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Description of key information

Skin irritation: the substance is non-irritant in the EPISKIN-SM™
Eye irritation :the substance is non-irritant in the EpiOcular™ test 

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:
16 December 2020 to 11 January 2021
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:
Official Journal of the European Union No. L142; Amended by EC No. 640/2012 OJ No. L193, 20 July 2012
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
June 2020
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen were cultured for 13 days leading to a highly differentiated and stratified epidermis model
Justification for test system used:
In the interest of sound science and animal welfare, a testing strategy is recommended that minimizes the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small Model™
- Tissue batch number(s): 21EKIN001 (for killed tissues three additional tissue batches were used that were kept in the freezer until use)
- Shipping date: 5 January 2021
- Expiry date: 11 January
- Date of initiation of testing: 4 January 2020 (pre-tests)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 36.7 - 37.3 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: rinsed with phosphate buffered saline , no further details, according to validated SOP
- Observable damage in the tissue due to washing: no data

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL in PBS
- Incubation time: 3 hours at 37 °C
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader.
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA: see attachment

NUMBER OF REPLICATE TISSUES: 3 tissues per treatment assessed twice

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: 3/3 per treatment (3 control tissues fresh/killed)
- Procedure used to prepare the killed tissues (if applicable): Living epidermis was transferred to 12 well plates and incubated with 2 mL Milli-Q for 48 ± 1 hours. After incubation, killed epidermis was stored at ≤ -15°C.
- N. of replicates : 3
- Method of calculation used:
True tissue viability is calculated as the difference between the living test item treated tissues incubated with MTT medium (ODlt_t+MTT) and the difference between killed tissues incubated with MTT (treated and untreated) ODkt_t+MTT and ODkt_u+MTT.
OD= ODlt_t+MTT – (ODkt_t+MTT-ODkt_u+MTT)
A correction for background absorption of 0.0044 was applied

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
≤ 50% of the mean viability of the negative controls --> Category 1 or Category 2 (additional information on corrosion needed)
> 50% of the mean viability of the negative controls --> No category
Control samples:
yes, concurrent no treatment
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied : 10.9 to 15.1 mg (moistened with 5 µL milli-Q water)

NEGATIVE CONTROL
- Amount(s) applied : 25 µL PBS

POSITIVE CONTROL
- Amount(s) applied : 25 µL 5% SDS
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 ± 1 hours at 37°C.
Number of replicates:
3 per treatment
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1, 2 and 3
Value:
109
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
see table
Irritation / corrosion parameter:
other: OD570
Run / experiment:
1
Value:
1.131
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
other: OD570
Run / experiment:
2
Value:
1.062
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
other: OD570
Run / experiment:
3
Value:
1.044
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Mean Absorption in the In Vitro Skin Irritation Test with Alkyl Ketene Dimer










































 



A


(OD570)



B


(OD570)



C


(OD570)



Mean


(OD570)



 



SD



Negative control



0.969



1.010



0.994



0.991



±



0.020



Test item(1)



1.131



1.062



1.044



1.079



±



0.046



Positive control



0.030



0.046



0.053



0.043



±



0.012



OD = optical density


SD = Standard deviation


(1) The test item values are corrected for the non-specific MTT reaction.


 


Mean Tissue Viability in the In Vitro Skin Irritation Test with Alkyl Ketene Dimer


























 



Mean tissue viability (percentage of control)



Standard deviation (percentage)



Negative control



100



2.1



Test item



109



4.6



Positive control



4.3



1.2



 


 

Interpretation of results:
GHS criteria not met
Conclusions:
In the Episkin SM test the substance was concluded non-irritating to the skin
Executive summary:

In a test according to OECD 439 the substance was tested for possible skin irritation through topical application for 15 minutes on a human three dimensional epidermal model (EPISKIN Small model (EPISKIN-SM).
Skin tissue was moistened with 5 µL of Milli-Q water and at least 10 mg of the test item was applied directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 ± 1 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.


The test item did interact with MTT. In addition to the normal procedure, three killed tissues treated with test item and three killed untreated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by the test item was 1.6% of the negative control tissues. The net OD of the treated killed tissues was subtracted from the ODs of the test item treated viable tissues.


Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 109%. Since the mean relative tissue viability for the test item was above 50% after 15 ± 0.5 minutes treatment the test item is considered to be non-irritant.


The positive control had a mean cell viability of 4.3% after 15 ± 0.5 minutes exposure. The absolute mean OD570  of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was ≤ 4.6%, indicating that the test system functioned properly.


In conclusion,the substance is non-irritant in the in vitro skin irritation test under the experimental conditions described.

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:
08-January 2021 to 28 January 2021
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:
2019
GLP compliance:
yes (incl. QA statement)
Species:
human
Details on test animals or tissues and environmental conditions:
The EpiOcular tissue construct is a nonkeratinized epithelium prepared from normal human keratinocytes. It models the cornea epithelium with progressively stratified, but not cornified cells (details see attachment).

