Ethyl salicylate

Endpoint summary

Description of key information

A key study is available assessing the in vitro skin irritation and one key study to assess the in vitro skin corrosion potential, and one key study is available to assess the in vitro eye irritation of the test substance. All studies have been performed according to the respective OECD guidelines and in compliance to GLP.

Skin irritation

In an vitro study performed according to OECD TG 439 and in compliance to GLP, the test item is considered irritant to skin according to EU CLP regulation.

In an vitro study performed according to OECD 431 and EU-Method B.40 and in compliance to GLP, the test item is considered as non corrosive to skin according to EU CLP regulation.

Eye irritation

In an assay performed according to OECD TG 492 and in compliance to GLP, the test item does not possess any eye irritating potential.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05.12.2016-19.01.2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 July 2016

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

30 May 2008

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: human-derived epidermal keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

TEST SYSTEM
- Source: MatTek Corporation
- Cell culture: The EpiDerm™ tissues (surface 0.63 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs, 10 mm Ø).
- Pre-incubation period: pre-incubation phase of the EpiDerm™ tissues started on the day of receipt.
- Treatment: EpiDerm tissues were treated with the test substance
- Amount/test concentration: 50 µL
- Duration of treatment: 3 minutes, 60 minutes

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure : room tempeature
- Temperature of post-treatment incubation (if applicable): 37+-1.5°C

CONTROL
- Negative Control: 50 µL deionised water was used as negative control per tissue;
- Positive Control: 50 µL 8.0 N potassium hydoxide (Sigma) was used a positive control per tissue

REMOVAL OF TEST MATERIAL AND CONTROLS
- Washing (if done): Tissues washed with DPBS at least 20 times in order to remove any residual test material. Exess DPBS was removed by gently shaking the tissue inserts and blotting the lower surface with blotting paper.

SCORING SYSTEM:
Viability measured using MTT assay
mean tissue viability < 50% after 3 minutes exposure: corrosive
mean tissue viability ≥ 50% after 3 minutes exposure AND < 15% after 60 minutes exposure: corrosive
mean tissue viability ≥ 50% after 3 minutes exposure AND = 15% after 60 minutes exposure: non-corrosive
Test Item Identified as Corrosive:
< 25% after 3 minutes exposure: Optional Sub-category 1A
≥ 25% after 3 minutes exposure: A combination of optional Sub-categories 1B and 1C

Historical data positive control:
Mean Viability: 21.60% (3 min), 7.02% (1 h); CV: 9.52% and 14.72%, respectively;
Range of Viabilities: 4.60 – 39.83%.
Historical data negative control:
Mean Absorption: 1.68 (3 min), 1.65 (1 h); CV:
4.87 and 3.19, respectively;
Range of Absorbance: 1.34 – 1.93 (3 min) and 1.32 – 1.85 (1h)

Acceptability of the assay:
An assay met the acceptance criteria if
- the mean OD of the tissue replicates treated with the negative control is ≥ 0.8 and ≤ 2.8 for every exposure time
- the mean viability of the tissue replicates treated with the positive control for 1 hour is <15% compared to the negative control
- the Coefficient of Variation (CV) in the range 20 – 100% viability between tissue replicates is ≤ 30%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

- Test item: 50 μL (79.4 μL/cm2 according to guideline) of undiluted test item
- Controls: each 50 μL

Duration of treatment / exposure:

3 minutes; 60 minutes

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min. incubation time

Value:

>= 111.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: not considered to be corrosive.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour incubation time

Value:

>= 117.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: not considered to be corrosive

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: test item did not reduce MTT
- Colour interference with MTT:it did not change colour
- After exposure to the negative control the absorbance values met the required acceptability criterion of a mean OD570 ≥ 0.8 and ≤ 2.8 for both treatment intervals thereby confirming the acceptable quality of the tissues.

Results after treatment with the test item and the controls

Dose Group	Exposure Interval (min)	Mean Absorbance (OD) of 2 tissues	Stand. Dev. [%]	Mean Rel. Absorbance [% of Negative Control]
Negative Control	3	1.577	3.1	100
Test Item		1.765	2.2	111.9
Positive Control		0.459	3.4	29.1
Negative Control	60	1.518	0.3	100
Test Item		1.782	1.9	117.4
Positive Control		0.153	12	10.1

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, it can be stated that in this study and under the reported experimental conditions, the test item is non corrosive to skin according to EU CLP and UN GHS.

