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Description of key information

Skin irritation

The test item was determined to be not irritant to skin in the in vitro Human Skin Model Test and it is not eye irritant in the in vitro Human Cornea Model Test.

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:
May - June 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)
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
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: 23341
- Delivery date: 14 June 2016
- Date of initiation of testing: 14 June 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 MATERIAL
- Amount applied: 30 μL (47 μL/cm2)
- Concentration: undiluted

NEGATIVE CONTROL
- Amount applied : 30 μL DPBS (MatTek)

POSITIVE CONTROL
- Amount applied: 30 μL
- Concentration: 5% SLS solution in deionised water
Duration of treatment / exposure:
1 hour
Duration of post-treatment incubation (if applicable):
approx 43 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean of 3 tissues
Value:
100.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
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 in the required range of the acceptability criterion of mean OD = 0.8 and = 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues.
- Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 60 minutes treatment interval, and thus assuring the validity of the test system; the mean relative absorbance value of the positive control was 4.5% compared to the negative control.
Interpretation of results:
GHS criteria not met
Conclusions:
The test item was determined to be not irritant to skin in the in vitro Human Skin Model Test.
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 item did not reduce MTT (test for direct MTT reduction), and it did not change colour when mixed with deionised water (test for colour interference). Also its intrinsic colour was not intensive. Consequently, additional tests with freeze-killed or viable tissues were not necessary.

Each three tissues of the human skin model EpiDerm™ were treated with the test item, the negative (DPBS) or the positive (5% SLS) control for 60 minutes. After treatment with the negative control the absorbance values were well within the required range of the acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues.

Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 60 minutes treatment interval, and thus assuring the validity of the test system.

After treatment with the test item the mean absorbance value did not decrease (100.4%) compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50%.

Therefore, the test item is not considered to possess an irritant potential.

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:
May-July 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)
GLP compliance:
yes (incl. QA statement)
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 cm2
- 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 (21h in the experiment).
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
- Volume: 50 μL
- Concentration: undiluted
Duration of treatment / exposure:
30 min
Duration of post- treatment incubation (in vitro):
120 min
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.: 23714
- 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 and then shaken for 3 hours at room temperature
- Data evaluation: the following was calculated:
mean of the blank control wells (ODBlk), ODBlk from each OD value of the same experiment (blank corrected values), mean of the two aliquots for each tissue (= corrected test item OD), mean of the two relating tissues for each control and test item (= corrected mean OD) (for further calculations only the corrected mean negative control OD value was needed), corrected OD value of the negative control corresponding to 100% viability (corrected negative control OD = Negative Control OD - ODBlk = 100% Viability)
- Description of evaluation criteria: If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is non-irritant and if the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is labelled irritant. A single test composed of at least 2 tissue replicates should be sufficient for a test chemical when the result is unequivocal. In cases of borderline results, such as non- concordant replicate measurements and/or mean percent tissue viability equal to 60±5%, a second test should be considered, as well as a third one in case of discordant results between the first two tests (according to OECD 492).
- 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
- Acceptability of the Assay: The results are acceptable 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 killed controls (items 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:
in vitro irritation score
Run / experiment:
Single test with two replicates
Value:
94.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The acceptance criteria were met.

Results of the pre-experiment:

The optical pre-experiment (colour interference pre-experiment) to investigate the test item's colour change potential in water or isopropanol did not lead to a change in colour.

Optical evaluation of the MTT-reducing capacity of the test item with MTT-reagent did not show blue color.

Results of the main experiment:

Results after treatment for 30 minutes

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

Rel. Absorbance [% of Negative Control]**

Blank

0.039

0.040

0.039

0.000

 

 

 

100.0

Negative Control

2.160

2.155

2.158

2.118

2.066

102.5

5.1

100.0

2.049

2.056

2.053

2.013

97.5

Positive Control

0.579

0.574

0.577

0.538

0.721

26.0

17.7

34.9

0.955

0.931

0.943

0.904

43.7

Test Item

1.966

1.931

1.949

1.910

1.945

92.4

3.4

94.1

2.014

2.025

2.019

1.980

95.8

* Mean of two replicate wells after blank correction


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

The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 76.0% (threshold for irritancy: <= 60%).

Therefore, the substance was found to be not irritant to the eye.

Interpretation of results:
GHS criteria not met
Conclusions:
The test item is not eye irritant.
Executive summary:

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

The test item did not prove to be an MTT reducer in the MTT pre-test. Also its intrinsic colour was not intensive and it did not prove to 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).

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% (94.1%).

In conclusion, it can be stated that in this study and under the experimental conditions reported, 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

Skin irritation

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

The test item did not reduce MTT (test for direct MTT reduction), and it did not change colour when mixed with deionised water (test for colour interference). Also its intrinsic colour was not intensive. Consequently, additional tests with freeze-killed or viable tissues were not necessary.

Each three tissues of the human skin model EpiDerm™ were treated with the test item, the negative (DPBS) or the positive (5% SLS) control for 60 minutes. After treatment with the negative control the absorbance values were well within the required range of the acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues.

Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 60 minutes treatment interval, and thus assuring the validity of the test system.

After treatment with the test item the mean absorbance value did not decrease (100.4%) compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50%.

Therefore, the test item is not considered to possess an irritant potential.

Eye irritation

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

The test item did not prove to be an MTT reducer in the MTT pre-test. Also its intrinsic colour was not intensive and it did not prove to 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).

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% (94.1%).

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

Justification for classification or non-classification

Skin Irritation

In the in vitro key study with the Human Skin Model Test, the mean relative absorbance value was 100.4% compared to the relative absorbance value of the negative control.

 

Eye Irritation

In the in vitro key study the relative mean absorption value corresponding to the viability of the tissues was 94.1%, which is above the cut off value of 60 %, indicating that the test item is not eye irritant.

Based on Regulation (EC) No 1272/2008, the substance is classified as not irritating to skin or eye.