute of Biosciences, Pune
- Sex: Females
- Age at study initiation: Young adult (8 to 10 weeks old) female rats
- Weight at study initiation: weight range of approximately 221.6 to 233.3 grams at initiation of dosing.
Body weights at the start :
Female
Mean : 227.60 g (= 100 %)
Minimum : 221.6 g (- 2.64 %)
Maximum : 233.3 g (+ 2.50 %)
Total No. of animals : 5
- Identification: Each rat was individually identified by the cage number.
- 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:All animals were acclimatized to laboratory conditions for minimum of 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): temperature was maintained at 19.0 to 22.5 degree centigrade
- Humidity (%): room humidity was maintained at 55.3% to 58.8%.
- Air changes (per hr): The animal room was independently provided with at least ten to fifteen air changes per hour of 100% fresh air that had been passed through the HEPA filters.
- Photoperiod (hrs dark / hrs light): An artificial light and dark cycle of 12 hours each was provided to the room.
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Controls:
not specified
Amount / concentration applied:
Dose Range Finding Study: 200, 1000, 2000 mg/kg bodyweight
Main study: 2000 mg/kg bodyweight
Duration of treatment / exposure:
24 hours
Observation period:
14 days
Number of animals:
5 female rats
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:
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 included general clinical signs, observations of eyes, mucous membranes, respiratory, circulatory system and behavior pattern.
Evaluation of Dermal Reaction:
Dermal reaction was observed daily for study period of 14 days.
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
Preparation of Animals:
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.
Irritation parameter:
erythema score
Basis:
mean
Time point:
14 d
Score:
0
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritation parameter:
edema score
Basis:
mean
Time point:
14 d
Score:
0
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
Evaluation of Dermal Reaction
Sex : Female
Dose Range Finding Study:
Group I : Animal treated at the dose level of 200 mg/kg body weight did not result in any skin reaction during the study period of 14 days.
Group I : Animal treated at the dose level of 1000 mg/kg body weight did not result in any skin reaction during the study period of 14 days.
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.
Main Study:
Group II : Animals 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:
Clinical Signs of Toxicity and Mortality
Sex : Female
Dose Range Finding Study:
Group I : Animal treated at the dose level of 200 mg/kg body weight did not result in any signs of toxicity during the study period of 14 days. The animal survived through the study period of 14 days.
Group I : Animal treated at the dose level of 1000 mg/kg body weight did not result in any signs of toxicity during the study period of 14 days. The animal survived through the study period of 14 days.
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. The animal survived through the study period of 14 days.
Main Study:
Group II : Animals 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.

Summary of Evaluation of Dermal Reaction

 

Laboratory Test Item Code :TAS/122/067

Test System : Sprague Dawley Rat

Sex : Female

Finding Study:

Group

 No.

Dose mg/kg

                          

Dermal Reaction

Total Number of

Animals

Animal Nos.

Period of signs

in days

 From - to

I

200

No dermal reaction observed

1

1

Day 0 - Day 14

 

Group

 No.

Dose mg/kg

                          

Dermal Reaction

Total Number of

Animals

Animal Nos.

Period of signs

in days

 From - to

I

1000

No dermal reaction observed

1

2

Day 0 - Day 14

 

Group

 No.

Dose mg/kg

                          

Dermal Reaction

Total Number of

Animals

Animal Nos.

Period of signs

in days

 From - to

I

2000

No dermal reaction observed

1

3

Day 0 - Day 14

 

Main Study:

Group

 No.

Dose mg/kg

                          

Dermal Reaction

Total Number of

Animals

Animal Nos.

Period of signs

in days

 From - to

II

2000

No dermal reaction observed

2

4, 5

Day 0 - Day 14

Summary of Clinical Signs of Toxicity and Mortality

 

Laboratory Test Item Code :TAS/122/067

Test System : Sprague Dawley Rat

Sex : Female

Finding Study:

Group

 No.

Dose mg/kg

                            Observed Signs

Total Number of

Animals

 

Animal Nos.

Period of signs in days

 From - to

 

Mortality

I

200

No clinical signs observed

1

1

Day 0 - Day 14

0/1

 

Group

 No.

Dose mg/kg

                            Observed Signs

Total Number of

Animals

 

Animal Nos.

Period of signs in days

 From - to

 

Mortality

I

1000

No clinical signs observed

1

2

Day 0 - Day 14

0/1

 

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

1

3

Day 0 - Day 14

0/1

 

Main Study:

Group

 No.

Dose mg/kg

                            Observed Signs

Total Number of

Animals

 

Animal Nos.

Period of signs in days

 From - to

 

Mortality

II

2000

No clinical signs observed

2

4, 5

Day 0 - Day 14

0/2

Individual Animal - Evaluation of Dermal Reaction

 

Laboratory Test Item Code :TAS/122/067

Test System : Sprague Dawley Rat

Sex : Female  

Finding Study:

Group : I                                                                      Dose  : 200 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

 

Group : I                                                                    Dose  : 1000 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

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

 

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

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

 

Main Study:

Group : II                                                                   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

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

 

 

 

Interpretation of results:
other: not irritating
Conclusions:
The overall irritation score of the substance was determined to be 0 and no erythema and edema (skin irritation) were found at the end of 14 days observation period after patch removal.
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:

A study was designed and conducted to determine the dermal reaction profile of the test chemical in Sprague Dawley rats. The study was performed as per OECD Guidelines 402 and complying to the GLP procedures. 5 females (nulliparous and non-pregnant) were used for the study.

