Registration Dossier

Administrative data

Description of key information

Skin Irritation

The dermal corrosion potential of the test chemical was determined by using a three-dimensional human epidermis model, according to OECD Test Guideline No. 431 “In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method”.

The mean of OD  for test chemical was determined to be 2.098 and 0.315 for 3 min. endpoint and 1 hour endpoint, respectively. The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 85.7 % and 13.4 % for 3 min. endpoint and 1 hour endpoint, respectively. Based on these values, the test chemical can be considered to corrosive to skin. It can be further classified under the category "Category 1" as per CLP Regulation.

16-Day cumulative irritation test was carried out on rabbits to assess the irritation potential of the test chemical.

Six New Zealand albino rabbits were used for each test.

The average cumulative irritation score was then calculated (maximum score = 64); applications of 10% and 1% produced scores of 3.6 and 0.3, respectively.

Hence, the test chemical was considered to be irritating to skin when subjected to repeated exposure.

Eye Irritation

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

The mean % tissue viability of test substance was determined to be 3.6%. Hence, under the experimental test conditions it was concluded that test substance was considered to be not irritating/ irritating to the human eyes and can thus be classified as ‘’Irritating to eyes in Category 2” as per CLP Regulation.

An eye irritation study was performed to assess the potential of the test chemical in rabbits following FHSLA Guidelines.

The test chemical was irritating at concentrations of 8 and 16% in DMP. Conjunctival Irritation was observed in the group treated with 8% test chemical in DMP, whereas conjunctival and corneal irritation was observed in the group treated with 16% test chemical

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

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: 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 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Principles of method if other than guideline:
The purpose of this study is to provide classification of dermal corrosion potential of a chemical by using a three-dimensional human epidermis model, according to OECD Test Guideline No. 431 “In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method”. The EpiDermTM SCT allows identification of non-corrosive and corrosive substances according to the United Nations (UN) Globally Harmonized System of Classification and Labeling of Chemicals (GHS).
GLP compliance:
no
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: MatTek In Vitro Life Science Laboratories
Source strain:
not specified
Details on animal used as source of test system:
Test System
The normal human-derived keratinocytes were cultured at the air-liquid interface in a chemically defined medium on a permeable polycarbonate insert (with a surface of 0.5 cm2). The keratinocytes 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 In Vitro Life Science Laboratories according to specific QC standards including: histology, tissue viability (MTT mean optical density), reproducibility (SD) and tissue thickness.
Vehicle:
water
Remarks:
the tissues were moistened with sterile water prior to application of the solid test chemical
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:
MatTek EpiDermTM tissue cultures

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure:
For the 3-minute exposure, the dosed tissues were placed in the sterile hood at room temperature. For the 60-minute exposure, the dosed tissues were placed in an incubator at 37deg C, 5% CO2 for the remainder of the 60-minute exposure period
- Temperature of post-treatment incubation (if applicable):
37deg C, 5% CO2

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

For the 3-minute exposure, four tissues at a time were dosed with an interval of 45 seconds. When a treatment of 3 minutes was reached for the set of four tissues, the dosed tissues were gently rinsed with a constant stream of sterile DPBS by filling and emptying the tissue inserts 20 times to remove any residual test article. The bottom of the inserts was gently botted on a sterile cotton compress and the tissues were then placed in 0.3 mL of fresh maintenance medium (in 24-well plates) until all tissues were treated and washed. After the 1-hour exposure, the tissues were gently rinsed with a constant stream of sterile DPBS by filling and emptying the tissue inserts 20 times to remove any residual test article. The bottom of the inserts was gently botted on a sterile cotton compress and the tissues were then placed in 0.3 mL of fresh maintenance medium (in 24-well plates) until all tissues were washed.
- Observable damage in the tissue due to washing:
none
- Modifications to validated SOP:
none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration:
24-well plates containing 300 microliters MTT medium (1.0 mg/mL).
- Incubation time:
3 hours of incubation at 37 deg C, 5% CO2 in a humified incubator
- Spectrophotometer:
Thermo Scientific™ Multiskan™ FC Microplate Photometer
- Wavelength:
570 nm
- Viability:
All data were background subtracted before analysis. MTT data are represented as % viable compared to negative control. Data was generated as follows:
Blanks:
The optical density (OC) 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) and the percent coefficient of variation (% CV)

