Registration Dossier

Administrative data

Description of key information

Skin Irritation:

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 for 24 hours under occlusion and observed for effects.

Undiluted test chemical was mildly irritating to the intact and abraded skin of rabbits after 24 hours exposure. Hence, the test chemical can be considered to be irritating to skin.

The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 2.3%.

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.

Eye Irritation:

The mean % tissue viability of test chemical was determined to be32.5%. 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

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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
data is from handbooks and peer reviewed journals
Qualifier:
according to
Guideline:
other: as mentioned below
Principles of method if other than guideline:
To assess the dermal irritation potential of the test chemical in rabbits
GLP compliance:
not specified
Species:
rabbit
Strain:
not specified
Type of coverage:
occlusive
Preparation of test site:
other: intact and abraded
Vehicle:
unchanged (no vehicle)
Controls:
not specified
Amount / concentration applied:
500 mg
Duration of treatment / exposure:
24 hours
Observation period:
24 hours
Details on study design:
No data available
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:
Mild irritation was observed
Interpretation of results:
other: Mild irritant
Conclusions:
Undiluted test chemical was mildly irritating to the intact and abraded skin of rabbits after 24 hours exposure. Hence, the test chemical can be considered to be irritating to skin.
Executive summary:

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 for 24 hours under occlusion and observed for effects.

 