Executive summary:

This in vitro study was performed to assess the corrosive potential of the test item by means of the Human Skin Model Test with EpiDerm™ tissues models. The test was performed according to OECD 431 and EU-Method B.40 and in compliance to GLP.

The test item passed the colour and the MTT interference pre-tests. Independent duplicate tissues of EpiDerm were exposed to the test item, the negative control (deionised water) or the positive control (8.0 N KOH) for 3 minutes and 1 hour, respectively.

Afterwards, the test and the control items were rinsed off the tissues, and a 3 hour incubation period (37 ± 1 °C, 5 ± 0.5 % CO2) with MTT solution followed. MTT solution was then aspirated from the wells and the wells were rinsed with DPBS. Inserts were transferred into new 24 well plates. The formazan salt was extracted for about 22.5 hours. The required acceptability criteria were met.

Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 minutes exposure period (29.1%) and for the 1 hour exposure period (10.1%) thus confirming the validity of the test system and the specific batch of tissue models.

After exposure to the test item the corrected relative absorbance value were not reduced both after 3 minutes exposure and after 1 hour exposure (111.9% and 117.4%, respectively). Therefore, the test item is not considered to be corrosive.

In conclusion, it can be stated that in this study and under the reported experimental conditions, the test item is non corrosive to skin according to EU CLP and UN GHS.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21.09.-10.10.2016

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:

28 July 2015

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

23 July 2009

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: human-derived epidermal keratinocytes

Source strain:

other: EpiDerm TM tissues (Epi-200-SIT Kit)

Justification for test system used:

Dermal irritation is generally defined as "the production of reversible inflammatory changes in the skin". The potential for chemical induced skin irritation is usually determined in vivo in the Draize rabbit skin irritation test as described in OECD guideline 404. However, because systemic reactions play a minor role in modulating local skin toxicity potential of chemicals, skin irritation potential may be predicted by in vitro systems, provided they are sufficiently complex to mimic human skin barrier and cell reactivity. In an international prevalidation study performed by ECVAM, the in vitro skin irritation test using the human skin model EpiDermTM and EpiSkinTM and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for skin irritancy potential.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: human skin model EpiDerm™
- Tissue batch number: 23361
- Delivery date: 14 June 2016
- Date of initiation of testing: 04 October 2016 (preincubation phase)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C, 5 ± 0.5% CO2
- Temperature of post-treatment incubation: 37 ± 1.5 °C, 5 ± 0.5 % CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: Rinsed with DPBS at least 15 times, after the rinsing the inserts were submerged in DPBS at least three times. Afterwards the inserts were once again rinsed with sterile DPBS from the inside and the outside.
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: (1 mg/mL)
- Incubation time: 3 hours
- Spectrophotometer: Versamax® Molecular Devices, Softmax Pro, version 4.7.1
- Wavelength: 570 nm
- Filter: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Positive: 4.64%, range 4.00 - 5.90 %

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Since the MTT solution did not turn blue/purple, the test item was not considered to reduce MTT and an additional test with freeze-killed tissues did not have to be performed.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the mean tissue viability is ≤ 50 %.
- The test substance is considered to be non-irritant to skin if the mean tissue viability is > 50 %

Acceptability of the Assay
Criterion 1
Negative control: The absolute OD 570 nm of the negative control tissues in the MTT test is an indicator of tissue viability obtained after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is meeting the acceptance criterion if the mean OD570 of the negative control tissues is ≥ 0.8 and ≤ 2.8.

Criterion 2
Positive control: An assay is meeting the acceptance criterion if mean relative tissue viability of the positive control is ≤ 20%.

Criterion 3
Standard deviation: The SD of 3 identical replicates should be < 18%. OD values should not be below historically established boundaries.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

- test item: 30 μL (47 μL/cm2 according to guideline)
- controls: Each 30 μL

Duration of treatment / exposure:

60 ± 1 minutes

Duration of post-treatment incubation (if applicable):

about 42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of three tissues

Value:

ca. 18.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

- Direct-MTT reduction: test item did not reduce MTT
- Colour interference with MTT: test item did not change colour in the colour interference test

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 (values between 1.781 and 2.036) for the 60 minutes treatment interval thus showing the quality of the tissues.
Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 4.8% thus ensuring the validity of the test system.

