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Description of key information

OECD 439: The test item is not irritating to skin.

OECD 492: The test item is not irritating to 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:
August 22, 2017 - October 16, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
Commission Regulation (EU) No. 640/2012 amending, for the purpose of its adaption to technical progress, Regulations (EC) No. 440/2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and the council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
July 28, 2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Skinethic skin irritation test -42bis Standard operating procedure (SOP) 2009
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
No pre-treatment ;The test item was applied neat to the tissues.
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from a single donor
Justification for test system used:
standard model
Vehicle:
unchanged (no vehicle)
Remarks:
No vehicle used in this study; The test item was applied neat to the tissues.
Details on test system:
CELL CULTURE
- Supplier: EpiSkin/SkinEthic Laboratories, Lyon, France)
- Source: human keratinocytes cultured on a polycarbonate filter in conditions which permit their terminal differentiation
- Format: 24 well plate
- Batch: 17-RHE-094
- Expires: September 18, 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment: room temperature
- Temperature of post-treatment incubation: 37°C

REMOVAL OF THE TEST MATERIAL AND CONTROL
After the end of the treatment interval, the residual test item was removed immediately by gently rinsing with a minimum volume of 25 mL DPBS using a pipette. Excess DPBS was removed by gently shaking the inserts and blotting the bottom with blotting paper.

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer:ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany at 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD > 0.7 (acceptance criteria); OD = 1.2 (result)
- Barrier function: 4.0h <= ET50 <= 10.0h (acceptance criteria); 5.1h (result)
- Morphology: Number of cell layers >= 4, absence of significant histological abnormalities, well differentiated epidermis consisting of basal, spinous, granular layers and a stratum corneum (acceptance criteria); 6 cell layers, absence of significant histological abnormalities, satisfactory (result)

NUMBER OF REPLICATE TISSUES: triplicates

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: three tissues, one independent experiment

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is identified as UN GHS Category 2 or Category 1 if the viability after exposure and post-exposure incubation is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability after exposure and post-treatment incubation is greater than 50%.
Control samples:
yes, concurrent negative control
Amount/concentration applied:
TEST MATERIAL: 16 mg of solid test material
NEGATIVE CONTROL: 16 µL (Dulbecco`s Phosphate-Buffered Saline)
POSITIVE CONTROL: 16 µL (5% aqueous solution of sodium dodecyl sulfate in deionised water)
Duration of treatment / exposure:
42 min (± 1 minute)
Duration of post-treatment incubation (if applicable):
42 hours (± 1 hour)
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Experiment 1 / Run 1
Value:
103.9
Vehicle controls validity:
not applicable
Remarks:
The test item was applied neat to the tissues
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: none
- Colour interference with MTT: none

ACCEPTANCE OF RESULTS:

Acceptability of the Quality Control Data of the Skin Model with Reference to Historical Batch Data:

Acceptance Criterion Result
Negative control OD ≥ 0.8 and ≤ 3.0 1.570 to 1.686

Acceptability of the Positive and Negative Control stated by Episkin/SkinEthic Laboratories:

Acceptance Criterion Result
Mean OD negative control ≥ 1.2 1.616
Mean viability positive control < 40% 1.7%
SD of group-mean value ≤ 18% 5.9% (positive control)
3.8% (negative control)

Acceptability of the Positive and Negative Control based on Historical Data of the Testing Laboratory:

Acceptance Criterion Result
Mean OD negative control ≥ 1.455 1.616
Mean viability positive control ≤ 2.97% 1.7%

Test Item Data Acceptance Criteria:

Acceptance Criterion Result
SD of group-mean value ≤ 18% 6.6%

The study met all acceptance criteria.


 Group Time / [min]  Mean OD  Mean Relative viability / [%]
 Negative Control 42  1.616 100 
 Positive Control 42

0.028

1.7

 Test Material

42

1.679

103.9

Interpretation of results:
GHS criteria not met
Remarks:
UN GHS: No Caterory (according to OECD TG 439)
Conclusions:
This study was performed according to GLP and the methods applied are fully compliant with OECD TG 439. Under the conditions of the present study, the test item is not considered to possess an irritant potential to skin (UN GHS: No Category).
Executive summary:

Objective

The objective of the present study was to investigate the potential of the test item to induce skin irritation in anin vitrohuman skin model.