In the dose range finding study a single dose of 200 mg/kg body weight of the test item was administered to 1 female animal. No death or clinical signs of toxicity was observed during first 48 hours, hence, additional 1 female animal was administered with the dose of 1000 mg/kg body weight. Administration of 1000 mg/kg body weight did not reveal any clinical signs of toxicity or death during first 48 hours, hence, additional 1 female animal was administered at the dose of 2000 mg/kg body weight. Administration of 2000 mg/kg body weight did not reveal any clinical signs of toxicity or death during first 48 hours.

As the dose range finding study revealed no mortality or clinical signs at the maximum dose of 2000 mg/kg, the main study was initiated with two additional animals. The animals were administered with a dose of 2000 mg/kg body weight in sequential manner at 48 hours intervals.

Animals from dose range finding study treated at the dose levels of 200 mg/kg, 1000 mg/kg and 2000 mg/kg and animals from main study treated at the dose level of 2000 mg/kg exhibited normal body weight gain and revealed no clinical signs of toxicity or mortality during the study period of 14 days.

Gross pathological examination did not reveal any abnormalities attributable to the treatment.

The overall irritation score of the substance was determined to be 0 and no erythema and edema (skin irritation) were found at the end of 14 days observation period after patch removal.

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from experimental study report
Qualifier:
according to
Guideline:
OECD Guideline 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 Assay

Following 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°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Twice
MTT 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 data

NUMBER OF REPLICATE TISSUES: 3

CALCULATIONS and STATISTICAL METHODS
All data were background subtracted before analysis. MTT data are presented as % viable compared to negative control. Data were generated as follows: MTT Assay Blanks:·        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: none
NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): neat

POSITIVE 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:
108.2
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, as the OD of the negative control tissues was between 1.195 and 1.430. Also, the positive control, 5% sodium dodecyl sulfate (SDS), reduced tissue viability to 4.5% of negative control and the standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates were passing the acceptance criteria.

Code N° Tissue  Raw data   Blank corrected data mean  % of viability
  n Aliq. 1 Aliq. 2 Aliq. 1 Aliq. 2 of aliquotes  
NC 1 2.2556 2.2412 2.220 2.206 2.213 104.0
  2 2.0192 2.0192 1.984 1.984 1.984 93.3
  3 2.1986 2.2399 2.163 2.204 2.184 102.7
PC 1 0.0837 0.0831 0.048 0.048 0.048 2.2
  2 0.1036 0.1037 0.068 0.068 0.068 3.2
  3 0.1101 0.1114 0.075 0.076 0.075 3.5
C2 1 2.1398 2.1475 2.104 2.112 2.108 99.1
  2 2.3716 2.4153 2.336 2.380 2.358 110.9
  3 2.467 2.4827 2.431 2.447 2.439 114.7

  mean SD mean of SD CV %
  of OD of OD viabilities [%] of viabilities [%]
NC 2.127 0.125 100.0 5.87 5.87
PC 0.064 0.014 3.0 0.67 22.29
C2 2.302 0.173 108.2 8.12 7.50
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 chemical was determined to be 108.2%. 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 followed 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. The objective of this study was to assess the dermal irritation potential of test article Tissues were exposed to 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, as the OD of the negative control tissues was between 1.195 and 1.430. Also, the positive control, 5% sodium dodecyl sulfate (SDS), reduced tissue viability to 4.5% of negative control and the standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates were passing the acceptance criteria.

The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 108.2%.

Hence, under the experimental test conditions it was concluded that test chemical was considered to be not irritating to the human skin and being 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

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from experimental study report.
Qualifier:
according to
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Principles of method if other than guideline:
The purpose of this study was to assess potential for the test article to be ocular irritants. The ocular irritation potential of a test article may be predicted by measurement of its cytotoxic effect, as reflected in the (MTT) assay, in the MatTek EpiOcular™ model
GLP compliance:
no
Species:
human
Strain:
other: Not applicable
Details on test animals or tissues and environmental conditions:
- Description of the cell system used:
The normal human-derived keratinocytes were cultured at the air-liquid interface in a chemically defined medium on a permeable polycarbonate insert (surface 0.5 cm2). They were cultured in chemically defined serum free medium to form a multi-layered epithelium similar to that found in native corneal mucosa. Each lot of tissues was Quality Assured by MatTek according to specific QC standards including: histology, tissue viability (MTT mean optical density), reproducibility (SD) and tissue thickness.