Undiluted test chemical was mildly irritating to the intact and abraded skin of rabbits after 24 hours exposure. Hence, the test chemical can be considered to be irritating to skin.
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.
GLP compliance:
no
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: as provided by MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
Source strain:
other: Not applicable
Details on animal used as source of test system:
- Description of the cell system used:The normal human-derived keratinocytes were cultured at the air-liquid interface in a chemically defined medium on a permeable polycarbonate insert (surface 0.5 cm2). They were cultured in chemically defined serum free medium to form a multi-layered epithelium similar to that found in native epidermis. Each lot of tissues was Quality Assured by MatTek according to specific QC standards including: histology, tissue viability (MTT mean optical density), reproducibility (SD) and tissue thickness.Test System IdentificationAll of the EpiDerm™ 3-dimensional human tissues used in this study were identified by the date of arrival and the lot number. Certificate of Analysis for the tissues are included in this report. Tissue plates were appropriately labeled with study information.
Justification for test system used:
The 3-Dimensional Human Dermal Epithelial Model (EpiDerm™, MatTek, In Vitro Life Science Laboratories, Bratislava, Slovakia) is made up of normal human keratinocytes in serum free medium. The cells form an epithelial tissue that consists of organized basal, spinous, granular, and cornified layers analogous to those found in vivo. The EpiDerm™ model also contains epidermis-specific differentiation markers such as pro-filaggrin, the K1/K10 cytokeratin pair, involucrin, and type I epidermal transglutaminase, as well as keratohyalin granules, tonofilament bundles, desmosomes, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns characteristic of in vivo epidermis. Each lot of tissues was Quality Assured by MatTek, Inc. according to specific QC standards including: histology (cell layers), tissue viability (MTT mean optical density) and reproducibility (SD). Tissue plates were appropriately labeled with study information. Bias was not a factor in this test system.
Vehicle:
unchanged (no vehicle)
Details on test system:
The tissues were exposed to the test article neat (undiluted) on April 25, 2018 (Run 1 of 1). EpiDerm™ tissues were purchased from MatTek. Quality control of the tissues was performed by MatTek and the Certificate of Analysis (CoA) for the tissues is provided and is kept in the study binder. Tissues were exposed for approximately 1 hour, with 35 minutes in an approximately 37°C, 5% CO2 humidified incubator and the remaining 25 minutes at room temperature. Following the exposure time, the tissues were rinsed and placed in fresh media for approximately 24 hours. The media was then changed again and the tissues were incubated in fresh media for another ~18 hours for a total of approximately 42 hour post-exposure recovery period. The tissue viability was then assessed by MTT assay. The tissue CoA was used instead of verification of barrier properties of the tissue.MTT and Color Pre-testsPretesting has actually been conducted for all chemicals, although the first intitial 8 test chemicals a pretesting was not conducted (for skin).MTT AssayFollowing the rinsing period, the MTT assay was performed by transferring the tissues to 24-well plates containing 300 µL MTT medium (1.0 mg/mL). After 3 hours MTT incubation at approximately 37°C, approximately 5% CO2 in a humidified incubator, the blue formazan salt was extracted by submerging tissues in 2 mL isopropanol in a 24-well plate. The extraction time was approximately 3 hours with gentle shaking. The optical density of the extracted formazan (200 µL/well of a 96-well plate) was determined using a Thermo Scientific Multiskan FC Microplate Photometer at 570 nm. Relative cell viability is calculated for each tissue as % of the mean negative control tissues.Evaluation of Test Article in the Cell Models:1. Cell system: Upon receipt, the MatTek EpiDerm™ tissue cultures were placed in 0.9 mL of fresh Maintenance medium (in a 6-well plate). The culture inserts are incubated for ~one hour. The tissues were then transferred to 6-well plates containing 0.9 mL fresh Maintenance medium and they were incubated overnight (18 ± 3 hrs) at ~37°C, 5% CO2 in a humidified incubator.2. Control and Test Article Exposures: On the day of dosing, the tissues are then removed from the incubator and the controls and the test article are applied topically to tissues by pipette(liquid) Tissues were exposed to controls and the test article for one hour, with ~35 minutes in a 37°C, 5% CO2 humidified incubator and the remaining 25 minutes at room temperature.a) Controls30 µL of negative control DPBS and 30 μL of the positive control 5% SDS was applied topically to the tissue and gently spread by placing a nylon mesh on the apical surface of each tissue, if necessary.b) Test Articles 30 µL of the test article was applied topically to the tissue 3. Post-exposure treatmentAfter the 1 hour exposure, the tissues were rinsed 15 times with sterile DPBS. After the 15th rinse from washing bottle, each insert wasw completely submerge 3 times in 150 ml DPBS. The apical surface was gently blotted with a cotton swab. The tissues were placed in 0.9 mL of fresh Maintenance medium (6-well plate) for 24 ± 2 hours. After this initial ~24 hour incubation, the tissues were placed in 6-well plates containing 0.9 mL fresh Maintenance medium and incubated for another 18 ± 3 hours, for a total of an approximately 42 hour post-exposure incubation.RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE- Model used: The EpiDerm™ 3 dimensional human tissue model- Tissue Lot number(s): 26459- Date of initiation of testing: 6/08/2017TEMPERATURE USED FOR TEST SYSTEM- Temperature used during treatment / exposure: 37°C- Temperature of post-treatment incubation (if applicable): 37°CREMOVAL OF TEST MATERIAL AND CONTROLS-Volume and number of washing steps: The test substance was rinsed from the tissues with sterile DPBS by filling and emptying the tissue insert 15 times to remove any residual test material. This was followed by completely submerge the insert 3 times in 150 ml DPBS.MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE- MTT concentration: 300 µL MTT medium (1.0 mg/mL).- Incubation time: After 3 hours- Spectrophotometer: Synergy H4 spectrophotometer - Wavelength: 570 nm- Filter: No data- Filter bandwidth: No data- Linear OD range of spectrophotometer: No dataNUMBER OF REPLICATE TISSUES: 3CALCULATIONS and STATISTICAL METHODSAll data were background subtracted before analysis. MTT data are presented as % viable compared to negative control. Data were generated as follows: MTT AssayBlanks:·        The optical density (OD) mean from all replicates for each plate (ODblank). Negative Controls (NC):·        The blank corrected value was calculated: ODNC= ODNCraw– ODblank. ·        The OD mean per NC tissue was calculated. ·        The mean OD for all tissues corresponds to 100% viability. ·        The mean, standard deviation (SD), standard error of the mean (SEM) and the percent coefficient of variation (% CV) was calculated. Positive Control (PC):·        Calculate the blank corrected value: ODPC= ODPCraw– ODblank. ·        The OD mean per PC tissue was calculated. ·        The viability per tissue was calculated: %PC = [ODPC/ mean ODNC] x 100. ·        The mean viability for all tissues was calculated: Mean PC = Σ %PC / number of tissues. ·        The standard deviation (SD), standard error of the mean (SEM) and the percent coefficient of variation (% CV) was calculated. Tested compound :·        Calculate the blank corrected value ODTT= ODTTraw– ODblank. ·        The OD mean per tissue was calculated. ·        The viability per tissue was calculated: %TT = [ODTT/ mean ODNC] x 100. ·        The mean viability for all tissues was calculated: Mean TT = Σ %TT / number of tissues. ·        The standard deviation (SD), standard error of the mean (SEM) and the percent coefficient of variation (% CV) was calculated. Data Correction Procedure for MTT Interfering Compounds (if applicable)True viability = Viability of treated tissue – Interference from test article = ODtvt– ODktwhere ODkt= (mean ODtkt– mean ODukt).ODtvt= optical density of treated viable tissueODkt= optical density of killed tissuesODtkt= optical density of treated killed tissueODukt= optical density of untreated killed tissue (NC treated tissue) Data Correction Procedure for Colored Compounds (if applicable)True viability = Viability of treated tissue incubated in MTT media – Viability of treated tissue incubated in media without MTT = ODtvt– ODvt.ODtvt= optical density of treated viable tissue incubated in MTT mediaODvt= optical density of viable tissues incubated in media alone - Evaluation of data The results of the assay was evaluated and compared to negative control. Table: Criteria for in vitro Interpretation: In VitroResults In VivoPredictionMean tissue viability ≤50% Irritant (I), R38Mean tissue viability >50% Non-irritant (NI)- Assay quality controls- Negative Controls (NC)The Dulbecco’s phosphate buffered saline (DPBS) was used as a NC. The assay passed all acceptance criteria if the ODs of the negative control exposed tissues were between ≥0.8 and ≤2.8.  - Positive Controls (PC)5% solution of sodium dodecyl sulfate was used as a PC. The assay is meeting the acceptance criteria if the viability of the PC is ≤20% of the negative control.   - Standard Deviation (SD)The standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates was ≤18.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µ
L- Concentration (if solution): neat
Duration of treatment / exposure:
The exposure times were approximately 1 hour, with ~35 minutes exposure in the incubator and ~25 minutes at room temperature.
Duration of post-treatment incubation (if applicable):
For a total of an approximately 42 hour post-exposure incubation.
Number of replicates:
3 tissues were used for test compound and control.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Run 1
Value:
2.3
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
The MTT data show the assay quality controls were met.

