1H-Benzimidazole, 6-bromo-4-fluoro-2-methyl-1-(1-methylethyl)-

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Laboratory phase of study: 12 November 2013 to 15 November 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

The protocol met the requirements of:OECD Guideline for the Testing of Chemicals (OECD TG 439). In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method. Adopted 26 July 2013

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

6-bromo-4-fluoro-2-methyl-1-(propan-2-yl)-1H-1,3-benzodiazole

EC Number:

807-019-0

Cas Number:

1231930-33-8

Molecular formula:

C11H12BrFN2

IUPAC Name:

6-bromo-4-fluoro-2-methyl-1-(propan-2-yl)-1H-1,3-benzodiazole

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL- Source and lot/batch No.of test material: RS0-H71422-089STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL- Storage condition of test material: Stored at room temperatureTREATMENT OF TEST MATERIAL PRIOR TO TESTING: NoneFORM AS APPLIED IN THE TEST: Applied directly to the tissue surface

In vitro test system

Test system:

human skin model

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Justification for test system used:

The EpiDerm model incorporates several features that make it advantageous in the study of potential dermal irritation. The test system uses a serum-free medium which eliminates the possiblity of serum protein and test material interaction. The target cells are epithelial, derived from human skin. Since the tissue has a functional stratum corneum, the test materials are applied directly to the tissue surface, at air interface, so that undiluted and/or end use dilutions can be tested directly.

Vehicle:

unchanged (no vehicle)

Details on test system:

TEMPERATURE USED FOR TEST SYSTEM- Temperature used during treatment / exposure: Room temperature- Temperature of post-treatment incubation: Standard culture conditions (37±1ºC in a humidified atmosphere of 5±1% CO2 in air) REMOVAL OF TEST MATERIAL AND CONTROLS- Number of washing steps: 15DYE BINDING METHOD- Dye used in the dye-binding assay: MTT (3-[4,5 - dimethylthiazol-2-yl] - 2,5 - diphenyltetrazolium bromide)- Spectrophotometer: Molecular Devices Vmax plate reader with the AUTOMIX function selected- Wavelength: 570nmNUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION:Test material, postive and negative controls were all tested in triplicatePREDICTION MODEL / DECISION CRITERIAThe assay was accepted when the following criteria were met:the positive control resulted in a mean tissue viability ≤20%,the mean optical absorbance (at 570nm) value of the negative control tissues was ≥ 1.000 and < 2.500the standard deviations of the positive and negative control calculated from individual percent tissue viabilities of the three identically treated replicates were <18%A test material was predicted to be an irritant (EU Classification R38) when the mean relative viability of the three treated tissues is less than or equal to 50% of the mean viability of the negative control.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL: 25 mgNEGATIVE CONTROL: 30 microlitres of sterile, CMF-DPBSPOSITIVE CONTROL: 30 microlitres of 5% Sodium Lauryl Sulfate

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

42 hour post exposure expression period

Number of replicates:

Tissue samples were treated in triplicate with both the test material and the controls

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

LSN2800269

Value:

ca. 92.7

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Positive Control

Value:

ca. 3.07

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:- Direct-MTT reduction: Test material was not observed to directly reduce MTT in the absence of viable cellsDEMONSTRATION OF TECHNICAL PROFICIENCY:MatTek determines the ET-50 value following exposure to Triton X-100 (1%) for each EpiDerm™ lot. The ET-50 must fall within a range established based on a historical database of results. Histology is provided upon request.ACCEPTANCE OF RESULTS: The assay is considered valid as the acceptance criteria were metthe positive control resulted in a mean tissue viability 3.07%the mean optical absorbance (at 570nm) value of the negative control tissues was 2.263the standard deviations of the positive and negative control calculated from individual percent tissue viabilities of the three identically treated replicates were <18%

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results of the in vitro skin irritation assay performed, EU CLP classification criteria are not met. The test material, LSN2800269 is classified as non-irritant as tissue viability obtained was 92.7%, which is >50%

Executive summary:

The test article, LSN2800269, was tested using the EpiDerm™ Skin Model for the Skin Irritation Test (SIT). The skin irritation potential was evaluated based upon measuring the relative conversion of MTT in the test material-treated tissues after exposure to each test article for a 60-minute exposure period, followed by a 42-hour post-exposure expression period. Skin irritation potential of the test article was predicted if the relative viability was less than or equal to 50%.

The assay was considered valid as the acceptance criteria were met: the positive control resulted in a mean tissue viability 3.07%; the mean optical absorbance (at 570nm) value of the negative control tissues was 2.263; the standard deviations of the positive and negative control calculated from individual percent tissue viabilities of the three identically treated replicates was <18%.

Based on the results of the in vitro skin irritation assay performed, EU CLP classification criteria are not met. The test material, LSN2800269 is classified as non-irritant as tissue viability obtained was 92.7%, which is >50%