- Test System Identification
All of the EpiOcular™ 3-dimensional human tissues used in this study were identified by the date of arrival and the lot number. Certificate of Analysis for the tissues is included in this report. Tissue plates were appropriately labeled with study information. Bias was not a factor in this test system.
- Justification of the test method and considerations regarding applicability
EpiOcularTM Eye Irritation (OCL) by MatTek In Vitro Life Science Laboratories, Bratislava, Slovakien

The test articles and controls were evaluated for potential ocular irritancy using the EpiOcular™ 3 dimensional human tissue model purchased from MatTek,In Vitro Life Science Lab. (Bratislava, Slovakia).The EpiOcular tissue construct is a nonkeratinized epithelium prepared from normal human keratinocytes (MatTek). It models the cornea epithelium with progressively stratified, but not cornified cells. These cells are not transformed or transfected with genes to induce an extended life span in culture. The “tissue” is prepared in inserts with a porous membrane through which the nutrients pass to the cells. A cell suspension is seeded into the insert in specialized medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test material to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo. Each lot of tissues was Quality Assured by MatTek, Inc. according to specific QC standards including: histology (cell layers), tissue viability (MTT mean optical density) and reproducibility (SD).
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 mg of solid test chemical
- Concentration (if solution): neat (undiluted)

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

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Concentration (if solution): neat
Duration of treatment / exposure:
Tissues were exposed for approximately 6 hrs ± 15 min for solid test articles, and controls, at approximately 37°C, 5% CO2 in a humidified incubator.
Observation period (in vivo):
Not applicable
Duration of post- treatment incubation (in vitro):
Following the washing and post soak, the tissues were rinsed and incubated at approximately 37°C, 5% CO2 in a humidified incubator for a post-exposure recovery time of 18 hours for solid test chemicals and controls
Number of animals or in vitro replicates:
2 tissues were used for test compound and control.
Details on study design:
- Details of the test procedure used
The tissues were exposed to the test article neat (undiluted). EpiOcular™ tissues were purchased from MatTek. Quality control of the tissues was performed by MatTek and the Certificate of Analysis (CoA)
for the tissues is provided and is kept in the study binder. Tissues were exposed for approximately 6 hrs ± 15 min for solid test articles and controls at approximately 37°C, 5% CO2 in a humidified incubator.
After the exposure, the test article was rinsed off the tissues and the tissues were soaked in media for ~25 minutes for solid test articles and controls.Following the washing and post soak, the tissues were rinsed and incubated at approximately 37°C, 5% CO2 in a humidified incubator for a post-exposure recovery time of 18 hours for solid test chemicals and controls.Tissue viability was assessed by 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

- MTT Auto reduction and colouring assessment
MTT Pre-test
The test article was assessed for the potential to interfere with the assay. Approximately 50 µL of liquid test article was added to 1 mL of MTT media (~1 mg/mL) and incubated in a humidified incubator at approximately 37°C and approximately 5% CO2 for 3 hours. 50 µL of ultrapure water was used as a negative control.
- Test Article Color Test
Approximately 50 µL of liquid test article was added to 1.0 mL of ultrapure water and 2.0 mL isopropanol and incubated in a humidified incubator at approximately 37°C and approximately 5% CO2 for 2 hours, 04 minutes and 35 seconds. Samples were then added to the wells of a clear 96-well plate and the plate was read on a Thermo Scientific Multiskan FC Microplate Photometer to 570 nm. Test articles that tested positive for excessive coloration (OD >0.08) were assessed on living-tissue controls that were incubated in both culture media and MTT media as well (n=3 for both conditions).

- MTT Assay:
Inserts are removed from the 24-well plate after 3 hrs of incubation and the bottom of the insert is blotted on absorbent material, and then transferred to a pre-labeled 6-well plate containing 1 ml isopropanol in each well so that no isopropanol is flowing into the insert. At the end of the non-submerged extraction inserts and tissues are discarded without piercing and 1 ml of isopropanol is added into each well. The extract solution is mixed and the optical density of the extracted formazan (200 μL/well of a 96-well plate) was determined using Thermo Scientific Multiskan FC Microplate Photometer at 570 nm. Relative cell viability was calculated for each tissue as % of the mean negative control tissues.

- Evaluation of Test Article in the cell Models
1. Cell System:
Upon receipt, the MatTek EpiOcular™ tissue cultures were placed in 1.0 mL of fresh Maintenance medium (in a 6-well plate) for 60 minutes. After the 60 minutes incubation, the Maintenance medium was exchanged with fresh medium and the tissues were incubated overnight (16-24 hrs) at approximately 37°C, approximately 5% CO2 in a humidified incubator.
2. Control and Test Article Exposures:
20 µL of calcium and magnesium free DPBS was added to each tissue and the tissues placed back into the incubator for 30 minutes. The controls and the test article will be applied topically to tissues by pipette.2 tissues will be used per test compound and control.
a)Controls: 50 µL of negative control sterile ultrapure water and positive control methyl acetate were added to the tissues. The tissues were placed into the ~37°C humidified incubator with 5% CO2 for the approximately 30 minute exposure time.
b)Test Article: When a solid was tested, 50 mg of the solid were added to the tissues. The tissues were placed into the ~37°C humidified incubator with 5% CO2 for the approximately 6 hrs ± 15 min.
3. Post exposure treatment:
After the exposure, the tissues were rinsed 20 times with sterile of DPBS to remove test material. The apical surface was gently blotted with a cotton swab and cultures were immediately transferred to a 12-well plate containing 5 mL of media per well. Tissues exposed to liquid test articles (and the respective control) were incubated, submerged in the media for ~12 minutes at room temperature.For liquid test articles, tissues, Tissuses were then transferred to 6-well plates containing 1.0 mL fresh Maintenance medium per well and incubated for a post-exposure recovery period for 2 hours at approximately 37 degC, 5% CO2 in a humidified incubator.
- Doses of test chemical and control substances used
Test Article:
When a solid was tested, 6 hours of the solid were added to the tissues. The tissues were placed into the ~37°C humidified incubator with 5% CO2 for the approximately 6 hrs ± 15 min.
Controls: 50 µL of negative control sterile ultrapure water, positive control methyl acetate were added to the tissues. The tissues were placed into the ~37°C humidified incubator with 5% CO2 for the approximately 30 minute exposure time.

