Registration Dossier

Administrative data

Description of key information

Skin Irritation:

The dermal irritation potential of test article was determined according to the OECD 439 In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method”.

The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 94.4%.

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

No general or local effects were noted at the application sites in any of the rabbits tested. Hence, the test chemical was considered to be not irritating to rabbit skin.

Eye Irritation:

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

The mean % tissue viability of test chemical was determined to be 86.3%.

Hence, under the experimental test conditions it was concluded that test chemical was not considered to be an eye irritant and being classified as “Not Classified” as per CLP Regulation.

Slight redness of the conjunctivae and very slight chemosis were detected in all rabbits. At 24 hours, only slight conjunctival redness was noted, which persisted in only one rabbit at the 48 hour reading. At 72 hours following the test chemical instillation, no ocular changes were noted. Fluorescein staining performed at 24 hours following test chemical instillation did not reveal any foreign body in the eye.

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

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
Study period:
December 04, 2017 to March 13, 2018
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 is to provide classification of dermal irritation potential of a chemical by using a three-dimensional human epidermis model, according to the OECD Test Guideline No. 439, “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method”.



GLP compliance:
yes
Test system:
human skin model
Remarks:
MatTek EpiDerm™ Tissue 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:
EpiDerm™ tissues, Lot No. 27646 Kits I and J, were received from MatTek on 12 Dec 2017, and Lot No. 27654 Kits O and P, were received from MatTek on 19 Dec 2017. All tissues were refrigerated at 2-8°C upon receipt. Before use, the tissues were incubated (37±1°C, 5±1% CO2) with assay medium (MatTek) for a one-hour equilibration. The tissues were then moved to new wells with fresh medium for an additional overnight equilibrium, for 18±3 hours. Equilibration medium was replaced with fresh medium before dosing.
Justification for test system used:
The EpiDerm™ Skin Model closely parallels human skin, thus providing a useful in vitro means to assess dermal irritancy and toxicology
Vehicle:
unchanged (no vehicle)
Details on test system:
EpiDerm™ Tissue Samples
EpiDerm™ tissues, Lot No. 27646 Kits I and J, were received from MatTek on 12 Dec 2017, and Lot No. 27654 Kits O and P, were received from MatTek on 19 Dec 2017 All tissues were refrigerated at 2-8°C upon receipt. Before use, the tissues were incubated (37±1°C, 5±1% CO2) with assay medium (MatTek) for a one-hour equilibration. The tissues were then moved to new wells with fresh medium for an additional overnight equilibrium, for 18±3 hours. Equilibration medium was replaced with fresh medium before dosing.

Mesh Compatibility
Five of the test articles supplied were liquids. These test articles were assessed for compatibility with pre-cut nylon mesh supplied with the tissues. The mesh was placed on a slide and 30 μl of a liquid test articles or PBS (negative control) were applied. After 60 minutes of exposure, the mesh was checked microscopically. If no damage or other interaction was observed, indicating that the mesh was compatible with the test article, the mesh was used as a spreading aid.

Tissue Viability (MTT Reduction)
At the end of the incubation period, each EpiDerm™ tissue was rinsed with PBS and transferred to a 24-well plate containing 300 μl of MTT solution (1 mg/ml MTT in DMEM). The tissues were then returned to the incubator for a three-hour MTT incubation period. Following the MTT incubation period, each EpiDerm™ tissue was rinsed with PBS and then treated with 2.0 ml of extractant solution (isopropanol) per well for at least two hours, with shaking, at room temperature. Two aliquots of the extracted MTT formazan were measured at 540 nm using a plate reader (μQuant Plate Reader, Bio-Tek Instruments, Winooski, VT).
For several tissues, the test article had stained the tissues. Therefore, the tissues were extracted with only 1.0 ml, allowing extraction to occur only through the bottom of the insert. After the extraction period, the tissue insert was removed and discarded and 1.0 ml of extraction solution were added to each well, bringing the volume to a total of 2.0 ml.