Results after treatment with the test item and the controls

Dose Group	Tissue	Absorbance 570 nm Well 1		Absorbance 570 nm Well 2		Absorbance 570 nm Well 3	Mean absorbance of 3 Wells	Mean absorbance of 3 Wells blank corrected	Mean absorbance of 3 tissues after blank correction*	Rel. Absorbance (%) Tissue 1,2 and 3**	Realtive Standard Deviation (%)	Mean Rel. Absorbance (% of Negative Control)***
NK	1	1.781		1.796		1.789	1.788	1.751	1.814	96.5	7.4	100
	2	1.997		2.036		1.987	2.006	1.969		108.6
	3	1.76		1.756		1.761	1.759	1.721		94.9
PC	1	0.144		0.141		0.131	0.139	0.101	0.087	5.6	14.1	4.8
	2	0.126		0.12		0.118	0.121	0.083		4.6
	3	0.114		0.123		0.108	0.115	0.077		4.3
TM	1	0.342		0.337		0.34	0.34	0.302	0.34	16.7	9.6	18.7
	2	0.398		0.393		0.401	0.397	0.359		19.8
	3	0.398		0.389		0.401	0.396	0.358		19.7

* Mean of three replicate wells after blank correction

** relative absorbance per tissue [rounded values]: 100*(absorbance tissue) / (mean absorbance negative control)

*** relative absorbance per treatment group [rounded values]: 100*(mean absorbance test item/positive control) / (mean absorbance negative control)

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is irritant to skin according to UN GHS and EU CLP regulation.

Executive summary:

This in vitro study was performed to assess the irritation potential of the test item by means of the Human Skin Model Test.

The test was performed according to OECD TG 439 and in compliance to GLP.

The test item passed the MTT- and the colour interference pre-tests. Each 30 μL of the test item, the negative control (DPBS), or the positive control (5% SLS) were applied to each tissue and spread to match the surface of triplicate tissue. The test item and the positive and negative controls were washed off the skin tissues after 60 minutes treatment. After further incubation for about 42 hours the tissues were treated with the MTT solution for 3 hours following about 70 hours extraction of the colorant from the

cells. The amount of extracted colorant was determined photometrically at 570 nm.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 (values between 1.781 and 2.036) for the 60 minutes treatment interval thus showing the quality of the tissues.

Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 4.8% thus ensuring the validity of the test system.

The relative standard deviations between the % variability values of the test item, the positive and negative controls in the main test were below 10% (threshold of the "OECD Guideline for the Testing of Chemicals 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”: < 18%), thus ensuring the validity of the study.

Compared to the relative absorbance value of the negative control the mean relative absorbance value was reduced to 18.7% after exposure of the skin tissues to the test item. This value is below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential.

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is irritant to skin according to UN GHS and EU CLP regulation.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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:

21.09.-06.10.2016

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:

July, 2015

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Species:

human

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

- Source: MatTek Corporation (82105 Bratislava, Slovakia).
- EpiOcularTM tissue: normal, human-derived epidermal keratinocytes cultured to form a stratified squamous epithelium
- Surface: 0.6 cm
- Shipment: at 2 - 8 °C on medium-supplemented agarose gels
- Equilibration step: 15 minutes at room temperature
- Inspection: each tissue was inspected for air bubbles between the agarose gel and insert. Cultures with air bubbles under the insert covering greater than 50% of the insert area were not used.
- Pre-incubation: standard culture conditions for 1 hour and after refreshing the Assay Medium at standard culture conditions overnight (about 19 h)

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

test item, negative control and positive control: 50 μL each

Duration of treatment / exposure:

30 minutes

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

2

Details on study design:

Details of the test procedure used:
- RhCE tissue construct used, including batch number: EpiOcular Kit Lot No.: 23737
- Conditions of exposure: 37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH
- Washing: extensively rinsing the tissues with Ca++Mg++-free DPBS
- Number of tissue replicates used per test chemical and controls: 2
- Wavelength and band pass used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): 570 nm (OD570), Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro, version 4.7.1. No reference wavelength measurement was used.
- MTT assay: incubation with 0.3 mL of MTT solution for 180 minutes at standard culture conditions, after incubation with MTT the inserts were incubated with isopropanol at 2-8°C overnight after which MTT was extracted for 2-3 hours at room temperature

