5-bromo-1,3-dichloro-2-fluorobenzene

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

5-bromo-1,3-dichloro-2-fluorobenzene

EC Number:

640-454-2

Cas Number:

17318-08-0

Molecular formula:

C6H2 BrCl2F

IUPAC Name:

5-bromo-1,3-dichloro-2-fluorobenzene

Test material form:

liquid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: MatTek In Vitro Life Science Laboratories, Bratislava - Slovakia

Vehicle:

unchanged (no vehicle)

Details on test system:

Pre-incubation of EpiDerm tissues
The assay medium was brought to room temperature before use. The assay medium (0.9 mL) was pipetted into the appropriate wells of two pre-labeled 6-well plates for both the 3 and 60-minute exposure periods. EpiDerm™ tissues were transferred into the 6-well plates containing the assay medium and were pre-incubated (37 °C, 5% CO2) for approximately
1 hour before dosing.

Main Test
Before pre-incubation was complete, a 24-well plate was prepared for use as a “holding plate” for both the 3 and 60-minute exposure periods. This plate was used to maintain the viability of the tissue inserts between rinsing following chemical exposure and MTT loading. Another 24-well plate was prepared for the MTT loading. 300 µL of either pre-warmed assay medium (holding plate) or MTT medium (MTT loading plate) was dispensed into each well. The two plates were placed into the incubator until required.
After pre incubation of the EpiDerm™ tissues, the medium was aspirated and replaced with 0.9 mL of fresh assay medium. Duplicate tissues were used for each treatment group. For the 60-minute exposure period, 50 µL of sterile distilled water (negative control) was added to the first two tissues. The tissues were dosed at regular intervals to allow for the time taken to rinse each tissue following exposure and to ensure that each tissue gets an equal exposure time. 50 µL of the test item and 50 µL of 8.0 N potassium hydroxide (positive control) were also applied to the corresponding tissues in turn. The plate was returned to the incubator
(37 °C, 5% CO2) for the 60 minute exposure period.
The same procedure was repeated for the 3 minute exposure period. Because the exposure time was only 3 minutes, the tissues were dosed at regular intervals to ensure that each tissue received an equal exposure time and to allow for the time taken to rinse each tissue following exposure. Rinsing was achieved by filling and emptying each tissue under a constant soft stream of Dulbecco’s Phosphate Buffered Saline (DPBS) to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper. Each tissue was placed into the prepared holding plate until all tissues were rinsed. Tissues were blotted and transferred to the 24-well plate prepared for MTT loading. The plate was incubated (37 °C, 5% CO2) for 3 hours. Once the 60-minute exposure period was complete, the same rinsing and MTT loading procedure was repeated.
After the 3-hour MTT incubation was complete, the inserts were blotted and transferred to labeled 24-well plates for MTT extraction. 2 mL of MTT extract (isopropanol) was used to completely immerse each insert and the plate was covered with plate sealer to prevent Isopropanol evaporation. The plates stood overnight at room temperature, to allow extraction to proceed.
After extraction, each tissue was pierced with a pipette fitted with a 1000 µL tip and the extraction solution was forced vigorously up and down to form a homogenous solution.
3 x 200 µL aliquots of the extract were transferred to the appropriate wells of a pre-labeled
96-well plate. 200 µL of isopropanol alone was added to the three wells designated as blanks. Absorbency at 570 nm (OD570) of each well was measured using the Labtech LT 4500 microplate reader.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50 µL of test substance

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

2

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes of exposure

Value:

121.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes of exposure

Value:

113.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:
Negative Control
The absolute OD570 of the negative control treated tissues in the MTT test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. The mean OD570 of the two negative control tissues should be ≥ 0.8 and ≤ 2.8 for each exposure time, which ensures that the tissue viability meets the acceptance criteria.
Positive control
Potassium Hydroxide 8.0N solution was used as a positive control. An assay meets the acceptance criteria if mean relative tissue viability of the 60 minute exposure for the positive control is < 15%.
Coefficient of Variation
In the range 20 and 100% viability, the coefficient of variation between tissue replicates should be ≤ 30%.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The mean percent cell viability for the substance at 3 and 60 minutes was 121.1% and 113.3%, respectively, therefore under the conditions of this assay the test item was considered to be non-corrosive to the skin.

Executive summary:

The purpose of this test is to evaluate the skin 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.

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. 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 three aliquots of 200 µL were transferred to the appropriate wells of a pre-labelled 96 well plate. The optical density (OD) was measured at 570 nm (OD570).

Data are presented in the form of percentage cell viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The relative mean percent viability for each treatment group was:

3 minutes exposure period: Negative control 100% cell viability; Positive control 2.9% cell viability; Test substance: 121.1% cell viability

60 minutes exposure period: Negative control 100% cell viability; Positive control 2.4% cell viability; Test substance: 113.3% cell viability

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