Trans-2-{4-[difluoro(3,4,5-trifluorophenoxy)methyl]-3,5-difluorophenyl}-5-(trans-4-propylcyclohexyl)-1,3-dioxane

Endpoint summary

Description of key information

Skin irritation in vitro (OECD 439): not irritating, reference 7.3.1 -1

Eye irritation in vitro (OECD 405): not irritating, reference 7.3.2 -1

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 6, 2011 - October 25, 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Council Regulation (EC) No. 761/2009 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and the council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Deviations:

no

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 system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

standard model

Vehicle:

unchanged (no vehicle)

Remarks:

No vehicle used in this study; The test item was applied neat to the tissues.

Details on test system:

CELL CULTURE
- Supplier: SkinEthic Laboratories (Nice, France)
- Source: human keratinocytes cultured on a polycarbonate filter in conditions which permit their terminal differentiation
- Format: 24 well plate
- Batch: 11 022A 0809


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

REMOVAL OF THE TEST MATERIAL AND CONTROL
After the end of the treatment interval, the residual test item was removed immediately by gently rinsing with a minimum volume of 25 mL PBS using a pipette. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper.

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer:ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany at 570 nm

Control samples:

yes, concurrent negative control

Amount/concentration applied:

TEST MATERIAL: 16 mg of solid test material
NEGATIVE CONTROL: 16 µL (Phosphate-Buffered Saline)
POSITIVE CONTROL: 16 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

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Experiment 1 / Run 1

Value:

100.34

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: none
- Direct-MTT reduction: none
- Colour interference with MTT: none

ACCEPTANCE OF RESULTS:
After treatment with the negative control the absorbance values reached a mean OD of 2.11 (SD 5.96 %). Therefore, the negative control fulfilled the validity criteria.
Treatment with the positive control revealed a mean relative viability of 1.36 % (SD 4.03 %), thus the positive control reached the validity criteria.

Group	Time / [min]	Mean OD	Mean Relative viability / [%]
Negative Control	42	2.111	100
Positive Control	42	0.029	1.36
Test Material	42	2.118	100.34

Interpretation of results:

GHS criteria not met

Conclusions:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 439. Under the experimental conditions reported, the test material is not irritating to the skin.

Executive summary:

This in vitro study was performed to assess the irritation potential of the test item by means of the Reconstructed Human Epidermis Test and in accordance with OECD TG 439. The test consisted of a topical exposure of the test material to a human reconstructed model followed by a cell viability test. Cell viability was quantitatively measured by dehydrogenase conversion of MTT into a blue formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict skin irritation potential. Triplicates of the human skin model RHE (Reconstructed Human Epidermis, SkinEthic) were treated with the test material, the negative or the positive control for 42 minutes (± 1 minute).
16 µL of either the negative control (PBS-buffer) or the positive control (aqueous solution of sodium dodecyl sulfate) were applied to each tissue. Before adding the solid test material, 10 µL of deionised water was spread to the epidermis surface to improve further contact between the test material and the epidermis. Afterwards, 16 mg of the test material were applied to each tissue. Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the treatment interval thus ensuring the validity of the test system. After treatment with the negative control the absorbance values reached the required acceptability criterion of a mean optical density (OD) ≥ 1.2 and ≤ 2.5 for the treatment interval thus showing the quality of the tissues.

The tissue viability after treatment with the test material was higher than 50 % (mean viability: 100.34 %). Therefore, the test material is not considered to possess an irritant potential to the skin. In conclusion, under the experimental conditions reported, the test material is not irritating to the skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

For this endpoint information from structural similar compounds is available. The studies for these similar compounds were performed according to GLP and the methods applied are fully compliant with OECD TG 405. See chapter 13 report for a more detailed justification.

Reason / purpose for cross-reference:

read-across source

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

3

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

3

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

3

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritant / corrosive response data:

No signs of irritation were observed at the cornea or iris after instillation of the test item. Two animals showed redness of the conjunctivae (score 1) 1 hour after treatment, that recovered completely 6 hours after administration. No abnormalities were detected in the untreated right eyes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation, in vitro

This in vitro study was performed to assess the irritation potential of the test item by means of the Reconstructed Human Epidermis Test and in accordance with OECD TG 439. The test consisted of a topical exposure of the test material to a human reconstructed model followed by a cell viability test. Cell viability was quantitatively measured by dehydrogenase conversion of MTT into a blue formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict skin irritation potential. Triplicates of the human skin model RHE (Reconstructed Human Epidermis, SkinEthic) were treated with the test material, the negative or the positive control for 42 minutes (± 1 minute). 16 µL of either the negative control (PBS-buffer) or the positive control (aqueous solution of sodium dodecyl sulfate) were applied to each tissue. Before adding the solid test material, 10 µL of deionised water was spread to the epidermis surface to improve further contact between the test material and the epidermis. Afterwards, 16 mg of the test material were applied to each tissue. Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the treatment interval thus ensuring the validity of the test system. After treatment with the negative control the absorbance values reached the required acceptability criterion of a mean optical density (OD) ≥ 1.2 and ≤ 2.5 for the treatment interval thus showing the quality of the tissues. The tissue viability after treatment with the test material was higher than 50 % (mean viability: 100.34 %). Therefore, the test material is not considered to possess an irritant potential to the skin. In conclusion, under the experimental conditions reported, the test material is not irritating to the skin.

Eye irritation, in vivo, read-across

There are no experimental data available on eye irritation with the registered substance. However, data with a structural analogue substance are available which are considered suitable and sufficient to conclude on this endpoint. For detailed read-across justification please refer to IUCLID section 13.

The objective of the present study was to investigate the eye irritating potential of the test item in rabbits. For this purpose, the test item was applied in a single dose to the left eye of rabbits, the untreated right eye served as control. The degree of eye irritation was evaluated by scoring lesions of conjunctiva, cornea and iris at specific intervals. The first examinations of the eyes followed 1 and 6 hours after treatment. Subsequently, the eyes were investigated twice daily for a further 4 days. In addition, potential systemic effects and the body weight development was determined. The study was performed initially with one animal, followed by the confirmatory test with two further animals. No mortality occurred and no clinical symptoms were seen during the experimental phase of the study. The body weight development of the rabbits was inconspicuous. No signs of irritation were observed at the cornea or iris after instillation of the test item. Two animals showed redness of the conjunctivae (score 1) 1 hour after treatment, that recovered completely 6 hours after administration. No abnormalities were detected in the untreated eyes. In conclusion, under the conditions of the present study, no eye irritating potential was detected for the test item.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data is reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance does not require classification for eye or skin irritation under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) No 2019/521.