Registration Dossier

Administrative data

Description of key information

Skin Irritation

The dermal irritation potential of test article was determined according to the OECD 439 test guideline for this study.

The mean of OD for test chemical was determined to be 2.285. The standard deviation of viabilities for test chemical were calculated to be 11.15.The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 108.0%. Hence, under the current experimental test conditions it was concluded that test chemical was considered to be non-irritating to human skin.

An in vivo Skin irritation study was conducted on guinea pigs to determine the irritation potential of test chemical. The mean irritation index of test chemical was 0.0 after 24 hours. Hence, the test chemical was considered to be not irritating to guinea pigs.

Eye Irritation

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from experimental study report
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Principles of method if other than guideline:
The purpose of this study was to assess potential for the test article to be dermal irritants. The dermal irritation potential of test article may be predicted by measurement of their cytotoxic effect, as reflected in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, in the MatTek EpiDerm™ model (MatTek Corp., Ashland, MA).
GLP compliance:
yes
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: EpiDerm™ 3-dimensional human tissues used in this study
Source strain:
other: Not applicable
Details on animal used as source of test system:
- Description of the cell system used:The normal human-derived keratinocytes were cultured at the air-liquid interface in a chemically defined medium on a permeable polycarbonate insert (surface 0.5 cm2). They were cultured in chemically defined serum free medium to form a multi-layered epithelium similar to that found in native epidermis. Each lot of tissues was Quality Assured by MatTek according to specific QC standards including: histology, tissue viability (MTT mean optical density), reproducibility (SD) and tissue thickness.- Test System IdentificationAll of the EpiDerm™ 3-dimensional human tissues used in this study were identified by the date of arrival and the lot number. Certificate of Analysis for the tissues are included in this report. Tissue plates were appropriately labeled with study information.
Justification for test system used:
The 3-Dimensional Human Dermal Epithelial Model (EpiDerm™, MatTek, Ashland, MA) is made up of normal human keratinocytes in serum free medium. The cells form an epithelial tissue that consists of organized basal, spinous, granular, and cornified layers analogous to those found in vivo. The EpiDerm™ model also contains epidermis-specific differentiation markers such as pro-filaggrin, the K1/K10 cytokeratin pair, involucrin, and type I epidermal transglutaminase, as well as keratohyalin granules, tonofilament bundles, desmosomes, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns characteristic of in vivo epidermis. Each lot of tissues was Quality Assured by MatTek, Inc. according to specific QC standards including: histology (cell layers), tissue viability (MTT mean optical density) and reproducibility (SD). Tissue plates were appropriately labeled with study information. Bias was not a factor in this test system.
Vehicle:
unchanged (no vehicle)
Details on test system:
The tissues were exposed to the test article neat (undiluted) on June 28, 2017 (Run 1 of 1). EpiDerm™ tissues were purchased from MatTek. Quality control of the tissues was performed by MatTek and the Certificate of Analysis (CoA) for the tissues is provided and is kept in the study binder. Tissues were exposed for approximately 1 hour, with 35 minutes in an approximately 37°C, 5% CO2 humidified incubator and the remaining 25 minutes at room temperature. Following the exposure time, the tissues were rinsed and placed in fresh media for approximately 24 hours. The media was then changed again and the tissues were incubated in fresh media for another ~18 hours for a total of approximately 42 hour post-exposure recovery period. The tissue viability was then assessed by MTT assay. The tissue CoA was used instead of verification of barrier properties of the tissue.MTT and Color Pre-testsPretesting for MTT auto-reduction and coloring was not performed for this study but was based on the results obtained from another study (CYP1690_R1b).MTT AssayFollowing the rinsing period, the MTT assay was performed by transferring the tissues to 24-well plates containing 300 µL MTT medium (1.0 mg/mL). After 2 hours, 57 minute and 25 second MTT incubation at approximately 37°C, approximately 5% CO2 in a humidified incubator, the blue formazan salt was extracted by submerging tissues in 2 mL isopropanol in a 24-well plate. The extraction time was approximately 2 hours 04 minutes and 11 seconds with gentle shaking. The optical density of the extracted formazan (200 µL/well of a 96-well plate) was determined using a Synergy H4 spectrophotometer at 570 nm. Relative cell viability is calculated for each tissue as % of the mean negative control tissues.Evaluation of Test Article in the Cell Models:1. Cell system: Upon receipt, the MatTek EpiDerm™ tissue cultures were placed in 0.9 mL of fresh Maintenance medium (in a 6-well plate). The culture inserts are incubated for ~one hour. The tissues were then transferred to 6-well plates containing 0.9 mL fresh Maintenance medium and they were incubated overnight at ~37°C, 5% CO2 in a humidified incubator.2. Control and Test Article Exposures: On the day of dosing, the tissues are then removed from the incubator and the controls and the test article are applied topically to tissues by pipette. Tissues were exposed to controls and the test articles for one hour, with ~35 minutes in a 37°C, 5% CO2 humidified incubator and the remaining 25 minutes at room temperature.a) Controls30 µL of negative control DPBS, positive control 5% SDS was applied topically to the tissue and gently spread by placing a nylon mesh on the apical surface of each tissue, if necessary.b)Test ArticleFor solid test article, the tissues were moistened with 25 μL of ultrapure water to improve contact of the tissue surface with the test article. Approximately 25 mg of each test article was evenly applied to the apical surface of each tissue (n=3). All the tissues were placed into the ~37°C incubator with 5% CO2. The exposure times were approximately 1 hour, with ~35 minutes exposure in the incubator and ~25 minutes at room temperature. 