[No public or meaningful name is available]

Endpoint summary

Description of key information

Skin irritation

reconstructed human epidermis (RhE) in vitro model EPISKIN^TM, OECD 439, GLP: negative

Eye irritation

reconstructed human cornea epidermis (RhCE) in vitro model EpiOcular^TM, OECD 492, GLP: negative

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 MAY 2021 - 20 JUL 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

26 June 2020

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

Commission Regulation (EU) No 2019/1390 of 31 July 2019 amending, for the purpose of its adaptation to technical progress, the Annex to Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Remarks:

EpiSkinTM Small Model (EpiSkinTMSM)

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: three-dimensional human skin model comprising a reconstructed epidermis with a functional stratum corneum

Justification for test system used:

The EPISKIN model has been validated for irritation testing in an international trial. After a review of scientific reports and peer reviewed publications on the EPISKIN method, it showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguishing between skin irritating and no-skin irritating test substances (STATEMENT ON THE VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007).

Vehicle:

unchanged (no vehicle)

Remarks:

The test item was applied in its original form, no formulation was required.

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTM Small Model (EpiSkinTMSM)
- Tissue batch number(s): 21-EKIN-022
- Expiry date: 07 June 2021

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: at room temperature (22.9-23.7 °C).
- Temperature of post-treatment incubation (if applicable): The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated overnight (18-24h) at 37±1 °C in an incubator with 5±1 % CO2, ≥95 % humidified atmosphere.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After the incubation time the EpiSkinTMSM units were removed and rinsed thoroughly with approximately 25 mL/each tissue 1x PBS solution to remove all of the test material from the epidermal surface. The rest of the 1x PBS was removed from the epidermal surface with suitable pipette tip linked to a vacuum source (care was taken to avoid damage of epidermis).

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: The MTT stock solution was diluted with pre-warmed (37 °C) “assay medium” to a final concentration of 0.3 mg/mL.
- Incubation time: After the 42 hours (± 1h) incubation, the EpiSkinTMSM units were transferred into the MTT solution filled wells (2 mL of 0.3 mg/mL MTT per well) and then incubated for 3 hours (± 5 min) at 37±1°C in an incubator with 5±1 % CO2 protected from light, ≥95% humidified atmosphere.
- Spectrophotometer: Varioskan™ LUX Type 3020
- Wavelength: 200 – 1000 nm

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Approximately 10 mg test item was added to 2 mL MTT 0.3 mg/mL solution and mixed. The mixture was incubated for three hours at 37±1 °C in an incubator with 5±1 % CO2, ≥ 95 % humidified atmosphere, protected from light and then any colour change observed (unaided eye assessment):
- Test items which do not interact with MTT: yellow
- Test items interacting with MTT: blue or purple
If the MTT solution colour becomes blue or purple, the test item interacts with the MTT. It is then necessary to evaluate the part of optical density (OD) due to the non-specific reduction of the MTT (i.e. by using killed epidermis).

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
3 replicate of negative control, positive control and test item.

PREDICTION MODEL / DECISION CRITERIA
-In case the test chemical is found to be non-corrosive, and the tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50 %, the test item is considered to be irritant to skin in accordance with UN GHS Category 2. The test item may be considered as non-irritant to skin in accordance with UN GHS No Category if the tissue viability after exposure and post-treatment incubation is more than (>) 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): The epidermal surface was first moistened with 5 μL deionised water in order to improve further contact between powder and epidermis. Subsequently, 10 mg of the test item was applied evenly to the epidermal surface

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): A volume of 10 μL negative control (1x PBS)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): A volume of 10 μL positive control (SDS 5 % aq.)

Additional control for dyes and chemicals able to colour the tissue:
In addition to the normal procedure, two additional test item-treated tissues were used for the non-specific OD evaluation (NSCliving).