Positive Controls (PC):
Calculation of 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 =%PC / number of tissues
The standard deviation (SD) and the percent coefficient of variation (% CV)

Tested Compound (TC):
Calculation of the blank corrected value: ODTC = ODTCraw – ODblank
The OD mean per treated tissue was calculated.
The viability per tissue was calculated: %TC = [ODTC / mean ODNC] x 100
The mean viability for all tissues was calculated: Mean TC = %TC / number of tissues
The standard deviation (SD) and the percent coefficient of variation (% CV)

Data Correction Procedure for MTT Interfering Compounds (if applicable)
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 tissue
ODtkt = optical density of treated killed tissue
ODukt = 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

Skin Corrosion Prediction
A chemical is regarded to be corrosive if the relative tissue viability after 3 min treatment with a test chemical is decreased below 50%. In addition, materials are regarded to be non-corrosive after 3 min (viability< 50%) are regarded to be corrosive if the relative tissue viability after 1-hour treatment with a test chemical is decreased below 15%.

Mean Tissue Viability Prediction

3 min < 50% Corrosive
3 min > 50% and 1 hour: < 15% Corrosive
3 min > 50% and 1 hour: > 15% Non-corrosive

Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
Before dosing, the tissues were moistened with 25 microliters of sterile water to improve the contact of the tissue surface to the test article. Approximately 25 mg of each test article was evenly applied to the apical surface of each tissue.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight):
50microliters

POSITIVE CONTROL
- Amount(s) applied (volume or weight):
50 microliters
Duration of treatment / exposure:
3 minute and 60 minute exposure
Duration of post-treatment incubation (if applicable):
37deg C, 5% CO2
Number of replicates:
duplicates
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Run 1
Value:
85.7
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: 3 min. endpoint; mean of OD: 2.098
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Run 2
Value:
13.4
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: 1 hrs. endpoint; mean of OD: 0.315
Other effects / acceptance of results:
The assay passed all acceptance criteria as the ODs of the negative control exposed tissues were > 0.8, the positive control exposed tissues were less than 15% viable and the standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates was <30 (except for where indicated as incompatible or with an asterisk).

Name CAS Tissue   3 min endpoint             1 hour endpoint          
      Aliquote 1 Aliquote 2 mean OD Mean viability Mean Diff  Diff /2 Aliquote 1 Aliquote 2 mean OD Mean viability Mean Diff 
              [%] [%] [%] [%]         [%] [%] [%]
NC N/A tissue 1 2.646 2.615 2.630 2.449 107.4 100.0 14.8 7.42 2.293 2.173 2.233 2.358 94.7 100.0 10.6
    tissue 2 2.266 2.268 2.267   92.6       2.499 2.468 2.483   105.3    
PC 1310-58-3 tissue 1 0.401 0.433 0.417 0.432 17.0 17.6 1.2 0.62 0.064 0.063 0.063 0.064 2.7 2.7 0.1
    tissue 2 0.447 0.448 0.447   18.3       0.064 0.065 0.065   2.7    
K1#4 4065-45-6 tissue 1 2.146 2.184 2.165 2.098 88.4 85.7 5.5 2.73 0.419 0.409 0.414 0.315 17.6 13.4 8.4
    tissue 2 1.999 2.064 2.031   82.9       0.221 0.213 0.217   9.2    

Chemical name CAS 3min Diff 1hour Diff
K1#4   4065-45-6 85.7 5.5 13.4 8.37

  Chemical name CAS Supplier  purity In vivo In vitro MTT pH Remark
4 K1#4 4065-45-6           1.63 Corrosive
Interpretation of results:
Category 1 (corrosive) based on GHS criteria
Conclusions:
The dermal corrosion potential of the test chemical was determined by using a three-dimensional human epidermis model, according to OECD Test Guideline No. 431 “In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method”. The mean of OD for test chemical was determined to be 2.098 and 0.315 for 3 min. enpoint and 1 hour endpoint, respectively. The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 85.7 % and 13.4 % for 3 min. endpoint and 1 hour endpoint,respectively. Based on these values, the test chemical can be considered to corrosive to skin. It can be further classified under the category "Category 1" as per CLP Regulation.
Executive summary:

The dermal corrosion potential of the test chemical was determined by using a three-dimensional human epidermis model, according to OECD Test Guideline No. 431 “In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method”.

