Registration Dossier

Administrative data

Description of key information

Skin Irritation

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 2.176. The standard deviation of viabilities for test chemical were calculated to be 17.58.The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 99.9%. Hence, under the current experimental test conditions it was concluded that test chemical was considered to be non-irritating to human skin.

A study with a design similar to OECD 404, 3 male and 3 female New Zealand White rabbits were exposed to the substance for 4 hours under semi-occlusive conditions. No signs of irritation were observed after 72 hours of observation. The substance was considered non-irritant.

Eye Irritation

In a study similar to OECD 405, 3 male and 3 female New Zealand White rabbits received 0.1 mL of the substance in the eye. Evaluation of the eyes over a period of 7 days did not show any signs of irritation. The substance was not irritating to the 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
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:
no
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:
99.9
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:
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 2.1422 2.1817 2.107 2.146 2.126 97.7
  2 2.2181 2.214 2.183 2.178 2.181 100.1
  3 2.2638 2.2585 2.228 2.223 2.226 102.2
PC 1 0.0956 0.0955 0.060 0.060 0.060 2.8
  2 0.0943 0.0956 0.059 0.060 0.059 2.7
  3 0.1508 0.1512 0.115 0.116 0.115 5.3

C2 1 2.1897 2.221 2.154 2.185 2.170 99.6
  2 1.8093 1.8534 1.774 1.818 1.796 82.5
  3 2.5781 2.6154 2.543 2.580 2.561 117.6

  mean SD mean of SD CV %
  of OD of OD viabilities [%] of viabilities [%]
NC 2.178 0.050 100.0 2.28 2.28
PC 0.078 0.032 3.6 1.48 41.12

C2 2.176 0.383 99.9 17.58 17.59
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 2.176.The standard deviation of viabilities for test chemical were calculated to be 17.58.The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 99.9%. Thus, 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 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 2.176. The standard deviation of viabilities for test chemical were calculated to be 17.58.The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 99.9%. 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
Study period:
7 to 10 April 1987
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Justification for type of information:
data is from experimental reports
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Qualifier:
equivalent or similar to
Guideline:
other: Federal Register Vol 50 no 188, Part II, 27 September 1985
Version / remarks:
section 798-4470-Primary Dermal Irritation
Principles of method if other than guideline:
A study was performed to assess the dermal irritation potential of 4-methyl-1-phenylpyrazolidin-3-one in rabbits
GLP compliance:
yes
Remarks:
QA statement and statement by study director in the report
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Buckmasters, Henham, Hertfordshire, England
- Age at study initiation: 9-13 weeks
- Weight at study initiation: 2.1-2.9 kg
- Housing: individually in metal cages with perforated floors
- Diet: SDS Standard Rabbit Diet ad libitum
- Water: tap water ad libitum
- Acclimation period: duration not indicated

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ca 19 °C
- Humidity (%): 30-70%
- Air changes (per hr): ca 19/hour
- Photoperiod (hrs dark / hrs light): 12/12
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Remarks:
moistened with 0.5 mL water
Controls:
not required
Amount / concentration applied:
0.5 mg moistened with 0.5 mL water
Duration of treatment / exposure:
4 hours
Observation period:
4 days
Number of animals:
3 males + 3 females
Details on study design:
TEST SITE
- Area of exposure: 2.5 cm2
- Type of wrap if used: elastoplast

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: after 4 h

OBSERVATION TIME POINTS: at 30 min after exposure, on day 2, 3 and 4

SCORING SYSTEM: Draize
Irritation parameter:
erythema score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritation parameter:
edema score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
No signs of irritation observed

Animal no

1

2

3

30 min

24

48

72

30 min

24

48

72

30 min

24

48

72

erythema

0

0

0

0

0

0

0

0

0

0

0

0

oedema

0

0

0

0

0

0

0

0

0

0

0

0

Animal no

4

5

6

30 min

24

48

72

30 min

24

48

72

30 min

24

48

72

erythema

0

0

0

0

0

0

0

0

0

0

0

0

oedema

0

0

0

0

0

0

0

0

0

0

0

0

 

Interpretation of results:
other: not irritating
Conclusions:
No signs of irritation were observed after 72 hours of observation. The substance was considered non-irritant.
Executive summary:

In a study with a design similar to OECD 404, 3 male and 3 female New Zealand White rabbits were exposed to the substance for 4 hours under semi-occlusive conditions. No signs of irritation were observed after 72 hours of observation. The substance was considered non-irritant.

