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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:
Experimental start date 10 August 2016 Experimental completion date 25 August 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:
Due to concern that the colored test item may interfere with MTT endpoint, a procedure using viable color correction tissues was performed to measure any possible interference. Negligible interference occurred, no affect on integrity/validity of study.
Qualifier:
according to
Guideline:
other: • 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 REACH
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Batch number 0708501022
Description Red crystalline solid
See certificate of analysis:
Purity 99.8%
Storage requirements Store under a nitrogen atmosphere in a cool, dry, well-ventilated area away from flammable materials and sources of heat or flame.
Expiration date September 1, 2012
Specific details on test material used for the study:
Identification: Dichlorobis(η-cyclopentadienyl)titanium (CAS# 1271-19-8)
Physical state/Appearance: Red crystalline powder
Batch Number: 0708501022
Purity (GLC): 99.3%
Expiry Date: 04 March 2019
Storage Conditions: Room temperature in the dark

In vitro test system

Test system:
human skin model
Remarks:
EPIDERM Reconstructed Human Epidermis model Kit
Source species:
other: EPIDERM Reconstructed Human Epidermis model Kit
Cell type:
other: Epidermal
Cell source:
other: EPIDERM Reconstructed Human Epidermis model Kit
Source strain:
other: EPIDERM Reconstructed Human Epidermis model Kit
Details on animal used as source of test system:
EpiDerm™ Reconstructed Human Epidermis Model Kit
Supplier : MatTek
Date received : 23 August 2016
EpiDermTM Tissues (0.63cm2) lot number : 23351
Assay Medium lot number : 081816TMA
Upon receipt of the EpidermTM tissues, the sealed 24 well plate was stored in a refrigerator until use.
Justification for test system used:
This model incorporates several features, which make it advantageous in the study of potential dermal corrosivity. The target cells are epithelial, derived from human skin, and formed into a stratified, cornified epithelium. Test items are applied to the culture surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly.
Vehicle:
unchanged (no vehicle)
Details on test system:
25 mg of the test item was added to 1 mL of a freshly prepared 1.0 mg/mL MTT solution. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 60 minutes. Untreated MTT solution was tested concurrently to act as a control.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
25 mg of the test item was added to 1 mL of a freshly prepared 1.0 mg/mL MTT solution.
Duration of treatment / exposure:
3 minutes and 60 minutes. The plate was returned to the incubator (37 °C, 5% CO2) for the 60 Minute exposure period
Duration of post-treatment incubation (if applicable):
Immediate observation following exposure
Number of replicates:
two x 24-well plates

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minute exposure
Value:
103.3
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Any other information on results incl. tables

Direct MTT Reduction

The MTT solution containing the test item did not turn blue/purple. This was taken to indicate the test item did not reduce MTT.

Assessment of Color Interference with the MTT endpoint

The solution containing the test item was a red color. This color was considered to have the potential to cause color interference and therefore additional tissues were incorporated into the test to correct for this interference. However, the results obtained showed a negligible amount of color interference occurred and it was therefore considered unnecessary to use the results of the color correction tissues for reporting purposes or for quantitative correction of the results.

Test Item, Positive Control Item and Negative Control Item

Mean OD562values and viabilities for the negative control, positive control and test item are given in theappendix.

The relative mean viabilities for each treatment group were as follows:

Exposure Period

Percentage Viability

Negative Control

Positive Control

Test Item

3 minute

100*

5.3

103.3

60 minute

100*

5.2

92.0

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

Quality Criteria

The mean OD562for the negative control treated tissues was 2.470 for the 3‑Minute exposure period and 2.463 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 5.2% 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.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was considered to be non-corrosive to the skin.
Executive summary:

 Summary

Introduction

The purpose of this test is 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. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium 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 the test item for exposure periods of 3 and 60 minutes. The test item was found to have the potential to cause color interference with the MTT end point and therefore additional 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 was rinsed from each tissue before each tissue was taken for MTT‑loading. After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction.

At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 mL samples were transferred to the appropriate wells of a pre-labeled 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

The relative mean viabilities for each treatment group were as follows:

Exposure Period

Percentage Viability

Negative Control

Positive Control

Test Item

3 minute

100*

5.3

103.3

60 minute

100*

5.2

92.0

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

Quality criteria: The quality criteria required for acceptance of results in the test were satisfied.

 

Conclusion

The test item was considered to be non-corrosive to the skin.