The EpiDermTMSCT allows discrimination between non-corrosive and corrosive chemicals according to the UN Globally Harmonized System of Classification and Labeling of Chemicals (GHS). The MatTek EpiDermTMtissue cultures were obtained from MatTek In Vitro Life Science Laboratories. The MatTek EpiDermTMtissue cultures were placed in the refrigerator at 5°C. Prior to use, the tissues were incubated (37±1°C, 5±1% CO2) in 0.9 mL of fresh maintenance medium in 6 -well plates for~one hour.

Before dosing, the tissues were moistened with 25microliter of sterile water to improve the contact of the tissue surface to the test article. Approximately 25 mg of solid test article was evenly applied to the apical surface of each tissue. Each treatment with test article or control was conducted in duplicate. The exposure period for the test articles and controls was 3 and 60 minutes. For the 60-minute exposure, the dosed tissues were placed in an incubator at 37±1°C, 5±1% CO2for the remainder of the 60-minute exposure period.

Following the rinsing period, the MTT assay was performed by transferring the tissues to 24-well plates containing 300microliter MTT medium (1.0 mg/mL). After 3 hours of incubation at 37±1°C, 5±1% CO2in a humified incubator, the blue formazan salt was extracted by submerging the tissues in 2 mL isopropanol (Lot 28677, kit A; provided by MatTek, Slovakia) in a 24-well plate. The extraction time was approximately 2 hours and 05 minutes with gentle shaking. The optical density of the extracted formazan (200mL/well if a 96-well plate) was determined using a Thermo Scientific™ Multiskan™ FC Microplate Photometer at 570 nm. Relative cell viability was calculated for each tissue as % of the mean negative control tissue. A chemical was regarded to be corrosive if the relative tissue viability after 3 min treatment with a test chemical was decreased below 50%. In addition, materials were regarded to be non-corrosive after 3 min (viability>50%) were regarded to be corrosive if the relative tissue viability after 1-hour treatment with a test chemical was decreased below 15%.

The mean of OD  for test chemical was determined to be 2.098 and 0.315 for 3 min. endpoint and 1 hour endpoint, respectively. The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 85.7 % and 13.4 % for 3 min. enpoint and 1 hour endpoint,respectively. Based on these values, the test chemical can be considered to corrosive to skin. It can be further classified under the category "Category 1" as per CLP Regulation.

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from peer reviewed journal
Qualifier:
according to
Guideline:
other: 16-Day cumulative irritation test
Principles of method if other than guideline:
16-Day cumulative irritation test was carried out on rabbits to assess the irritation potential of the test chemical
GLP compliance:
not specified
Species:
rabbit
Strain:
New Zealand White
Type of coverage:
open
Preparation of test site:
clipped
Vehicle:
other: Alcohol solution
Controls:
not specified
Amount / concentration applied:
0·05 ml of 10% and 1% test chemical in alcohol
Duration of treatment / exposure:
Each test material was applied 14 times, on Mondays to Fridays except for the last Friday
Observation period:
16 days
Number of animals:
6
Details on study design:
TEST SITE
- Area of exposure: four sites on the back, two anterior and two posterior, two being on each side of the dorsal midline

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) : Each test site was scored visually, together with one centrally located control site, and some were measured for skinfold thickness to the nearest 0.1 mm at 24hr and every subsequent day except Sundays (16 readings).

SCORING SYSTEM:
- Method of calculation:
The scores used were 0 for no erythema, I for barely perceptible erythema, 2 for well-defined erythema, 3 for moderate to severe erythema and 4 for beet-red erythema to slight eschar and/
or disruption of epidermal intactness. Scaliness was arbitrarily assigned a score of 1.
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
24 h
Score:
0.3
Max. score:
64
Reversibility:
not specified
Remarks on result:
positive indication of irritation
Remarks:
1% test chemical in alcohol
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
24 h
Score:
3.6
Max. score:
64
Reversibility:
not specified
Remarks on result:
positive indication of irritation
Remarks:
10% test chemical in alcohol
Irritant / corrosive response data:
The average cumulative irritation score was then calculated (maximum score = 64); applications of 10% and 1% produced scores of 3.6 and 0.3, respectivelya

Comparison of two skin-irritation parameters (skin/old thickness and mean cumulative irritation score) in·clipped albino rabbits tested for 16 days

Test material, concentration

Vehicle

Skin fold thickness**

[change from control]

Mean cumulative score*

4065-45-6, 1%

A

+0.17

3.6

Control site 2

 

+0.03

0.0

Control site 2`

 

+0.02

0.0

A = Alcohol (70%), *- Correlation hetween the two parameters was high: r = 0·87: P> 0·001 but <0·01.