Duration of treatment / exposure:

exposure time of 15 minutes (± 0.5 min)

Duration of post-treatment incubation (if applicable):

42 hours (± 1h) incubation

Number of replicates:

Three replicates were used for the test item and positive and negative controls, respectively. Furthermore, two replicates were used for the additional control.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1/ replicate 1

Value:

101

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1/ replicate 2

Value:

90

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1/ replicate 3

Value:

91

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

94

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: Colour change was not observed after three hours of incubation. Therefore, the test item was considered not to interact with the MTT, and additional controls and data calculations were not necessary. A false estimation of viability can be precluded.
- Colour interference with MTT: The test item showed no ability to become coloured in contact with water. The intrinsic colour of the test item is white and therefore considered not to be able to significantly stain the tissues and lead to a false estimate of viability. However, a white milky suspension was observed after the test item was mixed with isopropanol, so the test item showed interaction with isopropanol. Based on this information, two additional test item-treated tissues were used for the non-specific OD evaluation. Mean OD (measured at 570 nm) of these tissues was determined as 0.012. The Non Specific Colourliving % (NSCliving %) was calculated as 1 % (below 5 %). Therefore, additional data calculation was not necessary. A false estimation of viability can be precluded.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. The mean OD value of the three negative control tissues should be between 0.6 and 1.5 and the standard deviation value (SD) of the % viability should be ≤ 18.
- Acceptance criteria met for positive control: Yes. The acceptable mean percentage viability for positive controls is < 40% and the standard deviation value (SD) of the % viability should be ≤ 18.
- Acceptance criteria met for variability between replicate measurements: Yes. For test chemicals (test item and controls), the standard deviation value (SD) of the % viability should be ≤ 18.
The mean OD value of the three negative control tissues was 1.310. The mean OD value obtained for the positive control was 0.059 and this result corresponds to 4 % viability when compared to the results obtained from the negative control. Each calculated standard deviation value (SD) for the % viability was below 18. All validity criteria were within acceptable limits and therefore the study can be considered as valid.

Interpretation of results:

other: EU GHS criteria not met

Conclusions:

The test item did not show significantly reduced cell viability in comparison to the negative control (mean relative value: 94 %). All obtained test item viability results were far above 50 % when compared to the viability values obtained from the negative control.
Therefore, from this in vitro skin irritation test, using the EPISKIN model, indicate that the test item reveals no skin irritation potential under the utilised testing conditions. The test item is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category).

Executive summary:

The In Vitro Skin Irritation test was performed to predict its irritation potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD guideline 439 and under GLP compliance.

Disks of EPISKIN (three units) were treated with the test item and incubated for 15 minutes (± 0.5 min) at room temperature. Exposure to the test material was terminated by rinsing with 1x PBS solution. Epidermis units were then incubated at 37±1 °C for 42 hours (± 1 h) in an incubator with 5±1 % CO2, ≥95 % humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours (± 5 min) with MTT solution at 37±1 °C in 5±1 % CO2, ≥95 % humidified atmosphere, protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically.
SDS (5 % aq.) and 1×PBS treated epidermis (three units / positive and negative control) were used as positive and negative controls, respectively. For each treated tissue viability was expressed as a percentage relative to negative control.

The test item did not show significantly reduced cell viability in comparison to the negative control (mean relative value: 94 %). All obtained test item viability results were far above 50 % when compared to the viability values obtained from the negative control. Therefore, the test item was considered to be non-irritant to skin.
Positive and negative controls showed the expected OD and cell viability values within acceptable limits. Standard deviation of all calculated viability values (test item and controls) was below 18. The experiment was considered to be valid.

The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicate that the test item reveals no skin irritation potential under the utilised testing conditions. The test item is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 AUG 2021 - 27 SEP 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

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)

Version / remarks:

18 June 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Details on test animals or tissues and environmental conditions:

