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Description of key information

Eye irritation/corrosion: Non-irritant, EpiOcular Human Tissue model; pre-validated ocular Irritation Assay for Irritant/Non-Irritant classification using the EpiOcular Human Tissue Model guideline, Lehmeier 2012
Skin irritation/corrosion: Non-irritant, OECD 439, Commission Regulation (EC) No. 761/2009, Leimeier 2012

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:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to internationally recognised guideline; under GLP conditions.
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
In Vitro Skin Irritation: Reconstructed human Epidermis Test Method, 22 July 2010
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No. 761/2009, L 220, Annex III Part B.46. “ In vitro Skin Irritation: Reconstructed Human Epidermis Model Test”. 23-Jul-2009
Deviations:
no
Qualifier:
according to
Guideline:
other: L’Oréal Standard Operating Procedure: “EpiSkin Skin Irritation Test Method“; -ECVAM Skin Irritation Validation Study - Validation of the EpiSkin Test Method for the Prediction of Acute Skin 15 min - 42 hours Irritation of Chemicals. Version 1.8, Feb-2009
Deviations:
no
Qualifier:
according to
Guideline:
other: ECVAM Performance Standards for in vitro Skin Irritation Test Methods based on Reconstructed human Epidermis (RhE). Updated Version 24-Aug-2009
Deviations:
no
Qualifier:
according to
Guideline:
other: ISO/FDIS 10993-10, Annex D: 2009(E), “Biological evaluation of medical devices - Part 10: Tests for irritation and skin sensitization”
Deviations:
no
GLP compliance:
yes
Species:
other: in vitro: EPISKIN Standard model
Strain:
other: n/a
Details on test animals and environmental conditions:
This test uses the EPISKIN-SM recontructed human epidermis model (SKinEthic) which consists of human keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closey mimics the biochemical and physiological properties of the upper parts of the human skin i.e. the epidermis.
Type of coverage:
open
Preparation of test site:
other: No preparation of the test site.
Vehicle:
water
Controls:
other: additional tissues in triplicate were available for the positive and negative control groups.
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg (with 5ul distilled water)

VEHICLE
- name: distilled water
- - Amount(s) applied for moistening (volume or weight with unit): 5 ul

NEGATIVE CONTROL
- name: Phosphate buffered saline (PBS)
- Amount(s) applied (volume or weight with unit): 10 ul

POSITIVE CONTROL
- name: sodium dodecyl sulfate (SDS)
- Amount(s) applied (volume or weight with unit): 10 ul of 5% SDS solution
Duration of treatment / exposure:
15 minutes
Details on study design:
Pre-test: to check the MTT reducing ability of the test material, 10mg of the test material was mixed with 2 ml of MTT medium and incubated for 3 hours at 37 degrees C in the dark.

Main test: The test was performed with triplicate tissues for the test substance, positive and control groups. The test material was applied to directly on top of the skin tissue. After the exposure period of 15 minutes at room temperature, the tissues were washed with PBS to remove residual test substance. The skin tissues were transferred to new well plates containing maintainance medium incubated at 37 +/-1 degrees C, 5% CO2 for 42 hours. After the 42 hour incubation period, determination of cytotoxic effect was performed. Tissues were transferred into an MTT assay plate (containing prewarmed MTT solution) and incubated for 3 hours at 37 +/- 1 degrees C, 5% CO2. After incubation, a biopsy of each tissue were transferred to tube containing isopropanol and kept over the weekend in the dark at 2-8 degrees C. The OD of the extracts were measured using a spectrophotometer.

Pre-test: The test material did not cause the MTT to change colour to blue/purple indicating that the test material does not have MTT-reducing capability.

Results of the main test are shown in the following table:

Name Negative Control Positive Control Test Material
1 2 3 1 2 3 1 2 3
Absolute OD 550nm 1.325 1.338 1.225 0.144 0.135 0.138 1.267 1.398 1.161
1.260 1.311 1.196 0.143 0.131 0.144 1.233 1.379 1.111
Blank-corrected OD 550nm 1.282 1.295 1.182 0.102 0.092 0.095 1.224 1.355 1.118
1.217 1.268 1.153 0.100 0.089 0.101 1.190 1.336 1.068
Mean OD 550nm (blank corrected) 1.250 1.282 1.168 0.101 0.090 0.098 1.207 1.346 1.093
Total mean OD 550nm of 3 replicate tissues (blank-corrected) 1.233 0.096 1.215
SD OD 550nm 0.06 0.01 0.11
Relative tissue viabilities (%) 101.4 103.9 94.7 8.2 7.3 8.0 97.9 109.1 88.7
Mean tissue viability (%) 100 7.8 98.6
SD tissue viability (%) 4.8 0.4 10.3
Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study, the test material showed no irritant effects producing a mean relative tissue viability of 98.6%. Therefore the test material is considered to be a non-irritant. The positive and negative control groups achieved the expected result and confirm the validity of this study.
Executive summary:

