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Description of key information

Not irritating to skin or eye

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:
2018 February - 2018 April
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:
In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method, 28 July 2015.
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
“In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”. 06-Jul-2012.
Qualifier:
according to guideline
Guideline:
other: L’Oréal Standard Operating Procedure: “EpiSkin™ Skin Irritation Test Method"
Version / remarks:
ECVAM Skin Irritation Validation Study - Validation of the EpiSkin™ Test Method15 min - 42 hours for the Prediction of Acute Skin Irritation of Chemicals. Version 1.8, Feb-2009.
Qualifier:
according to guideline
Guideline:
other: ECVAM DB-ALM Protocol No. 131
Version / remarks:
“EpiSkin™ Skin Irritation Test15 min – 42 hours. 09-Jun-2012.
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
The test substance (Batch 0000140180) was provided by the study sponsor, Vertellus Holdings, and stored at room temperature. The test substance appeared as a clear oily liquid with no odour.
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Adult donors
Justification for test system used:
This test uses the EPISKIN-SM™ reconstructed human epidermis model (SkinEthic) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.

This test method is able to detect chemicals that cause skin irritation, i.e. produce reversible damage to the skin and allows for hazard identification in accordance with UN GHS “Category 2”. Depending on the regulatory framework it can also be used to identify non-classified chemicals.
Vehicle:
unchanged (no vehicle)
Details on test system:
The test was carried out with the reconstituted three-dimensional human skin model EPISKIN-SM™ (SkinEthic). This skin model consists of normal human epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell culture inserts. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: reconstituted three-dimensional human skin model EPISKIN-SM™ (SkinEthic)
- Tissue batch number(s): 18 EKIN 011
- Date of initiation of testing: 09 February 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature
- Temperature of post-treatment incubation (if applicable): 37 ± 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Once with DPBS. Excess DPBS was removed by blotting bottom with blotting paper.
- Observable damage in the tissue due to washing: None
- Modifications to validated SOP: None

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: Stock: 3 mg/mL MTT (Sigma, Lot No.: MKBZ5197V; VWR, Lot No.: 1017C385) in PBS (Gibco; Lot No.: 1909266). MTT medium: MTT stock solution was diluted 1 + 9 with DMEM-based medium (final concentration 0.3 mg/mL).
- Incubation time: 3 h
- Spectrophotometer: Plate spectrophotometer (acidic isopropanol as blank)
- Wavelength: 570 nm
- Filter bandwidth: ± 30 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Mean 11.28%
- Barrier function: 1.9 mg/mL
- Morphology: 7.2%
- Contamination: None
- Reproducibility: Fully reproducible

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues : The mixture of 10 µL test item per 2 mL MTT medium showed no reduction of MTT as compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%. The mixtures of 10 µL of the test item per 90 µL aqua dest. (distilled water) and per 90 µL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSCliving equalled 0%.
- Procedure used to prepare the killed tissues (if applicable): N/A
- N. of replicates : 3
- Method of calculation used: NSMTT [%] = [(ODKT - ODKU)/ODNK] * 100