EpiOcular™ (OCL-200-EIT)
The EpiOcular™ human cell construct (MatTek In Vitro Life Science Laboratories) is used in this assay. This three-dimensional human cornea model allows the identification of test items with the potential to induce eye irritation or serious eye damage by assessing cell viability after treatment.
- Source: MatTek In Vitro Life Science Laboratories, Mlynské Nivy 73, Bratislava, Slovakia
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The preparation of tissues for treatment was started after arrival in TOXI-COOP ZRT.’s Laboratory. Therefore, the storage of EpiOcular™ (OCL-200-EIT) units was not necessary. The assay medium supplied with the kits was stored in refrigerator (2-8 °C).
- Justification for selection : The EpiOcular™ Eye Irritation Test (EIT) was validated by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) and Cosmetics Europe between 2008 and 2013. From this validation study and its independent peer review it was concluded that the EpiOcular™ EIT is able to correctly identify chemicals (both substances and mixtures) not requiring classification and labelling for eye irritation or serious eye damage according to UN GHS, and the test method was recommended as scientifically valid for that purpose. The EpiOcular™ EIT is recommended to identify chemicals that do not require classification for eye irritation or serious eye damage according to UN GHS (UN GHS No Category) without further testing, however, a drawback of this test is the inability to distinguish between Category 1 (corrosive to eye) and Category 2 (eye irritant). Thus, in case of a positive result further testing is required.
- Quality check of the isolated corneas: The EpiOcular™ (OCL-200-EIT) kits are manufactured according to defined quality assurance procedures. All biological components of the EpiOcular™ tissue and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking an MTT cell viability test and a cytotoxicity test with Triton X-100 (100 μL of 0.3 % (v/v) Triton X-100). A certificate of quality as provided by the supplier is annexed to this report.

Vehicle:

other: MTT stock solution dissolved to a final concentration of 5 mg/mL in Ca++ and Mg++ free Dulbecco's phosphate buffered saline (DPBS).

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Approximately 50 mg test item was applied topically onto the EpiOcular™ tissues, approximately 83.3 mg/cm2 application.

VEHICLE
- Amount(s) applied (volume or weight with unit): Approximately 50 mg test item was added to 1 mL of 1 mg/mL MTT solution in a tube
- Lot/batch no. (if required): MKCL1832

Duration of treatment / exposure:

The plates with the treated tissue units were incubated for the exposure time of 6 hours (± 15 min) at standard culture conditions (37±2 °C in an incubator with 5±1 % CO2, ≥ 95 % humidified atmosphere).

Duration of post- treatment incubation (in vitro):

18 hours ± 15 minutes at standard culture conditions (Post-exposure Incubation)

Number of animals or in vitro replicates:

In this assay 2 replicates per test item, 2 replicates of the negative control and 2 replicates of the positive control were used.

Details on study design:

NUMBER OF REPLICATES: In this assay 2 replicates per test item, 2 replicates of the negative control and 2 replicates of the positive control were used.

NEGATIVE CONTROL USED: Sterile deionized water, Ultrapure water (prepared in the testing laboratory): Type: Synergy Smart UV HF ASTM Type 1: F8JA80461C, Batch Number: 20210811
Expiry date: August 18, 2021, Storage: Room temperature

POSITIVE CONTROL USED: Methyl Acetate (MA), 99.7% purity, Supplier: MatTek, Lot No.: 012621MJA, CAS No: 79-20-9, Expiration Date: January 26, 2022, Storage condition: Room temperature and dry place

APPLICATION DOSE AND EXPOSURE TIME: The plates with the treated tissue units were incubated for the exposure time of 6 hours (± 15 min) at standard culture conditions (37±2 °C in an incubator with 5±1 % CO2, ≥ 95 % humidified atmosphere).

POST-INCUBATION PERIOD: After the incubation time the EpiOcular™ units were removed and rinsed thoroughly with Ca++Mg++ Free-DPBS. After rinsing, the tissues were transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a pre-labeled 12-well plate for a 25±2 minutes immersion incubation (Post-Soak) at room temperature. This incubation in Assay Medium was intended to remove any test item absorbed into the tissue.
At the end of the Post-Soak immersion, each insert was removed from the Assay Medium, the medium was decanted off the tissue, and the insert was blotted on absorbent material, and afterwards transferred to the appropriate well of the pre-labeled 6-well plate containing 1 mL of warm (37 °C) Assay Medium.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation time the EpiOcular™ units were removed and rinsed thoroughly.
- POST-EXPOSURE INCUBATION: The tissues were incubated for 18 hours ± 15 minutes at standard culture conditions (Post-exposure Incubation). After the post-exposure incubation the EpiOcular™ units were transferred into the 24-well plate filled with MTT ready to use solution (300 μL of 1 mg/mL MTT per well) and then incubated for 3 hours (± 15 min) at 37±2 °C in an incubator with 5±1 % CO2, protected from light, ≥ 95 % humidified atmosphere.

