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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:
06 December 2016 - 13 January 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
Adopted July 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: European Community (EC). Commission regulation (EC) No. 440/2008, Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test"
Version / remarks:
Official Journal of the European Union No. L142, 31 May 2008.
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
white to brownish
Details on test material:
Batch 151222

In vitro test system

Test system:
human skin model
Remarks:
EpiDerm Skin Model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
Recommended test system in international guidelines (OECD and EC).
Vehicle:
unchanged (no vehicle)
Remarks:
The solid test item was applied directly on top of the skin tissue. CH02906 was spread to match the size of the tissue.
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:EpiDerm Skin Model (EPI-200)
- Tissue batch number(s):Lot no.: 24940 kit J and K (first experiment) 24956 kit Q and R (second experiment)
- Production date: /
- Shipping date: 7/12/2016 (first experiment); 11/12/2017 (second experiment)
- Delivery date: /
- Date of initiation of testing: 8/12/2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37.0 ± 1.0°C (actual range 35.1 - 37.0°C)
- Temperature of post-treatment incubation (if applicable): 37.0 ± 1.0°C (actual range 35.1 - 37.0°C)was

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: /
- Observable damage in the tissue due to washing: one of the replicates of the 3-minutes treatment time
- Modifications to validated SOP:

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT concentrate (5 mg/ml) was diluted (1:5) with MTT diluent (supplemented DMEM). Both supplied by MatTek Corporation.
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm
- Filter:
- Filter bandwidth:
- Linear OD range of spectrophotometer:

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD570 = 1.744 +- 0.093 (first experiment) and 1.407 +- 0.066 (second experiment), acceptance criteria: OD570= 1.0-3.0
- Barrier function: ET-50 = 5.21 hours (first experiment) and 8.09 hours (second experiment), acceptance criteria: 4.77-8.72hours
- Morphology:
- Contamination: Sterile based on long term antibiotic and antimycotic free culture
- Reproducibility:

NUMBER OF REPLICATE TISSUES: 4 (2 used after 3 minutes of exposure, 2 after 1 hour of exposure)

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: not needed
- Fresh tissues / killed tissues
- Procedure used to prepare the killed tissues (if applicable):
- N. of replicates :
- Method of calculation used:

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if 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: /
Amount/concentration applied:
At least 25 mg of CH02906 was added to 1 ml MTT (Sigma, Zwijndrecht, The Netherlands) solution (1 mg/ml) in phosphate buffered saline.
Duration of treatment / exposure:
3 minutes of exposure
1 hour of exposure
Number of replicates:
4

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
experiment 1: 3-minute exposure
Value:
ca. 72
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: results replicates different
Remarks:
one replicate was found to be corrosive (cell viability 45%), the other replicate non-corrosive (100% cell viability). The variation was most probably caused by problems during rinsing: the test item could not be fully removed from one replicate. Therefore, a second experiment was conducted.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
experiment 1: 1-hour exposure
Value:
ca. 83
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
experiment 2: 3-minutes exposure
Value:
ca. 104
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
experiment 2: 1-hour exposure
Value:
ca. 81
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: High variability
Remarks:
Although the variability between the two replicates was high (54%, cell viability of replicate 1 and 2 were 51% and 112%, respectively), both replicates were found to be non-corrosive.
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system:/
- Direct-MTT reduction:no direct MTT-reduction
- Colour interference with MTT: no colour interference

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes (0.8- Acceptance criteria met for positive control: yes (mean tissue viability <15% after 1 hour of exposure: 14% experiment 1, 7% experiment 2)
- Acceptance criteria met for variability between replicate measurements: yes for the 1-hour exposure in the first experiment (8.8%) and for the 3-minute exposure in the second experiment (19%)
- Range of historical values if different from the ones specified in the test guideline:

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
It is concluded that this test was valid and that CH02906 was not corrosive in the in vitro skin corrosion test under the experimental conditions described.
Executive summary:

In vitroskin corrosion test with CH02906 using a human skin model.

This report describes the ability of CH02906 to induce skin corrosion on a human three- dimensional epidermal model (EpiDerm (EPI-200)). The possible corrosive potential of CH02906 was tested through topical application for 3 minutes and 1 hour.

The study procedures described in this report were based on the most recent OECD431 and EC B.40 BIS guidelines.

Batch 151222 of CH02906 was a white to brownish powder with a purity of 99.9%. Skin tissue was moistened with 25 μl of Milli-Q water and at least 25 mg of CH02906 was applied directly on top of the skin tissue.

Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with CH02906 compared to the negative control tissues was 72% and 83%, respectively. At the 3-minutes treatment time the Coefficient of Variation between tissue replicates was 55% and the results of the individual skin tissues were 45 and 100%. Therefore one tissue had a corrosive result and the other tissue was not corrosive. This variation between replicates is most probably caused by the rinsing of the skin tissues after exposure. At the 3-minutes treatment time the test item could not be completely removed from one of the duplicate tissues during the rinsing process. The corrosion study was therefore repeated in a second experiment.

The relative mean tissue viability obtained after 3-minute and 1-hour treatments with CH02906 compared to the negative control tissues was 104% and 81%, respectively in the second experiment. Although at the 1-hour treatment time the Coefficient of Variation between tissue replicates was 54% in experiment 2, both individual skin tissues had a non-corrosive result of 51 and 112%. This non-corrosive result after 1-hour was also observed in the first experiment. Because the mean relative tissue viability for CH02906 was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment, CH02906 is considered to be not corrosive.

The positive control had a mean relative tissue viability of 14 and 7% after the 1-hour exposure in the first and second experiment, respectively. The absolute mean OD570(optical density at 570 nm) of the negative control tissues was within (1.046 - 1.658) the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit2.8) and the laboratory historical control data range in both experiments. In the range of 20 - 100% viability, the Coefficient of Variation between tissue replicates was20% for the control tissues, indicating that the test system functioned properly in both experiments.

Finally, it is concluded that this test was valid and that CH02906 was not corrosive in thein vitroskin corrosion test under the experimental conditions described in this report.