Data evaluation:
1) The mean OD value of the blank control wells (ODBlk) for each experiment were calculated
2) The mean ODBlk from each mean OD value of the same experiment (blank corrected values) were subtracted
3) The mean value of the two aliquots for each tissue (= corrected test item OD) were calculated
4) The mean value of the two relating tissues for each control and test item (= corrected mean OD) were calculated
For further calculations only the corrected mean negative control OD value was needed
5) The corrected OD value of the negative control corresponds to 100% viability
Corrected negative control OD = Negative Control OD - ODBlk = 100% Viability

Historical data positive control:
Mean Viability: 32.0%; Rel. Standard Deviation: 12.8%; Range of Viabilities: 6.90% - 40.4%; Mean Absorption: 0.538; Rel. Standard Deviation: 0.258; Range of Absorbance: 0.107- 0.849

Historical data negative control:
Mean Absorption: 1.65; Rel. Standard Deviation: 0.299; Range of
Absorbance: 1.27 – 2.05

Prediction Model
If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is labeled non-irritant.
If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is labeled irritant.

Acceptability of the Assay:
The results are acceptable according to MatTek Protocol, if:
1) The negative control OD is > 0.8 and < 2.5
2) The mean relative viability of the positive control is below 50% of the negative control viability
3) The difference of viability between the two relating tissues of a single test item is < 20% in the same run (for positive and negative control tissues and tissues of test items).
This applies also to the freeze-killed tissues (items and negative control) and the additional viable tissues (without MTT addition) which are calculated as percent values related to the viability of the relating negative control.

Irritation parameter:

other: tissue viability (%)

Run / experiment:

first replicate

Value:

78.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

other: tissue viability (%)

Run / experiment:

second replicate

Value:

82.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

other: tissue viability (%)

Run / experiment:

mean of two replicates

Value:

80.5

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:

Treatment with the positive control induced a decrease in the mean relative absorbance compared with the negative control to 43.4%, thus the validity of the test system is ensured.

Results after treatment for 30 minutes with the test item and the controls

Dose Group	Absorbance Well 1 (Tissue 1/2)	Absorbance Well 2 (Tissue 1/2)	Mean absorbance* (Tissue 1/2)	Mean Absorbance* Tissue 1 and 2	Mean absorbance of 2 Tissues*	Rel. Absorbance (%) Tissue 1 and 2**	Absolute Value of the Difference of the Rel. Absorbances (%) Tissue 1 and 2	Mean Rel. Absorbance (% of Negative Control)**
Blank	0.039	0.039	0.039	0
NC	2.11	2.057	2.084	2.045	2.023	101.1	2.2	100
	2.027	2.052	2.04	2.001		98.9
PC	0.943	0.921	0.932	0.893	0.878	44.2	1.6	43.4
	0.908	0.892	0.9	0.862		42.6
Test Item	1.637	1.615	1.626	1.588	1.628	78.5	4	80.5
	1.743	1.671	1.707	1.688		82.5

* Mean of two replicate wells after blank correction

** Relative absorbance [rounded values]: 100*(absorbance test item/positive control) / (mean absorbance negative control)

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item does not possess any eye irritating potential.

Executive summary:

This in vitro study was performed to assess the eye irritation potential of the test item by means of the Human Cornea Model Test.

The assay was performed according to OECD TG 492 and in compliance to GLP.

The colourless test item did not prove to be an MTT reducer in the MTT pre-test and it did not dye water or isopropanol in the colour interference pre-test. Therefore, additional tests with freeze-killed or viable tissues did not have to be performed.

Each 50 μL of the test item, the negative control (deionised water) or the positive control (methyl acetate) were applied to each of duplicate tissue for 30 minutes.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD > 0.8 and < 2.5 thus showing the quality of the tissues. Treatment with the positive control induced a decrease below 50% compared with the negative control value in the relative absorbance thus ensuring the validity of the test system. The difference of viability between the two relating tissues was < 20% in the same run (for test item tissues, positive and negative control tissues).

Irritating effects were not observed following incubation with the test item. Compared with the value of the negative control the relative mean absorption value corresponding to the viability of the tissues did not decrease below 60% (80.5%).