3.Post-exposure treatmentAfter the 1 hour exposure, the tissues were rinsed 20 to 25 times with 1 mL of DPBS. The apical surface was gently blotted with a cotton swab. The tissues were placed in 0.9 mL of fresh Maintenance medium (6-well plate) for either 25 hours, 38 minutes and 23 seconds or for 24 hours, 10 minutes and 09 seconds (as there were numerous tissues, they had to be broken down into 2 sets to complete dosing in a timely manner). After this initial ~24 hour incubation, the tissues were placed in 6-well plates containing 0.9 mL fresh Maintenance medium and incubated for another 17 hours, 03 minutes and 34 seconds prior to performing the MTT assay, for a total of an approximately 42 hour post-exposure incubation.RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE- Model used: The EpiDerm™ 3 dimensional human tissue model- Tissue Lot number(s): 26459- Date of initiation of testing: 6/08/2017TEMPERATURE USED FOR TEST SYSTEM- Temperature used during treatment / exposure: 37°C- Temperature of post-treatment incubation (if applicable): 37°CREMOVAL OF TEST MATERIAL AND CONTROLS-Volume and number of washing steps: TwiceMTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE- MTT concentration: 300 µL MTT medium (1.0 mg/mL).- Incubation time: After 2 hours, 57 minute and 25 second MTT incubation- Spectrophotometer: Synergy H4 spectrophotometer - Wavelength: 570 nm- Filter: No data- Filter bandwidth: No data- Linear OD range of spectrophotometer: No dataNUMBER OF REPLICATE TISSUES: 3CALCULATIONS and STATISTICAL METHODSAll data were background subtracted before analysis. MTT data are presented as % viable compared to negative control. Data were generated as follows: MTT AssayBlanks:·        The optical density (OD) mean from all replicates for each plate (ODblank). Negative Controls (NC):·        The blank corrected value was calculated: ODNC= ODNCraw– ODblank. ·        The OD mean per NC tissue was calculated. ·        The mean OD for all tissues corresponds to 100% viability. ·        The mean, standard deviation (SD), standard error of the mean (SEM) and the percent coefficient of variation (% CV) was calculated. Positive Control (PC):·        Calculate the blank corrected value: ODPC= ODPCraw– ODblank. ·        The OD mean per PC tissue was calculated. ·        The viability per tissue was calculated: %PC = [ODPC/ mean ODNC] x 100. ·        The mean viability for all tissues was calculated: Mean PC = Σ %PC / number of tissues. ·        The standard deviation (SD), standard error of the mean (SEM) and the percent coefficient of variation (% CV) was calculated. Tested compound :·        Calculate the blank corrected value ODTT= ODTTraw– ODblank. ·        The OD mean per tissue was calculated. ·        The viability per tissue was calculated: %TT = [ODTT/ mean ODNC] x 100. ·        The mean viability for all tissues was calculated: Mean TT = Σ %TT / number of tissues. ·        The standard deviation (SD), standard error of the mean (SEM) and the percent coefficient of variation (% CV) was calculated. Data Correction Procedure for MTT Interfering Compounds (if applicable)True viability = Viability of treated tissue – Interference from test article = ODtvt– ODktwhere ODkt= (mean ODtkt– mean ODukt).ODtvt= optical density of treated viable tissueODkt= optical density of killed tissuesODtkt= optical density of treated killed tissueODukt= optical density of untreated killed tissue (NC treated tissue) Data Correction Procedure for Colored Compounds (if applicable)True viability = Viability of treated tissue incubated in MTT media – Viability of treated tissue incubated in media without MTT = ODtvt– ODvt.ODtvt= optical density of treated viable tissue incubated in MTT mediaODvt= optical density of viable tissues incubated in media alone- Evaluation of data The results of the assay was evaluated and compared to negative control.  Table: Criteria for in vitro Interpretation: In VitroResults In VivoPredictionMean tissue viability ≤50% Irritant (I), R38Mean tissue viability >50% Non-irritant (NI)- Assay quality controls- Negative Controls (NC)The Dulbecco’s phosphate buffered saline (DPBS) was used as a NC. The assay passed all acceptance criteria if the ODs of the negative control exposed tissues were between ≥0.8 and ≤2.8.  - Positive Controls (PC)5% solution of sodium dodecyl sulfate was used as a PC. The assay is meeting the acceptance criteria if the viability of the PC is ≤20% of the negative control.   - Standard Deviation (SD)The standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates was ≤18.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL- Amount(s) applied (volume or weight with unit): 25 mg - Concentration (if solution): neat (undiluted)VEHICLE (Not used)- Amount(s) applied (volume or weight with unit): none- Concentration (if solution): none- Lot/batch no. (if required): none- Purity: noneNEGATIVE CONTROL- Amount(s) applied (volume or weight): 30 µL- Concentration (if solution): neatPOSITIVE CONTROL- Amount(s) applied (volume or weight): 30 µL- Concentration (if solution): 5% solution of sodium dodecyl sulfate
Duration of treatment / exposure:
The exposure times were approximately 1 hour, with ~35 minutes exposure in the incubator and ~25 minutes at room temperature.
Duration of post-treatment incubation (if applicable):
For a total of an approximately 42 hour post-exposure incubation.
Number of replicates:
3 tissues will be used per test compound and control.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Run 1
Value:
108
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: mean of OD: 2.285; Non-Irritant
Other effects / acceptance of results:
The MTT data show the assay quality controls were met.

