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Description of key information

The skin irritation and corrosion potential of the test substance was tested in two in vitro studies (OECD 431, 439). Experimental data (according to OECD 439) indicate an irritation potential to skin. In an in vitro skin corrosion study (OECD 431) a corrosive potential to skin was observed.

According to Annex VII, 8.2, Column 2, a study on eye irritating potential does not need to be conducted, because the substance is considered as a skin Corrosive Cat. 1.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-02-22 to 2016-06-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
2015
Deviations:
no
Qualifier:
according to
Guideline:
other: Council Regulation (EC) No 440/2008, Annex Part B, B.40Bis: “In Vitro Skin Corrosion: Human Skin Model Test”, Official Journal of the European Union No. L142, dated May 31st, 2008.
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
other: INVITTOX Protocol No. 118; “EPISKINTM Skin Corrosivity Test” updated December 2011 / February 2012 (ECVAM Database Service on Alternative Methods to Animal Experimentation).
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
other: reconstructed human epidermis
Cell type:
non-transformed keratinocytes
Justification for test system used:
The EPISKIN model has been validated for corrosivity testing in an international trial; it is considered to be suitable for this study (STATEMENT ON THE SCIENTIFIC VALIDITY OF THE EPISKINTM TEST (AN IN VITRO TEST FOR SKIN CORROSIVITY); ECVAM JRC Environment Institute, European Commission; Ispra; 03 April 1998).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTMSM, EPISKIN SNC Lyon, France
- Supplier: SKINETHIC Laboratories; 4, rue Alexander Fleming, 69007 – LYON, France
- Tissue batch number: first experiment: 16-EKIN-013; second experiment: 16-EKIN-018
- Expiry date: 04 April 2016 / 09 May 2016
- Date of initiating testing: 31 March 2016 / 4 May 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: After the incubation time, the units were removed and rinsed thoroughly with approximately 25 mL PBS 1 x solution to remove all of the test material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with a suitable pipette tip linked to a vacuum source (care was taken to avoid damaging to the epidermis).
- Observable damage in the tissue due to washing: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 mL of 0.3 mg/mL MTT
- Incubation time: 3 h
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues
- Procedure used to prepare the killed tissues: Water- killed epidermis preparation: Place the living epidermis in a 12 well plate with 2 mL of distilled water (replacing the culture medium). Incubate at 37 °C, 5% CO2, ≥95% humidified atmosphere for 48 hrs +/- 1 hour. At the end of the incubation, discard the water. Keep dead epidermis frozen (dry) in freezer at -18°C to -20°C (killed epidermis can be stored and used up to 6 months). Before use, the killed tissues are de-frozen at room temperature (app. 1 hour in 2 mL of maintenance medium). Further use of killed tissues is similar to living tissues.
- N. of replicates: 2
- Method of calculation used: Non specific MTT reduction calculation (NSMTT), according to guidelines.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2

PREDICTION MODEL / DECISION CRITERIA:
- The test substance is considered to be corrosive to skin (Cat. 1A) if the viability after 3 minutes exposure is less than 35%.
- The test substance is considered to be corrosive to skin (Cat 1B and 1C) if the viability after 3 minutes exposure is greater than or equal to 35% AND after 60 minutes exposure smaller than 35%; or if the viability after 60 minutes exposure is greater than or equal to 35% AND after 240 minutes exposure smaller than 35%.
- The test substance is considered to be non-corrosive to skin if the viability after 4 hours exposure is greater than or equal to 35%.
- Justification for the selection of the cut-off point: The cut-off value of 35 % and classification method was validated in an international validation of this kit (Fentem, 1998). The prediction model corresponds to the methods agreed by EU regulatory agencies in line with OECD 431 (OECD, 2015).
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 50 µL

NEGATIVE CONTROL
- Amount applied: 50 µL
- Concentration: 9 g/L

POSITIVE CONTROL
- Amount applied: 50 µL
Duration of treatment / exposure:
first experiment: 4 hours
second experiment: 4 hours, 1 hour, 3 minutes
Number of replicates:
2
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 (exposure: 4 h)
Value:
5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2 (exposure: 4 h)
Value:
5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2 (exposure: 1 h)
Value:
37
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2 (exposure: 3 min)
Value:
96
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: yes. During the check-method for possible direct MTT reduction, colour change was observed after three hours of incubation. The test item interacted with the MTT, therefore additional controls and data calculations were necessary. The non-specific MTT reduction (NSMTT) was determined to be 11.344 %. in first experiment and was determined to be 22.499 % at 4 hours exposure, 45.701 % at 1 hour exposure and 6.422 % at 3 minutes exposure in the additional experiment. As the NSMTT were below 50 % the true MTT metabolic conversion in all occasions and the correction of viability percentages were undertaken.
- Colour interference with MTT: no. The test item showed no ability to become coloured in contact with water therefore additional controls and data calculations were not necessary. A false estimation of viability can be excluded.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD value of the two negative control tissues was in the first experiment: 0.891 and in the second experiment: 1.455 at 4 hours exposure, 1.406 at 1 hour exposure and 1.446 at 3 minutes exposure.
- Acceptance criteria met for positive control: The positive control result showed in the first experiment 0 % viability, in the second experiment 2 % viability.
- Acceptance criteria met for variability between replicate measurements: first experiment: The difference of viability between the two tissue replicates: 0% to 4%; second experiment: The difference of viability between the two tissue replicates: 0% to 14%.
Conclusions:
In this in vitro skin corrosion test using the EPISKIN model with the test item the results indicate that the test item is corrosive to skin after 4 hours exposure and not corrosive after 1 hour and 3 min exposure. According to the UN GHS classification systems, the test item has been categorized as “Corrosive: Optional Sub- categories 1B and 1C”.
Executive summary:

The purpose of this study was to determine the skin corrosion potential of the test item on reconstituted human epidermis in the EPISKIN model in vitro. According to the results of the first experiment the test item was corrosive after 4 hours exposition, so the additional experiment was necessary with more exposure times (testing at 3 minutes and 1 hour). In this case, the 4 hour procedure was repeated during the additional experiment, but with an additional 3 minute and 1 hour exposure period. Disks of EPISKIN (two units) were treated with test item and incubated for 4 hours at room temperature on March 31, 2016 (first experiment). Furthermore, disks of EPISKIN (two units / exposure time) were treated with test item and incubated for 4 hours, 1 hour and 3 min at room temperature on May 04, 2016 (additional experiment). Exposure of test material was terminated by rinsing with PBS 1x solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in 5% CO2 protected from light. The formazan precipitated was then extracted using acidified isopropanol and quantified spectrophotometrically. The test item is a possible MTT-reducer, therefore additional controls (test item treated killed tissues and negative control treated killed tissues) were used to detect and correct for test substance interference with the viability measurement. NaCl (9 g/L saline) and glacial acetic acid treated epidermis were used as negative and positive controls respectively. For each treated tissue, viability was expressed as a % relative to negative control. The test item is considered to be non-corrosive to skin, if the mean relative viability after 4 hours of exposure is above or equal 35 % of the negative control. The test item showed significantly reduced cell viability (below 35 %) in comparison to the negative control after 4 hours of exposure in both experiments. In the first experiment (March 31, 2016) the average test item treated tissue viability (corrected value) was 5% and in the additional experiment (May 04, 2016) it was 5% at 4 hours of exposure. In the additional experiment the test item treated tissue viabilities (corrected value) were above 35 % of the mean negative control value after 1 hour and 3 min of exposure. The average test item treated tissue viabilities (corrected value) were 37 % at 1 hour and 96 % at 3 minutes of exposure. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-12-10 to 2016-02-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2012
Deviations:
no
Qualifier:
according to
Guideline:
other: EpiSkin™ SOP, Version 1.8 (February 2009), ECVAM Skin Irritation Validation Study: Validation of the EpiSkin™ test method 15 min - 42 hours for the prediction of acute skin irritation of chemicals.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
other: reconstructed human epidermis
Cell type:
non-transformed keratinocytes
Justification for test system used:
The EPISKIN model has been validated for irritation testing in an international trial. After a review of scientific reports and peer reviewed publications on the EPISKIN method, it showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguishing between skin irritating and no skin irritating test substances (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTMSM, EPISKIN SNC Lyon, France
- Tissue batch number: 15-EKIN-050
- Expiry date: 21 December 2015
- Date of initiation of testing: 16 December 2015

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After the incubation time, the EpiSkinTMSM units were removed and rinsed thoroughly with approximately 25 mL PBS 1 x solution to remove all of the test material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with a suitable pipette tip linked to a vacuum source (care was taken to avoid damaging to the epidermis).
- Observable damage in the tissue due to washing: None

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 mL of 0.3 mg/mL MTT per well
- Incubation time: 3 hours
- Spectrophotometer: Thermo Scientific; Multiscan FC
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test item showed no direct interaction with MTT. Using of additional control was not necessary.

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the viability after 15 minutes exposure and 42 hours post incubation is less than or equal to 50% of the negative control.
- The test substance is considered to be non-irritant to skin if the viability after 15 minutes exposure and 42 hours post incubation is greater than 50% of the negative control.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 10 µL