16-day uncovered test in the rahhit

CAS

Vehicle

Concentration (%)

Mean cumulative score

4065-45-6

A

1

0.3

10

3.6

  Maximum scores: man= 84; rabbit = 64.

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
The average cumulative irritation score was then calculated (maximum score = 64); applications of 10% and 1% produced scores of 3.6 and 0.3, respectively.
Hence, the test chemical was considered to be irritating to skin when subjected to repeated exposure.
Executive summary:

16-Day cumulative irritation test was carried out on rabbits to assess the irritation potential of the test chemical.

Six New Zealand albino rabbits were used for each test. The animals' backs were clipped on a Saturday, and on two subsequent Saturdays.

Each test material was applied 14 times, on Mondays to Fridays except for the last Friday. 0·05 ml of 10% and 1% test chemical in alcohol was was applied to one of four sites on the back of each clipped rabbit two anterior and two posterior, two being on each side of the dorsal midline. Each test site was scored visually, together with one centrally located control site, and some were measured for skinfold thickness to the nearest 0.1 mm at 24hr and every subsequent day except Sundays (16 readings).

The scores used were 0 for no erythema, I for barely perceptible erythema, 2 for well-defined erythema, 3 for moderate to severe erythema and 4 for beet-red erythema to slight eschar and/or disruption of epidermal intactness. Scaliness was arbitrarily assigned a score of 1.

The average cumulative irritation score was then calculated (maximum score = 64); applications of 10% and 1% produced scores of 3.6 and 0.3, respectively.

Hence, the test chemical was considered to be irritating to skin when subjected to repeated exposure.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from experimental study report.
Qualifier:
according to
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Principles of method if other than guideline:
The purpose of this study was to assess potential for the test article to be ocular irritants. The ocular irritation potential of a test article may be predicted by measurement of its cytotoxic effect, as reflected in the (MTT) assay, in the MatTek EpiOcular™ model
GLP compliance:
no
Species:
human
Strain:
other: Not applicable
Details on test animals or tissues and environmental conditions:
- Description of the cell system used:
The normal human-derived keratinocytes were cultured at the air-liquid interface in a chemically defined medium on a permeable polycarbonate insert (surface 0.5 cm2). They were cultured in chemically defined serum free medium to form a multi-layered epithelium similar to that found in native corneal mucosa. Each lot of tissues was Quality Assured by MatTek according to specific QC standards including: histology, tissue viability (MTT mean optical density), reproducibility (SD) and tissue thickness.