Code N° Tissue  Raw data   Blank corrected data mean  % of viability
  n Aliq. 1 Aliq. 2 Aliq. 1 Aliq. 2 of aliquotes  
NC 1 2.2655 2.2455 2.229 2.209 2.219 104.9
  2 2.0693 2.0371 2.033 2.001 2.017 95.4
  3 2.1138 2.1778 2.078 2.142 2.110 99.7
PC 1 0.0737 0.0743 0.037 0.038 0.038 1.8
  2 0.0784 0.0804 0.042 0.044 0.043 2.0
  3 0.0781 0.0805 0.042 0.044 0.043 2.0
C4 1 2.1521 2.1581 2.116 2.122 2.119 100.2
  2 2.2081 2.2246 2.172 2.188 2.180 103.1
  3 2.5979 2.5837 2.562 2.547 2.555 120.8

  mean SD mean of SD CV %
  of OD of OD viabilities [%] of viabilities [%]
NC 2.115 0.101 100.0 4.79 4.79
PC 0.041 0.003 2.0 0.15 7.48
C4 2.285 0.236 108.0 11.15 10.32
Interpretation of results:
other: not irritating
Conclusions:
The dermal irritation potential of test article was determined according to the OECD 439 test guideline followed for this study. The mean of OD for test chemical was determined to be 2.285.The standard deviation of viabilities for test chemical were calculated to be 11.15.The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 108.0%. Thus, test chemical was considered to be non irritating to the human skin.
Executive summary:

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

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

The mean of OD for test chemical was determined to be 2.285. The standard deviation of viabilities for test chemical were calculated to be 11.15.The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 108.0%. Hence, under the current experimental test conditions it was concluded that test chemical was considered to be non-irritating to human skin.

