Amines, C16-18 (even numbered)-alkyl , salts with phosphoric acid, mono- and di-C16-18 (even numbered) alkyl esters

Endpoint summary

Description of key information

In vitro testing was conducted in accordance with appropriate OECD Guidelines in order to investigate the skin irritation and eye irritation properties of the registered substance. It was concluded that the substance was not irritating or corrosive to the skin and the eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 September 2020 - 07 September 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

June 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Appearance: Beige solid
Storage conditions: At room temperature

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Adult donors

Details on animal used as source of test system:

EpiSkin TM Small/Human Epidermis
- Batch: 20-EKIN-036
- Cells are screened for potential biological contaminants (HIV1 and 2 antibodies, hepatitis C antibodies and hepatitis B antigen HBs, absence of bacteria, fungus and mycoplasma)
- Suggested expiration date: September 7, 2020

Justification for test system used:

Recommended test system in international guideline (OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test item. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours at 37°C
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: triplicate

NUMBER OF INDEPENDENT TEST SEQUENCES: One

ACCEPTABILITY CRITERIA
The in vitro skin irritation test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the acceptance limits of OECD439 (lower acceptance limit =0.6 and upper acceptance limit =1.5) and the Standard Deviation value (SD) of the % viability should be =18.
b) The mean relative tissue viability of the positive control should be =40% relative to the negative control and the Standard Deviation value (SD) of the % viability should be =18.
c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be =18.
d) The %NSCliving should be = 30% relative to the negative control OD.

PREDICTION MODEL / DECISION CRITERIA
A test item is considered irritant in the skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is = 50% of the mean viability of the negative controls.

A test item is considered non-irritant in the in vitro skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

Control samples:

yes, concurrent vehicle

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 10.4 to 21.0 mg

NEGATIVE CONTROL
- Amount(s) applied: 25 µL PBS

POSITIVE CONTROL
- Amount(s) applied: 25 µL 5% SDS

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

The test was performed on a total of 3 tissues per test item together with negative and positive controls.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Relative mean tissue viability

Value:

121

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

COLOR INTERFERENCE
- Instead of MTT solution these tissues were incubated with assay medium. The non-specific color by the test item was 4.46% of the negative control tissues. The OD of the tissues incubated with assay medium was subtracted from the ODs of the test item treated tissues incubated with MTT medium.
The test item was checked for possible direct MTT reduction and color interference. The solutions did not turn blue / purple, nor a blue / purple precipitate was observed. The OD for the test item solution was >0.08. Therefore, it was concluded that the test item did interfere with the MTT endpoint.

RESULTS
- The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 121%. Since the mean relative tissue viability for the test item was above 50% the test item is considered to be non-irritant.

ACCEPTANCE OF RESULTS
- The positive control had a mean cell viability of 4% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was = 6%, indicating that the test system functioned properly.

Interpretation of results:

GHS criteria not met

Conclusions:

The in vitro skin irritation test was conducted according to OECD 439 guideline and in accordance with GLP principles. It is concluded that this test is valid and that the test substance is not irritant in the in vitro skin irritation test under the experimental conditions described in this report. Therefore,
the substance does not need to be classified according to GHS and CLP criteria.

Executive summary:

In an in vitro skin irritation test using a human skin model (EPISKIN Standard Model), the influence of Amines, C16-18 (even numbered)-alkyl , salts with phosphoric acid, mono- and di-C16-18 (even numbered) alkyl esters on the viability of human skin was tested.

At least 10 mg of the test item was applied directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.
The test item induced color interference in aqueous conditions. In addition to the normal procedure, three tissues were treated with test item. Instead of MTT solution these tissues were incubated with assay medium. The non-specific color by the test item was 4.46% of the negative control tissues. The OD of the tissue incubated with assay medium was subtracted from the ODs of the test item treated tissues incubated with MTT medium.
Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 121%. Since the mean relative tissue viability for the test item was above 50% after 15 ± 0.5 minutes treatment the test item is considered to be non-irritant.
The positive control had a mean cell viability of 4% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was ≤ 6%, indicating that the test system functioned properly.