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

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

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

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

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

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

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

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

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

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

- Description of evaluation criteria used including the justification for the selection of the cut-off point for
the prediction model
Calculations and Statistical Methods
MTT Assay
Blanks:
· The OD mean from all replicates for each plate (ODblank).
Negative Controls (NC):
· The blank corrected value was calculated: ODNC= ODNCraw– ODblank.
· The OD mean per NC tissue was calculated.
· The mean OD for all tissues corresponds to 100% viability.
· The mean, standard deviation (SD), standard error of the mean (SEM) and the percent coefficient of variation (% CV) was calculated.
ODblank= optical density of blank samples (isopropanol alone).
ODNCraw= optical density negative control samples.
ODNC= optical density of negative control samples after background subtraction.
Positive Control (PC):
· Calculate the blank corrected value: ODPC= ODPCraw– ODblank.
· The OD mean per PC tissue was calculated.
· The viability per tissue was calculated: %PC = [ODPC/ mean ODNC] x 100.
· The mean viability for all tissues was calculated: Mean PC = Σ %PC / number of tissues.
· The standard deviation (SD), standard error of the meanthe mean (SEM) and the percent coefficient of variation (% CV) was calculated.
ODPCraw= optical density positive control samples.
ODPC= optical density of positive control samples after background subtraction.
Tested Articles:
· Calculate the blank corrected value ODTT= ODTTraw– ODblank.
· The OD mean per tissue is calculated.
· The viability per tissue is calculated: %TT = [ODTT/ mean ODNC] x 100.
· The mean viability for all tissues is calculated: Mean TT = Σ %TT / number of tissues.
· The standard deviation (SD) and the percent coefficient of variation (% CV)for the controls and the test articles will be calculated.
ODTTraw= optical density test article samples.
ODPC= optical density of test article samples after background subtraction.
Data Correction Procedure for MTT Interfering Compounds
True viability = Viability of treated tissue – Interference from test article = ODtvt – ODkt where ODkt =
(mean ODtkt – mean ODukt).
ODtvt = optical density of treated viable tissue
ODkt = optical density of killed tissues
ODtkt = optical density of treated killed tissue
ODukt = optical density of untreated killed tissue (NC treated tissue)
Data Correction Procedure for Colored Compounds
True viability = Viability of treated tissue incubated in MTT media – Viability of treated tissue incubated in
media without MTT = ODtvt – ODvt.
ODtvt = optical density of treated viable tissue incubated in MTT media
ODvt = optical density of viable tissues incubated in media alone.
Proposed Statistical methods
The mean, standard deviation (SD) and the percent coefficient of variation (% CV) for the controls and the test article will be calculated.
- Evaluation of data
The results of the assay was evaluated and compared to negative control.
Table: Irritancy Prediction
In VitroResults In VivoPrediction
Mean tissue viability ≤60% Irritant (I) – Category 1 or 2
Mean tissue viability >60% Non-irritant (NI) – No Category
- Assay quality controls
- Negative Controls (NC)
The assay is meeting the acceptance criterion if the mean viability of the NC in terms of Optical Density(OD570) of the NC tissues (treated with sterile ultrapure water) in the MTT assay are >0.8 to <2.5. This is an indicator of tissue viability following shipping and conditions under use.
- Positive Controls (PC)
Methyl acetate was used as a PC and tested concurrently with the test article. The assay is meeting the acceptance criteria if the viability of the PC is <50% of the negative control.
- Standard Deviation (SD)
Each test of ocular irritancy potential is predicted from the mean viability determined on 2 single tissues. The assay meets the acceptance criteria if SD calculated from individual percent tissue viabilities of the
replicates is <18% for three replicate tissues.
Irritation parameter:
other: mean % tissue viability
Run / experiment:
Run 1
Value:
3.6
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
The MTT data show the assay quality controls were met.

Code N° Tissue  Raw data Blank corrected data mean of OD % of viability
  n Aliq. 1 Aliq. 2 Aliq. 1 Aliq. 2
NC 1 2.0443 2.0409 2.010 2.006 2.008 102.3
  2 1.9538 1.9506 1.919 1.916 1.918 97.7
PC 1 0.6729 0.6902 0.638 0.656 0.647 33.0
  2 0.7611 0.7631 0.726 0.728 0.727 37.1

4065-45-6 1 0.10 0.1042 0.069 0.070 0.069 3.5
  2 0.106 0.1072 0.071 0.073 0.072 3.7

  mean Dif. mean of Dif. Dif./2 Classification
  of OD of OD viabilities [%] of viabilities      
NC 1.963 0.090 100.0 4.61 2.30 NI qualified
PC 0.687 0.081 35.0 4.10 2.05 I qualified

4065-45-6 0.071 0.003 3.6 0.14 0.07 I qualified
Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
The ocular irritation potential of test article was determined according to the OECD 492 test guideline followed for this study. The mean % tissue viability of test substance was determined to be 3.6%. Thus, the test chemical was considered to be irritating to the human eyes.
Executive summary:

The ocular irritation potential of test article was determined according to the OECD 492 test guideline for this study. The MatTek EpiOcular™ model was used to assess the potential ocular irritation of the test articles by determining the viability of the tissues following exposure to the test article via MTT. Tissues were exposed to 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 3.6%. Hence, under the experimental test conditions it was concluded that test substance was considered to be not irritating/ irritating to the human eyes and can thus be classified as ‘’Irritating to eyes in Category 2” as per CLP Regulation