Code N° Tissue  Raw data   Blank corrected data mean  % of viability
  n Aliq. 1 Aliq. 2 Aliq. 1 Aliq. 2 of aliquotes  
NC 1 1.865 1.8438 1.829 1.808 1.819 98.3
  2

C3 I qualified
1.8808
1.8835 1.845 1.848 1.846 99.8
  3 1.9193 1.925 1.884 1.889 1.886 101.9
PC 1 0.0795 0.0786 0.044 0.043 0.043 2.3
  2 0.0742 0.0747 0.039 0.039 0.039 2.1
  3 0.0853 0.085 0.050 0.049 0.049 2.7

C3 1 0.072 0.072 0.036 0.036 0.036 2.0
  2 0.0795 0.0805 0.044 0.045 0.044 2.4
  3 0.0852 0.0836 0.050 0.048 0.049 2.6

  of OD of OD viabilities [%] of viabilities [%]
NC 1.851 0.034 100.0 1.84 1.84
PC 0.044 0.005 2.4 0.29 12.23

C3 0.043 0.006 2.3 0.34 14.57

Classification

NC NI qualified
PC I qualified

C3 I qualified
Interpretation of results:
Category 2 (irritant) based on GHS criteria
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 2.3%. Thus, test chemical was considered to be irritating to the human skin.
Executive summary:

The dermal irritation potential of test article was determined according to the OECD 439 test guideline for this study. The MatTek EpiDerm™ model was used to assess the potential dermal irritation of the test article by determining the viability of the tissues following exposure to the test article via MTT. Tissues were exposed to the test article and controls for ~one hour, followed by a 42 hour post-exposure recovery period. The viability of each tissue was determined by MTT assay. 

The MTT data show the assay quality controls were met and passed the acceptance of criteria.

The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 2.3%.

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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (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:
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:
32.5
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
The MTT data show the assay quality controls were met.

Code N° Tissue  Raw data Blank corrected data mean of OD % of viability
  n Aliq. 1 Aliq. 2 Aliq. 1 Aliq. 2
NC 1 3.1865 2.9581 3.150 2.921 3.035 103.3
  2 2.815 2.9373 2.778 2.900 2.839 96.7
PC 1 0.9553 0.9523 0.918 0.915 0.917 31.2
  2 0.9687 0.9737 0.932 0.937 0.934 31.8

3558-60-9 1 0.8855 0.8642 0.849 0.827 0.838 28.5
  2 1.1031 1.1169 1.066 1.080 1.073 36.5

  mean Dif. mean of Dif. Dif./2 Classification
  of OD of OD viabilities [%] of viabilities      
NC 2.937 0.196 100.0 6.68 3.34 NI qualified
PC 0.926 0.017 31.5 0.59 0.30 I qualified