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods:
Tissues were exposed for approximately 6 hours for solid test articles and controls, at approximately37°C, 5% CO2 in a humidified incubator.
Following the post soak, the tissues were rinsed and incubated at approximately 37°C, 5% CO2 in a humidified incubator for a post-exposure recovery time totaling 18 hours for solid test articles and controls.

- Justification for the use of a different negative control than ultrapure H2O (Not applicable)
- Justification for the use of a different positive control than neat methyl acetate (Not applicable)
- Number of tissue replicates used per test chemical and controls: 2 tissues were used for test compound and control.
- Description of the method used to quantify MTT formazan
The blue formazan salt was extracted by placing the tissue insterts in 1 mL isopropanol in a 6-well plate. The extraction for solid exposed tissues was 3 hrs incubation. After an addition of 1 ml isopropanol and mixing, the optical density of the extracted formazan (200μL/well of a 96-well plate) was determined using a Thermo Scientific Multiskan FC Microplate Photometer at 570 nm.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for
the prediction model
Calculations and Statistical Methods
MTT Assay
Blanks:
· The OD mean from all replicates for each plate (ODblank).
Negative Controls (NC):
· The blank corrected value was calculated: ODNC= ODNCraw– ODblank.
· The OD mean per NC tissue was calculated.
· The mean OD for all tissues corresponds to 100% viability.
· The mean, standard deviation (SD), standard error of the mean (SEM) and the percent coefficient of variation (% CV) was calculated.
ODblank= optical density of blank samples (isopropanol alone).
ODNCraw= optical density negative control samples.
ODNC= optical density of negative control samples after background subtraction.
Positive Control (PC):
· Calculate the blank corrected value: ODPC= ODPCraw– ODblank.
· The OD mean per PC tissue was calculated.
· The viability per tissue was calculated: %PC = [ODPC/ mean ODNC] x 100.
· The mean viability for all tissues was calculated: Mean PC = Σ %PC / number of tissues.
· The standard deviation (SD), standard error of the meanthe mean (SEM) and the percent coefficient of variation (% CV) was calculated.
ODPCraw= optical density positive control samples.
ODPC= optical density of positive control samples after background subtraction.
Tested Articles:
· Calculate the blank corrected value ODTT= ODTTraw– ODblank.
· The OD mean per tissue is calculated.
· The viability per tissue is calculated: %TT = [ODTT/ mean ODNC] x 100.
· The mean viability for all tissues is calculated: Mean TT = Σ %TT / number of tissues.
· The standard deviation (SD) and the percent coefficient of variation (% CV)for the controls and the test articles will be calculated.
ODTTraw= optical density test article samples.
ODPC= optical density of test article samples after background subtraction.
Data Correction Procedure for MTT Interfering Compounds
True viability = Viability of treated tissue – Interference from test article = ODtvt – ODkt where ODkt =
(mean ODtkt – mean ODukt).
ODtvt = optical density of treated viable tissue
ODkt = optical density of killed tissues
ODtkt = optical density of treated killed tissue
ODukt = optical density of untreated killed tissue (NC treated tissue)
Data Correction Procedure for Colored Compounds
True viability = Viability of treated tissue incubated in MTT media – Viability of treated tissue incubated in
media without MTT = ODtvt – ODvt.
ODtvt = optical density of treated viable tissue incubated in MTT media
ODvt = optical density of viable tissues incubated in media alone.
Proposed Statistical methods
The mean, standard deviation (SD) and the percent coefficient of variation (% CV) for the controls and the test article will be calculated.
- Evaluation of data
The results of the assay was evaluated and compared to negative control.
Table: Irritancy Prediction
In VitroResults In VivoPrediction
Mean tissue viability ≤60% Irritant (I) – Category 1 or 2
Mean tissue viability >60% Non-irritant (NI) – No Category
- Assay quality controls
- Negative Controls (NC)
The assay is meeting the acceptance criterion if the mean viability of the NC in terms of Optical Density(OD570) of the NC tissues (treated with sterile ultrapure water) in the MTT assay are >0.8 to <2.5. This is an indicator of tissue viability following shipping and conditions under use.
- Positive Controls (PC)
Methyl acetate was used as a PC and tested concurrently with the test article. The assay is meeting the acceptance criteria if the viability of the PC is <50% of the negative control.
- Standard Deviation (SD)
Each test of ocular irritancy potential is predicted from the mean viability determined on 2 single tissues. The assay meets the acceptance criteria if SD calculated from individual percent tissue viabilities of the
replicates is <18% for three replicate tissues.
Irritation parameter:
other: mean % tissue viability
Run / experiment:
Run 1
Value:
103.3
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: mean of OD :2.185;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 OD % of viability
  n Aliq. 1 Aliq. 2 Aliq. 1 Aliq. 2
NC 1 2.1392 2.1045 2.104 2.070 2.087 98.7
  2 2.1925 2.1633 2.158 2.128 2.143 101.3
PC 1 0.692 0.715 0.657 0.680 0.669 31.6
  2 0.7927 0.7855 0.758 0.751 0.754 35.7
101-20-2 1 2.2617 2.3352 2.227 2.300 2.264 107.0
  2 2.1591 2.1219 2.124 2.087 2.106 99.6