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
data is from peer reviewed journals
Qualifier:
according to
Guideline:
other: FHSLA Guidelines
Principles of method if other than guideline:
To assess the eye irritation potential of the test chemical in rabbits following FHSLA Guidelines
GLP compliance:
not specified
Species:
rabbit
Strain:
not specified
Vehicle:
other: dimethyl phthalate
Controls:
yes, concurrent no treatment
Amount / concentration applied:
0.1 ml of 16, 8, 4% test chemical in DMP
Duration of treatment / exposure:
single
Observation period (in vivo):
1,2,3,4,7 days
Duration of post- treatment incubation (in vitro):
no data available
Number of animals or in vitro replicates:
6
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): not washed
- Time after start of exposure: no data available

SCORING SYSTEM: Draize. Maximum Score = 110
Irritation parameter:
other: cornea score & conjunctivae score
Basis:
mean
Time point:
7 d
Max. score:
110
Reversibility:
not specified
Remarks on result:
positive indication of irritation
Remarks:
Irritating (Cornea, conjunctiva- 16%)
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
7 d
Max. score:
110
Reversibility:
not specified
Remarks on result:
positive indication of irritation
Remarks:
Irritating (conjunctiva - 8%)
Irritant / corrosive response data:
The test chemical was irritating at concentrations of 8 and 16% in DMP
Conjunctival Irritation was observed in the group treated with 8% test chemical in DMP, whereas conjunctival and corneal irritation was observed in the group treated with 16% test chemical

Cas

Method

Number of rabbits

Eye wash

Y/N

Test concentration

Average score per day

Comments

1

2

3

4

5

6

7

4065-45-6

FHSLA

6

N

0.1ml, 16,8,4/ DMP

Conjunctival Irritation was observed in the group treated with 8% test chemical in DMP, whereas conjunctival and corneal irritation was observed in the group  treated with 16% test chemical

Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
The test chemical was irritating at concentrations of 8 and 16% in DMP. Conjunctival Irritation was observed in the group treated with 8% test chemical in DMP, whereas conjunctival and corneal irritation was observed in the group  treated with 16% test chemical
Hence, the test chemical can be considered to be irritating to eyes.
Executive summary:

An eye irritation study was performed to assess the potential of the test chemical in rabbits following FHSLA Guidelines. 0.1 ml of 16, 8, 4% test chemical in DMP was instilled into the eyes of 6 rabbits and the other eye served as an untreated control. The eyes remained unwashed through out the test. The reactions were scored at 1,2,3,4,7 days according to Draize method with a maximum score was 110.

The test chemical was irritating at concentrations of 8 and 16% in DMP. Conjunctival Irritation was observed in the group treated with 8% test chemical in DMP, whereas conjunctival and corneal irritation was observed in the group  treated with 16% test chemical

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

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin Irritation

Various studies have been summarized to determine the extent of dermal irritation caused by test chemical in living organisms. These results include in vivo experimental studies performed on rabbits, humans, as well as in vitro study for the test chemical.

The dermal corrosion potential of the test chemical was determined by using a three-dimensional human epidermis model, according to OECD Test Guideline No. 431 “In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method”.

The EpiDermTMSCT allows discrimination between non-corrosive and corrosive chemicals according to the UN Globally Harmonized System of Classification and Labeling of Chemicals (GHS). The MatTek EpiDermTMtissue cultures were obtained from MatTek In Vitro Life Science Laboratories. The MatTek EpiDermTMtissue cultures were placed in the refrigerator at 5°C. Prior to use, the tissues were incubated (37±1°C, 5±1% CO2) in 0.9 mL of fresh maintenance medium in 6 -well plates for~one hour.

Before dosing, the tissues were moistened with 25microliter of sterile water to improve the contact of the tissue surface to the test article. Approximately 25 mg of solid test article was evenly applied to the apical surface of each tissue. Each treatment with test article or control was conducted in duplicate. The exposure period for the test articles and controls was 3 and 60 minutes. For the 60-minute exposure, the dosed tissues were placed in an incubator at 37±1°C, 5±1% CO2for the remainder of the 60-minute exposure period.

