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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Study Initiation date: December 08, 2017 and Study Completion date : March 21, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Dark blue solid particulate/powder

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Reconstructed human epidermis tissues, SkinEthicTM RHE model, were procured from SkinEthic Laboratories, Episkin 4, Rue Alexander Fleming, 69366 Lyon Cedex 07, France; was used in the study (Lot N° 17-RHE-127)
Source strain:
other: Reconstructed human epidermis tissues, SkinEthicTM RHE model
Details on animal used as source of test system:
Not Applicable
Justification for test system used:
This study addresses the human health endpoint skin corrosion. It makes use of reconstructed human epidermis (RHE) (human derived non-transformed epidermal keratinocytes) which closely mimics the histological, morphological, biochemical and physiological properties of the upper parts of the human skin, i.e., the epidermis. Use of reconstructed human epidermis (RHE) is also recommended by OECD and other regulatory authorities. SkinEthicTM RHE model has been validated and is part of OECD validated reference methods (VRMs) and is also a recommended model for conducting in vitro skin corrosion studies. The results of the study are believed to be of value in predicting the potential of inducing skin corrosiveness by the test item in human.

Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Reconstructed human epidermis tissues, SkinEthicTM RHE model
- Tissue batch number(s): Lot N° 17-RHE-127
- Date of initiation of testing: December 12, 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: at room temperature for 3 minute exposure and at 37±1 °C and 5±1% CO2 in a
humidified incubator for 60 minute exposure.
- Temperature of post-treatment incubation (if applicable): Not Applicable

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Treated tissues were rinsed 20 times in a constant soft stream of 1 mL DPBS from the 5-8cm distance from the insert to remove all residual test item from the epidermal surface. Mesh was removed by washing for all the tissues.
- Observable damage in the tissue due to washing: No damage
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT (1.0 mg/mL)
- Incubation time: 180 minutes at 37±1 °C and 5±1% CO2 in a 95% humidified incubator
- Spectrophotometer: SynergyHT Microplate Reader : BioTek
- Wavelength: 570 nm.
- Filter: Monochromator base
- Filter bandwidth: Monochromator base
- Linear OD range of spectrophotometer:

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: O.D 1.1
- Barrier function: 4.5 h
- Morphology: Well differentiated Epidermis consisting of basal, spinous, granular layers and stratum corneum
- Contamination: No
- Reproducibility: Yes

NUMBER OF REPLICATE TISSUES: Three replicates were used per exposure time for test item, positive and negative control.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- killed tissues : two freeze killed tissues were maintained for negative and positive control. Both the control tissues were treated for the exposure period of 60 minutes. For the treatment of freeze killed negative and positive control tissues

- Procedure used to prepare the killed tissues (if applicable): Killed tissues were prepared by incubating the viable tissues at -20 ± 5°C for 48 hours
- N. of replicates : 2
- Method of calculation used: For positive control (exposure period of 60 minutes) and Test item (exposure period of 03 and 60 minutes), NSMTT was < 0% relative to the negative control, hence true MTT metabolic conversion of treated tissue was not determined.

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

PREDICTION MODEL / DECISION CRITERIA
-- The test substance is considered to be non-corrosive to skin if [complete, e.g. the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.]
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439: No deviation from TG 431
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20 mg
- Concentration (if solution): Not applicable

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): 40 μL/0.5 cm2 of sterile distilled water
- Concentration (if solution): N.A

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 40 μL/0.5 cm2
- Concentration (if solution): 8N KOH
Duration of treatment / exposure:
At room temperature for 3 minute exposure and at 37±1 °C and 5±1% CO2 in a
humidified incubator for 60 minute exposure
Duration of post-treatment incubation (if applicable):
Not applicable
Number of replicates:
3

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Percent relative viability in the tissues treated with the test item was 106.5% at 3 minute exposure
Value:
106.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: non-corrosive
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Percent relative viability in the tissues treated with the test item was 109.7% at 60 minute exposure period
Value:
109.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: non-corrosive
Other effects / acceptance of results:
The tissues were exposed to test item and sterile distilled water (negative control) for 3 minutes at room temperature and 60 minutes at 37±1 °C and 5±1% CO2 using three replicates/time point and positive control tissues were exposed for 60 minutes 37±1 °C and 5±1% CO2. Since potassium hydroxide is direct MTT reducer, adapted control with two freeze killed tissues were used and treated with 8N KOH for exposure
of 60 minutes. To evaluate the non-specific OD due to the residual test item staining (OD due to color -unrelated to any mitochondrial activity), adapted control with two live tissues were used for test item and negative control for exposure of 3 minutes and 60 minutes.

Applicant's summary and conclusion

Interpretation of results:
other: non-corrosive
Conclusions:
From the results of this study, it is concluded that test item is non-corrosive in accordance with the United Nations Globally Harmonized System of Classification and Labelling of Chemicals as indicated in OECD Test Guideline 431 under specified conditions of this study.
Executive summary:

This study was performed to evaluate the non-corrosive and corrosive potential of the test substance (test item) using reconstructed human epidermis (RHE) tissue in accordance with the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS).

The tissues were exposed to test item and sterile distilled water (negative control) for 3 minutes at room temperature and 60 minutes at 37±1 °C and 5±1% CO2 using three replicates/time point and positive control tissues were exposed for 60 minutes 37±1 °C and 5±1% CO2. Since potassium hydroxide is direct MTT reducer, adapted control with two freeze killed tissues were used and treated with 8N KOH for exposure of 60 minutes. To evaluate the non-specific OD due to the residual test item staining (OD due to color - unrelated to any mitochondrial activity), adapted control with two live tissues were used for test item and negative control for exposure of 3 minutes and 60 minutes.

Percent relative viability in the tissues treated with the test item was 106.5% at 3 minute exposure period and 109.7% at 60 minute exposure period. Significant reduction in percent cell viability was not observed either 3 minute or 60 minute exposure period in the treated tissues when compared with the concurrent negative control. Differences between the viability of treated tissues was ≤ 3.77 % i.e. %CV. For adapted controls to correct colour interference due to test item, %NSC (non-specific color) was between 0.3 to 0.5% relative to the negative control, hence TOD (True MTT metabolic conversion) and relative viability calculations were not required.

All the OD values (corrected OD) for the negative control replicates were between 2.271 to 2.404, against guideline requirement of ≥ 0.8 and ≤ 3.0 (the acceptance criteria for SkinEthicTM RHE model). Positive control showed 5.31% cell viability, against guideline requirement of <15%, compared to concurrent negative control, which demonstrate the efficiency of the test system, SkinEthicTM RHE model.

 

All criteria for a valid study were met as described in the study plan. From the results of this study, under the specified experimental conditions, test item is concluded to be non-corrosive in in vitroskin corrosion test using reconstructed human epidermis (RHE) tissues.