  mean Dif. mean of Dif. Dif./2 Classification
  of OD of OD viabilities [%] of viabilities      
NC 2.115 0.056 100.0 2.65 1.33 NI qualified
PC 0.711 0.086 33.6 4.05 2.02 I qualified
101-20-2 2.185 0.158 103.3 7.47 3.73 NI qualified
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 of OD for test chemical was determined to be 2.185. The mean % tissue viability of test chemical was determined to be 103.3%. Thus, 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 liquid test articles and controls for ~30 minutes, followed by a ~12 minute post-soak and approximately 2 hour recovery after the post-soak. The viability of each tissue was determined by MTT assay.

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

The mean of OD for test chemical was determined to be 2.185.The mean % tissue viability of test chemical was determined to be 103.3%. Hence, under the experimental test conditions it was concluded that test chemical was considered to be not irritating to the human eyes.

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:
guideline study with acceptable restrictions
Justification for type of information:
data is from experimental report
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Principles of method if other than guideline:
To evaluate the acute eye irritation index of the test chemical in New Zealand White rabbit.
GLP compliance:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Age :10 to 12 weeks
-Sex: Female
- Weight :2.0kg ±200g
- Diet (e.g. ad libitum):Pelleted feed
- Water (e.g. ad libitum):Community tap water , ad libitum.
- Acclimation period:The rabbit was acclimatized to standard laboratory condition for 24 hours in experimental room before study

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22-250C
- Humidity (%):40-60%
- Air changes (per hr):10-15 air changes per hour
- Photoperiod (hrs dark / hrs light):to 12 hours artificial fluorescent light and 12 hours dark

IN-LIFE DATES: From: To:
Vehicle:
other: Dstilled water
Controls:
yes
Amount / concentration applied:
0.1 gm
Duration of treatment / exposure:
24 hours
Observation period (in vivo):
21 days
The eyes were examined at 1, 24, 48 and 72 hours after test substance application. The grades of ocular reaction (conjunctiva, cornea and iris) were recorded at each observation. To determine the reversibility of the effect the animal was observed normally for 21 days
Number of animals or in vitro replicates:
Three female rabbits
Details on study design:
TEST SITE
- Area of exposure:conjunctival sac of one eye of each animal
REMOVAL OF TEST SUBSTANCE
- Washing (if done): not washed
SCORING SYSTEM:Scale of weighted scores for grading the severity of ocular lesions developed by //Draize et al
TOOL USED TO ASSESS SCORE: hand-slit lamp / biomicroscope / fluorescein: hand-slit lamp
Examination of reactions was facilitated by use of biomicroscope and hand slit lamp. After recording the observations at 24 hours, the eyes were further examined with the aid of fluorescein.
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
110
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
The test compound when instilled into the eyes of New Zealand white rabbit at the dose level of 0.1 gm did not produce any eye irritation throughout the observation period of 72 hours. After that the animals were observed for 21 days for any eye irritation and eye discharge
Other effects:
The test compound when instilled into the conjunctival sac of rabbits did not show any observable clinical signs throughout the observation period of 21 days.

TABLE-     GRADING OF OCULAR LESIONS

 

S.NO/

SEX

 

OBSERVATION

Score

Total

Total Score

1/F

 

1 hr

24hrs

48 hrs

72 hrs

Cornea

A.       Opacity-Degree of Density

0

0

0

0

0

 

0×0×5=00

B.       Area of Cornea Involved

0

0

0

0

0

Iris

A.       Values

0

0

0

0

0

0×5=00

Conjunctivae

A.       Redness

0

0

0

0

0

 

0+0+0×5=0

B.       Chemosis

0

0

0

0

0

C.       Discharge 

0

0

0

0

0

2/F

Cornea

A.       Opacity-Degree of Density

0

0

0

0

0

 

0×0×5=00

B.       Area of Cornea Involved

0

0

0

0

0

Iris

A.       Values

0

0

0

0

0

0×5=00

Conjunctivae

A.       Redness

0

0

0

0

0

 

0+0+0×5=0

B.       Chemosis

0

0

0

0

0

C.       Discharge 

0

0

0

0

0

3/F

Cornea

A.       Opacity-Degree of Density

0

0

0

0

0

 

0×0×5=00

B.       Area of Cornea Involved

0

0

0

0

0

Iris

A.       Values

0

0

0

0

0

0×5=00

Conjunctivae

A.       Redness

0

0

0

0

0

 

0+0+0×5=0

B.       Chemosis

0

0

0

0

0

C.       Discharge 

0

0

0

0

0

Grand total

0

Mean

0.0

Eye Irritation Scoring index

0.0

Interpretation of results:
other: not irritating
Conclusions:
In the confirmatory test, the test compound when applied to the conjunctival sac of the rabbits in the amount of 0.1 gm did not produce any eye irritation or inflammation during the entire observation period.. However, some blood vessels observed hyperemic upto 24 hours after the application of test compound. There were no other signs observed throughout the observation period of 21 days.