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 3

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritating to skin if the viability after 3 minutes exposure is less than or equal to 50% relative to negative control (Irritant (I): UN GHS “Category 2”).
- The test substance is considered to be non-irritating to skin if the viability after 3 minutes exposure is greater than 50% (Non-Irritant (NI): UN GHS “No Category”).
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439: N/A
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
10 µL
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Negative control (mean)
Value:
100
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Positive control (mean)
Value:
5.6
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Test item (mean)
Value:
115.6
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
not applicable
Interpretation of results:
GHS criteria not met
Conclusions:
The test substance was evaluated for skin irritation in vitro by assessing its effect on a reconstituted three-dimensional human epidermis model according to OECD guideline 439. The test substance was observed to be non-irritating at doses up to 10 µL.
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:
2018 January 25 - 2018 May 31
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)
Qualifier:
according to guideline
Guideline:
other: EURL ECVAM DB-ALM Method Summary No. 164: EpiOcular™ Eye Irritation Test
Qualifier:
according to guideline
Guideline:
other: EpiOcular™ Eye Irritation Test (OCL-200-EIT)
Version / remarks:
For the prediction of acute ocular irritation of chemicals For use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Vertellus, 0000140180
- Expiration date of the lot/batch: 15 June 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: assumed to be stable
Details on test animals or tissues and environmental conditions:
The test was carried out with the EpiOcular™ reconstructed human cornea-line epithelium (RhCE) model (MatTek). The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified, highly differentiated squamous epithelium morphologically similar to that found in a human cornea. The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinised surface, showing a cornea-like structure analogous to that found in vivo.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
50 mg
Duration of treatment / exposure:
6 hr
Duration of post- treatment incubation (in vitro):
18 hr
Number of animals or in vitro replicates:
2/dose
Details on study design:
- Details of the test procedure used : RhCE tissue was treated with test substance for 6 hrs. After an 18 hr post-treatment incubation, tissue samples were evaluated for viability using an MTT reduction assay.
- RhCE tissue construct used, including batch number : EpiOcular™ tissues on agarose (Lot No.: 27019 for NSMTT control, 27039 for main experiment)
- Doses of test chemical and control substances used : Negative control: 50 µL. Positive control: 50 µL. Test item: 50 mg. The test item was ground into a fine powder using a mortar and pestle. Approximately 50 mg (83.3 mg/cm^2) of the test item were applied directly to the EpiOcular TM tissue using an application spoon, avoiding compression of the test item. The test item was spread to match size of the tissue.
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable) : Exposure: 6 hr, 37 °C. Post-exposure immersion: 25 min, room temperature. Post-exposure incubation: 18 hr, 37 °C.
- Justification for the use of a different negative control than ultrapure H2O (if applicable) : N/A.
- Justification for the use of a different positive control than neat methyl acetate (if applicable) : N/A
- Description of any modifications to the test procedure : N/A
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable) : The mixture of 50 mg test item per 1 mL MTT medium showed reduction of MTT as compared to the solvent. The mixture turned blue/purple. Since the mean relative tissue viability of the test item treated tissues (TM) was above the 60% threshold value killed tissue controls were performed for quantitative correction of results.
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable) : 2
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer) : 570
- Description of the method used to quantify MTT formazan : Absorbance (OD570)
- Description of the qualification of the HPLC/UPLC-spectrophotometry system (if applicable) : N/A
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model : Ocular irritation potential of the test item was predicted from the relative mean tissue viabilities obtained after treatment compared to the negative control tissues concurrently treated with Aqua dest. (distilled water). The test item is considered to be irritant to the eye but it cannot be differentiated between UN GHS “Category 1” or “Category 2”, if the relative tissue viability is less or equal to 60%. The test item is considered to be non-irritant in accordance with UN GHS “No Category” if relative tissue viability is higher than 60%.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria : Yes
- Complete supporting information for the specific RhCE tissue construct used : Yes
- Reference to historical data of the RhCE tissue construct: Yes
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals : Yes
- Positive and negative control means and acceptance ranges based on historical data : Yes
- Acceptable variability between tissue replicates for positive and negative controls: Yes
- Acceptable variability between tissue replicates for the test chemical: Yes
Irritation parameter:
other: Tissue Viability [%]
Run / experiment:
Negative Control
Value:
100
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
other: Tissue Viability [%]
Run / experiment:
Positive Control
Value:
20.3
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
not applicable
Remarks on result:
positive indication of irritation
Irritation parameter:
other: Tissue Viability [%]
Remarks:
NSMTT corrected
Run / experiment:
Test item
Value:
98.4
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Range of historical values if different from the ones specified in the test guideline: Observed values in range of historical values
Interpretation of results:
GHS criteria not met
Conclusions:
The test substance was evaluated for eye irritation potential according to OECD Guideline 492. In this study under the given conditions the test item showed no irritant effects. The test item is classified as “non-irritant“ in accordance with UN GHS “No Category”.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In vitro, the registered substance was found to be non-irritating, not classified, to skin or eye under the conditions of the OECD 439 and 492 protocols.

Justification for classification or non-classification

The substance is not irritating to skin or eye, and is not classified according to Regulation EC No. 1272/2008.