3558-60-9 0.956 0.235 32.5 8.01 4.00 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 32.5%. Thus, the test chemical was considered to be irritating to the human eyes.
Executive summary:

The ocular irritation potential of test article was determined according to the OECD 492 test guideline for this study. The MatTek EpiOcular™ model was used to assess the potential ocular irritation of the test articles by determining the viability of the tissues following exposure to the test article via MTT. Tissues were exposed to 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 be32.5%. 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:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin Irritation

Various studies were reviewed to determine the level of dermal irritation caused by the test chemical in living organisms. These include in vivo asa well in vitro experimental results for the target. The results are summarized below:

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 for 24 hours under occlusion and observed for effects.

Undiluted test chemical was mildly irritating to the intact and abraded skin of rabbits after 24 hours exposure. Hence, the test chemical can be considered to be irritating to skin.

This is supported by the results of an in vitro study performed according to the OECD 439 test guideline to evaluate the dermal irritation potential of test article. 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 2.3%.

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.

 

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.

The estimated results is supported by a study performed on humans to assess the irritation potential of the test chemical. The test chemical 8% in petrolatum was applied to skin of human volunteers in a 48 hours closed patch test.

The test chemical was not irritating to humans after 48 hours exposure. Hence, the test chemical can be considered not irritating to skin.

The above results are lent support by a study where the dermal irritation potential evaluation of the test chemical was evaluated as a part of an acute dermal LD50 toxicity study in rabbits.

Doses were 1.25 (n = 6), 2.5 (n = 4) and 5.0 (n = 4) g/kg. All animals dosed at 5 g/kg died within 24 hours, before skin evaluation could be performed. Erythema was slight in 3/6 and 1/4 rabbits and moderate in 3/6 and 3/4 rabbits exposed to 1.25 and 2.5 g/kg, respectively of 1-phenylethanol. Edema was moderate in 5/6 and 4/4 animals exposed to 1.25 and 2.5 g/kg, respectively. No edema score was reported for the remaining animal at 1.25 g/kg.

Since, erythema and edema was observed in most of the animals tested, the test chemical can be considered to be moderately irritating to rabbit skin.

These results are further supported by a study performed to evaluate the dermal irritation potential of the test chemical in rabbits. The test chemical applied full strength to the intact and abraded skin of rabbits under occlusion. The effects were observed for 24 hours. The test chemical was mildly irritating to intact and abraded skin of rabbits.

Hence, the test chemical can be considered to be mildly irritating to rabbit skin.

Even thought the results of the estimated data and human in vivo data for the test chemical indicate a possibility that the test chemical can indeed be not irritating to skin. But the in vitro Guideline study and the rabbit data suggest otherwise. Taking all these factors into consideration, the test chemical can be considered to be irritating to skin. Comparing the above annotations with the criteria of CLP Regulation, the test chemical can be classified under the category “Category 2”.

Eye Irritation

Various studies have been reviewed to determine the extent of ocular damage caused by the test chemical to living organisms. These include in vivo experiments on rabbits as well as in vitro experimental results for the test chemical. The studies are summarized below:

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 be32.5%. 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 the results of an OECD 405 Guideline study performed for the test chemical to evaluate the eye irritant nature of the test chemical. 3 female New Zealand White rabbits were used for the study.

Rabbits free from injury of eye were selected for the study. The eyes of all the rabbits were examined 24 hours prior to treatment. One eye of each rabbit served as control and other as treated. Control eye was left untreated whereas; 0.1 ml of test item was instilled in the other (treated) eye of each rabbit. The eye was observed at 1, 24, 48, 72 hour, day 7, day 14 and day 21 for animal no. 1 and 3 whereas at 1, 24, 48, 72 hour, day 7 and day 14 for animal no. 2 after test item instillation. Ophthalmoscope was used for scoring of eye lesions. In the initial test, 0.1 ml of test item (as such) 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 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. Untreated eye of all the three rabbits were normal throughout the experimental period. Observation at 1 hour after instillation of test item revealed: Cornea- No ulceration or opacity was seen in all the animals; Area of Opacity- Zero in all the animals; Iris: Normal in all the animals; Conjunctivae - Diffuse, crimson color; individual vessels not easily discernible in all the animals; Chemosis: Some swelling above normal (includes nictitating membranes) in animal no. 2 and obvious swelling with partial eversion of lids in animal no. 1 and 3.

