Registration Dossier

Toxicological information

Skin irritation / corrosion

Currently viewing:

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 April 2014 to 02 July 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD test guidelines in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Qualifier:
according to guideline
Guideline:
other: EPA 40 CFR 158.340
GLP compliance:
yes

Test material

Constituent 1
Test material form:
other: liquid
Details on test material:
Identity: H2925 (CAS No 68130-53-0), Lot# 2013090401
Provided by: Chemtura Corporation
Date Received: 18 Mar 2014
Storage: Room temperature and humidity.
Description: Clear yellow liquid
Sample Preparation: Used as received.

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Not specified
Source strain:
not specified
Details on animal used as source of test system:
EpiDerm™ tissues, Lot 19981 Kit D, were received from MatTek Corporation (Ashland, MA) on 08 Apr 2014 and refrigerated at approximately 4°C. Before use, tissues were incubated (37°C ± 1°C, 5% ± 1% CO2) with assay medium (MatTek) for a one-hour equilibration. Equilibration medium was replaced with fresh medium before dosing.
Justification for test system used:
In accordance with test guidelines.
Vehicle:
unchanged (no vehicle)
Details on test system:
Test Article Reduction of MIT: 100 µI of the test article were mixed with 1 ml of MTT solution (1 mg/ml Methyl thiazole tetrazolium diluted in Dulbecco's Modified Eagle's Medium (DMEM)), A Negative Control, 100 µI of tissue culture water (TCH2O), was tested concurrently, The solutions were incubated at room temperature in the dark for 60 minutes, After incubation, the solutions were visually inspected for purple coloration, which indicates that the test article reduced MTT Since tissue viability is based on MTT reduction, direct reduction by a test article can exaggerate viability, making a test article seem less irritating than it really is. The test article did not reduce MTT and the assay continued as per the protocol.

Mesh Compatibility: Pre-cut nylon meshes supplied with the tissues were placed on a slide and 30 µI of the test article or TCH2O Negative Control were applied. After 60 minutes exposure, the mesh was checked microscopically, No interaction between the test article or TCH2O and the mesh was observed so the test article was dosed using the mesh as a spreading aid,

Dosing:
30 µI of the test article or control articles were applied to the EpiDerm ™ tissue. The nylon mesh was then placed on top to facilitate even distribution of the test material. A Negative Control (phosphate buffered saline (PBS)) and a Positive Control (5% sodium dodecyl sulfate (SDS) solution, MatTek) were tested concurrently, with a nylon mesh placed on top to facilitate even distribution of the material. Each treatment with test article or control was conducted in triplicate.
The exposure period for the test article and controls was 60 minutes. The dosed tissues were placed in an incubator at 37° ± 1°C, 5% ± 1% CO2 for 35 ± 1 minutes, then returned to the sterile hood for the remainder of the 60-minute exposure period.
After dosing and incubation, the tissues were rinsed with PBS, blotted to remove the test substance and dry the tissue, and transferred to fresh medium. The rinsed EpiDerm™ tissues were returned to the incubator for 24 ± 2 hours. Medium was changed at 24 ± 2 hours. Tissues were returned to the incubator for an additional 18 ± 2 hours.

Tissue Viability (MTT Reduction): At the end of the incubation period, each EpiDerm™ tissue was rinsed with phosphate buffered saline (PBS) and transferred to a 24-well plate containing 300 µI of MTT solution (1 mg/ml MTT in DMEM). The tissues were then returned to the incubator for a three-hour MTT incubation period. Following the MTT incubation period, each EpiDerm TM tissue was rinsed with PBS and then treated with 2.0 ml of extractant solution (isopropanol) per well for at least two hours, with shaking, at room temperature. Two aliquots of the extracted MTT formazan were measured at 540 nm using a plate reader (µQuant Plate Reader, BioTek Instruments, Winooski, VT).

Analysis of Data: The mean absorbance value for each time point was calculated from the optical density (OD) of the duplicate samples and expressed as percent viability for each sample using the following formula:
% viability =100 X (OD sample/OD Negative Control)

Quality Controls: The assay meets the acceptance criterion if the mean OD540 of the Negative Control tissues is ≥ 1.0 and ≤ 2.5, and the mean viability of Positive Control tissues, expressed as percentage of the Negative Control tissues, is ≤ 20%. In addition, the standard deviation (SD) calculated from individual percent tissue viabilities of the three identically-treated replicates is < 18%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
30 µI of the test article were applied to the top of each EpiDerm™ tissue.
Duration of treatment / exposure:
60 minutes
Duration of post-treatment incubation (if applicable):
Not specified
Number of replicates:
Tests were repeated in triplicate.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
96.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 60 minutes. Max. score: 100.0. Remarks: Viability score are reported as %. The lower the score the more harmful a substance is expected to be. . (migrated information)

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
H2925 (CAS No. 68130-53-0), Lot# 2013090401 is considered to be a non-irritant and therefore it is not classified in accordance with the CLP Regulation.
Executive summary:

OBJECTIVE: To predict dermal irritation potential of test articles in the context of identification and classification of skin irritation hazard according to the European Union (EU) classification, United Nations Globally Harmonized System of Classification and Labelling of Chemicals (GHS) classification system (Category 2 and non-irritants), and OECD Guideline for the Testing of Chemicals No. 439 - In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method. This study is designed based on MatTek protocol in vitro EpiDerm™ Skin Irritation Test.

METHOD SYNOPSIS: MatTek EpiDerm™ tissue samples were treated in triplicate with the test article, Negative Control and Positive Control for 60 minutes. Following treatment and subsequent incubation time, the viability of the tissues was determined using Methylthiazole tetrazolium (MTT) uptake and reduction. The absorbance of each sample was measured at 540 nm. The viability was then expressed as a percent of control values. If the mean tissue viability was ≤ 50%, the test material was classified as an irritant; if the mean tissue Viability was >50%, the test material was classified as a non-irritant.

SUMMARY/CONCLUSION:

Test and Control Article Identity

Mean Tissue Viability

Irritancy Classification

H2925 (CAS No. 68130-53-0), Lot# 2013090401

96.4%

Non-Irritant

Phosphate Buffered Saline (Negative Control)

100.0%

Non-Irritant

5% Sodium Dodecyl Sulfate (Positive Control)

2.4%

Irritant