Registration Dossier

Diss Factsheets

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 female young adult, non-pregnant rats 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 2000 mg/kg bw 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 female Sprague Dawley rats under the experimental conditions tested and classified as “Category- Not Classified” as per CLP Classification.

Eye Irritation:

Based on the available in vitro and in vivo data, it was concluded that the test chemical was not observed to be an ocular irritant and thus under the experimental conditions tested and classified as “Category- Not Classified” as per CLP Classification.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from an experimental report.
Qualifier:
according to guideline
Guideline:
other: Acute Dermal toxicity (OECD 402 Guidelines)
Principles of method if other than guideline:
According to Acute Dermal toxicity (OECD 402 Guidelines)
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
- Age at study initiation: Young adult (8 to 10 weeks old)
- Sex: Female. Females were nulliparous and non-pregnant
- Weight at study initiation: weight range of approximately 222.6 to 242.1 grams at initiation of dosing.
Body weights at the start :
Female
Mean : 232.68 g (= 100 %)
Minimum : 222.8 g (- 4.25 %)
Maximum : 242.1 g (+ 4.05 %)
Total No. of animals : 5
- Housing: The rats were individually housed in polycarbonate cages with paddy husk as bedding.
- Identification: Each rat was individually identified by the cage number.
- Diet (e.g. ad libitum): Rodent feed supplied by the Nutrivet Life Sciences, Pune, was provided ad libitum
- 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): 19.2 to 21.8 degree centigrade
- Humidity (%): 56.0% to 59.1%.
- Air changes (per hr): 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 roomDose Range Finding Study: 200, 1000, 2000 mg/kg bodyweight
Main study: 2000 mg/kg bodyweight
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Controls:
not required
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 females
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 for 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 days. 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
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 reactions
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/122/065

Test System : Sprague Dawley Rat

Sex : Female

Dose 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

Interpretation of results:
other: Non-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, as no signs of erythema and oedema was observed at limit dose of 2000 mg/kg bw. Hence, it was concluded that the test chemical was not 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 female young adult, non-pregnant rats were used for the 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 applied directly 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. A single dose of 200 mg/kg, 1000 mg/kg and 2000 mg/kg body weight of the test item were applied to 1 female animal respectively in the dose range finding study. Since no signs of irritation were observed at the maximum dose of 2000 mg/kg in the test animals, 2 additional animals were tested in the main study with 2000 mg/kg.The animals were applied with a dose of 2000 mg/kg body weight in sequential manner at 48 hours intervals. Dermal reaction was observed daily for study period of 14 days. 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 female Sprague Dawley rats under the experimental conditions tested and classified as “Category- Not Classified” as per CLP Classification.

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

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from experimental study report.
Qualifier:
according to guideline
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Principles of method if other than guideline:
The purpose of this study was to assess potential for the test article to be ocular irritants. The ocular irritation potential of a test article may be predicted by measurement of its cytotoxic effect, as reflected inthe 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, in the MatTek EpiOcular™ model
GLP compliance:
no
Species:
human
Strain:
other: Not applicable
Details on test animals or tissues and environmental conditions:
- Description of the cell system used:
The normal human-derived keratinocytes were cultured at the air-liquid interface in a chemically defined medium on a permeable polycarbonate insert (surface 0.5 cm2). They were cultured in chemically defined serum free medium to form a multi-layered epithelium similar to that found in native corneal mucosa. Each lot of tissues was Quality Assured by MatTek according to specific QC standards including: histology, tissue viability (MTT mean optical density), reproducibility (SD) and tissue thickness.

