Registration Dossier

Administrative data

Description of key information

Skin irritation

A study was performed according to OECD TG 439. Under the conditions of this study, the test item is not considered to possess an irritant potential to skin (reference 7.3.1 -1).

Eye irritation

In a study performed according OECD TG 492, the test item did not show an eye hazard potential (reference 7.3.2 -1).

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:
December 5, 2017 - February 2, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
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:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
July 28, 2015
Deviations:
no
Qualifier:
according to
Guideline:
other: Skinethic skin irritation test -42bis Standard operating procedure (SOP) 2009
Deviations:
no
GLP compliance:
yes (incl. certificate)
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: 18-RHE-001
- Expires: January 15, 2018

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


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany
- Wavelength: 570 nm
- Linear OD range of spectrophotometer: 0 - 3.0

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA

Acceptance Criteria Result
Cell viability (570 nm optical density, MTT test) OD >0.7 OD 1.2 (CV = 6.8 %)

Barrier function integrity test
(Exposure time inducing 50%
viability using Triton X-100 1%) 4.0h ≤ ET50 ≤ 10.0h 4.7h

Histological observation
(HES stained vertical paraffin sections) Number of cell layers ≥ 4 6 cell layers
Absence of significant
histological abnormalities. Absence of significant histological abnormalities.

Well differentiated epidermis Satisfactory
consisting of basal, spinous,
granular layers and a stratum c
orneum.


NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1 experiment

PREDICTION MODEL / DECISION CRITERIA:
A test item is considered as non-irritant to skin (UN GHS No Category) if the tissue viability after exposure and post-treatment incubation is > 50%.
A test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post-treatment incubation is ≤ 50%. Since the in vitro skin irritation test according to OECD 439 cannot resolve between UN GHS Categories 1 and 2, further information on skin corrosion is required to decide on its final classification.
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:
94.3
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 1.714 to 1.890

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

Acceptance Criterion Result
Mean OD negative control ≥ 1.2 1.821
Mean viability positive control < 40% 1.2%
SD of group-mean value ≤ 18% 8.3% (positive control)
5.2% (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 1.821
Mean viability positive control ≤ 2.97% 1.2%

Test Item Data Acceptance Criteria:

Acceptance Criterion Result
SD of group-mean value ≤ 18% 4.3%

The study met all acceptance criteria.


 Group Time / [min]  Mean OD  Mean Relative viability / [%]
 Negative Control 42  1.821 100 
 Positive Control 42

0.021

1.2

 Test Material

42

1.817

94.3

Interpretation of results:
GHS criteria not met
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:

A study was performed according to GLP and the methods applied are fully compliant with OECD TG 439. 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.

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.

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 94.3% 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).

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:
November 27, 2017 - January 31, 2018
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)
Version / remarks:
July 28, 2015
Deviations:
no
Qualifier:
according to
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:
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. certificate)
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
Number of animals or in vitro replicates:
in vitro: duplicate design
Details on study design:
Controls
Negative/Vehicle Control: Sterile deionized water

Positive Control: Methyl acetate, MatTek In Vitro Life Science Laboratories, Lot-No 032817ISA, Expiration date March 28, 2018

Number of Replicates
The test item as well as the positive and negative control were tested in batch-duplicates. Therefore, a total number of six tissues was used in this study.

Preparation
On day of receipt, the tissues were equilibrated in their 24-well shipping container to room temperature for about 15 minutes. Afterwards the tissues were removed from the shipping container using sterile forceps and transferred to 6-well plates containing 1 mL pre-warmed (37°C) assay medium. Any agarose adhering to the inserts was removed by gentle blotting on gauze or paper towel. Afterwards, the tissues were incubated at 37°C and 5% CO2 overnight (16-24 hours) without medium exchange.

Pre-Treatment
After the overnight incubation, the tissues were pre-wetted with 20 µL DPBS and incubated at 37°C and 5% CO2 for 30 minutes (± 2 minutes).

Treatment
After the 30 minute DPBS pre-treatment, the negative and the positive control were tested by applying 50 µL topically on the EpiOcular tissues. The solid test item was tested by evenly applying 50 mg topically on the EpiOcular tissues. The tissues were placed back into the culture medium after dosing and incubated at 37°C and 5% CO2 for 6 hours (± 15 minutes).

At the end of the 6 hours treatment time, the positive control, negative control and the test item were removed by extensively rinsing the tissues with pre-warmed (room temperature) DPBS. Three clean beakers, containing a minimum of 100 mL each of DPBS were used per group. The inserts containing the tissue were lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. To assure throughput, the two tissues per group were rinsed simultaneously by holding the replicate inserts together by their collars using forceps. The test item or control articles were decanted from the tissue surface onto a clean absorbent material and the cultures dipped into the first beaker of DPBS, swirled in a circular motion in the liquid for approximately 2 seconds, lifted out so that the inserts were mostly filled with DPBS, and the liquid was decanted back into the container. This process was performed at least two additional times in the first beaker. The culture was then rinsed in the second and third beakers of DPBS at least three times each in the same fashion. Finally, any remaining liquid as decanted onto the absorbent material.

After rinsing, the tissues were immediately transferred in 5 mL of pre-warmed (room temperature) assay medium in a 12-well plate for 25 minutes (± 2 minutes) at room temperature.

Post-Treatment Incubation
After the 25 minutes incubation, each insert was removed from the assay medium, the medium was decanted off the tissue, and the insert were blotted on absorbent material and transferred in 6-well plates filled with 1 mL of pre-warmed (37°C) assay medium for 18 hours (± 15 minutes) at 37°C and 5% CO2.

MTT-Assay
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.

Evaluation of Results
All formulas for the calculation of the relative viability were given by the supplier of the human in vitro eye model (MatTek In Vitro Life Science Laboratories).
The calculation was performed with the MS-Excel-Sheet “InVitroEyeIrritation_V01”. The mean OD (optical density) of the two negative control tissues as calculated. This value corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability was calculated according to the following formula:

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).


Acceptance Criteria
The results are acceptable if:

The 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

The difference of viability between the two relating tissues of a single chemical is <20% in the same run (for positive and negative control tissues and tissues of single chemicals). This applies also to the killed controls (single chemicals 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:
other: Viability %
Run / experiment:
Run 1 / Experiment 1
Value:
97.8
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 (1.403 and 1.615).
2. The mean relative viability of the positive control is below 50% of the negative control viability (47.0%).
3. The difference of viability between the two relating tissues of a single chemical is <20% (values between 1.1% to 14.0%) 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 1.509 100.0% 
 Positive Control 0.709 47.0%
 Test Item 1.475 97.8%
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:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 492.The objective 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 1.509 (study acceptance criterion: >0.8 and <2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 47.0% (study acceptance criterion: <50%). Thus, the acceptance criteria were met.

Following treatment with the test item, the tissue viability was 97.8% 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

Skin irritation

A study was performed according to GLP and the methods applied are fully compliant with OECD TG 439. 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.

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.

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 94.3% 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).

Under the conditions of the present study, the test item is not considered to possess an irritant potential to skin (reference 7.3.1 -1).

Eye irritation

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 492.The objective 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 1.509 (study acceptance criterion: >0.8 and <2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 47.0% (study acceptance criterion: <50%). Thus, the acceptance criteria were met.

Following treatment with the test item, the tissue viability was 97.8% 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 (reference 7.3.2 -1).

Justification for classification or non-classification

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

The available data for skin and eye irritation are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on this data, the substance is not considered to be classified for skin or eye irritation under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) 2019/521.