Quality Controls
The assay meets the acceptance criteria if the mean OD540 of the negative control tissues is between 1.0 and 2.5, inclusive, and the mean viability of positive control tissues, expressed as percentage of the negative control tissues, is at least 20%. In addition, the standard deviation (SD) calculated from individual percent tissue viabilities of the three identically-treated replicates must be less than 18%.
Note: Chemicals that provide tissue viabilities in a range of 30% to 70% may provide high SD. If the high SD (above acceptance limits) is typical for the chemical and the classification of the chemical is consistent in all independent runs, MatTek recommends that this result be accepted, although it did not meet the Assay Acceptance Criterion.

Analysis of Data
See Table 1 for Experimental Data. The mean absorbance value for each time point was calculated from the optical density (OD) of the duplicate samples and expressed as percent viability for each sample using the following formula:
% viability = 100 X (OD sample/OD negative control)

Skin Irritation Prediction
According to the EU1,2 and GHS3 classification (R38 / Category 2 or no label), an irritant is predicted if the mean relative tissue viability of three individual tissues exposed to the test substance is 50% or less of the mean viability of the negative controls.

In vitro result In vivo Classification
Mean tissue viability ≤ 50% Category 2
Mean tissue viability > 50% Non-irritant (NI)


Assessment of direct MTT reduction and assessment of coloring or staining materials was not performed. Therefore, it cannot be fully assessed if the test articles interfered with MTT viability measurements.

Retention of Data
Upon signing the final report, all raw data, supporting documentation and reports are submitted to the Archivist by the Study Director. The raw data are filed at MB Research by project number. The final report is filed at MB Research by Sponsor name and MB project number.
All data generated during the conduct of this study will be archived at MB Research for at least one year from the date of the final report and optionally longer at additional cost. The Sponsor will be contacted in writing to determine final disposition of the records.
Any remaining test article will be discarded upon submission of the report.

Amendment to the Protocol
There were no amendments to the protocol. See Appendix C for the protocol in its entirety
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) Controls
30 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 Article
For 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 treatment
After 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: 6/08/2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): 37°C

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

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 µL MTT medium (1.0 mg/mL).
- Incubation time: After 2 hours, 57 minute and 25 second MTT incubation
- Spectrophotometer: Synergy H4 spectrophotometer
- Wavelength: 570 nm
- Filter: No data
- Filter bandwidth: No data
- Linear OD range of spectrophotometer: No data

NUMBER OF REPLICATE TISSUES: 3

CALCULATIONS and STATISTICAL METHODS
All data were background subtracted before analysis. MTT data are presented as % viable compared to negative control. Data were generated as follows:

MTT Assay
Blanks:
·        The optical density (OD) mean from all replicates for each plate (ODblank).

Negative Controls (NC):
Identity: Phosphate-Buffered Saline (PBS), Lot No. AC10239794
Provided by:MatTek
Date Received:12 Dec 2017 and 19 Dec 2017
Expiration Date:18 Jul 2018
Storage:Room temperature and humidity
Description:Clear colorless liquid
Sample Preparation:Used as received

Positive Control (PC):
Identity: 5% Sodium Dodecyl Sulfate (SDS), Lot No. 071817MAB
Provided by:MatTek
Date Received:12 Dec 2017 and 19 Dec 2017
Expiration Date:18 Jul 2018
Storage:Room temperature and humidity
Description:Clear colorless liquid
Sample Preparation:Used as received

- Assay quality controls
- Negative Controls (NC)
The Dulbecco’s phosphate buffered saline (DPBS) was used as a NC. The assay passed all acceptance criteria if the ODs of the negative control exposed tissues were between ≥0.8 and ≤2.8.
 
- Positive Controls (PC)
5% solution of sodium dodecyl sulfate was used as a PC. The assay is meeting the acceptance criteria if the viability of the PC is ≤20% of the negative control.
 