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

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

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

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Concentration (if solution): neat
Duration of treatment / exposure:
Tissues were exposed for approximately 30 minutes for liquid test article and controls, at approximately 37°C, 5% CO2 in a humidified incubator.
Observation period (in vivo):
Not applicable
Duration of post- treatment incubation (in vitro):
Following the washing step and the post-soak, the tissues were incubated at approximately 37°C, 5% CO2 in a humidified incubator for a post-exposure recovery time of ~2 hours for liquid test articles , or 18 hrs for solid test articles, and controls.
Number of animals or in vitro replicates:
2 tissues were used for test compound and control.
Details on study design:
- Details of the test procedure used
The tissues were exposed to the test article neat (undiluted). EpiOcular™ tissues were purchased from MatTek. Quality control of the tissues was performed by MatTek and the Certificate of Analysis (CoA) for the tissues is provided and is kept in the study binder. Tissues were exposed for approximately 30 minutes for liquid test articles and controls, at approximately 37°C, 5% CO2 in a humidified incubator. After the exposure, the test article was rinsed off the tissues and the tissues were soaked in media for ~12 minutes for liquid test articles and controls. Following the washing step and the post-soak, the tissues were incubated at approximately 37°C, 5% CO2 in a humidified incubator for a post-exposure recovery time of ~2 hours for liquid test articles and controls. Tissue viability was assessed by 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

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

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

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

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

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

Data Correction Procedure for Colored Compounds
True viability = Viability of treated tissue incubated in MTT media – Viability of treated tissue incubated in media without MTT = ODtvt – ODvt.
ODtvt = optical density of treated viable tissue incubated in MTT media
ODvt = optical density of viable tissues incubated in media alone.
Proposed Statistical methods
The mean, standard deviation (SD) and the percent coefficient of variation (% CV) for the controls and the test article will be calculated.
- Evaluation of data
The results of the assay was evaluated and compared to negative control.
Table: Irritancy Prediction
In VitroResults In VivoPrediction
Mean tissue viability ≤60% Irritant (I) – Category 1 or 2
Mean tissue viability >60% Non-irritant (NI) – No Category
- Assay quality controls
- Negative Controls (NC)
The assay is meeting the acceptance criterion if the mean viability of the NC in terms of Optical Density (OD570) of the NC tissues (treated with sterile ultrapure water) in the MTT assay are >0.8 to <2.5. This is an indicator of tissue viability following shipping and conditions under use.
- Positive Controls (PC)
Methyl acetate was used as a PC and tested concurrently with the test article. The assay is meeting the acceptance criteria if the viability of the PC is <50% of the negative control.
- Standard Deviation (SD)Each test of ocular irritancy potential is predicted from the mean viability determined on 3 single tissues. The assay meets the acceptance criteria if SD calculated from individual percent tissue viabilities of the replicates is <18% for three replicate tissues.
Irritation parameter:
other: mean % tissue viability
Run / experiment:
Run 1
Value:
28.4
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
mean of OD:0.797, 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 3.1117 2.9793 3.074 2.942 3.008 107.2
  2 2.6914 2.5897 2.654 2.552 2.603 92.8
PC 1 1.0574 1.0313 1.020 0.994 1.007 35.9
  2 1.1066 1.0689 1.069 1.032 1.050 37.4
705-86-2 1 0.9778 0.9734 0.940 0.936 0.938 33.4
  2 0.7037 0.6804 0.666 0.643 0.655 23.3

  of OD of OD viabilities [%] of viabilities      
NC 2.806 0.405 100.0 14.43 7.22 NI qualified
PC 1.029 0.043 36.7 1.55 0.77 I qualified
705-86-2 0.797 0.284 28.4 10.11 5.05 I qualified
Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
The ocular irritation potential of test article was determined according to the OECD 492 test guideline followed for this study. The mean % tissue viability of test chemical was determined to be 28.4%. Thus, 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 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 % tissue viability of test chemical was determined to be 28.4%. Hence, under the experimental test conditions it was concluded that test chemical 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:

The in vivo and in vitro skin irritation study of the test chemical is as follows:

Study 1:

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 female young adult, non-pregnant rats were used for the 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 applied directly 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. A single dose of 200 mg/kg, 1000 mg/kg and 2000 mg/kg body weight of the test item were applied to 1 female animal respectively in the dose range finding study. Since no signs of irritation were observed at the maximum dose of 2000 mg/kg in the test animals, 2 additional animals were tested in the main study with 2000 mg/kg.The animals were applied with a dose of 2000 mg/kg body weight in sequential manner at 48 hours intervals. Dermal reaction was observed daily for study period of 14 days. 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 female Sprague Dawley rats under the experimental conditions tested and classified as “Category- Not Classified” as per CLP Classification.

