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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:
08 January 2018 - 12 January 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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
29 July 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EC Guideline 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". Official Journal of the European Union No. L142,
Version / remarks:
31 May 2008
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Appearance: Off-white powder
Test item storage: At room temperature

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
skin obtained from plastic surgery from multiple donors
Justification for test system used:
Recommended test system in international guidelines (OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model
- Tissue batch number(s): 27667
- The skin tissues were kept in the refrigerator the day they were received. The next day, at least 1 hour before the assay was started the tissues were transferred to 6-well plates containing 0.9 mL DMEM per well. The plates were incubated for approximately 2.5 hours at 37.0 ± 1.0ºC.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 36.0-37.3°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: after exposure and after incubation tissues were washed with phosphate buffered saline (once)
- Observable damage in the tissue due to washing: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 1.708 +/- 0.053
- Barrier function: 8.47 hours
- Contamination: sterile (no contamination)

NUMBER OF REPLICATE TISSUES: 2

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1 experiment with 3 independent OD570 measurements per replicate.

ACCEPTABILITY CRITERIA
- The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range.
- The mean relative tissue viability following 1-hour exposure to the positive control should be <15 %.
- In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be ≤ 30%.

DECISION CRITERIA
A test item is considered corrosive in the in vitro skin corrosion test if:
- The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
- In addition, a test item considered non-corrosive (viability ≥ 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test item is decreased below 15%.
A test item is considered non-corrosive in the in vitro skin corrosion test if:
- The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
- In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 26.20 - 27.44 mg
- The test item was applied directly on top of the skin tissues (pre-wetted with 25 μl Milli-Q water)

NEGATIVE CONTROL
- Amount applied: 50 μL

POSITIVE CONTROL
- Amount applied: 50 μL
Duration of treatment / exposure:
3 minutes and 1 hour
Duration of post-treatment incubation (if applicable):
3 hours in MTT medium
Number of replicates:
2 tissues for the test item, 2 for the negative control and 2 for the positive control for both the 3-minute and 1-hour time point

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean of 2 replicates
Run / experiment:
3-minute exposure
Value:
101
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
mean tissue viability: 15%
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean of 2 replicates
Run / experiment:
1-hour exposure
Value:
104
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
mean tissue viability: 8.9%
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: the results of the positive control data were within the historical data range and thereby showing the test system functioned properly.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the absolute mean OD570 of the two tissues of the negative control was within the laboratory historical control data range.
- Acceptance criteria met for positive control: yes, the mean relative tissue viability following 1-hour exposure to the positive control was <15 % (i.e., 8.9%).
- Acceptance criteria met for variability between replicate measurements: yes, the Coefficient of Variation (CV) between tissue replicates was ≤ 30% (i.e., ≤ 27%)

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
Not classified according to Regulation (EC) 1272/2008
Conclusions:
In the in vitro skin corrosion test, D6-Noreth was found to be not corrosive to the skin (mean tissue viability of 101% and 104% after a 3-minute and a 1-hour exposure period, respectively). The test item is not classified according to GHS and according to Regulation (EC) 1272/2008.