Following the rinsing period, the MTT assay was performed by transferring the tissues to 24-well plates containing 300microliter MTT medium (1.0 mg/mL). After 3 hours of incubation at 37±1°C, 5±1% CO2in a humified incubator, the blue formazan salt was extracted by submerging the tissues in 2 mL isopropanol (Lot 28677, kit A; provided by MatTek, Slovakia) in a 24 -well plate. The extraction time was approximately 2 hours and 05 minutes with gentle shaking. The optical density of the extracted formazan (200mL/well if a 96-well plate) was determined using a Thermo Scientific™ Multiskan™ FC Microplate Photometer at 570 nm. Relative cell viability was calculated for each tissue as % of the mean negative control tissue. A chemical was regarded to be corrosive if the relative tissue viability after 3 min treatment with a test chemical was decreased below 50%. In addition, materials were regarded to be non-corrosive after 3 min (viability>50%) were regarded to be corrosive if the relative tissue viability after 1-hour treatment with a test chemical was decreased below 15%.

The mean of OD  for test chemical was determined to be 2.098 and 0.315 for 3 min. endpoint and 1 hour endpoint, respectively. The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 85.7 % and 13.4 % for 3 min. endpoint and 1 hour endpoint, respectively. Based on these values, the test chemical can be considered to corrosive to skin. It can be further classified under the category "Category 1" as per CLP Regulation.

16-Day cumulative irritation test was carried out on rabbits to assess the irritation potential of the test chemical.

Six New Zealand albino rabbits were used for each test. The animals' backs were clipped on a Saturday, and on two subsequent Saturdays.

Each test material was applied 14 times, on Mondays to Fridays except for the last Friday. 0·05 ml of 10% and 1% test chemical in alcohol was applied to one of four sites on the back of each clipped rabbit two anterior and two posterior, two being on each side of the dorsal midline. Each test site was scored visually, together with one centrally located control site, and some were measured for skin fold thickness to the nearest 0.1 mm at 24hr and every subsequent day except Sundays (16 readings).

The scores used were 0 for no erythematic, I for barely perceptible erythema, 2 for well-defined erythema, 3 for moderate to severe erythema and 4 for beet-red erythema to slight eschar and/or disruption of epidermal intactness. Scaliness was arbitrarily assigned a score of 1.

The average cumulative irritation score was then calculated (maximum score = 64); applications of 10% and 1% produced scores of 3.6 and 0.3, respectively.

Hence, the test chemical was considered to be irritating to skin when subjected to repeated exposure.

These results are supported by a 21-Day cumulative irritation test performed in humans (covered test site) to evaluate the irritation potential of the test chemical.

Six adult white test subjects were used for the study. 0·05 ml of 1,10% test chemical in alcohol was applied to the backs of the subjects under occlusion (Non-woven Webril® pad (Kendall Co. Boston, Mass.) with acrylic tape, B1enderm® (Minnesota Mining Co., St. Paul, Minn.). Each patch was removed at 24 hours. The skin was scored 30 min later and new material and a fresh patch were applied.

The scores used were 0 for no erythema, ± for questionable erythema, I for well-defined erythema, 2 for erythema and induration, 3 for vesiculation and 4 for a bullous

reaction. The cumulative score was obtained for each test material and the mean cumulative score for six subjects was used as an index of irritation. When a given site showed a bullous response (grade 4), no further applications were made and the site continued to receive a score of 4 for the remaining period. The cumulative irritation scores after 21 days for 1,10% test chemical in alcohol were 8.6 and 53.1 respectively.

The test chemical was apparently more irritating to human skin than to rabbits when subjected to repeated exposure.

Hence, the test chemical was considered to be irritating to skin of humans.

These results are also supported by the Procedures outlined by the Federal Hazardous Substances Labeling Act (FHSLA) to test the test chemical for acute skin irritation.

An occlusive patch containing 0.5 mg of 16, 8,4 % of the test chemical in DMP was applied to the intact and abraded skin of albino rabbits. Patches remained in place for 24 hours and were then removed and scored for irritation according to the Draize method. Sites were again scored 24 hours after patch removal.

The test chemical was minimally irritating (PI1 = 0.25-0.50) when applied as 16% solutions in dimethyl phthalate (DMP), however it was non-irritating at 4,8% in DMP.