This study protocol is based on OECD 439 guideline; Commission Regulation (EC) No. 761/2009, L 220, Annex III Part B.46; L’Oréal Standard Operating Procedure: “EpiSkin Skin Irritation Test Method“; -ECVAM Skin Irritation Validation Study - Validation of the EpiSkin Test Method for the Prediction of Acute Skin 15 min - 42 hours Irritation of Chemicals. Version 1.8; ECVAM Performance Standards for in vitro Skin Irritation Test Methods based on Reconstructed human Epidermis (RhE); ISO/FDIS 10993-10, Annex D: 2009(E); and was performed under GLP conditions. EPISKIN tissue were exposed to 10 mg of test material (applied directly to the tissue with 5ul distilled water) for 15 minutes. After exposure tissues were rinsed and further incubated for a 42 hours at 37 degrees C. After this incubation period, determination of the cytotoxic effect was performed by transferring tissues to plates containing MTT medium and incubated for 3 hours. Tissue biopsies were taken and extraction of contents were performed by placing tissues in isopropanol. The OD of the extracts were measured using a spectrophotometer to determine cytotoxicity. Under the conditions of this study, the test material showed no irritant effects producing a mean relative tissue viability of 98.6%. The test material is considered to be a non-irritant. The positive and negative control groups achieved the expected result and confirm the validity of this study.

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:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to internationally recognised guideline; under GLP conditions.
Qualifier:
according to
Guideline:
other: Ocular Irritation Assay for Irritant/Non-Irritant classification using the EpiOcular Human Tissue Model.
Deviations:
no
Principles of method if other than guideline:
This method has been designed to predict and classify the occular irritation potential of a chemical by assessment on its effect on EpiOcular, a non-ocular human epithelial cell model. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after test material exposure. The EpiOcular Eye Irritation Test (EIT) protocol has been pre-validated by COLIPA and is currently under validation by ECVAM (validation still ongoing).
GLP compliance:
yes
Species:
other: in vitro: EpiOcular human tissue model
Strain:
other: n/a
Details on test animals or tissues and environmental conditions:
The test was performed with the EpiOcular corneal model (MatTek). The model consists of normal, human-derived epidermal keratinocytes that have been cultured to form a stratified, squamous epithelium similar to that found in the cornea. The epidermal cells, which are cultured on specially prepared cell culture inserts using serum free medium differentiate to form a multi-layered structure which closely parallels the corneal epithelium.
Vehicle:
unchanged (no vehicle)
Controls:
other: additional tissue was available for negative and positive control groups.
Amount / concentration applied:
Test Material:
50 mg of test material was applied directly to the surface of the EpiOcular tissue.

Positive control:
50 ul methyl acetate.

Negative control:
50 ul of distilled water.
Duration of treatment / exposure:
90 minutes
Details on study design:
Pre-test: to check the MTT reducing ability of the test material, 50mg of the test material was mixed with 500 ul of MTT medium and incubated for 2 hours at room temperature.

Main test: The test was performed on each dose group in duplicate, together with negative and positive controls. After dosing, the tissues were incubated for 90 +/- 5 minutes at 37 +/- 1 degrees C, 5% CO2 / 95% air. After the exposure period, tissues were rinsed with DPBS to remove test material. Tissues were then transferred to an post-soak plate (with prewarmed assay medium) and incubated for 12 minutes at room temperature. These tissues were further transferred to a post treatment plate (containing prewarmed assay medium) and incubated for 18 +/- 0.25 h at 37 +/- 1 degrees C, 5% CO2 / 95% air. After this incubation period, determination of the cytotoxic effect was performed. Tissues were transferred into an MTT assay plate (containing prewarmed MTT solution) and incubated for 180 +/- 10 minutes at 37 +/- 1 degrees C, 5% CO2 / 95% air. After incubation, tissues were transferred to 'extraction plates' containing 2ml of isopropanol and kept overnight (in sealed bags to reduce evaporation of isopropanol) in the dark at 2-8 degrees C. Tissue were then discarded and the OD of the extracts were measured using a spectrophotometer.

Pre-test: The test material did not cause the MTT to change colour to blue/purple indicating that the test material does not have MTT-reducing capability.