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

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 to 20 April 1987
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Justification for type of information:
data is from experimental reports
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Qualifier:
equivalent or similar to
Guideline:
other: Federal Register Vol 50 no 188, Part II, 27 September 1985
Version / remarks:
section 798-4500-Primary Eye Irritation
Principles of method if other than guideline:
A study was performed to determine the degree of ocular damage caused by the test chemical in rabbits
GLP compliance:
yes
Remarks:
report includes a statement by the study director and a QA statement
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Buckmasters, Henham, Hertfordshire, England
- Age at study initiation: 10-11 weeks
- Weight at study initiation: 2.2-2.6 kg
- Housing: individually in metal cages with perforated floors
- Diet: SDS Standard Rabbit Diet ad libitum
- Water: tap water ad libitum
- Acclimation period: duration not indicated

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ca 19 °C
- Humidity (%): 30-70%
- Air changes (per hr): ca 19/hour
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
not specified
Controls:
not specified
Amount / concentration applied:
0.5 mL (70 mg)
Duration of treatment / exposure:
NA
Observation period (in vivo):
after 1 hour and on day 1, 2, 3, 4 and 7
Number of animals or in vitro replicates:
3 males + 3 females
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): no

SCORING SYSTEM: Draize

TOOL USED TO ASSESS SCORE: hand-slit lamp
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
1
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
1
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
Evaluation of the eyes over a period of 7 days did not show any signs of irritation

Animal no

1

2

3

1

24

48

72

4

7

1

24

48

72

4

7

1

24

48

72

4

7

 

cornea

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

iris

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

conjunctivae

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

redness

1

0

0

0

0

0

1

0

0

0

0

0

1

0

0

0

0

0

 

chemosis

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

Animal no

4

5

6

 

1

24

48

72

4

7

1

24

48

72

4

7

1

24

48

72

4

7

 

cornea

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

iris

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

conjunctivae

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

redness

1

0

0

0

0

0

1

0

0

0

0

0

1

0

0

0

0

0

 

chemosis

1

0

0

0

0

0

1

0

0

0

0

0

0

0

0

0

0

0

 

Interpretation of results:
other: not irritating
Conclusions:
Evaluation of the eyes over a period of 7 days did not show any signs of irritation. The substance was not irritating to the eyes.
Executive summary:

In a study similar to OECD 405, 3 male and 3 female New Zealand White rabbits received 0.1 mL of the substance in the eye. Evaluation of the eyes over a period of 7 days did not show any signs of irritation. The substance was not irritating to the eyes.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 06, 2017 to March 24, 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 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 is to provide classification of chemicals concerning their eye irritation potential using an alternative to the Draize Rabbit Eye Test, according to the OECD Test Guideline No. 492, “Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage”. The EpiOcular™ EIT is intended to differentiate those materials that are UN GHS No Category (i.e., do not meet the requirements for UN GHS classification) from those that would require labeling as either UN GHS Category 1 or 2. This assay is not intended to differentiate between UN GHS Category 1 / Hazard
Code 318 and UN GHS Category 2 / Hazard Codes 319 and 320.
GLP compliance:
yes
Species:
other: MatTek EpiOcular Tisssue Model OCL-200
Strain:
other: Not applicable
Details on test animals or tissues and environmental conditions:
-Test System: MatTek EpiOcular™ Tissue Model OCL-200

Storage:EpiOcular™ tissues and assay medium will be refrigerated at approximately 2-8°C upon arrival and until use.

Supplier:MatTek Corporation, Ashland, MA

- Justification of the test method and considerations regarding applicability
The EpiOcular™ Tissue Model closely parallels human ocular tissue, thus providing a useful in vitro means to assess ocular irritancy and toxicology
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

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 will be topically exposed to the test article and control articles for 6 hours ± 15 minutes.



Observation period (in vivo):
Not applicable
Duration of post- treatment incubation (in vitro):
Following the post soak,Tissues will be incubated in 1 ml fresh assay medium in a humidified 37±1°C, 5±1% CO2 incubator.