- Standard Deviation (SD)
The standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates was ≤18.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg
- Concentration (if solution): neat (undiluted)

VEHICLE (Not used)
- Amount(s) applied (volume or weight with unit): none
- Concentration (if solution): none
- Lot/batch no. (if required): none
- Purity: none

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): neat

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5% solution of sodium dodecyl sulfate
Duration of treatment / exposure:
Tissues will be topically exposed to the test article and control articles for 60 minutes.
Duration of post-treatment incubation (if applicable):
After dosing, the tissues will be returned to the incubator for 35 ±1 minute, and then returned to the sterile hood for the remainder of the 60-minute exposure period.
Number of replicates:
All treatments with test articles and controls will be dosed in triplicate EpiDerm™ tissues.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Run 1
Value:
94.4
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
All treatments with test articles and controls will be dosed in triplicate EpiDerm™ tissues.

In vitro result In vivo Classification
Mean tissue viability ≤ 50% Category 2
Mean tissue viability > 50% Non-irritant
Interpretation of results:
other: not irritating
Conclusions:
The dermal irritation potential of test article was determined according to the OECD 439 test guideline followed for this study. The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 94.4%. Thus, the test chemical was considered to be not irritating to the human skin.
Executive summary:

The dermal irritation potential of test article was determined according to the OECD 439 In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method”. The MatTek EpiDerm™ model was used to assess the potential dermal irritation of the test article by determining the viability of the tissues following exposure to the test article via MTT. The objective of this study was to assess the dermal irritation potential of test article Tissues were exposed to test article and controls for ~one hour, followed by a 42 hour post-exposure recovery period. The viability of each tissue was determined by MTT assay. 

The MTT data shows that the assay quality controls were met. The mean tissue viabilities for the Positive control, Methyl acetate were 6.5%, 10.7% respectively in the first and second run, whereas the tissue viabilities of the negative control, Tissue culture water remained at 100% in the both the runs.

The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 94.4%.

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

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Justification for type of information:
Experimental result using the standard OECD protocol.
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Principles of method if other than guideline:
Skin irritation potential of the test chemical was investigated in rabbits using the standard OECD protocol.
GLP compliance:
not specified
Species:
rabbit
Strain:
New Zealand White
Type of coverage:
semiocclusive
Preparation of test site:
not specified
Vehicle:
not specified
Controls:
not specified
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): single 500 mg doses of the test material were applied.


Duration of treatment / exposure:
Three minutes and one hour.
Observation period:
1 hour
Number of animals:
Three
Details on study design:
TEST SITE
- Area of exposure: skin of the trunk
- % coverage: no data available
- Type of wrap if used: semi-occlusive dressing

REMOVAL OF TEST SUBSTANCE
- Washing (if done): no data available
- Time after start of exposure: no data available

OBSERVATION TIME POINTS
(indicate if minutes, hours or days) : no data available

SCORING SYSTEM:
- Method of calculation:no data available
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
other: 1 hours
Score:
0
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
No general or local effects were noted at the application sites in any of the rabbits tested.
Other effects:
No clinical signs, either general or local (at the application sites), were noted in any rabbit.
Interpretation of results:
other: non-irritating
Conclusions:
No general or local effects were noted at the application sites in any of the rabbits tested.Hence, the test chemical was considered to be not irritating to rabbit skin.
Executive summary:

Skin irritation potential of the test chemical was investigated in rabbits using the standard OECD protocol.

Single 500 mg doses of the test material were applied under semi-occlusive dressing to the skin of the trunk of three New Zealand albino rabbits for three minutes and one hour.

No general or local effects were noted at the application sites in any of the rabbits tested.Hence, the test chemical was considered to be not irritating to rabbit skin.

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

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 05, 2017 to July 12, 2017
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 chemical may be predicted by measurement of its cytotoxic effect, as reflected in the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, in the MatTek EpiOcular™ model (MatTek Corp., Ashland, MA).