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 peer reviewed journals
Qualifier:
according to
Guideline:
other: As mentioned below
Principles of method if other than guideline:
Skin irritation test was conducted on guinea pigs to determine the irritation potential of the test chemical
GLP compliance:
not specified
Species:
guinea pig
Strain:
other: Pirbright- White
Details on test animals and environmental conditions:
TEST ANIMALS
- Sex: female
- Age at study initiation: No Data available
- Weight at study initiation:280-350 g
- Housing: animals were kept 3 to a cage
- Diet (e.g. ad libitum):altromin ad libitum
- Water (e.g. ad libitum): water ad libitum
- Acclimation period: No Data available

Or Test Tissue
- Source: No Data available
- Type: No Data available
- Acclimation : No Data available
- Housing conditions No Data available

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22-24⁰C
- Humidity (%):50-55%
- Air changes (per hr): No Data available
- Photoperiod (hrs dark / hrs light): Under artificial illumination for 10 hours a day
Type of coverage:
open
Preparation of test site:
other: clipped or shaved
Vehicle:
other: FCA and Physiological saline
Controls:
not specified
Amount / concentration applied:
10%, 3% and 1 %
Duration of treatment / exposure:
24 hours
Observation period:
24 hours
Number of animals:
10
Details on study design:
TEST SITE
- Area of exposure: clipped or shaved shoulder area (4*6 cm)

OBSERVATION TIME POINTS
(indicate if minutes, hours or days) : The results were read 24 hours after challenge exposure with the test chemical

SCORING SYSTEM:
- Method of calculation:A (+) reaction was considered as threshold of irritation
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
24 h
Score:
0
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
no irritation observed

Table 1: Results of the study

Test chemical

Concentration

Irritation index

7-diethylamino-4-methyl coumarin (coumarin 1)

10%

0.0

The mean irritation index is the average of 3 readings

Interpretation of results:
other: not irritating
Conclusions:
The mean irritation index of test chemical was 0.0 after 24 hours.
Hence, the test chemical was considered to be not irritating to guinea pigs
Executive summary:

Skin irritation test was conducted on guinea pigs to determine the irritation potential of test chemical. 10 female Pirbright-White guinea pigs were treated with an emulsion prepared from FCA and physiological saline but without the test chemical and applied on a semicircular arc on the clipped or shaved shoulder area (4*6 cm) from left to right in such a way that whole emulsion was used up for the animals.1 day before the challenge exposure, teh guinea pigs were treated by applying different concentrations of the test chemical (10%, 3% and 1%) to the right flank.The results of the challenge exposure were observed after 24 hours. A (+) reaction was considered as threshold of irritation. The mean irritation index of test chemical was 0.0 after 24 hours. Hence, the test chemical was considered to be not irritating to guinea pigs

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:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Principles of method if other than guideline:
The purpose of this study was to assess potential for the test article to be ocular irritants. The ocular irritation potential of a test article may be predicted by measurement of its cytotoxic effect, as reflected in the (MTT) assay, in the MatTek EpiOcular™ model
GLP compliance:
no
Specific details on test material used for the study:

RADIOLABELLING INFORMATION (Not applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature / Fridge storage
- Stability under test conditions: No data available
- Solubility and stability of the test substance in the solvent/vehicle: No data available
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No data available

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test article tested as provided neat (undiluted).
- Preliminary purification step (if any): No data available
- Final dilution of a dissolved solid, stock liquid or gel: No data available
- Final preparation of a solid: No data available

FORM AS APPLIED IN THE TEST:solid
Species:
human
Strain:
other: Not applicable
Details on test animals or tissues and environmental conditions:
- Description of the cell system used:
The normal human-derived keratinocytes were cultured at the air-liquid interface in a chemically defined medium on a permeable polycarbonate insert (surface 0.5 cm2). They were cultured in chemically defined serum free medium to form a multi-layered epithelium similar to that found in native corneal mucosa. Each lot of tissues was Quality Assured by MatTek according to specific QC standards including: histology, tissue viability (MTT mean optical density), reproducibility (SD) and tissue thickness.

