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Description of key information

Based on the results of the OECD 439 study, the test item is not irritant to skin.

Based on the results of the OECD 492, 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
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 September 2016 - 11 January 2017
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:
2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2009
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Cell type:
non-transformed keratinocytes
Details on animal used as source of test system:
Epi-200 SIT kits and MTT-100 assays diluent were purchased from MatTek Corporation (82105 Bratislava, Slovakia). EpiDerm™ tissues were shipped with cool packs on medium-supplemented agarose gels in a 24-well plate. On day of receipt the pre-incubation phase of the EpiDerm™ tissues started.
Justification for test system used:
The EpiDerm TM is an in vitro system recommended by the corresponding guideline.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200-SIT Kit
- Tissue batch number: 23356
- Date of initiation of testing: 20 September 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C for 35 minutes; room temperature for 25 minutes
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: rinsed with DPBS at least 15 times
- Observable damage in the tissue due to washing: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: Versamax Molecular Devices, Softmax Pro
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

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 (Category 2) if the mean relative tissue viability of three individual tissues is reduced ≤ 50% of the negative control
- The test substance is considered to be non-irritant to skin if the mean relative tissue viability of three individual tissues is reduced > 50% of the negative control
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 30 µL
Duration of treatment / exposure:
35 min at 37 °C + 25 min at room temperature
Duration of post-treatment incubation (if applicable):
25 h + 18 h
Number of replicates:
3
Irritation / corrosion parameter:
other: relative absorbance (%)
Run / experiment:
Tissue 1
Value:
100.5
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
other: relative absorbance (%)
Run / experiment:
Tissue 2
Value:
127.2
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
other: relative absorbance (%)
Run / experiment:
Tissue 3
Value:
108.5
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
Interpretation of results:
GHS criteria not met
Conclusions:
The test item is not 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 item passed the pre-tests for colour interference and direct MTT reduction. Consequently, additional tests with freeze-killed or viable tissues were not necessary.

The test met the required acceptability criteria.

After treatment with the test item the mean relative absorbance value did not decrease (112.1%) compared to the relative absorbance value of the negative control. This value does not affect 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:
13 September 2016 - 04 January 2017
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:
2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
other: Reconstructed human Cornea-like Epithelium (RhCE)
Details on test animals or tissues and environmental conditions:
EpiOcular™ kits and MTT-100 kits are purchased from MatTek Corporation (82105 Bratislava, Slovakia). EpiOcular™ tissues were shipped at 2 - 8 °C on medium-supplemented agarose gels in a 24-well plate. On day of receipt of the EpiOcular™ tissues, the equilibration step started.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
50 μL of the test item
Duration of treatment / exposure:
30 min
Duration of post- treatment incubation (in vitro):
12 min + 120 min
Number of animals or in vitro replicates:
Duplicate tissues
Details on study design:
- Details of the test procedure used: MTT Assay

- RhCE tissue construct used, including batch number: EpiOcular Kit, Lot No.: 23737

- Doses of test chemical and control substances used: 50 µL undiluted test chemical / control substances

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods:
Exposure: 30 min, 37 °C
Post-exposure immersion: 12 min, room temperature
Post-exposure incubation: 120 min, 37 °C

- Number of tissue replicates used per test chemical and controls: 2 per test chemical, 2 per positive control and 2 per negative control

- Wavelength used for quantifying MTT formazan: 570 nm

- Description of the method used to quantify MTT formazan: measurement of absorbance at 570 nm (OD570) with a plate reader reader
(Versamax® Molecular Devices, Softmax Pro)

- Description of evaluation criteria used:
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.

- Acceptance criteria
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
Irritation parameter:
other: relative absorbance (%)
Run / experiment:
Tissue 1
Value:
70.2
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: relative absorbance (%)
Run / experiment:
Tissue 2
Value:
61.8
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Interpretation of results:
GHS criteria not met
Conclusions:
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 test item did not prove to be an MTT reducer in the MTT pre-test, it is colourless 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.

The test met the acceptance criteria. Irritating effects were not observed following incubation with 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% (66.0%). Therefore, the test item is not considered to possess an eye irritant potential.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation study:

An 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 passed the pre-tests for colour interference and direct MTT reduction. Consequently, additional tests with freeze-killed or viable tissues were not necessary.

The test met the required acceptability criteria. After treatment with the test item the mean relative absorbance value did not decrease (112.1%) compared to the relative absorbance value of the negative control. This value does not affect the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

Eye irritation study:

An 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 item did not prove to be an MTT reducer in the MTT pre-test, it is colourless 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.

The test met the acceptance criteria. Irritating effects were not observed following incubation with 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% (66.0%).

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

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. Based on this data, the substance is not considered to be skin corrosive and eye irritating under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EC) No 2017/776.