Observation at 24 hours after instillation of test item revealed: Cornea- No ulceration or opacity was seen in all the animals; Area of Opacity- Zero in all the animals; Iris: Normal in all the animals; Conjunctivae - Diffuse, crimson color; individual vessels not easily discernible was seen in all the animals; Chemosis: Some swelling above normal (includes nictitating membranes) was seen in all the animals.

At 24 hours observation the rabbits were examined for corneal epithelium cell damage using sodium fluorescein strips and noticed 30%, 30% and 20% damage in animal no. 1, 2 and 3 respectively.

Observation at 48 hours after instillation of test item revealed: Cornea- No ulceration or opacity was seen in all the animals; Area of Opacity- Zero in all the animals; Iris: Normal in all the animals; Conjunctivae - Diffuse, crimson color; individual vessels not easily discernible was observed in animal no. 1 and 3 whereas some blood vessels definitely hyperaemic (injected) was observed in animal no. 2; Chemosis: Some swelling above normal (includes nictitating membranes) was observed in all 3 animals.

Observation at 72 hours after instillation of test item revealed: Cornea- No ulceration or opacity was seen in all the animals; Area of Opacity- Zero in all the animals; Iris: Normal in all the animals; Conjunctivae - Diffuse, crimson color; individual vessels not easily discernible was observed in animal no. 1 and 3 whereas some blood vessels definitely hyperaemic (injected) was observed in animal no. 2; Chemosis: Some swelling above normal (includes nictitating membranes) was observed in all 3 animals.

Observation on day 7 after instillation of test item revealed: Cornea- No ulceration or opacity was seen in all the animals; Area of Opacity- Zero in all the animals; Iris: Normal in all the animals; Conjunctivae - Some blood vessels definitely hyperaemic (injected) was observed in all the animals; Chemosis: Some swelling above normal (includes nictitating membranes) was observed in animal no. 1 and 3 and no swelling (Normal) in animal no. 2.

Observation on day 14 after instillation of test item revealed: Cornea- Scattered or diffuse areas of opacity (other than slight dulling of normal lustre); details of iris clearly visible was observed in animal no.1 and 3 whereas no ulceration or opacity in animal no. 2; Area of Opacity- One quarter (or less) but not zero was observed in animal no. 1 and 3 whereas zero was observed in animal no.2; Iris: Normal in all the animals; Conjunctivae - Some blood vessels definitely hyperaemic (injected) was observed in animal no.1 and 3 whereas blood vessels normal was observed in animal no. 2; Chemosis: Some swelling above normal (includes nictitating membranes) was observed in animal no. 1 and 3 whereas no swelling (Normal) was observed in animal no. 2. Animal no. 2 recovered on day 14.

Observation on day 21 after instillation of test item revealed: Cornea- No ulceration or opacity was observed in animal no. 1 and 3; Area of Opacity- Zero was observed in animal no. 1 and 3; Iris: Normal in all the animals; Conjunctivae - Blood vessels normal was observed in animal no. 1 and 3; Chemosis: No swelling (Normal) was observed in animal no. 1 and 3.

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, 0.00, 2.00, 1.00; 0.00, 0.00, 1.33, 1.00, and 0.00, 0.00, 2.00, 1.00, respectively.

Under the experimental conditions tested, all the three animals were fully reversible within an observation period of 14 for animal no. 2. and 21 days for animal no. 1 and 3. Hence, under the experimental test conditions, the test chemical was considered to be an eye irritant. It was further classified under the category "Category 2".

These results are further supported by another OECD 405 Guideline study performed to determine the eye irritation potential of the test chemical.

Rabbits free from injury of eye were selected for the study. The eyes of all the rabbits were examined 24 hours prior to treatment. One eye of each rabbit served as control and other as treated. Control eye was left untreated whereas; 0.1 gof test itemwas instilled in the other (treated) eye of each rabbit. The eye was observed at 1, 24, 48, 72 hour, day 7, day 14 and day 21 for all the three animals post test item instillation.Ophthalmoscope was used for scoring of eye lesions. In the initial test,100 mg of test itemwas instilled into the conjunctival sac of the right eye of animal no.1 whereas the left eye of the rabbit served as the control. As animal no. 1 showed no severe ocular lesions; hence a confirmatory test was conducted on additional two rabbits (animal no. 2 and 3); 100 mgof test itemwas instilled into the conjunctival sac of right eye of both the rabbits and left eye served as the control. All the animals were observed till day 21 post test item instillation. Untreated eye of all the three rabbits were normal throughout the experimental period.