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

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

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

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

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

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

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

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

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

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

- Description of evaluation criteria used including the justification for the selection of the cut-off point for
the prediction model
Calculations and Statistical Methods
MTT Assay
Blanks:
· The OD mean from all replicates for each plate (ODblank).
Negative Controls (NC):
· The blank corrected value was calculated: ODNC= ODNCraw– ODblank.
· The OD mean per NC tissue was calculated.
· The mean OD for all tissues corresponds to 100% viability.
· The mean, standard deviation (SD), standard error of the mean (SEM) and the percent coefficient of variation (% CV) was calculated.
ODblank= optical density of blank samples (isopropanol alone).
ODNCraw= optical density negative control samples.
ODNC= optical density of negative control samples after background subtraction.
Positive Control (PC):
· Calculate the blank corrected value: ODPC= ODPCraw– ODblank.
· The OD mean per PC tissue was calculated.
· The viability per tissue was calculated: %PC = [ODPC/ mean ODNC] x 100.
· The mean viability for all tissues was calculated: Mean PC = Σ %PC / number of tissues.
· The standard deviation (SD), standard error of the meanthe mean (SEM) and the percent coefficient of variation (% CV) was calculated.
ODPCraw= optical density positive control samples.
ODPC= optical density of positive control samples after background subtraction.
Tested Articles:
· Calculate the blank corrected value ODTT= ODTTraw– ODblank.
· The OD mean per tissue is calculated.
· The viability per tissue is calculated: %TT = [ODTT/ mean ODNC] x 100.
· The mean viability for all tissues is calculated: Mean TT = Σ %TT / number of tissues.
· The standard deviation (SD) and the percent coefficient of variation (% CV)for the controls and the test articles will be calculated.
ODTTraw= optical density test article samples.
ODPC= optical density of test article samples after background subtraction.
Data Correction Procedure for MTT Interfering Compounds
True viability = Viability of treated tissue – Interference from test article = ODtvt – ODkt where ODkt =
(mean ODtkt – mean ODukt).
ODtvt = optical density of treated viable tissue
ODkt = optical density of killed tissues
ODtkt = optical density of treated killed tissue
ODukt = optical density of untreated killed tissue (NC treated tissue)
Data Correction Procedure for Colored Compounds
True viability = Viability of treated tissue incubated in MTT media – Viability of treated tissue incubated in
media without MTT = ODtvt – ODvt.
ODtvt = optical density of treated viable tissue incubated in MTT media
ODvt = optical density of viable tissues incubated in media alone.
Proposed Statistical methods
The mean, standard deviation (SD) and the percent coefficient of variation (% CV) for the controls and the test article will be calculated.
- Evaluation of data
The results of the assay was evaluated and compared to negative control.
Table: Irritancy Prediction
In VitroResults In VivoPrediction
Mean tissue viability ≤60% Irritant (I) – Category 1 or 2
Mean tissue viability >60% Non-irritant (NI) – No Category
- Assay quality controls
- Negative Controls (NC)
The assay is meeting the acceptance criterion if the mean viability of the NC in terms of Optical Density(OD570) of the NC tissues (treated with sterile ultrapure water) in the MTT assay are >0.8 to <2.5. This is an indicator of tissue viability following shipping and conditions under use.
- Positive Controls (PC)
Methyl acetate was used as a PC and tested concurrently with the test article. The assay is meeting the acceptance criteria if the viability of the PC is <50% of the negative control.
- Standard Deviation (SD)
Each test of ocular irritancy potential is predicted from the mean viability determined on 2 single tissues. The assay meets the acceptance criteria if SD calculated from individual percent tissue viabilities of the
replicates is <18% for three replicate tissues.
Irritation parameter:
other: mean % tissue viability
Run / experiment:
Run 1
Value:
68.8
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: mean of OD :1.319;Non- Irritant
Other effects / acceptance of results:
The MTT data show the assay quality controls were met.

Tissue 2 (well C1 and D1) of the negative control did not look 100% okay after the treatment, which may be due to some kind of damage of the tissue. This gives us a SD of D>20, however, the negative control can still be used since the SD error bar is above the cut-off-limit of the assay (i.e. 60%). Chemical #8 (CAS 91-44-1) remained somewhat stuck to the tissues after the washing step but as seen by the results, the tissues has not been affected by this.

Code N° Tissue  Raw data Blank corrected data mean of OD % of viability
  n Aliq. 1 Aliq. 2 Aliq. 1 Aliq. 2
NC 1 2.1668 2.2422 2.131 2.207 2.169 113.2
  2 1.6825 1.7137 1.647 1.678 1.663 86.8
PC 1 0.7964 0.7961 0.761 0.761 0.761 39.7
  2 0.7495 0.7464 0.714 0.711 0.713 37.2
91-44-1 1 1.2858 1.4028 1.250 1.367 1.309 68.3
  2 1.3774 1.3506 1.342 1.315 1.329 69.3

  mean Dif. mean of Dif. Dif./2 Classification
  of OD of OD viabilities [%] of viabilities      
NC 1.916 0.506 100.0 26.43 13.22 NI D>20
PC 0.737 0.048 38.5 2.52 1.26 I qualified
91-44-1 1.319 0.020 68.8 1.03 0.51 NI qualified
Interpretation of results:
other: irritating
Conclusions:
The ocular irritation potential of test article was determined according to the OECD 492 test guideline followed for this study. The mean of OD for test chemical was determined to be 1.319.Tissue 2 (well C1 and D1) of the negative control did not look 100% okay after the treatment, which may be due to some kind of damage of the tissue. This gives us a SD of D>20, however, the negative control can still be used since the SD error bar is above the cut-off-limit of the assay (i.e. 60%). The mean % tissue viability of test chemical was determined to be 68.8%. Thus, test chemical was considered to be non-irritating to the human eyes.
Executive summary:

