Registration Dossier

Administrative data

Description of key information

The test item is skin irritant, but not skin corrosive and eye irritant, but not eye corrosive.

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:
27.06.2016 - 18.07.2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Source strain:
other: not applicable
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: Epi-200-SITKit
- Tissue batch number(s): 23345

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 35 minutes at 37 °C and 25 minutes at room temperature
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: at least 15 times
- 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: 42 hours
- Spectrophotometer: Versamax® Molecular Devices, Softmax Pro, version 4.7.1
- Filter: 570 nm

NUMBER OF REPLICATE TISSUES: 3

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The substance is considered skin irritant category 2 according to UN GHS (published 2003, last (6th) revision 2015) if the mean relative tissue viability of three individual tissues is reduced ≤ 50% of the negative control.

ACCEPTANCE CRITERIA
- 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.
- Positive control: An assay is meeting the acceptance criterion if mean relative tissue viability of the positive control is ≤ 20%.
- Standard deviation: The rel. SD of 3 identical replicates should be < 18%. OD values should not be below historically established boundaries.
- Historical data and the quality certificate of the supplier of the test kit demonstrated the robustness of the test system / test kit.
Control samples:
yes, concurrent negative control
yes, concurrent no treatment
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL
- Concentration (if solution): undiluted; 47 µL/cm2

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): undiluted

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5% in deionised water
Duration of treatment / exposure:
60 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
3.7
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not led to a change in colour.
Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.
The acceptance criteria were met.

Results

* Mean of three replicate wells after blank correction

** Relative absorbance per tissue [rounded values]: 100×(absorbancetissue) / (mean absorbancenegative control)

*** Relative absorbance per treatment group [rounded values]:100×(mean absorbancetest item/positive control) /(mean absorbancenegative control

 

Dose Group

Tissue No.

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 + 3**

Relative Standard Deviation [%]

Mean Rel. Absorbance [% of Negative Control]***

Blank

 

0.038

0.047

0.037

0.041

 

 

 

 

 

Negative Control

1

1.972

1.935

1.892

1.933

1.892

1.810

104.5

4.9

100.0

2

1.881

1.831

1.874

1.862

1.821

100.6

3

1.788

1.738

1.746

1.757

1.717

94.8

Positive Control

1

0.093

0.091

0.091

0.092

0.051

0.050

2.8

7.6

2.8

2

0.093

0.094

0.094

0.093

0.053

2.9

3

0.087

0.086

0.086

0.086

0.046

2.5

Test Item

1

0.110

0.115

0.114

0.113

0.072

0.067

4.0

16.1

3.7

2

0.096

0.096

0.095

0.096

0.055

3.0

3

0.115

0.118

0.114

0.116

0.075

4.1

 

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
The test item is to be considered as irritant to skin.
Executive summary:

In this in vitro study the irritation potential of the test item was assessed by means of the Human Skin Model Test. The study was according to OECD 439 and GLP.

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

The main study consists of topical exposure of the test item to the human reconstructed epidermis model followed by a cell viability test. The cell viability is measured by dehydrogenase conversion of MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazoliumbromide], in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison of untreated negative controls is used to predict skin irritation potential and used for the purpose of classification as irritating or non-irritating. The test chemical is considered to be irritant to skin in accordance with UN GHS and EU CLP Category 2 if the tissue viability after exposure and post-treatment incubation is ≤ 50%.

Three tissues of the human skin model EpiDermTM were treated with the test item, the negative (DPBS) or the positive (5% SLS) control for 60 minutes.

Hereafter the skin tissues are washed and further incubated for about 42 hours, whereafter the tissues were treated with MTT for 3 hours following about 2.7 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, thus showing the quality of the tissues. Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control to 2.8 %, 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 17% (threshold of the OECD TG: < 18%), thus ensuring the validity of the study.

After treatment with the test item the mean relative absorbance value decreased to 3.7% compared to the relative absorbance value of the negative control. This value is below the threshold for irritancy of ≤ 50% and 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 to be considered as irritant to skin.

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:
09.06.2016 - 17.06.2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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 (21h in the 1st experiment, 18h in the 2nd 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: 83.3 μL/cm2
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.: 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 after which MTT was extracted for 2-3 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:
- Historical data positive control: Mean Viability: 32.3%; Rel. Standard Deviation: 11.1%; Range of Viabilities: 6.90% - 43.4%; Mean Absorption: 0.566; Rel. Standard Deviation: 0.283; Range of Absorbance: 0.107- 0.943
- Historical data negative control: Mean Absorption: 1.65; Rel. Standard Deviation: 0.295; Range of Absorbance: 1.27 – 2.16

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:
Test Item
Value:
16.6
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
The acceptance criteria were met.

Results


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

 

 

 

 

 

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

0.308

0.297

0.302

0.263

0.343

12.7

7.7

16.6

0.465

0.459

0.462

0.423

20.5

 

* Mean of two replicate wells after blank correction


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

Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
In this study and under the experimental conditions reported, the test item is eye irritant.
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 test was according to OECD 492 and GLP.

In the pre-tests, the test item did not reduce MTT and the intrinsic colour of the substance was not intensive and did not color water or isopropanol. Therefore, additional tests with freeze-killed or viable tissues did not have to be performed.

For the main test, 50 μL of the test item, the negative control (deionised water) or the positive control (methyl acetate) was applied to tissue for 30 minutes and the test was performed in duplicate. The absorbance values after treatment with the negative control were well within the required acceptability criterion showing the quality of the tissues, while treatment with the positive control induced a decrease below 50% compared to the negative control ensuring the validity of the test system.