GLP compliance:
yes
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
Human Corneal Epithelia (HCE) by MatTek, Inc.:
The test article and controls were evaluated for potential ocular irritancy using the EpiOcular™ 3 dimensional human tissue model purchased from MatTek Corporation (Ashland, MA). This model consists of normal human keratinocytes cultured on a permeable synthetic membrane at the air-liquid interface in a chemically defined medium. The cells form a stratified, squamous corneal epithelium resembling the corneal mucosa of the human eye. This model has been used with several common tests of cytotoxicity including MTT and interleukin 1-alpha (IL-1α). A growing body of evidence indicates that EpiOcular™ effectively provides a non-animal means to assess potential irritancy. The EpiOcular™ model closely mimics the human corneal mucosa and thus provides an important in vitro approach in the evaluation of ocular irritancy and toxicity. 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
- 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 hours for 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 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 test article and controls.




Number of animals or in vitro replicates:
3 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 chemical 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 hours for test article 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 test article and controls. 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 article 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
~50 mg of test chemical 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 2 hours, 55 minutes and 02 seconds. 50 µL of ultrapure water was used as a negative control.

- Test Article Color Test
Approximately 50 mg of test chemical 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 BioTek Synergy H4 (or equivalent) plate reader set to 570 nm. Test article 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 2 hours 55 minutes and 00 seconds (solids) of MTT incubation at approximately 37°C, approximately 5% CO2 in a humidified incubator, the tissues were rinsed twice with DPBS. The blue formazan salt was extracted by submerging tissues in 2 mL isopropanol in a 24-well plate. The extraction time was 2 hours 00 minutes with gentle shaking for solid exposed tissues. 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 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 59 minutes and 00 seconds. The tissues were not incubated overnight.

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 31 minutes 00 seconds. 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, 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 6 hour exposure time.

b)Test Article:
Approximately 50 mg of the test article 3-nitrobenzoic acid were added to the tissues as a fine powder. The tissues were placed into the ~37°C humidified incubator with 5% CO2 for the approximately 6 hour exposure time.

3. Post exposure treatment
After the exposure, the tissues were rinsed 20 to 25 times with ~1 mL 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 solid test article (and the respective control) were incubated, submerged in the media for ~25 minutes at room temperature. 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 17 hours 48 minutes 01 seconds at approximately 37 degC, 5% CO2 in a humidified incubator.

- Doses of test chemical and control substances used
a)Test Article:
Approximately 50 mg of the test chemical were added to the tissues as a fine powder. The tissues were placed into the ~37°C humidified incubator with 5% CO2 for the approximately 6 hour exposure time.

b)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 6 hour exposure time.

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods:
Tissues were exposed for approximately ~6 hours for test article and controls, at approximately 37°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 test article 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: 3 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 time was 2 hours 00 minutes with gentle shaking for solid exposed tissues. 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.

- 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:
1
Value:
86.3
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The MTT data show the assay quality controls were met, as the OD of the negative control tissues was between 0.981 and 1.284 in run 1. Also, the positive control, methyl acetate, reduced tissue viability to be 11.3% of negative control (for 6 hour exposures with solids) in run one and the standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates was 0.3 passing the acceptance criteria.

Test material identity

Tissue Viability

Irritancy Classification

GHS Category

 

Mean

SD

CAS No. 67-71-0

86.3%

19.38%

Non –irritant

Not Classified

 

Test and control article identity

Tissue no.

Raw data

Blank corrected data

Mean of aliquots

% viability

OD

Viabilities (%)

MEAN

SD

Mean

SD

 

Aliq 1

Aliq 2

 

Aliq 1

Aliq 2

 

 

 

 

67-71-0

1

 

1.593

1.552

1.546

1.505

1.526

96.0

1.372

0.308

86.3

19.38

2

1.264

1.265

1.217

1.218

1.218

76.6

Interpretation of results:
other: not irritating
Conclusions:
The ocular irritation potential of test chemical was determined according to the OECD 492 test guideline followed for this study. The mean % tissue viability of test chemical was determined to be 86.3%. Thus, the test chemical wasnot considered to be an eye irritant.
Executive summary:

The ocular irritation potential of test chemical was determined according to the OECD 492 test guideline followed 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. The objective of this study was to assess the ocular irritation potential of test article. Tissues were exposed to test article and controls for ~6 hours, followed by a ~25 minute post-soak and approximately 18 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, as the OD of the negative control tissues was between 0.981 and 1.284 in run 1. Also, the positive control, methyl acetate, reduced tissue viability to be 11.3% of negative control (for 6 hour exposures with solids) in run one and the standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates was 0.3 passing the acceptance criteria.