Study 2:

An acute Dermal Irritation/corrosion Study of test chemical was performed as per OECD guideline No. 404 using three healthy young adult female. Rabbits with good intact skin were selected for the study. The hairs of all the rabbits were clipped at contralateral sites, approximately 24 hours prior to treatment. A dose of 0.5 ml of test item (as such) was applied to the skin, over an area of approximately 6 x 6 cm clipped of hair on one side of rabbits. The other untreated side was kept as control area and 0.5 ml of distilled water was applied at this site. At the end of 4 hours, the gauze patch was removed and test item application site was wiped with water without altering the integrity of the epidermis.  Initially, the test item was applied to the clipped area of skin of one rabbit. The test site was covered with gauze patch. After 4 hours of exposure, animal no. 1 revealed very slight erythema (barely perceptible) and edema at 1, 24, 48 and 72 hours observation. Hence the confirmatory test was conducted on additional two animals (no. 2 and 3) to confirm the non-irritant nature of the test item. After 4 hours of exposure, animal no. 2 and 3 revealed very slight erythema (barely perceptible) and no edema at 1, 24, 48 and 72 hours observation. The patch was removed after 4 hours and animals were observed for erythema and oedema at 1, 24, 48 and 72 hours after patch removal, evaluated and graded as per Draize method. The individual mean score at 24, 48 and 72 hours for Animal Nos. 1, 2 and 3 were 0.33, 0.33, 0.33 and 0.00, 0.00, 0.00, for erythema and oedema formation, respectively. Hence, it was concluded that the test chemical was Non-Irritating to the skin of female New Zealand White rabbits under the experimental conditions tested and Classified as “Category- Not Classified as Skin Irritant” as per CLP Classification.

Study 3:

The dermal irritation potential of test article was determined according to the OECD 439 test guideline for this study. The MatTek EpiDerm™ model was used to assess the potential dermal irritation of the test article by determining the viability of the tissues following exposure to the test article via MTT. Tissues were exposed to the test article and controls for ~one hour, followed by a 42 hour post-exposure recovery period. The viability of each tissue was determined by MTT assay. The MTT data show the assay quality controls were met and passed the acceptance of criteria. The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 29.8%. Hence, under the current experimental test conditions it was concluded that test chemical was considered to be irritating to human skin.

Study 4:

The dermal irritation potential of test article was determined according to the OECD 439 test guideline for this study. The MatTek EpiDerm™ model was used to assess the potential dermal irritation of the test article by determining the viability of the tissues following exposure to the test article via MTT. Tissues were exposed to the test article and controls for ~one hour, followed by a 42 hour post-exposure recovery period. The viability of each tissue was determined by MTT assay.  The MTT data show the assay quality controls were met and passed the acceptance of criteria. The Mean % tissue viability compared to negative control (n=3) of the test chwmical was determined to be 2.4 %. Hence, under the current experimental test conditions it was concluded that test chemical was considered to be irritating to human skin.

Eye Irritation Study:

The in vivo and in vitro eye irritation study of the test chemical is as follows:

Study 1:

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 % tissue viability of test chemical was determined to be 28.4%. Hence, under the experimental test conditions it was concluded that test chemical is likely to be considered as irritating to the human eyes.