The test was also valid as the difference in viability between the two relating tissues was < 20% in the same run (for test item tissues, positive and negative control tissues).

Treatment with the test item indicated irritating effects. Compared with the negative control the relative mean absorption value corresponding to the viability of the tissues was 16.6%. This is below the cut off value of 60% and according to the criteria the substance should be classified as eye irritant.

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is eye irritant.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation / corrosion

For this endpoint there are 2 studies available.

In the first study the irritation potential of the test item was assessed according to OECD 439 and GLP. The test item did not reduce MTT (test for direct MTT reduction), or change colour when mixed with deionised water (test for colour interference) and its intrinsic colour was not intensive (colourless to yellow). Consequently, no additional tests with freeze-killed or viable tissues were necessary.

Three tissues were treated with the test item, the negative (DPBS) or the positive (5% SLS) control for 60 minutes, washed and further incubated for about 42 hours. Hereafter the tissues were treated with MTT and the amount of extracted colorant was determined photometrically at 570 nm. The acceptance criteria for the negative control, positive control and the variability between related tissues were met, thus ensuring the validity of the test system.

After treatment with the test item the mean relative absorbance value decreased to 3.7% compared to the negative control. This value is below the threshold for irritancy of ≤ 50% and therefore, the test item is considered to be skin irritant.

In the second study the corrosive potential of the test item was assessed according to OECD 431 and GLP. In the pre-tests the test item did not reduce MTT (test for direct MTT reduction) or change colour when mixed with deionised water (test for colour interference). Consequently, additional tests with freeze-killed or viable tissues were not necessary.

The tissues were exposed to the undiluted test item, the negative control (deionised water) or the positive control (8.0 N KOH) for 3 minutes or 1 hour, washed and incubated with MTT.

The acceptance criteria for the negative control, positive control and the variability between related tissues were met, thus ensuring the validity of the test system.

Exposure to the test item did not decrease the relative absorbance (101.8%) after 3 minutes. After 1 hour exposure the relative absorbance was reduced to 33.4%, however these values did not exceed the threshold for corrosivity (50% after the 3 minutes exposure and 15% after 1 hour exposure). Therefore, the test item is not corrosive to the skin.

Eye irritation / corrosion

For this endpoint there are 2 studies available.

In the first study the eye irritant potential of the test item was assessed according to OECD 492 and GLP. In the pre-tests, the test item did not reduce MTT or show an intensive intrinsic colour and did not color water or isopropanol. Therefore, additional tests with freeze-killed or viable tissues did not have to be performed.

50 μL of the test item, the negative control (deionised water) or the positive control (methyl acetate) was applied to the tissue for 30 minutes. The acceptance criteria for the negative control, positive control and the variability between related tissues were met, thus ensuring the validity of the test system. The test item showed irritating effects as, compared to the negative control, the relative viability of the tissues was 16.6%. This is below the cut off value of 60% and according to the criteria the substance should be classified as eye irritant.

In the second study to potential of the test item to cause damage to the cornea was assessed according to OECD 437 and GLP. In this study the opacity of the cornea and the permeability after treatment is assessed to determine the damaging effect of the test item. The undiluted test item, the positive (2-Ethoxyethanol), or negative control (saline) was applied to corneae for 10 minutes. After washing the cornea were post-incubated for 120 minutes after which the opacity was measured. The permeability of the corneae was by transfer of sodium fluorescein after incubation of 90 minutes. The negative (mean IVIS = 1.00) and the positive control (mean IVIS = 94.25) met the criteria and ensured the validity of the test. The test item did not cause an increase of the corneal opacity or permeability (mean IVIS = 0.65). As the threshold for serious eye damage is IVIS ≥ 55, the test item is not considered to cause serious damage to the eyes according to OECD 437.

Justification for classification or non-classification

Skin irritation / corrosion

For this endpoint there are 2 studies available. Both studies are needed for classification purposes.

In the first study the irritation potential of the test item was assessed according to OECD 439 and GLP. After treatment with the test item the mean relative absorbance value decreased to 3.7% compared to the negative control. This value is below the threshold for irritancy of ≤ 50% and therefore, the test item is considered to be skin irritant.

In the second study the corrosive potential of the test item was assessed according to OECD 431 and GLP. Exposure to the test item did not decrease the the relative absorbance (101.8%) after 3 minutes, but was reduced to 33.4% after 1 hour. However these values did not exceed the threshold for corrosivity (50% after the 3 minutes exposure and 15% after 1 hour exposure). Therefore, the test item is not corrosive to the skin.

In conclusion, the substance is classified as a Skin Irritant Cat. 2.

Eye irritation / corrosion

For this endpoint there are 2 studies available. Both studies are needed for classification purposes.

In the first study the irritation potential of the test item was assessed according to OECD 492 and GLP. After treatment with the test item the relative viability was 16.6% compared to the negative control. This value is below the cut off value of 60% and therefore, the test item is considered to be eye irritant.

In the second study the corrosive potential of the test item was assessed according to OECD 437 and GLP. Exposure to the test item did not increase the corneal opacity or permeability (mean IVIS = 0.65). This result is below the threshold for corrosivity (IVIS ≥ 55) and therefore, the test is not corrosive to the eye.

In conclusion, the substance is classified as Eye Irritant Cat. 2.