Number of animals or in vitro replicates:
2 tissues were used for test compound and control.
Details on study design:
-Plate Reader Linearity Check:
The linearity of the plate reader or spectrophotometer used for optical density (OD) determination will be verified prior to its use the same week the EIT assay is being
performed.
A dilution series of trypan blue or thiazolyl blue tetrazolium bromide (MTT) formazan will be prepared and 200 μl aliquots will be pipetted into a 96-well plate.
The optical density of the plate wells will be measured at a wavelength of 570 nm (OD570), with no reference wavelength.
A regression line and an R-squared value will be generated using Microsoft Excel®. Verification will be considered acceptable if the R-squared value is >0.999.

Assessment of Direct MTTReduction:No assessment of the direct MTT (methyl thiazole tetrazolium) reduction potential of each test article will be made.

-Assessment of Coloring or Staining Materials:
No assessment of each test article’s ability to absorb light at the wavelength (570 nm) used for MTT determination will be made.

- Pre-Incubation:
EpiOcular™ tissues will be placed in six-well plates containing warmed assay media and will be equilibrated in a humidified 37±1°C, 5 ±1% CO2 incubator for at least one hour. The media will then be changed and the tissues incubated overnight (16-24 hours).
Any tissues not being incubated the same day will be allowed to re-equilibrate at 37±1°C, 5±1% CO2 and will be stored at approximately 2-8°C..

-Pre-Treatment:After the overnight incubation, the tissues will be moistened with 20 μl of phosphatebuffered saline (PBS) and incubated at 37±1°C, 5±1% CO2 for 30±2 minutes.

-Dosing:Whenever possible, solids should be ground to a fine powder before application. 50 mg of a solid test article will be applied topically to duplicate tissues
and incubated at 37±1°C, 5±1% CO2 for 6 hours ± 15 minutes.
After dosing and incubation, the tissues will be thoroughly rinsed with PBS and soaked in 5 ml of room-temperature assay medium in a 12-well plate for the appropriate amount of time.
Tissues will be soaked for 25±2 minutes.

-MTT Extraction:
Following the three-hour MTT incubation period, each tissue will be removed individually and gently rinsed with PBS to remove any residual MTT solution.
The extraction plate will be covered and sealed to reduce evaporation of extractant.
For solid, colored, or staining test articles, 2.0 ml of extractant solution will be used in a six-well plate, allowing extraction to occur through the bottom of the insert.
 
Extraction Conditions:The extraction will be allowed to proceed overnight at room temperature in the dark.
Alternatively, the extraction can proceed for at least two hours, with shaking, at room temperature.

-Decant Extractant:
Tissues immersed in extractant solution in a 24-well plate: After the extraction period is complete, the liquid within each tissue insert will be decanted back into the well
from which it was taken, i.e., the solution will be mixed with the extractant in the well.
The tissue inserts will then be discarded.

-Transferring to 96-Well Plate:Two 200 μl aliquots from each well of the extraction plate(s) will be pipetted into a 96- well microtiter plate.

-Measuring Optical Density:The optical density of the extracted samples will be determined at a single wavelength of 570 nm and using eight 200 μl aliquots of the Extractant as blanks.

Calculating Percent Viability:
The percent viability of the test tissues will be determined using the following formula:
% Viability = 100 x (ODsample / ODNegative Control)

Quality Controls:
Negative Controls: The assay meets the acceptance criterion if the OD570 of the Negative Control is greater than 0.8 and less than 2.5.
Positive Controls: The assay meets the acceptance criterion if the mean relative viability of the positive control is below 50% of negative control viability.
Tissue Variability: The difference in viability between identically treated tissues must be less than 20%. This applies to tissues treated with the same test article as well as living and killed controls.

Ocular Irritation Potential:
An irritant is predicted if the mean relative tissue viability of two individual tissues exposed to the test substance is less than or equal to 60% of the mean viability of the
negative control-treated tissue viability.

In Vitro Result In Vivo Prediction (GHS3)
Mean tissue viability ≤ 60% Category 1 / Hazard Code 318, or
Category 2 / Hazard Codes 319 and 320
Mean tissue viability > 60% No Category (Non-Irritating)

Borderline Results:
If the test article-treated tissue viability is 60±5%, a second EIT should be performed. If the results of the second test disagree with the first, then a third test should be performed. The conclusion will be based on the agreement of two of the three tests.