 

The mean % tissue viability of test chemical was determined to be 86.3%.

 

Hence, under the experimental test conditions it was concluded that test chemical was not considered to be an eye irritant and being classified as “Not Classified” as per CLP Regulation.

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Justification for type of information:
Experimental result following standard method.
Qualifier:
according to
Guideline:
other: as mentioned below
Principles of method if other than guideline:
The primary ocular irritation potential of the test chemical was tested in rabbits
GLP compliance:
not specified
Species:
rabbit
Strain:
New Zealand White
Vehicle:
unchanged (no vehicle)
Controls:
not specified
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Single 100 mg doses of the test material was applied.
Duration of treatment / exposure:
24 h
Observation period (in vivo):
24, 48 and 72 hours
Number of animals or in vitro replicates:
Three (two male and one female)
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): no data available
- Time after start of exposure: no data available

SCORING SYSTEM: no data available

TOOL USED TO ASSESS SCORE: hand-slit lamp / biomicroscope / fluorescein:Fluorescein staining was performed at 24 hours following test chemical instillation
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
72 h
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
mean
Time point:
72 h
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
Slight redness of the conjunctivae and very slight chemosis were detected in all rabbits. At 24 hours, only slight conjunctival redness was noted, which persisted in only one rabbit at the 48 hour reading. At 72 hours following the test chemical instillation, no ocular changes were noted. Fluorescein staining performed at 24 hours following test chemical instillation did not reveal any foreign body in the eye.
Interpretation of results:
other: not irritating
Conclusions:
Slight redness of the conjunctivae and very slight chemosis were detected in all rabbits. At 24 hours, only slight conjunctival redness was noted, which persisted in only one rabbit at the 48 hour reading. At 72 hours following the test chemical instillation, no ocular changes were noted. Fluorescein staining performed at 24 hours following test chemical instillation did not reveal any foreign body in the eye.
Hence, the test chemical can be considered to not irritating to rabbit eyes.
Executive summary:

The primary ocular irritation potential of the test chemical was tested in rabbits. Three (two male and one female) New Zealand White rabbits were used for the study. Single 100 mg doses of the test material was instilled in to the eyes of rabbits and observed for effects. Fluorescein staining was performed at 24 hours following test chemical instillation. The treated eyes were observed and scored at 24, 48 and 72 hours post instillation of the test chemical.

Slight redness of the conjunctivae and very slight chemosis were detected in all rabbits. At 24 hours, only slight conjunctival redness was noted, which persisted in only one rabbit at the 48 hour reading. At 72 hours following the test chemical instillation, no ocular changes were noted. Fluorescein staining performed at 24 hours following test chemical instillation did not reveal any foreign body in the eye.

Hence, the test chemical can be considered to not irritating to rabbit 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 on rabbtis as well as in vitro experimental study for the test chemical. The results are summarized below:

The dermal irritation potential of test article was determined according to the OECD 439 In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method”. The MatTek EpiDerm™ model was used to assess the potential dermal irritation of the test article by determining the viability of the tissues following exposure to the test article via MTT. The objective of this study was to assess the dermal irritation potential of test article Tissues were exposed to test article and controls for ~one hour, followed by a 42 hour post-exposure recovery period. The viability of each tissue was determined by MTT assay. 

The MTT data shows that the assay quality controls were met. The mean tissue viabilities for the Positive control, Methyl acetate were 6.5%, 10.7% respectively in the first and second run, whereas the tissue viabilities of the negative control, Tissue culture water remained at 100% in the both the runs.

The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 94.4%.

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

This in vitro result is supported by an OECD 404 Guideline study performed to evaluate the skin irritation potential of the test chemical in rabbits.

Single 500 mg doses of the test material were applied under semi-occlusive dressing to the skin of the trunk of three New Zealand albino rabbits for three minutes and one hour.

