Registration Dossier

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 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

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

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

An in vivo study performed to assess the potential of the test chemical in New Zealand White Rabbits.

The test chemical produced a mild corneal injury in of five of six New Zealand White rabbits, and it produced mild conjunctival injury in six out of six rabbits.

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

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Justification for type of information:
data is from experimental reports
Qualifier:
according to
Guideline:
other: Acute Dermal toxicity (OECD 402 Guidelines)
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
- 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 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 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 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: 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 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

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 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:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Justification for type of information:
Data is from secondary source
Qualifier:
according to
Guideline:
other: As mentioned below
Principles of method if other than guideline:
An Eye irritation study was conducted on New Zealand White rabbits to determine eye irritant potency of chemical
GLP compliance:
not specified
Species:
rabbit
Strain:
New Zealand White
Vehicle:
unchanged (no vehicle)
Controls:
not specified
Amount / concentration applied:
0.1 mL aliquot
Duration of treatment / exposure:
24 hours
Observation period (in vivo):
24 hours
Number of animals or in vitro replicates:
6
Details on study design:
Single 24—hour application of 0.1 ml technical grade compound, one eye of each of six New Zealand White rabbits
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
24 h
Reversibility:
not specified
Remarks on result:
positive indication of irritation
Irritant / corrosive response data:
The test chemical produced a mild corneal injury in of five of six New Zealand White rabbits , and it produced mild conjunctival injury in six out of six rabbits.

Test

Results

interpretation

Single 24—hour application of 0.1 ml technical grade compound, one eye of each of six New Zealand White rabbits

The test chemical produced a mild corneal injury in of five of six New Zealand White rabbits , and it produced mild conjunctival injury in six out of six rabbits.

Irritating

Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
The test chemical produced a mild corneal injury in of five of six New Zealand White rabbits , and it produced mild conjunctival injury in six out of six rabbits.
Hence, the test chemical can be considered to be irritating to eyes.
Executive summary:

eye irritation study performed to assess the potential of the test chemical in New Zealand White Rabbits.

0.1ml technical grade test chemical was instilled into the eyes of 6 New Zealand White rabbits for 24 hours. The treated eyes were observed for effects through out the study.

The test chemical produced a mild corneal injury in of five of six New Zealand White rabbits, and it produced mild conjunctival injury in six out of six rabbits.

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

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin Irritation

Various studies have been summarized to determine the level of dermal irritation in living organisms. These include in vitro and in vivo experimental studies along with estimated study for the test chemical.

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.

The dermal irritation potential of test article was also 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 and can thus be classified as ''Irritating to skin in Category 2” as per CLP Regulation.

The in vivo result is supported by a study performed to assess the dermal irritation potential of the test chemical in rabbits. Six New Zealand White rabbits were used for the study.

0.5 ml technical grade compound was applied to intact and abraded skin of 6 rabbits for 24 hours. The test animals were observed for signs of irritation.

The test chemical produced no primary irritation of intact skin or to the skin surrounding an abrasion.

Hence, the test chemical can be considered to be not irritating to skin.

The above studies are supported by a skin irritation study performed in humans to assess the irritation potential of the test chemical. The test chemical was tested 1% in petrolatum on human volunteers in a 48 hours closed patch test.

The test chemical was not irritating to humans after 48 hours exposure.

In addition to the above results, a skin irritation study was performed in rabbits to assess the irritation potential of the test chemical.

Undiluted test chemical was applied to the intact and abraded skin of rabbits under occlusion for 24 hours and observed for effects.

Slight irritation was observed after 24 hours of exposure to the test chemical. Hence, the test chemical can be considered to be irritating to rabbit skin.

Skin irritation effects were also estimated by four different models i.e, Battery, Leadscope, SciQSAR and CASE Ultra used within Danish QSAR database for the test chemical. Based on estimation, no severe skin irritation effects were known when the test chemical was exposed to rabbit skin. Hence, the test chemical can be considered not irritating to skin.

 

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 can be considered to be not irritating to skin. Comparing the above annotations with the criteria of CLP Regulation, the test chemical can be classified under the category “Not Classified”.

Eye Irritation

Various studies have been reviewed to determine the extent of ocular damage caused by the test chemical in living organisms. These include in vivo experimental studies performed on rabbits along with in vitro study for the test chemical.

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

This is supported by an in vivo study performed to assess the potential of the test chemical in New Zealand White Rabbits.

0.1ml technical grade test chemical was instilled into the eyes of 6 New Zealand White rabbits for 24 hours. The treated eyes were observed for effects through out the study.

The test chemical produced a mild corneal injury in of five of six New Zealand White rabbits, and it produced mild conjunctival injury in six out of six rabbits.

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

The in vivo and in vitro results are in mutual agreement with each other indicating a strong possibility that the test chemical is indeed irritating to eyes. Comparing the above annotations with the criteria of CLP Regulation, the test chemical can be classified under the category “Category 2”.

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 can be considered to be not irritating to skin. Comparing the above annotations with the criteria of CLP Regulation, the test chemical can be classified under the category “Not Classified”.

The in vivo and in vitro results are in mutual agreement with each other indicating a strong possibility that the test chemical is indeed irritating to eyes. Comparing the above annotations with the criteria of CLP Regulation, the test chemical can be classified under the category “Category 2”.