Duration:The duration of the EpiOcularTM Eye Irritation Test is approximately five days.
Irritation parameter:
other: mean % tissue viability
Run / experiment:
Run 1
Value:
52.9
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:
Negative Controls: The assay meets the acceptance criterion if the OD570 of the Negative Control is greater than 0.8 and less than 2.5.
Positive Controls: The assay meets the acceptance criterion if the mean relative viability of the positive control is below 50% of negative control viability.
Tissue Variability: The difference in viability between identically treated tissues must be less than 20%.

Test and Control Article Identity

 

Tissue Viability

Irritancy Classification

GHS Category

 

Mean

SD

2654-57-1

52.9

23.36

Irritant

Category 1 or 2

 

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

 

 

 

 

2654-57-1

1

 

0.710

0.765

0.665

0.720

0.692

41.2

0.888

0.393

52.9

23.

36*

2

1.129

1.131

1.084

1.086

1.085

64.6

*Standard deviation greater than 20%.

 

CAS No. 2654-57-1 had tissue viabilities of 41.2% and 64.6%, with an SD of 23.36%. Due to the mean tissue viability of 52.9%, it was judged that

the test chemical was classified as Category 1/ Hazard Code 318, or Category 2/Hazard Codes 319 and 320.

Interpretation of results:
other: Category 1 or 2
Conclusions:
The ocular irritation potential of test article was determined according to the OECD 492 test guideline. The test chemical had tissue viabilities of 41.2% and 64.6%, with an SD of 23.36%. Due to the mean tissue viability of 52.9%, it was judged that the test chemical was classified as Category 1/ Hazard Code 318, or Category 2/Hazard Codes 319 and 320.
Executive summary:

The ocular irritation potential of test article was determined according to the OECD 492 test guideline. The MatTek EpiOcular™ model was used to assess the potential ocular 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 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 0.8 to 2.5 in run 1. Also, the positive control, methyl acetate, reduced tissue viability to be below 50% 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 23.36% passing the acceptance criteria.

The test chemical had tissue viabilities of 41.2% and 64.6%, with an SD of 23.36%. Due to the mean tissue viability of 52.9%, it was judged that the test chemical was classified as Category 1/ Hazard Code 318, or Category 2/Hazard Codes 319 and 320.

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 reviewed to determine the level of dermal irritation caused by the test chemical in living organisms. These include in vivo as well as in vitro experimental results for the test chemical performed on rats, rabbits. The studies are summarized below:

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 2.176. The standard deviation of viabilities for test chemical were calculated to be 17.58.The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 99.9%. Hence, under the current experimental test conditions it was concluded that test chemical was considered to be non-irritating to human skin.

This result is supported by a study with a design similar to OECD 404, 3 male and 3 female New Zealand White rabbits were exposed to the substance for 4 hours under semi-occlusive conditions. No signs of irritation were observed after 72 hours of observation. The substance was considered non-irritant.

These results are supported by a study designed and conducted to determine the dermal reaction profile of the test chemical in Sprague Dawley rats. This study was performed as per OECD guideline No. 402. Ten rats (5 male and 5 female) were used for conducting dermal irritation/ corrosion study.

The animals were kept in their cages for at least 5 days prior to administration for acclimatization to the laboratory condition and after acclimatization period, animals were randomly selected. Approximately 24 hours before application, the hair of each rat was closely clipped from the trunk (dorsal surface and sides from scapular to pelvic area) with an electric clipper, so as to expose at least 10% of the body surface area. The test item was moistened with distilled water. The test item was applied onto the exposed skin of the animal, taking care to spread the test item evenly over the entire area of approximately 10% of the total body surface area or as much of the area as can reasonably be covered. The test item was held in contact with the skin using a porous gauze dressing and non irritating tape around the animal to cover the exposure site for first 24 hours exposure period. Elizabethan collar was placed on each animal for first 24 hours after application of the test item. These collars prevent ingestion of test item. Following 24 hours of exposure, the wrapping was removed and the test site wiped free of excess test item. Distilled water was used to remove residual test item.

The test chemical was applied to shorn skin of 5 male and 5 female animals at 2000 mg/kg body weight. Administration of the test item at 2000 mg/kg did not result in any skin reaction at the site of application during the study period of 14 days. Also, the erythema and edema score of rats was calculated as 0. Administration of the test item did not result in any signs of toxicity and mortality during the study period of 14 days.  Animals exhibited normal body weight gain through the study period of 14 days. Gross pathological examination did not reveal any abnormalities attributable to the treatment.