Eventhough the results of the one in vivo test suggest that the test chemical was not irritating to skin, but the results from other studies indicate otherwise. The results from the other in vivo studies are further supported by the in vitro study, which clearly indicates that the test chemical indeed has the potential to cause irritation to skin. Based on the observations, the test chemical can be considered to be a skin irritant. Comparing the above annotations with the criteria of CLP Regulation, the test chemical was classified under the category “Category 2”.

Eye irritation

Various studies have been summarized to ascertain the level of ocular irritation caused by test chemical in living organisms. These results include in vivo experimental studies performed on rabbits, as well as in vitro study for the test chemical.

The ocular irritation potential of test article was determined according to the OECD 492 test guideline for this study. The MatTek EpiOcular™ model was used to assess the potential ocular irritation of the test articles by determining the viability of the tissues following exposure to the test article via MTT. Tissues were exposed to 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 3.6%. Hence, under the experimental test conditions it was concluded that test substance was considered to be not irritating/ irritating to the human eyes and can thus be classified as ‘’Irritating to eyes in Category 2” as per CLP Regulation.

This in vitro result is supported by an eye irritation study was performed to assess the potential of the test chemical in rabbits following FHSLA Guidelines. 0.1 ml of 16, 8, 4% test chemical in DMP was instilled into the eyes of 6 rabbits and the other eye served as an untreated control. The eyes remained unwashed throughout the test. The reactions were scored at 1,2,3,4,7 days according to Draize method with a maximum score was 110.

The test chemical was irritating at concentrations of 8 and 16% in DMP. Conjunctival Irritation was observed in the group treated with 8% test chemical in DMP, whereas conjunctival and corneal irritation was observed in the group treated with 16% test chemical

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

A similar study was performed following FHSLA Guidelines to assess the potential of the test chemical in rabbits. 0.1 ml of 16, 8, 4% test chemical in petrolatum was instilled into the eyes of 6 rabbits and the other eye served as an untreated control. The eyes remained unwashed throughout the test. The reactions were scored at 1,2,3,4,7 days according to Draize method with a maximum score was 110.

The test chemical was irritating at concentrations of 8 and 16% in petrolatum. Conjunctival Irritation was observed in the group treated with 8% test chemical in petrolatum, whereas conjunctival and corneal irritation was observed in the group treated with 16% test chemical. Hence, the test chemical can be considered to be irritating to eyes.

These results are further supported by another Draize method employed to determine the eye irritation potential of the test chemical in rabbits. 0.1 ml of 5% test chemical in water was instilled into the eyes of 9 rabbits, and the other eye served as an untreated control. The eyes of 3 rabbits were washed with water four seconds after instillation of the test chemical.

The reactions were scored at 1, 2, 3, 4, 7 days according to Draize method with a maximum score was 110.

The overall irritation score for the test chemical at 1,2,3,4 and 7 days was 0.0. The test chemical was considered to be not irritating to eyes.

Eventhough the results of the Draize test suggest that the test chemical was not irritating to eyes, but the results from other studies indicate otherwise. The results from the other in vivo studies are further supported by the in vitro study, which clearly indicates that the test chemical indeed has the potential to cause irritation to eyes. Based on the observations, the test chemical can be considered to be an eye irritant. Comparing the above annotations with the criteria of CLP Regulation, the test chemical was classified under the category “Category 2”.

Justification for classification or non-classification

Eventhough the results of the one in vivo test suggest that the test chemical was not irritating to skin, but the results from other studies indicate otherwise. The results from the other in vivo studies are further supported by the in vitro study, which clearly indicates that the test chemical indeed has the potential to cause irritation to skin. Based on the observations, the test chemical can be considered to be a skin irritant. Comparing the above annotations with the criteria of CLP Regulation, the test chemical was classified under the category “Category 2”

Eventhough the results of the Draize test suggest that the test chemical was not irritating to eyes, but the results from other studies indicate otherwise. The results from the other in vivo studies are further supported by the in vitro study, which clearly indicates that the test chemical indeed has the potential to cause irritation to eyes. Based on the observations, the test chemical can be considered to be an eye irritant. Comparing the above annotations with the criteria of CLP Regulation, the test chemical was classified under the category “Category 2”.