Study 2:

An acute eye irritation/corrosion study was conducted in rabbits to evaluate the eye irritant nature of the test chemical. The study was performed as per OECD 405 Guidelines using three male New Zealand White rabbits. Rabbits without any eye injuries were selected for the study. The eyes of all the rabbits were examined 24 hours prior to treatment. One eye of each rabbit served as control and other was treated with the test chemical. Control eye was left untreated whereas; 0.1 ml of test item was instilled in the other (treated) eye of each rabbit. The eye was observed at 1, 24, 48 and 72 hours after test item instillation. Ophthalmoscope was used for scoring of eye lesions. In the initial test, 0.1 ml of test item was applied into the conjunctival sac of the right eye of animal no.1 whereas the left eye of the rabbit served as the control. As animal no. 1 showed no severe ocular lesions till 24 hour observation hence a confirmatory test was conducted on additional two rabbits (animal no. 2 and 3); 0.1 ml of test item was instilled into the conjunctival sac of right eye of both the rabbits and left eye served as the control. Ocular lesions were seen in animal no. 2 and 3 which were recovered till 72 hours observation. Untreated eye of all the three rabbits was normal throughout the experimental period of 72 hours. Observation at 24 hours after instillation of test item revealed, no ulceration or opacity in cornea of all the animals; Area of Opacity-Zero in all the animals; Iris was found to be normal in all the animals. No damage to the conjunctivae was observed, blood vessels was normal in all the animals. No chemosis and swelling (normal) was seen in all the animals. At 24 hours observations, the rabbits were examined for corneal epithelium cell damage using sodium fluorescein strips and noticed 0% damage in animal no. 1, 2 and 3. Observation at 48 hour after instillation of test item revealed no ulceration or opacity in cornea of all the animals; Area of opacity was zero in all the animals. Iris was found to be normal in all the animals. Conjunctivae - Blood vessels was normal in all the animals; Chemosis: No swelling (normal) was seen in all the animals. No damage to the conjunctivae was observed, blood vessels was normal in all the animals. Similar effects were observed after the end of 72 hours. The individual mean score for animal nos. 1, 2 and 3 at 24, 48, 72 hours for corneal opacity, iris, conjunctiva and chemosis were found 0.00 for all the parameters (Iris, Cornea, respectively. Under the experimental conditions tested, no eye irritation on eyes of rabbits was observed at 72 hours. Hence, the test chemical was considered as “Non Irritant” to New Zealand White male rabbit eyes and being classified as “Not Classified as an Eye Irritant” as per the criteria of CLP regulation.

Study 3:

An in vivo eye irritation study using the test chemical was performed. Three albino rabbits were administered 0.1 ml of a 0.5% solution of the read-across analogue in propylene glycol. Evaluations were made every 24 h for 4 days and again on day 7 according to the Draize procedure. There was no evidence of irritation due to administration of the test chemical in the eye of the animals. Based on all the available observations and results, it was concluded that the test chemical was not irritant to the eyes of the albino rabbits after ocular administration.

Study 4:

The ocular irritation potential of test article was determined according to the OECD 492 test guideline for this study. The MatTek EpiOcular™ model was used to assess the potential ocular irritation of the test articles by determining the viability of the tissues following exposure to the test article via MTT. Tissues were exposed to solid test articles and control for approx.6 hours, followed by a 25 minute post-soak and approximately 18 hours recovery after the post-soak. The viability of each tissue was determined by MTT assay. The MTT data show the assay quality controls were met, passing the acceptance criteria. The mean % tissue viability of test substance was determined to be 52.0%. Hence, under the experimental test conditions it was concluded that test substance was considered to be not irritating/ irritating to the human eyes.

Justification for classification or non-classification

Even though the in vitro Guideline study and one study in rabbit claims that the test chemical was irritating to skin, but other in vivo studies suggest otherwise. Also when tested at the highest possible dose of 2000mg/kg in rats, the test chemical failed to show any signs of erythema or edema.

Taking all these factors into consideration, the test chemical is likely to be considered as 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”.

Also, in eye irritation endpoint, the in-vivo studies suggest that the test chemical is not likely to have any adverse effect on the eyes of the rabbits. Thus, comparing the above annotations with the criteria of CLP Regulation, the test chemical is not likely to classify as an 'Eye Irritant'

Categories Display