No general or local effects were noted at the application sites in any of the rabbits tested. Hence, the test chemical was considered to be not irritating to rabbit skin.

These results are supported by a Range finding study performed to determine the potential of the test chemical to cause dermal irritation in rabbits.

2 male New Zealand White rabbits were used for the study. 0.5 g of the test substance, prepared as a paste in saline (1:1), was held under an impervious patch in continuous 24-hr contact with the closely clipped intact and abraded skin. The rabbits were observed for signs of erythema and edema and graded at 24,72hours and 7 days. The primary irritation score is the sum of the mean values of 24 and 72 hours divided by 4.

At 24 hr on abraded skin erythema was observed in both rabbits. The Primary Irritation Score was calculated to be 0.3. The scores were zero for erythema and edema at 72 hours and 7 days.

Since the effects were reversible the test chemical can be considered to be not irritating to rabbit skin.

The results of the in vitro and in vivo experimental studies are in mutual agreement with each other indicating a very strong possibility that the test chemical is indeed not irritating to skin. Comparing the above annotations with the criteria of the CLP regulation the test chemical can be classified under the category “Not Classified”.

Eye irritation

Various studies have been reviewed to determine the extent of ocular damage caused by the test chemical 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 chemical was determined according to the OECD 492 test guideline followed 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. The objective of this study was to assess the ocular irritation potential of test article. Tissues were exposed to test article and controls for ~6 hours, followed by a ~25 minute post-soak and approximately 18 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, as the OD of the negative control tissues was between 0.981 and 1.284 in run 1. Also, the positive control, methyl acetate, reduced tissue viability to be 11.3% of negative control (for 6 hour exposures with solids) in run one and the standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates was 0.3 passing the acceptance criteria.

The mean % tissue viability of test chemical was determined to be 86.3%.

Hence, under the experimental test conditions it was concluded that test chemical was not considered to be an eye irritant and being classified as “Not Classified” as per CLP Regulation.

The in vitro result is supported by an in vivo study performed to assess the primary ocular irritation potential of the test chemical in rabbits. Three (two male and one female) New Zealand White rabbits were used for the study. Single 100 mg doses of the test material was instilled in to the eyes of rabbits and observed for effects. Fluorescein staining was performed at 24 hours following test chemical instillation. The treated eyes were observed and scored at 24, 48 and 72 hours post instillation of the test chemical.

Slight redness of the conjunctivae and very slight chemosis were detected in all rabbits. At 24 hours, only slight conjunctival redness was noted, which persisted in only one rabbit at the 48 hour reading. At 72 hours following the test chemical instillation, no ocular changes were noted. Fluorescein staining performed at 24 hours following test chemical instillation did not reveal any foreign body in the eye.

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

The above results are supported by the Range finding studies conducted to determine the level of irritation potential of the test chemical in rabbits. 2 male New Zealand White rabbits were used for the study. 0.1g of the ground test substance was introduced in to the conjunctival sac of the eyes of two rabbits. The eyes remained unwashed throughout the test. The eyes were stained with fluorescein before the observation period. The treated eyes were scored at 4, 24,48 and 72 hours till 7 days for corneal opacity, iris, conjunctival irritatiion.

In second animal after 24 hr, No corneal opacity was observed, fluorescein instillation resulted in an area < 25% of the cornea staining. This uptake of dye suggests that a superficial, transient irritation of the corneal epithelium occurred. This staining of the cornea did not occur at the next scheduled examination at 48 hr after dosing. All irritation was reversible within 7 days but not within 24 h after dosing.

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

The results of the in vitro and in vivo experimental studies are in mutual agreement with each other indicating a very strong possibility that the test chemical is indeed not irritating to eyes. Comparing the above annotations with the criteria of the CLP regulation the test chemical can be classified under the category “Not Classified”.

 

Justification for classification or non-classification

The results of the in vitro and in vivo experimental studies are in mutual agreement with each other indicating a very strong possibility that the test chemical is indeed not irritating to skin and eyes. Comparing the above annotations with the criteria of the CLP regulation the test chemical can be classified under the category “Not Classified”.