Based on above findings, it can be concluded that the test compound can be considered as practically non-irritant when applied in the amount of 0.1 gm in the conjunctival sac of the rabbits under the test condition.
 
Executive summary:

Acute Eye Irritation/Corrosion Study of test chemical in Rabbits was performed as per OECD guideline no. 405.

3 young adult female New Zealand White rabbits were used for the study. One healthy rabbit of body weight 2.13 kg was selected for study after acclimatization. Both eyes of rabbit were examined for any abnormal discharge such as eye irritation, ocular defects or pre-existing corneal injury from eye 24 hours prior to application of test compound.

The test compound was applied in the conjunctival sac of rabbit after gently pulling the lower lid away from the eyeball at the dose rate of 0.1gm. The other eye which remain untreated, served as a control. The acute irritation to eye conjunctiva, cornea and iris was evaluated at 1, 24, 48 and 72 hours after the treatment. The grades of ocular reaction (conjunctiva, cornea and iris) were recorded at each observation. To determine the reversibility of the effect the animal was observed normally for 21 days. Any other lesions in the eye viz pannus, staining were observed and scored accordingly. Examination of reactions was facilitated by use of biomicroscope and hand slit lamp. Individual animal weight before and during the study was observed.

The test compound did not produce any eye irritation or any eye discharge. Furthermore, no other clinical signs were recorded after application of test compound such as cage side activity, pain etc. The result obtained from the initial test was confirmed in additional two animals of same sex and same dose level.

In the confirmatory test the test compound was applied in the amount of 0.1 gm in the conjunctival sac of rabbit after gently pulling the lower lid away from the eyeball. The other eye which remain untreated, served as a control. The acute irritation to eye conjunctiva, cornea and iris was evaluated at 1, 24, 48 and 72 hours after the treatment. The grades of ocular reaction (conjunctiva, cornea and iris) were recorded at each observation. To determine the reversibility of the effect the animals were observed normally for 21 days. Any other lesions in the eye viz pannus, staining were observed and scored accordingly. Examination of reactions was facilitated by use of biomicroscope and hand slit lamp. Individual animal weight before and during the study was observed.

In the confirmatory test, the test compound when applied to the conjunctival sac of the rabbits in the amount of 0.1 gm did not produce any eye irritation or inflammation during the entire observation period.. However, some blood vessels observed hyperemic upto 24 hours after the application of test compound. There were no other signs observed throughout the observation period of 21 days.

Based on above findings, it can be concluded that the test compound can be considered as practically non-irritant when applied in the amount of 0.1 gm in the conjunctival sac of the rabbits under the test condition.

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 dermal irritation/corrosion potential of the test chemical in living organisms. These include in vivo as well as in vitro experimental studies for the test chemical. The results are mentioned as follows:

A study was designed and conducted to determine the dermal reaction profile of the test chemical in Sprague Dawley rats. The study was performed as per OECD Guidelines 402 and complying to the GLP procedures. 5 females (nulliparous and non-pregnant) were used for the study.

In the dose range finding study a single dose of 200 mg/kg body weight of the test item was administered to 1 female animal. No death or clinical signs of toxicity was observed during first 48 hours, hence, additional 1 female animal was administered with the dose of 1000 mg/kg body weight. Administration of 1000 mg/kg body weight did not reveal any clinical signs of toxicity or death during first 48 hours, hence, additional 1 female animal was administered at the dose of 2000 mg/kg body weight. Administration of 2000 mg/kg body weight did not reveal any clinical signs of toxicity or death during first 48 hours.

As the dose range finding study revealed no mortality or clinical signs at the maximum dose of 2000 mg/kg, the main study was initiated with two additional animals. The animals were administered with a dose of 2000 mg/kg body weight in sequential manner at 48 hours intervals.

Animals from dose range finding study treated at the dose levels of 200 mg/kg, 1000 mg/kg and 2000 mg/kg and animals from main study treated at the dose level of 2000 mg/kg exhibited normal body weight gain and revealed no clinical signs of toxicity or mortality during the study period of 14 days.

Gross pathological examination did not reveal any abnormalities attributable to the treatment.

The overall irritation score of the substance was determined to be 0 and no erythema and edema (skin irritation) were found at the end of 14 days observation period after patch removal.

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 to determine the irritation potential of 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. The objective of this study was to assess the dermal irritation potential of test article Tissues were exposed to 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, as the OD of the negative control tissues was between 1.195 and 1.430. Also, the positive control, 5% sodium dodecyl sulfate (SDS), reduced tissue viability to 4.5% of negative control and the standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates were passing the acceptance criteria.

The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 108.2%.

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

The in vitro study is supported by an in vivo study conducted on New Zealand white rabbits in accordance with OECD 404 to assess the irritation parameter of the test chemical.

