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Description of key information

The test material was investigated for skin and eye irritation in in vitro methods. The GLP compliant studies were fully compliant with OECD TG 439, 437, and 492.

The follwing results have been obtained:

Skin irritation in vitro: OECD 439: negative

Eye irritation in viitro: OECD 437: inconclusive; OECD 492: negative

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:
Nov 14 - Dec 11, 2018
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:
July 26, 2013
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Council Regulation (EC) No. 440/2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006. B.40.bis. In vitro skin corrosion: human skin model test
Deviations:
no
GLP compliance:
yes (incl. QA statement)
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-135
- Expires: Nov 26, 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
At the end of the exposure periods, the test item, positive and negative control was removed immediately by gently rinsing with a minimum volume of 20 mL DPBS using a pipette. Excess DPBS was removed by gently shaking the tissue inserts and blotting the bottom of the tissue inserts with blotting paper.

Control samples:
yes, concurrent negative control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 mg ± 2 mg of solid test material
- Concentration (if solution): n/a

VEHICLE
- Amount(s) applied (volume or weight with unit): n/a
- Concentration (if solution): n/a
- Lot/batch no. (if required): n/a
- Purity: n/a

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL ± 0.5 µl (deionised water )
- Concentration (if solution): n/a

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL ± 0.5 µl
- Concentration (if solution): A dodecyl sulfate sodium salt solution dissolved deionised water pure was used as positive control.
Duration of treatment / exposure:
42 min
Number of replicates:
3
Controls:
yes, concurrent negative control
Amount / concentration applied:
16 mg
Duration of treatment / exposure:
42 min
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean relative viability in %
Value:
91.3
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:
The negative control OD values were 1.749, 1.681 and 1.853 and, thus, in the range of ≥0.8 and ≤3.0

-Acceptability of the Positive and Negative Control:
After treatment with the negative control (DPBS-buffer) the mean OD was 1.761 (standard deviation: 4.9%) and, thus, higher than the historically established boundary of 1.424.
After treatment with the positive control (5% aqueous solution of sodium dodecyl sulfate) the mean viability value was 1.30% (standard deviation: 7.7%) and, thus, lower than the historically established boundary of 2.82%.
The standard deviation of the negative control and the positive control was ≤18%, respectively.


-Test Substance Data Acceptance Criteria:
The standard deviation of the three tissues treated with the test item was 6.7% and, thus, ≤18%.
Therefore, the study fulfilled the validity criteria.




 Group Time / [min]  Mean OD  Mean Relative viability / [%]
 Negative Control 42 1.761 100.00 

 Positive Control

42

0.022

1.30
 Test Material 42 1.608 91.30
Interpretation of results:
GHS criteria not met
Remarks:
Non-corrosive
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 be irritant to skin.
Executive summary:

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 be irritant to skin.

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

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Nov 01 - Dec 11, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
2017
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
Commission Regulation (EU) 2017/735 of 14 February 2017 amending, for the purpose of its adaption to technical progress, the Annex to Regulation (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
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
PREPARATION OF THE TEST MATERIAL
The test item was prepared as a 20% (w/v) solution in a 0.9% sodium chloride solution. The stability in the vehicle was not investigated. The test item preparation was administered within <1 hour after preparation.
Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL: 750 µL (i.e. 150mg/750µL)

NEGATIVE / VEHICLE CONTROL: 750 µL

Designation: 0.9% sodium chloride solution
Supplier: B. Braun Melsungen AG, Germany
Batch: 17361013
Storage: 2 to 8°C
Released until: August 2020


POSITIVE CONTROL: 750 µL

Designation: Art. 814223
Synonym: Imidazole
Supplier: Merck KGaA, Germany
Batch: S5798623
Purity (GC) 99.8% (a/a)
Storage: At room temperature
Released until: April 30, 2023

Imidazole was dissolved with 0.9% sodium chloride solution to a concentration of 20% (w/v).

Duration of treatment / exposure:
240 minutes
Number of animals or in vitro replicates:
in vitro: triplicate design
Irritation parameter:
in vitro irritation score
Run / experiment:
Run 1 / Experiment 1
Value:
5.8
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
Remarks:
According to OECD 437 no prediction can be made regarding the eye hazard potential of the test item.
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was 0.1 and, thus, within three standard deviations of the current historical mean of the negative control (IVIS: -1.4 – 3.1).
- Acceptance criteria met for positive control: After treatment with the positive control (20% Imidazole) the calculated IVIS was 117.0 and, thus, also within two standard deviations of the current historical mean of the positive control (IVIS: 82.3 – 132.4).

Therefore, the study fulfilled the acceptance criteria.

Results

The IVIS obtained after treatment with the test material was 5.8 and, thus higher than 3 and lower than 55, i.e. according to OECD 437 no prediction can be made regarding the eye hazard potential of the test item.

The results are given in the following table:

 

Opacity

Permeability

IVIS

per cornea

per group (mean value)

Standard deviation

Negative
control

0.9% sodium chloride solution

0.0

-0.001

-0.015

0.1

0.4

0.6

-0.003

0.555

-0.1

-0.002

-0.130

Positive
control

Imidazole
(20%)

68.44

1.928

97.320

117.0

17.1

86.2

2.630

125.650

70.4

3.846

128.090

Test item

Test material preparation

4.6

-0.001

4.585

5.8

4.4

2.1

-0.002

2.070

10.7

-0.001

10.685


No observations (e.g. tissue peeling, residual test chemical, non-uniform opacity patterns) were seen in a visually inspection of the corneas after treatment.

Study Acceptance Criteria


After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was 0.1 and, thus, within three standard deviations of the current historical mean of the negative control (IVIS: -1.4 – 3.1). After treatment with the positive control (20% Imidazole) the calculated IVIS was 117.0 and, thus, also within two standard deviations of the current historical mean of the positive control (IVIS: 82.3 – 132.4). Therefore, the study fulfilled the acceptance criteria.
The resulting classification of the test item in this study is unequivocal and no borderline results were obtained. Therefore, a single testing run composed of three corneas per group was considered sufficient.


Interpretation of results:
study cannot be used for classification
Conclusions:
Under the conditions of the present study, the eye hazard potential of the test item cannot be predicted.
Executive summary:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 437. Under the conditions of the present study, the eye hazard potential of the test item cannot be predicted.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Dec 05, 2018 - Feb 25, 2019
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:
October 9, 2017
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: Art. 8.09711
Synonym: Methyl acetate
Supplier: Merck KGaA
CAS No.: 79-20-9
Batch No.: S7451611
Appearance: Liquid
Assay (GC, area%): 99.6 % (a/a)
Minimum shelf life: June 30, 2022
Storage conditions: Tightly closed, dark, at room temperature (15 – 25°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
Irritation parameter:
other: Viability %
Run / experiment:
Run 1 / Experiment 1
Value:
106.3
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 (2.187 and 1.840).
2. The mean relative viability of the positive control is below 50% of the negative control viability (29.7%).
3. The difference of viability between the two relating tissues of a single chemical is <20% (values between 1.6% to 17.2%) 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 2.014 100.0% 
 Positive Control 0.599 29.7%
 Test Item 2.140 106.3%
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 identified as not requiring classification and labeling according to UN GHS (No Category).
Executive summary:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 492. Under the conditions of the present study, the test item did not show an eye hazard potential. The test item  is identified as not requiring classification and labeling according to 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

Justification for classification or non-classification

The information provided shows that the test material does not need to classified for skin or eye irritation according to the EU Regulation (EC) No 1272/2008 on Classification, Labelling and Packaging of Substances and Mixtures.