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

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

The mean of OD for test chemical was determined to be 1.319.Tissue 2 (well C1 and D1) of the negative control did not look 100% okay after the treatment, which may be due to some kind of damage of the tissue. This gives us a SD of D>20, however, the negative control can still be used since the SD error bar is above the cut-off-limit of the assay (i.e. 60%).The mean % tissue viability of test chemical was determined to be 68.8%. Hence, under the experimental test conditions it was concluded that test chemical was considered to be non-irritating to the human eyes.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin Irritation

Various studies have been reviewed to evaluate the dermal irritation potential of the test chemical in living organisms. These include in vivo experimental studies performed on guinea pigs, rabbits as well as in vitro experiments for the test chemical. The experimental studies are also compared with estimated data for the test chemical.

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

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

The mean of OD for test chemical was determined to be 2.285. The standard deviation of viabilities for test chemical were calculated to be 11.15.The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 108.0%. Hence, under the current experimental test conditions it was concluded that test chemical was considered to be non-irritating to human skin.

This is supported by an in vivo Skin irritation study conducted on guinea pigs to determine the irritation potential of test chemical. 10 female Pirbright-White guinea pigs were treated with an emulsion prepared from FCA and physiological saline but without the test chemical and applied on a semicircular arc on the clipped or shaved shoulder area (4*6 cm) from left to right in such a way that whole emulsion was used up for the animals.1 day before the challenge exposure, teh guinea pigs were treated by applying different concentrations of the test chemical (10%, 3% and 1%) to the right flank.The results of the challenge exposure were observed after 24 hours. A (+) reaction was considered as threshold of irritation. The mean irritation index of test chemical was 0.0 after 24 hours. Hence, the test chemical was considered to be not irritating to guinea pigs

These results are further supported by another skin irritation study was performed on rabbit skin to observe the irritation potential of test chemical. The animal was treated with technically pure compound under an occlusive bandage for the observation period of 24 hours. No known irritation effects were observed, hence the test chemical can be considered as non irritant to the rabbit skin.

Skin irritation effects were also estimated by four different model i.e, Battery, leadscope, SciQSAR and CASE Ultra used within Danish QSAR database for test chemical. Based on estimation, No severe skin irritation effects were known when test chemical was exposed to rabbit skin.

The estimated and experimental results are in mutual agreement with each other indicating a strong possibility of the test chemical being not irritating to skin. Hence, the test chemical can be classified under the category “Not Classified” as per CLP Regulation.

Eye Irritation

Various studies have been reviewed to determine the level of ocular damage/corrosion caused by the test chemical in living organisms. These include in vivo as well as in vitro experimental studies 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 of OD for test chemical was determined to be 1.319.Tissue 2 (well C1 and D1) of the negative control did not look 100% okay after the treatment, which may be due to some kind of damage of the tissue. This gives us a SD of D>20, however, the negative control can still be used since the SD error bar is above the cut-off-limit of the assay (i.e. 60%).The mean % tissue viability of test chemical was determined to be 68.8%. Hence, under the experimental test conditions it was concluded that test chemical was considered to be non-irritating to the human eyes.

The in vitro study is supported by the results of an in vivo study performed according to the rabbit eye test protocol. The test chemical was not irritating to rabbit eyes.

The above results are supported by another study ocular irritation potential of the test chemical was assessed by performing rabbit eye test.

Undiluted test chemical did not induce any irritation to rabbit eyes. Hence, it can be considered to be not irritating to eyes.

The experimental in vitro as well as in vivo results are in mutual agreement with each other indicating a very strong possibility that the test chemical was indeed not irritating to eyes. Hence, the test chemical can be classified under the category “Not Classified” as per CLP Regulation.

Justification for classification or non-classification

The in vivo and in vitro experimental results are in mutual agreement with each other indicating a very strong possibility that the test chemical was indeed not irritating to eyes and skin.

Hence, the test chemical can be classified under the category “Not Classified” as per CLP Regulation.