3 young adult female New Zealand White rabbits were used for the study. In the initial test one healthy rabbit of body weight 1.89 kg was selected for study after acclimatization. The test compound in the amount of 0.5 gm was applied uniformly at the different sites on the shaven back skin of animal. The hairs of back sides were removed (approximately 6 cm2) one day earlier before the treatment. The site of application was covered with impervious dressing secured in position with adhesive tape. The first patch was applied on the shaven back skin of rabbit and removed after three minutes. No serious reaction was observed at the site of application. The second patch was applied on the different shaven back side and removed after one hour. There were no signs of skin reaction observed at this site of application. Finally, a third patch was applied at a different site and was removed after four hour. No skin reaction was observed after four hours patch removal. Finally, the animal was observed for 14 days, for any irritation and corrosion.

Because no corrosive effect was observed in the initial test, a confirmatory test was done in order to confirm the irritant or negative response of the test substance by taking two additional animals. In the confirmatory test, the test compound in the amount of 0.5 gm was applied on the shaven back skin (approximately 6 cm2) of two animals each with one patch, for an exposure period of four hours. After four hours the patch was removed and the skin reactions were graded according to Draize’s method.

0.5 gm of the test chemical did not produce any clinical signs of dermal irritation at the treated sites during the observation period. The dermal irritation index of the test chemical was calculated to be 0.0. Hence, the test chemical was considered to be not irritating to skin. It can be classified under the category "Not Classified" as per CLP Regulation.

These results are supported by an open patch test method followed to determine the primary irritation potential of the test chemical on guinea pigs.

10 male albino guinea pigs of the Hartley strain per dose group weighing 400—500 g were used for the study.

During the test, hair on the back of the animal was cut with electric air clippers. A depilatory containing calcium thioglycolate was immediately applied to this area for depilation. Twenty-four hours after depilation, various concentrations of the test chemical in acetone was applied to the depilated area. The test chemical (0.03 ml) was applied to a circle on the back of the animals 1.5 cm in diameter at concentration of 0.5, 1 and 3%. The sites of application were observed for presence or absence of the skin reactions at 24 and 48 hours after application.

The test chemical did not caused skin reactions during the observation period. Hence the test material was considered to be not irritating to the skin of guinea pigs.

In the same study,an open patch test was performed on 5 white male albino rabbits using the same procedure. The test chemical did not caused skin reactions during the observation period. Hence the test material was considered to be not irritating to the rabbits’ skin.

The above results are supported by an initial 21-day cumulative irritation assay conducted to determine the irritancy potential of test chemical.

1,3 ,6 and 9% test chemical in white petrolatum and petrolatum control were applied daily to the same site on the backs of 10 white male human subjects for 21 days. Patches of non-woven fabric, 2cmx2cm, were impregnated with 0.2ml of test material and then occluded with impermeable tape.  

Each patch was removed at 24 Hrs. intervals & respective test sites rest 30 min later prior to re-patching.

Skin reactions were graded according to the following scheme: 0= no reaction; ±= questionable erythema; 1= definite erythema; 2= erythema and induration; 3= vesiculation, 4= bullous reaction. A ± reaction was given a score of 0.5.

One of the 10 subjects showed a positive response to 3% test chemical beginning 10 days after initiation of patching; this subject did not react to either of the higher (6% & 9%) concentrations. In no instance did the severity of the observed reaction exceed a grade of 1. The significance of the effect noted with 3% test chemical was questionable as it was not dose related. Based on these results, 9% concentration was considered to be of minimal irritancy potential in a 21days cumulative irritation study.

Based on the above findings the test chemical was considered to be not irritating at concentration of 1 and 6% of test chemical.

These results are further supported by a challenge patch test conducted on para-spinal skin of 213 white male human subjects in order to confirm the irritation potential of test chemical.

The test chemical was applied at concentrations of1, 3 and 9% in petrolatum to A-1 test patches held on the upper back for 48 hours under occlusive condition.

Skin reactions were graded after 48 hours of patch removal according to the following scheme: 0= no reaction; ±= questionable erythema; 1= definite erythema; 2= erythema and induration; 3= vesiculation, 4= bullous reaction. A ± reaction was given a score of 0.5.

No positive reactions were observed in any of the 213 test subjects following treatment with 1, 3 and 9% of test chemical. The lack of irritation confirmed that these concentrations are not likely to be irritating.  

Therefore the test chemical was considered to be not irritating on skin of treated human skin.

Based on the available in vivo and in vitro results, the test chemical indeed lacks the potential to cause irritation to skin. Hence, the test chemical can be considered to be not irritating to skin. Comparing the above annotations to 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 extent of ocular irritation/corrosion potential of the test chemical in living organisms. These include in vivo as well as in vitro experimental studies for the test chemical. The results are mentioned as follows:

The ocular irritation potential of test article was determined according to the OECD 492 test guideline for this study. The MatTek EpiOcular™ model was used to assess the potential ocular irritation of the test articles by determining the viability of the tissues following exposure to the test article via MTT. Tissues were exposed to liquid test articles and controls for ~30 minutes, followed by a ~12 minute post-soak and approximately 2 hour recovery after the post-soak. The viability of each tissue was determined by MTT assay.