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item does not possess any eye irritating potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

There are three in vitro studies available that assessed the possible skin irritation/corrosion and the eye irritation potential of the test substance. They were performed according to GLP and internationally accepted guidelines.

In vitro skin corrosion:

This in vitro study was performed to assess the corrosive potential of the test item by means of the Human Skin Model Test with EpiDerm™ tissues models. The test was performed according to OECD 431 and EU-Method B.40 and in compliance to GLP.

The test item passed the colour and the MTT interference pre-tests. Independent duplicate tissues of EpiDerm were exposed to the test item, the negative control (deionised water) or the positive control (8.0 N KOH) for 3 minutes and 1 hour, respectively. Afterwards, the test and the control items were rinsed off the tissues, and a 3 hour incubation period (37 ± 1 °C, 5 ± 0.5 % CO2) with MTT solution followed. The formazan salt was extracted for about 22.5 hours.

The required acceptability criteria were met. Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 minutes exposure period (29.1%) and for the 1 hour exposure period (10.1%) thus confirming the validity of the test system and the specific batch of tissue models.

After exposure to the test item the corrected relative absorbance value were not reduced both after 3 minutes exposure and after 1 hour exposure (111.9% and 117.4%, respectively).

Therefore, the test item is not considered to be corrosive.

In conclusion, it can be stated that in this study and under the reported experimental conditions, the tes item is non corrosive to skin according to EU CLP regulation.

In vitro skin irritation:

This in vitro study was performed to assess the irritation potential of the test item by means of the Human Skin Model Test. The test was performed according to OECD TG 439 and in compliance to GLP.

The test item passed the MTT- and the colour interference pre-tests. Each 30 μL of the test item, the negative control (DPBS), or the positive control (5% SLS) were applied to each tissue and spread to match the surface of triplicate tissue.

The test item and the positive and negative controls were washed off the skin tissues after 60 minutes treatment. After further incubation for about 42 hours the tissues were treated with the MTT solution for 3 hours following about 70 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 (values between 1.781 and 2.036) for the 60 minutes treatment interval thus showing the quality of the tissues. Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 4.8% thus ensuring the validity of the test system.

The relative standard deviations between the % variability values of the test item, the positive and negative controls in the main test were below 10% (threshold of the "OECD Guideline for the Testing of Chemicals 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”: < 18%), thus ensuring the validity of the study.

Compared to the relative absorbance value of the negative control the mean relative absorbance value was reduced to 18.7% after exposure of the skin tissues to the test item.

This value is below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential.

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is irritant to skin according to EU CLP regulation.

In vitro eye irritation:

This in vitro study was performed to assess the eye irritation potential of the test item by means of the Human Cornea Model Test. The assay was performed according to OECD TG 492 and in compliance to GLP.

The colourless test item did not prove to be an MTT reducer in the MTT pre-test and it did not dye water or isopropanol in the colour interference pre-test. Therefore, additional tests with freeze-killed or viable tissues did not have to be performed.

Each 50 μL of the test item, the negative control (deionised water) or the positive control (methyl acetate) were applied to each of duplicate tissue for 30 minutes.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD > 0.8 and < 2.5 thus showing the quality of the tissues. Treatment with the positive control induced a decrease below 50% compared with the negative control value in the relative absorbance thus ensuring the validity of the test system. The difference of viability between the two relating tissues was < 20% in the same run (for test item tissues, positive and negative control tissues). Irritating effects were not observed following incubation with the test item. Compared with the value of the negative control the relative mean absorption value corresponding to the viability of the tissues did not decrease below 60% (80.5%).

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item does not possess any eye irritating potential.

Conclusions:

Taken together, the test substance under the reported experimental conditions, is non corrosive to skin according to EU CLP and UN GHS, is irritant to skin and therefore Category 2 (irritant) based on GHS criteria, and does not possess any eye irritating potential according to EU CLP and UN GHS.

Justification for classification or non-classification

All three key studies are well documented and according to GLP and internationally accepted guidelines.

The test substance under the reported experimental conditions, is non corrosive to skin and does not possess any eye irritating potential according to EU CLP.

As skin irritation is observed in the key in vitro skin irritation study, the test item is categorized as irritant to skin, Category 2 (irritant) based on EU CLP criteria.