It is concluded that the test substance is not irritant in the in vitro skin irritation test under the experimental conditions in this report. Therefore, the substance does not need to be classified according to GHS and CLP criteria. 

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8 June 2020 - 3 July 2020

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

other: EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test". Official Journal of the European Union No. L142

Version / remarks:

31 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

18 June 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: One or multiple donors (MatTek Corporation, USA)

Details on animal used as source of test system:

EpiDerm™ Reconstructed Human Epidermis (Lot number 33059)
- All cells used to produce Epiderm™ are purchased or derived from tissue obtained by MatTek Corporation, USA from acredited institutions.
- Cells are screened for potential biological contaminants (HIV-1, Hepatitis B, Hepatitis C, bacteria, yeast and fungi)

Justification for test system used:

Recommended test system in international guideline (OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model (EPI-200)
- Tissue batch number(s): 33059 kit C
- Date of initiation of testing: 8 June 2020

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37.0 ± 1.0ºC
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test item. The skin inserts were carefully dried. Rinsed tissues were kept in 24 well plates on 300 µL DMEM until 6 tissues (= one application time) were dosed and rinsed.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: Duplicates

DECISION CRITERIA
- The test substance is considered to be non-corrosive to skin if the relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50% and the the relative tissue viability after the 1-hour treatment is not decreased below 15%
- The test substance is considered to be corrosive to skin if the relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50% and if the relative tissue viability after the 1-hour treatment is not decreased below 15%.

ACCEPTANCE CRITERIA
The in vitro skin corrosion test is considered acceptable if it meets the following criteria:
- The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range and the acceptance limits of OECD431 (lower acceptance limit =0.8 and upper acceptance limit = 2.8).
- The mean relative tissue viability following 1-hour exposure to the positive control should be <15 %.
- In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be = 30%.
- The %NSC should be = 30% relative to the negative control OD.
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

Control samples:

yes, concurrent vehicle

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 53.13 to 61.46 mg

VEHICLE CONTROL - Milli-Q
- Amount(s) applied: 50 µL

POSITIVE CONTROL - 8N KOH
- Amount(s) applied: 50 µL

Duration of treatment / exposure:

3 minutes or 1 hour

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1-hour exposure

Value:

101

Vehicle controls validity:

valid

Remarks:

100%

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

5.0%

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-minute exposure

Value:

95

Vehicle controls validity:

valid

Remarks:

100%

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

9.0%

Other effects / acceptance of results:

COLOR INTERFERENCE
- The test item showed color interference in aqueous conditions. In addition to the normal procedure, two tissues were treated with test item. Instead of MTT solution these tissues were incubated with DMEM. The color interference by the test item was 0.11% and 0.10% of the negative control tissues after 3 minutes and 1 hour respectively. The OD of the treated tissues without MTT assay was subtracted from the ODs of the test item treated viable tissues with MTT assay.

RESULTS
- The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 95% and 101% respectively. Because the mean relative tissue viability for the test item was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment the test item is considered to be not corrosive.

ACCEPTANCE OF RESULTS:
- The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit =0.8 and upper acceptance limit =2.8) and the laboratory historical control data range. The mean relative tissue viability following the 1-hour exposure to the positive control was 5%.
In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was < 16%, indicating that the test system functioned properly.

Interpretation of results:

GHS criteria not met

Conclusions:

The in vitro skin corrosion test was conducted according to OECD 431 guideline and in accordance with GLP principles. It is concluded that this test is valid and that the test substance is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report. Therefore, the substance does not need to be classified according to GHS and CLP criteria.