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

The mean of OD for test chemical was determined to be 2.185.The mean % tissue viability of test chemical was determined to be 103.3%. Hence, under the experimental test conditions it was concluded that test chemical was considered to be not irritating to the human eyes.

This in vitro result is supported by an in vivo study performed as per OECD 404 to determine the acute Eye Irritation/Corrosion potential of the test chemical in Rabbits. 3 young adult female New Zealand White rabbits were used for the study. One healthy rabbit of body weight 2.13 kg was selected for study after acclimatization. Both eyes of rabbit were examined for any abnormal discharge such as eye irritation, ocular defects or pre-existing corneal injury from eye 24 hours prior to application of test compound.

The test compound was applied in the conjunctival sac of rabbit after gently pulling the lower lid away from the eyeball at the dose rate of 0.1gm. The other eye which remain untreated, served as a control. The acute irritation to eye conjunctiva, cornea and iris was evaluated at 1, 24, 48 and 72 hours after the treatment. The grades of ocular reaction (conjunctiva, cornea and iris) were recorded at each observation. To determine the reversibility of the effect the animal was observed normally for 21 days. Any other lesions in the eye viz pannus, staining were observed and scored accordingly. Examination of reactions was facilitated by use of biomicroscope and hand slit lamp. Individual animal weight before and during the study was observed.

The test compound did not produce any eye irritation or any eye discharge. Furthermore, no other clinical signs were recorded after application of test compound such as cage side activity, pain etc. The result obtained from the initial test was confirmed in additional two animals of same sex and same dose level.

In the confirmatory test the test compound was applied in the amount of 0.1 gm in the conjunctival sac of rabbit after gently pulling the lower lid away from the eyeball. The other eye which remain untreated, served as a control. The acute irritation to eye conjunctiva, cornea and iris was evaluated at 1, 24, 48 and 72 hours after the treatment. The grades of ocular reaction (conjunctiva, cornea and iris) were recorded at each observation. To determine the reversibility of the effect the animals were observed normally for 21 days. Any other lesions in the eye viz pannus, staining were observed and scored accordingly. Examination of reactions was facilitated by use of biomicroscope and hand slit lamp. Individual animal weight before and during the study was observed.

In the confirmatory test, the test compound when applied to the conjunctival sac of the rabbits in the amount of 0.1 gm did not produce any eye irritation or inflammation during the entire observation period.. However, some blood vessels observed hyperemic upto 24 hours after the application of test compound. There were no other signs observed throughout the observation period of 21 days.

 

Based on above findings, it can be concluded that the test compound can be considered as practically non-irritant when applied in the amount of 0.1 gm in the conjunctival sac of the rabbits under the test condition.

This is supported by the results of Draize test performed to assess the irritation potential of the test chemical. Undiluted test chemical was instilled in the eyes of 3rabbit 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 test chemical at 24, 48 and 72hours was 0. Based on the scores, the test chemical was considered to be not irritating to eyes.

The above results are supported by an eye irritation study was conducted on three New Zealand white albino rabbits to observe the presence or absence of ocular lesions caused by the test chemical.

A single dose of 0.1 mL equalling approximately 42 mg of test chemical (purity 98.8%) was placed in the conjunctival sac of one eye of each of three New Zealand white albino rabbits. Following application, the lids were gently held together for about 1 second to limit loss of test material. After 24 hr, treated eyes were rinsed with saline solution and evaluated at 1, 24, 48 and 72 hr and 7, 14 and 21 days for irritation according to the Draize eye irritation grading scale.

No effects in cornea, iris, conjunctivae or aqueous humour were observed in any rabbit at any time. Under the test conditions, the test material was therefore considered to be not irritating to the rabbit eye.

These results are further supported by an OECD 405 Guideline study performed for a liquid hand soap formulation containing 0.15% of test chemical to evaluate the eye irritation potential of the test chemical in six New Zealand rabbits. 

In this study, 10 uL of the undiluted test substance was applied directly to the cornea of the right eye in six rabbits. The eyes of 3 rabbits were rinsed four seconds after application by spraying 20 mL of lukewarm water gently into the eye.

In the un-rinsed group one day after treatment clear discharge, petechiae and corneal dulling over 40% of the eye were noted in all three rabbits. These symptoms were cleared after 3 days in two rabbits and by the fourth day in the third rabbit. In the rinsed group redness of the conjunctivae was noted in only one rabbit one day after application this had cleared by the second day. The other rabbits in the treated rinsed group were without irritation. The maximum average score (MAS) in the un-rinsed group was 16.7 and in the rinsed group was 0.7.

Since all the observed effects were not persisted, the test chemical was considered to be not irritating to the eyes of treated rabbits.

Based on the available in vivo and in vitro results, the test chemical indeed lacks the potential to cause irritation to eyes. Hence, the test chemical can be considered to be not irritating to eyes. Comparing the above annotations to the criteria of CLP Regulation, the test chemical can be classified under the category “Not Classified”.

Justification for classification or non-classification

Available data for the test chemical suggests that it is not likely to cause any irritation to eyes and skin.

The test chemical can be considered to be not irritating to eyes and skin and can be classified under the category “Not Classified” as per CLP regulation.