NEGATIVE CONTROL
- Amount applied: 10 µL
- Concentration: 1 x PBS

POSITIVE CONTROL
- Amount applied: 10 µL
- Concentration: 5% aq. Solution
Duration of treatment / exposure:
15 min
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Remarks:
(mean value, n=3)
Run / experiment:
Test item
Value:
43
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: No colour change was observed after three hours of incubation. The test material did not interact with the MTT, therefore additional controls and data calculations were not necessary. A false estimation of viability can be excluded.
- Colour interference with MTT: The test item showed no ability to become coloured in contact with water therefore additional controls and data calculations were not necessary. A false estimation of viability can be excluded.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Prior to routine use of the method Toxi-Coop ZRT. demonstrated the technical proficiency in a separate study (study no.: 392.554.2938) using the ten Proficiency Chemicals according to OECD Test Guideline No. 439.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD value of the three negative control tissues was 0.861.
- Acceptance criteria met for positive control: The mean OD value obtained for the positive control was 0.078 and this result corresponds to 9 % viability when compared to the results obtained from the negative controls.
- Acceptance criteria met for variability between replicate measurements: Each calculated standard deviation value (SD) for the % viability was below 18. All validity criteria were within acceptable limits and therefore the study can be considered as valid.
Interpretation of results:
other: Results indicated that the test item is Irritant (UN GHS Category 2) and/or Corrosive (UN GHS Category 1).
Conclusions:
In this in vitro skin irritation test in the EPISKIN model with test item the results indicated that the test item is Irritant (UN GHS Category 2) and/or Corrosive (UN GHS Category 1). However, this test method (OECD 439) cannot resolve between UN GHS Categories 1 and 2, further information on skin corrosion will be required to decide on its final classification.
Executive summary:

The purpose of this study was to determine the skin irritation potential of the test item on reconstituted human epidermis in the EPISKIN model in vitro. Disks of epidermal units (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test material was terminated by rinsing the epidermal units with 1x PBS solution. Epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5 % CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in 5 % CO2 and protected from light. The resulting formazan chrystals were extracted with acidified isopropanol and quantified with the optical densities (OD) recorded spectrophotometrically. SDS 5 % aq. and 1 x PBS treated (three units / positive and negative control) epidermis units were used as positive and negative controls, respectively. For each treated tissue, viability was expressed as a percentage relative to negative control. The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control. The test item showed significantly reduced cell viability (corrected value) in comparison to the negative control (mean value: 43 %). All obtained test item viability results were below 50 % when compared to the viability values obtained from the negative control. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (corrosive)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the study need not be conducted because the available information indicates that the criteria are met for classification as corrosive to the skin or irritating to eyes
Endpoint conclusion
Endpoint conclusion:
no study available

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation in vitro:

The purpose of this study was to determine the skin irritation potential of the test item on reconstituted human epidermis in the EPISKIN model in vitro. Disks of epidermal units (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test material was terminated by rinsing the epidermal units with 1x PBS solution. Epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5 % CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in 5 % CO2 and protected from light. The resulting formazan chrystals were extracted with acidified isopropanol and quantified with the optical densities (OD) recorded spectrophotometrically. SDS 5 % aq. and 1 x PBS treated (three units / positive and negative control) epidermis units were used as positive and negative controls, respectively. For each treated tissue, viability was expressed as a percentage relative to negative control. The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control. The test item showed significantly reduced cell viability (corrected value) in comparison to the negative control (mean value: 43 %). All obtained test item viability results were below 50 % when compared to the viability values obtained from the negative control. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

Skin corrosion in vitro:

The purpose of this study was to determine the skin corrosion potential of the test item on reconstituted human epidermis in the EPISKIN model in vitro. According to the results of the first experiment the test item was corrosive after 4 hours exposition, so the additional experiment was necessary with more exposure times (testing at 3 minutes and 1 hour). In this case, the 4 hour procedure was repeated during the additional experiment, but with an additional 3 minute and 1 hour exposure period. Disks of EPISKIN (two units) were treated with test item and incubated for 4 hours at room temperature on March 31, 2016 (first experiment). Furthermore, disks of EPISKIN (two units / exposure time) were treated with test item and incubated for 4 hours, 1 hour and 3 min at room temperature on May 04, 2016 (additional experiment). Exposure of test material was terminated by rinsing with PBS 1x solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in 5% CO2 protected from light. The formazan precipitated was then extracted using acidified isopropanol and quantified spectrophotometrically. The test item is a possible MTT-reducer, therefore additional controls (test item treated killed tissues and negative control treated killed tissues) were used to detect and correct for test substance interference with the viability measurement. NaCl (9 g/L saline) and glacial acetic acid treated epidermis were used as negative and positive controls respectively. For each treated tissue, viability was expressed as a % relative to negative control. The test item is considered to be non-corrosive to skin, if the mean relative viability after 4 hours of exposure is above or equal 35 % of the negative control. The test item showed significantly reduced cell viability (below 35 %) in comparison to the negative control after 4 hours of exposure in both experiments. In the first experiment (March 31, 2016) the average test item treated tissue viability (corrected value) was 5% and in the additional experiment (May 04, 2016) it was 5% at 4 hours of exposure. In the additional experiment the test item treated tissue viabilities (corrected value) were above 35 % of the mean negative control value after 1 hour and 3 min of exposure. The average test item treated tissue viabilities (corrected value) were 37 % at 1 hour and 96 % at 3 minutes of exposure. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on irritating or corrosive properties, the test item is classified and labelled as skin corrosive Category 1B or 1C (H314: "Causes severe skin burns and eye damage"), as well as eye corrosive Category 1 (H318: "Causes serious eye damage"), according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighth time in Regulation (EU) No 2016/918.