Executive summary:

In an in vitro skin irritation test using a human skin model (EPISKIN Standard Model), the influence of Amines, C16-18 (even numbered)-alkyl , salts with phosphoric acid, mono- and di-C16-18 (even numbered) alkyl esters on the viability of human skin was tested. Skin tissue was moistened with 25 μL of Milli-Q water and at least 25 mg of the test item was applied directly on top of the skin tissue. The positive control had a mean relative tissue viability of 5% after the 1-hour exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ¿2.8) and the laboratory historical control data range. In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was < 16, indicating that the test system functioned properly.
The test item showed color interference in aqueous conditions. In addition to the normal procedure, two tissue were treated with test item. Instead of MTT solution these tissues were incubated with DMEM. The color interference by the test item was 0.11% and 0.10% of the negative control tissues after 3 minutes and 1 hour respectively. The OD of the treated tissues without MTT assay was subtracted from the ODs of the test item treated viable tissues with MTT assay.
Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 95% and 101%, respectively. Because the mean relative tissue viability for the test item was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the test item is considered to be not corrosive.

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:

07 Sep 2020 - 20 Sep 2020

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Details on test animals or tissues and environmental conditions:

Non-keratinized epithelium (0.6 cm2) prepared from normal human keratinocytes.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 50.1 to 55.9 mg

Duration of treatment / exposure:

6 hours ± 15 minutes

Duration of post- treatment incubation (in vitro):

18 hours

Number of animals or in vitro replicates:

The test was performed on a total of 2 tissues per test item together with a negative control
and positive control

Details on study design:

- Control substances used:
Negative control: Sterile Milli-Q water
Positive control: Methyl Acetate [CAS Number 79-20-9]

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: After the exposure period with the test item (6 hours ± 15 minutes at 37.0 ± 1.0°C), the tissues were thoroughly rinsed with Ca2+Mg2+-free D-PBS (brought to room temperature) to remove residual test item. After rinsing the cell culture inserts were each dried carefully and immediately 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 minute immersion incubation at room temperature (Post-Soak). After the Post-Soak period cell culture inserts were each dried carefully and transferred to the 6-well plate containing 1.0 mL of warm Assay Medium and were incubated for 18 hours ± 15 minutes at 37°C.

- Number of tissue replicates used per test chemical and controls: The test was performed on a total of 2 tissues per test item together with a negative control and positive control

- Wavelength and band pass used for quantifying MTT formazan: The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

- Supporting information for the specific RhCE tissue construct used: EpiOcular™ (OCL-200-EIT MatTek Corporation, Lot: 31755, kit A.

Irritation parameter:

percent tissue viability 

Run / experiment:

Mean Tissue Viability

Value:

94

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

GHS criteria not met

Conclusions:

The test item is non-irritant in the EpiOcular™ test under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate the eye hazard potential of the test item. For this purpose, the test item was topically applied on the Reconstructed Human EpiOcular™ Model.
The possible eye hazard potential of the test item was tested through topical application for 6 hours.
The study procedures described in this report were based on the most recent OECD guideline.
Batch AEK-20-106 of the test item was a beige solid. The test item (50.1 to 55.9 mg) was applied directly on top of the tissue for 6 hours ± 15 minutes.
After exposure, the cornea epithelial construct was thoroughly rinsed to remove the test item and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 18 hours at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect.
The positive control had a mean cell viability of 9.8% after 6 hours ± 15 minutes exposure.
The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The difference between the percentage of viability of two tissues treated identically was less than 7%, indicating that the test system functioned properly.
The test item showed color interference in aqueous conditions. In addition to the normal procedure, two tissues were treated with test item. Instead of MTT solution, these tissues were incubated with assay medium. The non-specific colour of the test item was 0.30% of the negative control tissues. The OD of the treated tissues without MTT assay was subtracted from the ODs of the test item treated viable tissues with MTT assay.
Eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 6 hours ± 15 minutes treatment with the test item compared to the negative control tissues was 94%. Since the mean relative tissue viability for the test item was above 60% after 6 hours ± 15 minutes treatment, the test item is considered to be non-irritant.
In conclusion, the test item is non-irritant in the EpiOcular™ test under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro skin corrosion

