1H-Benzimidazole, 6-(2-chloro-5-fluoro-4-pyrimidinyl)-4-fluoro-2-methyl-1-(1-methylethyl)-

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Laboratory phase of study: 22 October 2013 to 24 October 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 431 (In Vitro Skin Corrosion: Human Skin Model Test)

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-2-methyl-1-(propan-2-yl)-1H-1,3-benzodiazole

EC Number:

941-461-6

Cas Number:

1231930-42-9

Molecular formula:

C15H13CIF2N4

IUPAC Name:

6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-2-methyl-1-(propan-2-yl)-1H-1,3-benzodiazole

Specific details on test material used for the study:

- Source and lot/batch No.of test material: RSO-H71422-082- Storage condition of test material: Stored at room temperature- Treatment of test material prior to testing: None- Form as applied in the test: Applied directly to the tissue surface

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Details on animal used as source of test system:

Not applicable

Justification for test system used:

Justification for test system used The EpiDerm model incorporates several features that make it advantageous in the study of potential dermal corrosivity. The target cells are epithelial derived from human skin. The tissue posses a functional stratum corneum to model barrier properties of skin. Finally test articles 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: 37oC, except tissues exposed for 3 minutes which were held at room temperature during dosing.- Temperature of post-treatment incubation: Post rinsing tissue samples are incubated at standard culture conditions (37±1ºC in a humidified atmosphere of 5±1% CO2 in air) for 180 minutesREMOVAL OF TEST MATERIAL AND CONTROLS- Number of washing steps: One. After the appropriate exposure time, the EpiDerm™ tissues were extensively rinsed with warm (approximately 37ºC) Calcium and Magnesium-Free Dulbecco's Phosphate Buffered Saline.- Observable damage in the tissue due to washing: During the rinsing of the 60-minute tissues treated with the positive control, 8N potassium hydroxide, the first tissue detached from the tissue insert and was lostDYE 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- Wavelength: 550 nmNUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION:For the test material and each control two tissue samples will be used to assess viability after 3-minute exposure and two will be used to assess viability after 60 minute exposurePREDICTION MODEL / DECISION CRITERIA- Evaluation of test results:If the test material reduced tissue viability to <50% of the negative control value after a 3-minute exposure, the test material was considered corrosive. In addition, test materials which resulted in tissue viability ≥ 50% after a 3-minute exposure but <15% after a 60-minute exposure were also classified as corrosive. Test materials which resulted in tissue viability ≥ 50% after 3-minute exposure and ≥ 15% after 60-minute exposure would be classified as non-corrosive.- Criteria for Determination of a Valid Test:The assay was accepted if the positive control resulted in a corrosive classification (i.e., <50% cell viability compared to negative controls, after a 3-minute exposure and/or <15% cell viability compared to negative controls after a 60-minute exposure).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL- Amount(s) applied (volume or weight with unit): 25 mg sample of test material was applied as dry powder directly to tissue surface followed by 25 microliters of sterile de-ionised waterNEGATIVE CONTROL- Amount(s) applied (volume or weight): 50 microliters of sterile de-ionised waterPOSITIVE CONTROL- Amount(s) applied (volume or weight): 50 microliters of 8 normal potassium hydroxide

Duration of treatment / exposure:

3-minute exposure for a corrosive classification and a 60-minute confirmatory exposure for materials found to be non-corrosive by the 3-minute exposure

Duration of post-treatment incubation (if applicable):

Post rinsing of test article tissue samples were held at standard culture conditions for 180 minutes (37±1ºC in a humidified atmosphere of 5±1% CO2 in air)

Number of replicates:

Two tissues were used to assess viability after the 3-minute exposure, and two were used to assess viability after the 60-minute exposure.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test Article - 3 minutes exposure time

Value:

ca. 104.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

Non-corrosive

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test Article - 60 minutes exposure time

Value:

ca. 103.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

non-corrosive

In vivo

Other effects:

OTHER EFFECTS:- Direct-MTT reduction: The test material was not observed to directly reduce MTT in the absence of viable cells. The results of the killed control experiment (performed using 8N potassium hydroxide) showed that there was significant direct MTT reduction in the positive control-treated killed controlsDEMONSTRATION 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 analysis of the data generated in this study showed that the assay was valid based on the 3-minute exposure data obtained for the positive control (corrosive prediction based on a 13.2% viability value; prediction model cut-off value is <50% viability for the 3-minute exposure time); furthermore, the corrosive prediction was also confirmed by the 60-minute exposure (single tissue) (corrosive prediction based on a 8.2% viability value; prediction model cut-off value is <15% viability for the 60 minute exposure time).

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results of the in vitro skin corrosion assay performed, EU CLP classification criteria are not met. The test material, LSN2833975 is classified as non-corrosive as tissue viability obtained after 3 minute exposure time was 104.6% which is ≥50%60 minute exposure time was 103.8% which is ≥15%

Executive summary:

An in vitro skin corrosion assay was performed on LSN2833975 using the EpidermTM Skin model. The method utilizes a 3-minute exposure for a corrosive classification and a 60-minute confirmatory exposure for materials found to be non-corrosive by the 3-minute exposure. Viable cells reduce the yellow, soluble, oxidized form of the MTT to the reduced blue-black insoluble form. The reduced dye is extracted from the tissue with isopropanol and the amount of reduced dye determined spectrophotometrically. The relative viability of the treated tissues is calculated as a percentage of the control viability. Test materials that reduce tissue viability to <50% within 3 minutes are classified corrosive by this method. In addition, test materials which result in tissue viability of ≥50% after a 3-minute exposure, but result in tissue viability of <15% after a 60 -minute exposure are also classified as corrosive. Test materials which result in tissue viabilities of ≥50% after a 3 minute exposure, and ≥15% after a 60 -minute exposure are classified non-corrosive.

The analysis of the data generated in this study showed that the assay was valid based on the 3-minute exposure data obtained for the positive control (corrosive prediction based on a 13.2% viability value; prediction model cut-off value is <50% viability for the 3 -minute exposure time); furthermore, the corrosive prediction was also confirmed by the 60-minute exposure (single tissue) (corrosive prediction based on a 8.2% viability value; prediction model cut-off value is <15% for the 60 -minute exposure time.

The test material, LSN2833975 is classified as non-corrosive as tissue viability obtained after

3 minute exposure time was 104.6% which is≥50%

60 minute exposure time was 103.8% which is≥15%