METHODS FOR MEASURED ENDPOINTS:
In the present study, the irritancy potential of test items is predicted by the mean tissue viability of tissues exposed to the test item.

DECISION CRITERIA:
Assay Acceptance Criteria
-The mean OD value of the two negative control tissues should be >0.8 and <2.8
- The acceptable percentage viability for positive control (mean of two tissues) is: 6 hours exposure: below 50 % of negative control viability (solids)
- The difference of viability between the two relating tissues of a single chemical should be < 20 % in the same run (for positive and negative control tissues and tissues of single chemicals). This applies also to the killed controls (single chemicals and negative killed control) and the colorant controls which are calculated as percent values related to the viability of the relating negative control.

Irritation parameter:

mean percent tissue viability 

Run / experiment:

mean

Value:

105

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

percent tissue viability 

Run / experiment:

1/ replicate 1

Value:

101

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

percent tissue viability 

Run / experiment:

1/ replicate 2

Value:

108

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: Prior to routine use of the method TOXI-COOP ZRT. demonstrated the technical proficiency in a separate study (Study No.: 392-492-1722) using the fifteen Proficiency Chemicals according to OECD Test Guideline No. 492.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD value of the two negative control tissues was: 2.098
- Acceptance criteria met for positive control: The positive control result showed 3 % viability after 6 hours exposure.
All validity criteria were within acceptable limits and therefore the study can be considered as valid.

Interpretation of results:

other: EU GHS criteria not met

Conclusions:

The test item did not show significantly reduced cell viability in comparison to the negative control (mean viability: 105 % relative to the negative control). All obtained test item viability results were above 60 % when compared to the viability values obtained from the negative control. Therefore, this in vitro eye irritation test, using the EpiOcular™ Model, indicated that the test item reveals no eye irritation potential under the applied testing conditions and is considered as non-irritant to eye (UN GHS No Category).

Executive summary:

The In Vitro Eye Irritation test was performed to determine the acute ocular irritation potential of the test item on three-dimensional RhCE tissue in the EpiOcular™ model in vitro, according to the OECD guideline 492 and under GLP compliance.

Before the treatment the tissues were pre-wetted with 20 μL of Ca++Mg++ Free- Dulbecco's phosphate buffered saline (DPBS) and incubated at standard culture conditions (37±2 °C in an incubator with 5±1 % CO2, ≥ 95 % humidified atmosphere) for 30±2 minutes. Disks of EpiOcular™ (two units) were treated with the test item (approx. 50 mg, equal to 83.3 mg/cm²) and incubated for 6 hours (± 15 min) at standard culture conditions.

Exposure to the test item was terminated by rinsing with Ca++Mg++ Free-DPBS solution. After rinsing, the tissues were incubated for a 25±2 minutes immersion incubation (Post-Soak) at room temperature. At the end of the Post-Soak immersion, the test item-treated tissues were incubated for 18 hours ± 15 minutes at standard culture conditions (Post-exposure incubation). Fresh Assay Medium was used during the Post-Soak and Post-exposure incubation. Subsequently, the viability of each disk was assessed by incubating the tissues for 3 hours (± 15 min) with MTT solution at 37±2 °C in an incubator with 5±1 % CO2, protected from light, ≥ 95 % humidified atmosphere. The precipitated formazan was then extracted using isopropanol and quantified spectrophotometrically.

Sterile deionized water and methyl acetate treated tissues were used as negative and positive controls, respectively. The disks of EpiOcular™ (two units / control) were treated with positive and negative control and incubated for 6 hours ± 15 minutes. The post-exposure procedure was conducted identically to the test item-treated tissues, as described above.

The test item did not show significantly reduced cell viability in comparison to the negative control (mean viability: 105 % relative to the negative control). All obtained test item viability results were above 60 % when compared to the viability values obtained from the negative control.