Hence, it was concluded that the test chemical was Non-Irritating to the skin of Sprague Dawley rats under the experimental conditions tested and Classified as “Category- Unclassified” as per CLP Classification.

The above in vivo result is also supported by another in vitro study performed according to the OECD 439 In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method” to determine the dermal irritation potential of the test chemical. 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, as the OD of the negative control tissues was between 1.195 and 1.430. Also, the positive control, 5% sodium dodecyl sulfate (SDS), reduced tissue viability to 4.5% of negative control.

The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 97.9%.

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.

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

Eye Irritation

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

In a study similar to OECD 405, 3 male and 3 female New Zealand White rabbits received 0.1 mL of the substance in the eye. Evaluation of the eyes over a period of 7 days did not show any signs of irritation. The substance was not irritating to the eyes.

The in vitro eye irritation /potential of test article was determined according to the OECD 492 test guideline. The MatTek EpiOcular™ model was used to assess the potential ocular 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 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 0.8 to 2.5 in run 1. Also, the positive control, methyl acetate, reduced tissue viability to be below 50% 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 23.36% passing the acceptance criteria.

The test chemical had tissue viabilities of 41.2% and 64.6%, with an SD of 23.36%. Due to the mean tissue viability of 52.9%, it was judged that the test chemical was classified as Category 1/ Hazard Code 318, or Category 2/Hazard Codes 319 and 320.

The above in vitro study is supported by a similar study performed according to OECD 492 test guideline to evaluate the ocular irritation potential of the test chemical. The MatTek EpiOcular™ model was used to assess the potential ocular irritation of the test articles by determining the viability of the tissues following exposure to the test article via MTT. Tissues were exposed to solid test articles and control for approx.6 hours, followed by a 25 minute post-soak and approximately 18 hours recovery after the post-soak. The viability of each tissue was determined by MTT assay.

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

The mean % tissue viability of test substance was determined to be 52.9%. Hence, under the experimental test conditions it was concluded that test substance 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.

 

In an eye irritation study conducted as per OECD 405 Guidelines, 6 White Vienna rabbits (four male and two female) were used to assess the ocular effects of the test chemical.

A volume of 0.1 mL of the unchanged test substance was instilled into the right eye of each rabbits and the animals were scored for corneal changes, iris effects and conjunctival reaction, 24, 48 and 72 hours after test substance administration.

There were no effects on cornea and iris. Shortly after instillation grade 2 redness and discharge grade 1 of the conjunctivae was noted. After 24 hours only grade 1 redness was observed and after 48 and 72 hours no such findings were recorded.

Since the chemical did not induce any ocular adverse effects within 72 hours the study was terminated. Hence the test chemical was considered to be not irritating to the White Vienna rabbits’ eye.

This in vivo result is further supported by another eye irritation study performed in six albino (3/sex) rabbits to assess the ocular irritation index of test chemical.  

The test substance was instilled into the conjunctival sac of the right eye of each rabbit at volume of 0.1 ml (0.66% solution of PMP in propylene glycol) and the left eye served as control. The eyes were not rinsed and examined for ocular lesions after 24 hours and on days 2, 3, 4, and 7 using a cap lamp and also examined with a fluorescent lamp after injection of 2% sodium fluorescein solution.

The ocular irritation index was observed to be 0.66/110 on day 1 and 0.00/110 on the remaining days. As the ocular lesions were not persisted until 7 days observation period with ocular irritation index of 0.00, the test chemical was considered to be not irritating to the eyes of albino rabbits.

Even though in -vitro Guideline studies for the test chemical claims that there is a possibility of test chemical to cause eye irritation, but it is strongly negated by the results of the invivo studies for the test chemicals. Hence, the test chemical can be considered to be not irritating to eyes. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Not Classified.”

Justification for classification or non-classification

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

Even though in -vitro Guideline studies for the test chemical claims that there is a possibility of test chemical to cause eye irritation, but it is strongly negated by the results of the invivo studies for the test chemicals. Hence, the test chemical can be considered to be not irritating to eyes. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Not Classified.”