Study Design

The test item was applied topically to a human reconstructed skin model followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the skin irritation potential.

Triplicates of the human skin RHE-model were treated with the test item, the negative or the positive control for 42 minutes (± 1 minute). 16 µL of either the negative control (DPBS-buffer) or the positive control (5% aqueous solution of sodium dodecyl sulfate) were applied to the tissues. Before application of 16 mg of the solid test item, 10 µL of deionised water was spread to the epidermis surface to improve the contact between the test item and the epidermis.

Results

All acceptability criteria after treatment with the negative control (DPBS-buffer) and the positive control (5% aqueous solution of sodium dodecyl sulfate) were met.

Following treatment with the test item, the tissue viability was 103.9% and, thus, higher than 50%,i.e.according to OECD 439 the test item is considered as non-irritant to skin (UN GHS: No Category).

Conclusion

Under the conditions of the present study, the test item is not considered to possess an irritant potential to skin (UN GHS: No Category).

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:
August 22, 2017 - November 7, 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:
July 28, 2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: DB-ALM Protocol No. 164: Ocular Irritation Assay for Chemicals using EpiOcular™ EIT,
Version / remarks:
September 14, 2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EpiOcular Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; For use with MatTek Corporation`s Reconstructed Human EpiOcular Model; MatTek Corporation
Version / remarks:
June 29, 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No pre-treatment, the test item was applied neat to the tissues.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL: 50 mg per tissue

NEGATIVE / VEHICLE CONTROL: 50 µL per tissue

Sterile deionized water was used as negative control.


POSITIVE CONTROL: 50 µL per tissue

Designation: Methyl acetate
Supplier: MatTek In Vitro Life Science Laboratories
Lot-No.: 032817ISA
Catalog #: TC-MA
Purity (GC): 99.7%
Appearance: Colorless liquid
Expiration date: March 28, 2018
Storage: 15 to 30°C
Duration of treatment / exposure:
6 hours
Duration of post- treatment incubation (in vitro):
18 hours
Number of animals or in vitro replicates:
in vitro: duplicate design
Details on study design:
- Details of the test procedure used
- RhCE tissue construct used, including batch number: EpiOcular Tissues (OCL-200, OCL-212) (Lot No: 27005) was obtained from MatTek In Vitro Life Science Laboratories
- Doses of test chemical and control substances used: undiluted
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: 6 hours at 37°C (exposure), 25 min at room temperature (post-exposure immersion), 18 hours at 37°C (post-exposure incubation)
- Number of tissue replicates used per test chemical and controls: two tissues (test item, negative and positive control)
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): 570 nm wavelength
- Description of the method used to quantify MTT formazan:

After the post-treatment incubation period, the treated tissues were transferred in a 24-well plate filled with 300 µL MTT solution (1.0 mg/mL MTT). Once all the tissues were placed into the 24-well plate, the plate was incubated for 180 minutes (± 10 minutes) at 37°C and 5% CO2. The inserts were removed from the 24-well plate after 180 minutes (± 10 minutes). The bottom of the inserts was blotted on absorbent material, and then transferred to a 6-well plate containing 2 ml isopropanol so that no isopropanol was flowing into the inserts. The plate was sealed with a standard plate sealer. To extract the MTT, the plates was placed on an orbital plate shaker and shaken for 2 to 3 hours at room temperature. The corresponding negative and positive controls were treated identically. The extract solution was mixed and 2 x 200 µL were transferred into a 96-well plate. The OD was read using a spectrophotometer at 570 nm wavelength. A functional test of the microplate reader was performed using a filter test plate. The results of the functional test and the printout of the measurement were included in the raw data of the study.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
Viabililty (%) = (test item OD/mean negative control OD) x 100
If the test item-treated tissue viability is >60.0% relative to negative control-treated tissue viability, the test item is labeled non-irritant (UN GHS No Category).
If the test item-treated tissue viability is <=60.0% relative to negative control-treated tissue viability, the test item is labeled irritant (UN GHS Category 1 or Category 2).
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria:
1. The negative control OD is > 0.8 and < 2.5 (0.945 and 1.025).
2. The mean relative viability of the positive control is below 50% of the negative control viability (33.9%).
3. The difference of viability between the two relating tissues of a single chemical is < 20% (values between 1.1% to 8.2%) in the same run (for positive and negative control tissues and tissues of single chemicals).
- Reference to historical data of the RhCE tissue construct
1. Tissue viability: OD (540- 570 nm) [1.1 – 3.0] (acceptance criteria); 1.419 +/- 0.105 (result)
2. Barrier function: ET-50 [12.2 – 37.5 min] (acceptance criteria); 15.97 min (result)
3. Sterility: No contamination (acceptance criteria); sterile (result)
- Positive and negative control means and acceptance ranges based on historical data
Negative control: OD >0.8 and <2.5
The mean relative viability of the positive control is:
a) 30 minute exposure: below 50% of control viability
b) 6 hour exposure: below 50% of control viability
- Acceptable variability between tissue replicates for positive and negative controls: The difference of viability between the two relating tissues of a single chemical is <20% in the same run.
- Acceptable variability between tissue replicates for the test chemical: The difference of viability between the two relating tissues of a single chemical is <20% in the same run.
Irritation parameter:
other: Viability %
Run / experiment:
Run 1 / Experiment 1
Value:
111.4
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:
OTHER EFFECTS:
- Visible damage on test system: No observations