Results of the main test are shown in the table below:

Name Negative Control Positive Control Test Material
Replicate 1 Replicate 2 Replicate 1 Replicate 2 Replicate 1 Replicate 2
Mean OD 550nm (blank corrected) 0.997 1.173 0.015 0.013 0.738 0.754
Total mean OD 550nm of 2 replicate tissues (blank corrected) 1.085 0.014 0.746
SD OD 550nm 0.1 0 0.01
Relative tissue viability (%) 91.9 108.1 1.4 1.2 68 69.5
SD tissue viability (%) 11.5 0.1 1.1
Mean tissue viability (%) 100 1.3 68.8
Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: expert judgment
Conclusions:
Under the conditions of this study, the test material showed no irritant effects producing a mean relative tissue viability of 68.8%. Therefore the test material is considered to be a non-irritant. The positive and negative control groups achieved the expected result and confirm the validity of this study.
Executive summary:

This study protocol is based on pre-validated ocular Irritation Assay for Irritant/Non-Irritant classification using the EpiOcular Human Tissue Model guideline. EpiOcular tissue were exposed to 50 mg of test material (applied directly to the tissue without vehicle) for 90 minutes. After exposure tissues were rinsed and further incubated for an additional 12 minutes at room temperature then 180 minutes at 37 degrees C. After this incubation period, determination of the cytotoxic effect was performed by transferring tissues to plates containing MTT medium and incubated for 2 hours. Extraction of tissue contents were performed by placing tissues in isopropanol overnight. Tissues were discarded and the OD of the extracts were measured using a spectrophotometer to determine cytotoxicity. Under the conditions of this study, the test material showed no irritant effects producing a mean relative tissue viability of 68.8%. The test material is considered to be a non-irritant. The positive and negative control groups achieved the expected result and confirm the validity of this study.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Eye irritation/corrosion:

This study protocol is based on pre-validated ocular Irritation Assay for Irritant/Non-Irritant classification using the EpiOcular Human Tissue Model guideline. EpiOcular tissue were exposed to 50 mg of test material (applied directly to the tissue without vehicle) for 90 minutes. After exposure tissues were rinsed and further incubated for an additional 12 minutes at room temperature then 180 minutes at 37 degrees C. After this incubation period, determination of the cytotoxic effect was performed by transferring tissues to plates containing MTT medium and incubated for 2 hours. Extraction of tissue contents were performed by placing tissues in isopropanol overnight. Tissues were discarded and the OD of the extracts were measured using a spectrophotometer to determine cytotoxicity. Under the conditions of this study, the test material showed no irritant effects producing a mean relative tissue viability of 68.8%. The test material is considered to be a non-irritant. The positive and negative control groups achieved the expected result and confirm the validity of this study.

Skin irritation/corrosion:

This study protocol is based on OECD 439 guideline; Commission Regulation (EC) No. 761/2009, L 220, Annex III Part B.46; L’Oréal Standard Operating Procedure: “EpiSkin Skin Irritation Test Method“; -ECVAM Skin Irritation Validation Study - Validation of the EpiSkin Test Method for the Prediction of Acute Skin 15 min - 42 hours Irritation of Chemicals. Version 1.8; ECVAM Performance Standards for in vitro Skin Irritation Test Methods based on Reconstructed human Epidermis (RhE); ISO/FDIS 10993-10, Annex D: 2009(E); and was performed under GLP conditions. EPISKIN tissue were exposed to 10 mg of test material (applied directly to the tissue with 5ul distilled water) for 15 minutes. After exposure tissues were rinsed and further incubated for a 42 hours at 37 degrees C. After this incubation period, determination of the cytotoxic effect was performed by transferring tissues to plates containing MTT medium and incubated for 3 hours. Tissue biopsies were taken and extraction of contents were performed by placing tissues in isopropanol. The OD of the extracts were measured using a spectrophotometer to determine cytotoxicity. Under the conditions of this study, the test material showed no irritant effects producing a mean relative tissue viability of 98.6%. The test material is considered to be a non-irritant. The positive and negative control groups achieved the expected result and confirm the validity of this study.


Justification for selection of skin irritation / corrosion endpoint:
One study available; GLP compliant and has a Klimisch score 1.

Justification for selection of eye irritation endpoint:
One study available; GLP compliant and has a Klimisch score 1.

Justification for classification or non-classification

The substance does not meet classification criteria under EU Directive 67/548/EEC for dermal irritation.

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for dermal irritation.

The substance does not meet classification criteria under EU Directive 67/548/EEC for eye irritation.

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for eye irritation.