In the interest of sound science and animal welfare, a testing strategy is recommended minimizing the need of in vivo testing. One of the validated in vitro eye irritation tests is the Reconstructed Human EpiOcular™ Model., which is recommended in international guidelines
Controls:
yes, concurrent positive control
other: negative control treated with 50 µL Milli-Q water
Amount / concentration applied:
50.9 to 54.2 mg
Duration of treatment / exposure:
6 hours ± 15 minutes at 37.0 ± 1.0°C
Duration of post- treatment incubation (in vitro):
18 hours ± 15 minutes at 37°C
Number of animals or in vitro replicates:
2 replicates per treatment (Batch : OCL-200-EIT MatTek Corporation, Lot: 31771, Kit M); 2 freeze-killed tissues were treated with substance and one freeze-killed non treated tissue (tissues from from an additional EpiOcular batch that was stored in the freezer)
Details on study design:
Before the assay was started the entire tissues were pre-wetted with 20 μL of Ca2+Mg2+-Free-DPBS.
The substance was added into the 6-well plates on top of the tissues. After the exposure period with the test item (6 hours ± 15 minutes at 37.0 ± 1.0°C), the tissues were thoroughly rinsed with Ca2+Mg2+-free D-PBS (brought to room temperature) to remove residual test item. Due to the physical properties of the test item, the test item could not be removed completely. After rinsing the cell culture inserts were each dried carefully and immediately transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a pre-labeled 12-well plate for a 25 ± 2 minute immersion incubation at room temperature (Post-Soak). After the Post-Soak period cell culture inserts were each dried carefully and transferred to the 6-well plate containing 1.0 mL of warm Assay Medium and were incubated for 18 hours ± 15 minutes at 37°C.
After incubation, cell culture inserts were dried carefully to remove excess medium. The cell culture inserts were transferred into a 24-wells plate prefilled with 0.3 mL MTT-medium
(1.0 mg/mL). The tissues were incubated for 180 ± 10 minutes at 37°C.
After incubation with MTT-medium the tissues were placed on blotting paper to dry the tissues and then transferred to a pre-labeled 6-well plate containing 2 mL isopropanol in each well so that no isopropanol is flowing into the insert. Formazan was extracted with 2 mL isopropanol for 2 - 3 hours at room temperature with gentle shaking.
The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
Irritation parameter:
mean percent tissue viability 
Run / experiment:
mean
Value:
78
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Mean Absorption in the EpiOcular™ Test with Alkyl Ketene Dimer






































 



A


(OD570)



B


(OD570)



Mean


(OD570)



 



SD



Negative control



1.720



1.779



1.749



±



0.042



Test item



1.326



1.419



1.372



±



0.066



Positive control



0.373



0.307



0.340



±



0.047



OD = optical density


SD = Standard deviation


Duplicate exposures are indicated by A and B.


In this table the values are corrected for background absorption (0.043). Isopropanol was used to measure the background absorption.


Mean Tissue Viability in the EpiOcular™ Test with Alkyl Ketene Dimer


























 



Mean tissue viability (percentage of control)



Difference between two tissues
 (percentage)



Negative control



100



3.4



Test item



78



3.8



Positive control



19



5.3



 


 

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the results of the in vitro EpiOcular™ test , the substance is considered non-irritant
Executive summary:

In a test according to OECD 492, the substance (50.9 to 54.2 mg) was applied directly on top of the tissue for 6 hours ± 15 minutes.


After exposure the cornea epithelial construct was thoroughly rinsed to remove the test item and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 18 hours at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect.


The positive control had a mean cell viability of 19% after 6 hours ± 15 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The difference between the percentage of viability of two tissues treated identically was less than 6%, indicating that the test system functioned properly.


The test item did interact with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). In addition to the normal procedure, two freeze-killed tissues treated with test item and one freeze-killed negative control treated tissue were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by the test item was 0.003% of the negative control tissues. The ODs of the test item treated viable tissues was corrected using he OD of the freeze-killed tissues.


Eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 6 hours ± 15 minutes treatment with the test item compared to the negative control tissues was 78%. Since the mean relative tissue viability for the test item was above 60% after 6 hours ± 15 minutes treatment the test item is considered to be non-irritant.


In conclusion, the substance is non-irritant in the EpiOcular™ test under the experimental conditions described in this report.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation


In a test according to OECD 439 the substance was tested for possible skin irritation through topical application for 15 minutes on a human three dimensional epidermal model (EPISKIN Small model (EPISKIN-SM™).
Skin tissue was moistened with 5 µL of Milli-Q water and at least 10 mg of the test item was applied directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 ± 1 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.


The test item did interact with MTT. In addition to the normal procedure, three killed tissues treated with test item and three killed untreated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by the test item was 1.6% of the negative control tissues. The net OD of the treated killed tissues was subtracted from the ODs of the test item treated viable tissues.


Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 109%. Since the mean relative tissue viability for the test item was above 50% after 15 ± 0.5 minutes treatment the test item is considered to be non-irritant.


The positive control had a mean cell viability of 4.3% after 15 ± 0.5 minutes exposure. The absolute mean OD570  of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was ≤ 4.6%, indicating that the test system functioned properly.


In conclusion,the substance is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.


Eye irritation


In a test according to OECD 492, the substance (50.9 to 54.2 mg) was applied directly on top of the tissue for 6 hours ± 15 minutes.


After exposure the cornea epithelial construct was thoroughly rinsed to remove the test item and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 18 hours at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect.


The positive control had a mean cell viability of 19% after 6 hours ± 15 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The difference between the percentage of viability of two tissues treated identically was less than 6%, indicating that the test system functioned properly.


The test item did interact with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). In addition to the normal procedure, two freeze-killed tissues treated with test item and one freeze-killed negative control treated tissue were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by the test item was 0.003% of the negative control tissues. The ODs of the test item treated viable tissues was corrected using he OD of the freeze-killed tissues.


Eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 6 hours ± 15 minutes treatment with the test item compared to the negative control tissues was 78%. Since the mean relative tissue viability for the test item was above 60% after 6 hours ± 15 minutes treatment the test item is considered to be non-irritant.


In conclusion, the substance is non-irritant in the EpiOcular™ test under the experimental conditions described in this report.

Justification for classification or non-classification

Based on the results of the available studies the substance should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations or according to EU regulation 1272/2008 (CLP).

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