ACCEPTANCE OF RESULTS:
1. The negative control OD is >0.8 and <2.5 (0.945 and 1.025).
2. TThe mean relative viability of the positive control is below 50% of the negative control viability (33.9%).
3. The difference of viability between the two relating tissues of a single chemical is <20% (values between 1.1% to 8.2%) in the same run (for positive and negative control tissues and tissues of single chemicals).

The study met all acceptance criteria

   Mean OD  Mean Viability
 Negative Control  0.985 100.0% 
 Positive Control  0.334 33.9%
 Test Item  1.098 111.4%
Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the present study, the test item did not show an eye hazard potential. The test item is labeled non-irritant (UN GHS: No Category).
Executive summary:

The objective of the present study was to investigate the potential of the test item to induce eye irritation in an in vitro human cornea model.

The test item was applied topically to a reconstructed human cornea-like epithelium model (EpiOcular) followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the eye irritation potential. Duplicates of the EpiOcular-model were treated with the test item, the negative or the positive control for 6 hours (± 15 minutes). 50 mg of the test item and 50 µL of either the negative control (sterile deionized water) or the positive control (methyl acetate) were applied to the tissues.

After treatment with the negative control (sterile deionized water) the mean OD was 0.985 (study acceptance criterion: >0.8 and <2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 33.9% (study acceptance criterion: <50%). Thus, the acceptance criteria were met.

Following treatment with the test item, the tissue viability was 111.4% and, thus, higher than 60%, i.e. according to OECD 492 the test item is labeled non-irritant (UN GHS: No Category).

Under the conditions of the present study, the test item did not show an eye hazard potential. The test item is labeled non-irritant (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 skin irritation study

The objective of the present study was to investigate the potential of the test item to induce skin irritation in an in vitro human skin model according to OECD 439.

All acceptability criteria after treatment with the negative control (DPBS-buffer) and the positive control (5% aqueous solution of sodium dodecyl sulfate) were met.

Following treatment with the test item, the tissue viability was 103.9% and, thus, higher than 50%, i.e. according to OECD 439 the test item is considered as non-irritant to skin (UN GHS: No Category).

In vitro eye irritation study

The objective of the present study was to investigate the potential of the test item to induce eye irritation in an in vitro human cornea model according to OECD Guideline 492.

After treatment with the negative control (sterile deionized water) the mean OD was 0.985 (study acceptance criterion: >0.8 and <2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 33.9% (study acceptance criterion: <50%). Thus, the acceptance criteria were met.

Following treatment with the test item, the tissue viability was 111.4% and, thus, higher than 60%, i.e. according to OECD 492 the test item is labeled non-irritant (UN GHS: No Category).

 

Justification for classification or non-classification

Based on the provided information there is no need for classification according to the EU Regulation (EC) No 1272/2008 on Classification,Labelling and Packaging of Substances and Mixtures, as amended for the 10th time in Regulation (EU) No 2017/776.