The positive and negative controls showed the expected values within acceptable limits. The experiment is considered to be valid.

The results obtained from this in vitro eye irritation test, using the EpiOcular™ Model, indicated that the test item reveals no eye irritation potential under the applied testing conditions and is considered as non-irritant to eye (UN GHS No Category).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation in vitro

OECD 439

The In Vitro Skin Irritation test was performed to predict its irritation potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD guideline 439 and under GLP compliance.

Disks of EPISKIN (three units) were treated with the test item and incubated for 15 minutes (± 0.5 min) at room temperature. Exposure to the test material was terminated by rinsing with 1x PBS solution. Epidermis units were then incubated at 37±1 °C for 42 hours (± 1 h) in an incubator with 5±1 % CO2, ≥95 % humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours (± 5 min) with MTT solution at 37±1 °C in 5±1 % CO2, ≥95 % humidified atmosphere, protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically.
SDS (5 % aq.) and 1×PBS treated epidermis (three units / positive and negative control) were used as positive and negative controls, respectively. For each treated tissue viability was expressed as a percentage relative to negative control.

The test item did not show significantly reduced cell viability in comparison to the negative control (mean relative value: 94 %). All obtained test item viability results were far above 50 % when compared to the viability values obtained from the negative control. Therefore, the test item was considered to be non-irritant to skin.
Positive and negative controls showed the expected OD and cell viability values within acceptable limits. Standard deviation of all calculated viability values (test item and controls) was below 18. The experiment was considered to be valid.

The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicate that the test item reveals no skin irritation potential under the utilised testing conditions. The test item is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category).

 

Eye irritation in vitro

OECD 492

The In Vitro Eye Irritation test was performed to determine the acute ocular irritation potential of the test item on three-dimensional RhCE tissue in the EpiOcular™ model in vitro, according to the OECD guideline 492 and under GLP compliance.

Before the treatment the tissues were pre-wetted with 20 μL of Ca++Mg++ Free- Dulbecco's phosphate buffered saline (DPBS) and incubated at standard culture conditions (37±2 °C in an incubator with 5±1 % CO2, ≥ 95 % humidified atmosphere) for 30±2 minutes. Disks of EpiOcular™ (two units) were treated with the test item (approx. 50 mg, equal to 83.3 mg/cm²) and incubated for 6 hours (± 15 min) at standard culture conditions.

Exposure to the test item was terminated by rinsing with Ca++Mg++ Free-DPBS solution. After rinsing, the tissues were incubated for a 25±2 minutes immersion incubation (Post-Soak) at room temperature. At the end of the Post-Soak immersion, the test item-treated tissues were incubated for 18 hours ± 15 minutes at standard culture conditions (Post-exposure incubation). Fresh Assay Medium was used during the Post-Soak and Post-exposure incubation. Subsequently, the viability of each disk was assessed by incubating the tissues for 3 hours (± 15 min) with MTT solution at 37±2 °C in an incubator with 5±1 % CO2, protected from light, ≥ 95 % humidified atmosphere. The precipitated formazan was then extracted using isopropanol and quantified spectrophotometrically.

Sterile deionized water and methyl acetate treated tissues were used as negative and positive controls, respectively. The disks of EpiOcular™ (two units / control) were treated with positive and negative control and incubated for 6 hours ± 15 minutes. The post-exposure procedure was conducted identically to the test item-treated tissues, as described above.

The test item did not show significantly reduced cell viability in comparison to the negative control (mean viability: 105 % relative to the negative control). All obtained test item viability results were above 60 % when compared to the viability values obtained from the negative control.

The positive and negative controls showed the expected values within acceptable limits. The experiment is considered to be valid.

The results obtained from this in vitro eye irritation test, using the EpiOcular™ Model, indicated that the test item reveals no eye irritation potential under the applied testing conditions and is considered as non-irritant to eye (UN GHS No Category).

Justification for classification or non-classification

Based on the results of the OECD in vitro skin and eye irritation guideline studies following GLP, the registered substance is not classified in accordance with Regulation (EC) No. 1272/2008.