The following grading scores were observed in treated eye of tested rabbits.

Observation at 1 hour after instillation of test item revealed: Cornea-No ulceration or opacity was seen in all the animals;Area of Opacity-Zero inall the animals;Iris:Normal in all the animals;Conjunctivae - Some blood vessels definitely hyperaemic (injected) was seen in all the animals;Chemosis:Some swelling above normal (includes nictitating membranes) was seen in all the animals.

Observation at 24 hours after instillation of test item revealed: Cornea-Scattered or diffuse areas of opacity (other than slight dulling of normal lustre); details of iris clearly visible was seen in all the animals;Area of Opacity- One quarter (or less) but not zerowas seen in all the animals;Iris:Normal in all the animals;Conjunctivae -Diffuse, crimson color; individual vessels not easily discernible was seen in all the animals;Chemosis:Some swelling above normal (includes nictitating membranes) was seen in animal no. 1 and 3 whereas obvious swelling with partial eversion of lids was seen in animal no 2.

At 24 hours observation the rabbits were examined for corneal epithelium cell damage using sodium fluorescein strips and noticed 40%, 50% and 40% damage in animal no. 1, 2 and 3 respectively.Observation at 48 hours after instillation of test item revealed: Cornea- Scattered or diffuse areas of opacity (other than slight dulling of normal lustre); details of iris clearly visible was seen in all the animals;Area of Opacity-One quarter (or less) but not zero was seen inall the animals;Iris:Normal in all the animals;Conjunctivae - Diffuse, crimson color; individual vessels not easily discernible in animal no. 3;Chemosis:Some swelling above normal (includes nictitating membranes) was seen in animal no. 1 and 2 whereas obvious swelling with partial eversion of lids in animal no. 3.Observation at 72 hours after instillation of test item revealed: Cornea- Scattered or diffuse areas of opacity (other than slight dulling of normal lustre); details of iris clearly visible was seen in all the animals;Area of Opacity-One quarter (or less) but not zero was seen inall the animals;Iris:Normal in all the animals;Conjunctivae - Diffuse, crimson color; individual vessels not easily discernible in all the animals;Chemosis:Some swelling above normal (includes nictitating membranes) was seen in all the animals. Observation on day 7 after instillation of test item revealed: Cornea- Scattered or diffuse areas of opacity (other than slight dulling of normal lustre); details of iris clearly visible was seen in all the animals;Area of Opacity-One quarter (or less) but not zero was seen inall the animals;Iris:Normal in all the animals;Conjunctivae-Some blood vessels definitely hyperaemic (injected) was seen in animal no.1 and diffuse, crimson color; individual vessels not easily discernible was observed in animal no. 2 and 3;Chemosis:Some swelling above normal (includes nictitating membranes) was seen in all the animals.Observation on day 14 after instillation of test item revealed: Cornea- Scattered or diffuse areas of opacity (other than slight dulling of normal lustre); details of iris clearly visible was seen in animal no. 1 and 2 whereas no ulceration or opacity was seen in animal no. 3;Area of Opacity-One quarter (or less) but not zero was seen in animals no. 1 and 2 whereas zero was seen inanimal no. 3;Iris:Normal in all the animals;Conjunctivae -Some blood vessels definitely hyperaemic (injected) was seen in all the animals;Chemosis:Some swelling above normal (includes nictitating membranes) was seen in all the animals. Observation on day 21 after instillation of test item revealed: Cornea-No ulceration or opacity in all the animals;Area of Opacity-Zero in all the animals;Iris:Normal in all the animals;Conjunctivae -Blood vessels normal in all the animals;Chemosis:No swelling (Normal) in all the animals.

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 1.00, 0.00, 2.00, 1.00; 1.00, 0.00, 2.00, 1.33, and 1.00, 0.00, 2.00, 1.33, respectively. The effects observed in all the animals were fully reversible within an observation period of 21 days.  

Hence under the experimental test conditions, the test chemical was “An Eye Irritant (Irritating to Eyes)” of New Zealand White Female rabbit eyes.

Available results for the test chemical indicate a very strong possibility that the test chemical can cause irritation to eyes. Hence the test chemical can be considered to be irritating to eyes. It can be classified under the category "Category 2" as per CLP Regulation.

Justification for classification or non-classification

Available results for the test chemical indicate a very strong possibility that the test chemical can cause irritation to eyes and skin.Hence the test chemical can be considered to be irritating to eyes and skin. It can be classified under the category "Category 2" as per CLP Regulation.