In an in vitro skin irritation test using a human skin model (EPISKIN Standard Model), the influence of Amines, C16-18 (even numbered)-alkyl , salts with phosphoric acid, mono- and di-C16-18 (even numbered) alkyl esters on the viability of human skin was tested. Skin tissue was moistened with 25 μL of Milli-Q water and at least 25 mg of the test item was applied directly on top of the skin tissue. The positive control had a mean relative tissue viability of 5% after the 1-hour exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ¿2.8) and the laboratory historical control data range. In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was < 16, indicating that the test system functioned properly.

The test item showed color interference in aqueous conditions. In addition to the normal procedure, two tissue were treated with test item. Instead of MTT solution these tissues were incubated with DMEM. The color interference by the test item was 0.11% and 0.10% of the negative control tissues after 3 minutes and 1 hour respectively. The OD of the treated tissues without MTT assay was subtracted from the ODs of the test item treated viable tissues with MTT assay.

Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 95% and 101%, respectively. Because the mean relative tissue viability for the test item was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the test item is considered to be not corrosive.

 

In vitro skin irritation

In an in vitro skin irritation test using a human skin model (EPISKIN Standard Model), the influence of Amines, C16-18 (even numbered)-alkyl , salts with phosphoric acid, mono- and di-C16-18 (even numbered) alkyl esters on the viability of human skin was tested.

At least 10 mg of the test item was applied directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

The test item induced color interference in aqueous conditions. In addition to the normal procedure, three tissues were treated with test item. Instead of MTT solution these tissues were incubated with assay medium. The non-specific color by the test item was 4.46% of the negative control tissues. The OD of the tissue incubated with assay medium was subtracted from the ODs of the test item treated tissues incubated with MTT medium.

Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 121%. Since the mean relative tissue viability for the test item was above 50% after 15 ± 0.5 minutes treatment the test item is considered to be non-irritant.

The positive control had a mean cell viability of 4% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was ≤ 6%, indicating that the test system functioned properly.

It is concluded that the test substance is not irritant in the in vitro skin irritation test under the experimental conditions in this report. Therefore, the substance does not need to be classified according to GHS and CLP criteria. 

 

In vitro eye irritation

The objective of this study was to evaluate the eye hazard potential of the test item. For this purpose, the test item was topically applied on the Reconstructed Human EpiOcular™ Model.

The possible eye hazard potential of the test item was tested through topical application for 6 hours.

The study procedures described in this report were based on the most recent OECD guideline.

Batch AEK-20-106 of the test item was a beige solid. The test item (50.1 to 55.9 mg) was applied directly on top of the tissue for 6 hours ± 15 minutes.

After exposure, the cornea epithelial construct was thoroughly rinsed to remove the test item and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 18 hours at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect.

The positive control had a mean cell viability of 9.8% after 6 hours ± 15 minutes exposure.

The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The difference between the percentage of viability of two tissues treated identically was less than 7%, indicating that the test system functioned properly.

The test item showed color interference in aqueous conditions. In addition to the normal procedure, two tissues were treated with test item. Instead of MTT solution, these tissues were incubated with assay medium. The non-specific colour of the test item was 0.30% of the negative control tissues. The OD of the treated tissues without MTT assay was subtracted from the ODs of the test item treated viable tissues with MTT assay.

Eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 6 hours ± 15 minutes treatment with the test item compared to the negative control tissues was 94%. Since the mean relative tissue viability for the test item was above 60% after 6 hours ± 15 minutes treatment, the test item is considered to be non-irritant.

In conclusion, the test item is non-irritant in the EpiOcular™ test under the experimental conditions described in this report.

Justification for classification or non-classification

In vitro testing was conducted on the registered substance to investigate its skin irritation and eye irritation properties. It was concluded that it does not meet the criteria for classification in accordance with Regulation (EC) No 1272/2008.