4-chloromethyl-4''-ethyl-2'-fluoro-[1,1':4',1''-terphenyl]

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-08-16 to 2017-08-18

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

2012

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 28, 2015

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Skinethic skin irritation test -42bis Standard operating procedure (SOP) 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-chloromethyl-4''-ethyl-2'-fluoro-[1,1':4',1''-terphenyl]

Cas Number:

1115233-52-7

Molecular formula:

C21 H18 Cl F

IUPAC Name:

4-chloromethyl-4''-ethyl-2'-fluoro-[1,1':4',1''-terphenyl]

Test material form:

solid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: not specified

Justification for test system used:

The reconstructed human epidermis model in vitro method is an accepted in vitro method to replace animal testing. The human skin RHE™ model closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e the epidermis, and has been validated by the ECVAM in 2008.

Vehicle:

unchanged (no vehicle)

Remarks:

10 µL deionised water were spread in epidermis surface before test item application to improve contact of test item and epidermis.

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic™ RHE-model RHE/S/17 from Episkin/SkinEthic Laboratories, Lyon, France
- Tissue batch number: 17-RHE-086
- Production date: not specified
- Shipping date: not specified
- Delivery date: 2017-08-15
- Date of initiation of testing: 2017-08-16

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: minimum of 25 mL DPBS were used for rinsing
- Observable damage in the tissue due to washing: not specified
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: treatment: 3 h +/- 5 min, extraction: 2 h +/- 5 min
- Spectrophotometer: ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany
- Wavelength: 570 nm
- Filter: not specified
- Filter bandwidth: not specified
- Linear OD range of spectrophotometer: not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD > 0.7
- Barrier function: 4.0 h <= ET50 <= 10.0 h
- Morphology: Number of cell layers 4. Absence of significant histological abnormalities. Well differentiated epidermis consisting of basal, spinous, granular layers and a stratum corneum.
- Contamination: not specified
- Reproducibility: not specified

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test item was no MTT reducer, thus, no controls were used.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive or irritant to skin if the mean tissue viability is less or equal to 50 %.
- The test substance is considered to be non-corrosive and non-irritant to skin if the mean tissue viability is greater than 50 %.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL: 16 +/- 2 mg of solid test material
NEGATIVE CONTROL: 16 +/- 0.5 µL (Dulbecco`s Phosphate-Buffered Saline)
POSITIVE CONTROL: 16 +/- 0.5 µL (5 % aqueous solution of sodium dodecyl sulfate in deionised water)

Duration of treatment / exposure:

42 min (± 1 minute)

Duration of post-treatment incubation (if applicable):

42 hours (± 1 hour)

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

98.2

Vehicle controls validity:

not applicable

Remarks:

The test item was applied neat to the tissues

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: The pre-test for direct MTT-reducing capacity of the test item did not result in blue color, i.e. the test item is not a direct MTT reducer.
- Colour interference with MTT: In the pre-test medium coloration by the test item was observed, but no tissues were stained during the study. Therefore, no additional tissues for color control were treated and the test item caused no colour interferences.

DEMONSTRATION OF TECHNICAL PROFICIENCY: No direct information provided. However, laboratory has established a historical database for the study.

ACCEPTANCE OF RESULTS:
Please refer to “Any other information on results”.

Any other information on results incl. tables

 

Group	Tissue 1		Tissue 2		Tissue 3		Mean		SD
	OD	Viability [%]	OD	Viability [%]	OD	Viability [%]	OD	Viability [%]	Viability [%]
Negative Control	2.127	105.3	1.930	95.6	2.001	99.1	2.019	100.0	4.9
Positive Control	0.021	1.0	0.019	0.9	0.022	1.1	0.021	1.0	100
Test item	1.946	96.4	2.003	99.2	1.996	98.9	1.982	98.2	1.5

 

Acceptability of the Quality Control Data of the Skin Model with Reference to Historical Batch Data:

	Acceptance Criterion	Result
Negative control OD	≥ 0.8 and ≤ 3.0	1.930 to 2.127

 

Acceptability of the Positive and Negative Control stated by Episkin/SkinEthic Laboratories:

	Acceptance Criterion	Result
Mean OD negative control	≥ 1.2	2.019
Mean viability positive control	< 40 %	1.0 %
SD of group-mean value	≤ 18 %	10.0 % (positive control) 4.9 % (negative control)

Acceptability of the Positive and Negative Control based on Historical Data of the Testing Laboratory:

	Acceptance Criterion	Result
Mean OD negative control	≥ 1.463	2.019
Mean viability positive control	≤ 2.98 %	1.0 %

 

Test Item Data Acceptance Criteria:

	Acceptance Criterion	Result
SD of group-mean value	≤ 18 %	1.5 %

The study met all acceptance criteria.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item is not considered to possess an irritant potential to skin.

Executive summary:

A study according to OECD TG 439 was conducted to investigate the potential of the test item to induce skin irritation in an in vitro human skin model. The test item was applied topically to a human reconstructed skin model followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the skin irritation potential. Triplicates of the human skin RHE-model were treated with the test item, the negative or the positive control for 42 minutes (± 1 minute). 16 µL of either the negative control (DPBS-buffer) or the positive control (5 % aqueous solution of sodium dodecyl sulfate) were applied to the tissues. Before application of 16 mg of the solid test item, 10 µL of deionised water was spread to the epidermis surface to improve the contact between the test item and the epidermis. All acceptability criteria after treatment with the negative control (DPBS-buffer) and the positive control (5 % aqueous solution of sodium dodecyl sulfate) were met. Following treatment with the test item, the tissue viability was 98.2 % and, thus, higher than 50 %, i.e. according to OECD 439 the test item is considered as non-irritant to skin (UN GHS: No Category). Under the conditions of the present study, the test item is not considered to possess an irritant potential to skin.