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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:
17 May 2016 to 09 June 2016
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)
Deviations:
yes
Remarks:
deviations not affecting integrity or validity of the study (see below)
Qualifier:
according to
Guideline:
other: Method B.40bis (in Vitro Skin Corrosion Human Skin Model Test)
Deviations:
yes
Remarks:
deviations not affecting integrity or validity of the study (see below)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Appearance/physical state: Brown solid
- Storage conditions: Room temperature in the dark

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from a single donor
Source strain:
other: neonatal
Vehicle:
unchanged (no vehicle)
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
Approximately 180 mg
Duration of treatment / exposure:
3 minutes and 60 minutes
Duration of post-treatment incubation (if applicable):
3 hours
Number of replicates:
Two

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minute exposure
Value:
91.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minute exposure
Value:
99.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
DIRECT MTT REDUCTION
- An assessment found the test item was able to directly reduce MTT. Therefore, an additional procedure using freeze-killed tissues was performed. However, the results obtained showed that negligible interference due to direct reduction of MTT occurred.
- It was therefore considered unnecessary to use the results of the freeze-killed tissues for quantitative correction of results or for reporting purposes.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
- The solution containing the test item was a beige colour.
- This was considered to not have the potential to cause colour interference with the MTT endpoint.

TEST ITEM, POSITIVE CONTROL ITEM AND NEGATIVE CONTROL ITEM
- Mean OD562 values and viabilities for the negative control, positive control and test item are given in Appendix 1 (attached).
- The relative mean viabilities for each treatment group are shown in the table below.

QUALITY CRITERIA
- The mean OD562 for the negative control treated tissues was 1.896 for the 3-Minute exposure period and 1.874 for the 60-Minute exposure period. The negative control acceptance criteria were therefore satisfied.
- The relative mean tissue viability for the positive control treated tissues was 3.0% relative to the negative control following the 60-minute exposure period. The positive control acceptance criterion was therefore satisfied.
- In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

Any other information on results incl. tables

RELATIVE MEAN VIABILITIES FOR EACH TREATMENT GROUP

Exposure period

Percentage viability negative control*

Percentage viability positive control

Percentage viability test item

3 minute

100

3.7

91.6

60 minutes

100

3.0

99.4

* Mean viability of the negative control tissues is set at 100%

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The relative mean viability of the test item treated tissues was 91.6% after 3 minutes exposure and 99.4% after 60 minutes exposure.
Executive summary:

GUIDELINE

The study was performed in compliance with the OECD Guideline for the Testing of Chemicals No 431 In Vitro Skin Corrosion: Reconstructed Human EpiDermis (RHE) Test Method (28 July 2015) and Method B.40bis of Commission Regulation (EC) No 440/2008 of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH). The purpose of the test was to evaluate the corrosivity potential of the test item using the EpiDerm Human Skin Model after treatment periods of 3 and 60 minutes. Corrosion is directly related to cytotoxicity in the EpiDerm tissue and is determined by reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) to formazan by viable cells in the test item-treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.

 

METHODS

Duplicate tissues were treated with discs of the test item for exposure periods of 3 and 60 minutes. The test item was found to directly reduce MTT and therefore additional non-viable tissues were incorporated into the testing for correction purposes. Negative and positive control groups were treated for each exposure period. At the end of the exposure period the test item discs were removed from each tissue. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density (OD) was measured at 562 nm (OD562). Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

 

RESULTS

Mean viability of the negative control tissues was set at 100 % and quality criteria for acceptance of results were satisfied. Relative mean viabilities after 3 minutes exposure were determined to be 100 % (negative control), 3.7 % (positive control and 91.6 % (test item). Relative mean viabilities after 60 minutes exposure were determined to be 100 % (negative control), 3.0 % (positive control) and 99.4 % (test item).

 

CONCLUSION

The relative mean viability of the test item treated tissues was 91.6% after 3 minutes exposure and 99.4% after 60 minutes exposure.