4''-Butoxy-4-ethyl-2',2'',3''-trifluoro-[1,1':4',1'']-terphenyl

Endpoint summary

Description of key information

OECD TG 439: Not Irritating

OECD TG 437: Not Irritating

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:

2016-12-07 to 2017-02-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

07-2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: The SkinEthic(TM) RHE-model RHE/S/17 (Batch No: 17-RHE-004) was obtained from Episkin/SkinEthic Laboratories, Lyon, France

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)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic(TM) RHE-model RHE/S/17
- Tissue batch number(s): 17-RHE-004
- Production date: not specified
- Shipping date: not specified
- Delivery date: not specified
- Date of initiation of testing: 2017-01-11

Assessment for Direct MTT Reduction
Two wells of a 24-well plate were filled with 300 μL of a MTT-solution (final concentration: 1.0 mg/mL) per well. In one well 16 mg of the test item was added and mixed. Deionised water (16 μL) was used as negative control in the second well. The solution was incubated for 3 hrs +/- 5 minutes at 37°C and 5% CO2 protected from light. A direct interaction with the MTT was detected if the solution clearly turned blue or purple.
Assessment of Coloured or Staining Test Items
One well of a 24-well plate was filled with 300 μL of deionised water and one well of an additional 24-well plate was filled with 1500 μL isopropanol. In both wells 16 mg of the test item were added and incubated at room temperature with gentle agitation about 150 rpm. The well filled with test item and deionised water was incubated for 42 minutes and the well filled with test item and isopropanol was incubated for 2 hours.
Pre-Incubation
On the day of receipt, the tissues were transferred into 6-well plates containing 1 mL of pre-warmed (room temperature) growth medium under sterile conditions using sterile forceps. Afterwards, the tissues were incubated at 37°C and 5% C02 until test item application on the next day.
Treatment
For the test item as well as the positive (dodecyl sulfate sodium salt) and negative (Dulbecco’s Phosphate-Buffered Saline) controls, three tissues each were treated for 42 +/- 1 minutes. For treatment the tissues were transferred into 24-well plates containing 300 μL of pre-warmed (room temperature) maintenance medium. The negative and positive controls were applied directly to the surface of the epidermises, a nylon mesh was carefully applied to the whole surface as spreading aid. Before adding 16 mg of the test item, 10 μL of deionised water were spread to the epidermis surface to improve further contact between the test item and the epidermis. After application of the test item, the 24-well plates were incubated at room temperature for 42 +/- 1 minutes. At the end of the exposure period, the test item, negative and positive control were removed immediately by gently rinsing with a minimum volume of 25 mL DPBS using a pipette. Excess DPBS was removed by gently shaking the tissue inserts and blotting the bottom of the tissue inserts with blotting paper. The inserts were placed in 6-well plates with 2 mL fresh pre-warmed (room temperature) growth medium. The tissues were incubated for 42 +/- 1 hrs at 37°C and 5% C02.
MTT-Assay
For the MTT-Assay the stock solution was diluted with pre-warmed (room temperature) maintenance medium to a concentration of 1 mg/mL. The treated tissues were transferred to a 24-well plate filled with 300 μL MTT solution per well and incubated at 37°C and 5% C02 for 3 hrs +/- 5 minutes. To extract the precipitated blue formazan salt from the tissues, the tissues were transferred to 24-well plates filled with 800 μL isopropanol per well. 700 μL isopropanol were added on the top of each tissue to ensure that the tissues were completely covered with isopropanol. To prevent isopropanol from evaporation, the plates were covered with a self-adhesive film and the lid of the plate was added. The plates were put on a shaker with gentle agitation of about 150 rpm at room temperature for 2 hrs +/- 5 min. After extraction, the tissues were pierced with a needle in order to get the whole extraction solution in the corresponding well. The formazan solution was homogenized by pipetting 3 times up and down. Afterwards, 3*200 μL aliquots of the extraction solution from each well were transferred to a 96-well plate. The concentration of the formazan was measured by determining the OD spectrophotometrically at 570 nm with a microplate reader, which was calibrated with a filter test plate.

Amount/concentration applied:

Solid test item: 16 +/- 2 mg per tissue
Negative control: 16 +/- 0.5 μL per tissue
Positive control: 16 +/- 0.5 μL per tissue

Duration of treatment / exposure:

see above

Duration of post-treatment incubation (if applicable):

see above

Number of replicates:

see above

Irritation / corrosion parameter:

% tissue viability

Value:

101.37

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Results
The pre-test for direct MTT-reducing capacity of the test item did not result in blue colour, i.e. the test item is not a direct MTT reducer and the test item has no colorant properties. The tissue viability after treatment with the test item was higher than 50% (mean viability: 101.37%). Therefore, the test item is not considered to possess an irritant potential to skin.

Acceptability of the Test
The negative control OD values were 2.010, 1.974 and 1.889 and, thus, in the range of >= 0.8 and <= 3.0
After treatment with the positive control (5% aqueous solution of sodium dodecyl sulfate) the mean viability value was 1.22% (standard deviation: 12.96%) and, thus, lower than the historically established threshold of 3 .09%.
After treatment with the negative control (DPBS-buffer) the mean OD was 1.958 (standard deviation: 3.16%) and, thus, higher than the historically established threshold of 1.441.
The standard deviation between the three tissues replicates treated with the test item was 7.22% and, thus, <= 18%. The standard deviations between the three tissue replicates of the negative control and the positive control were 3.16% and 12.96%, respectively, and, thus, <= 18%.

The study met all acceptance criteria.

Study Design

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 +/- 1 minutes. 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.

 

Results

After treatment with the negative control (DPBS-buffer) the mean OD was 1.958 (study acceptance criterion: > 1.441). Treatment with the positive control (5% aqueous solution of sodium dodecyl sulfate) revealed a mean viability value of 1.22% (study acceptance criterion: <3.09%). Thus, the acceptance criteria were met. Following treatment with the test item, the tissue viability was 101.37 % and, thus, higher than 50%, i.e. according to OECD 439 the test item is considered as non-irritant to skin.

 

Conclusion

Under the conditions of the present study, the test item was not considered to possess an irritant potential to skin.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the present study, the test item was not considered to possess an irritant potential to skin.

Executive summary:

Study Design

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 +/- 1 minutes. 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.

 

Results

After treatment with the negative control (DPBS-buffer) the mean OD was 1.958 (study acceptance criterion: > 1.441). Treatment with the positive control (5% aqueous solution of sodium dodecyl sulfate) revealed a mean viability value of 1.22% (study acceptance criterion: <3.09%). Thus, the acceptance criteria were met. Following treatment with the test item, the tissue viability was 101.37 % and, thus, higher than 50%, i.e. according to OECD 439 the test item is considered as non-irritant to skin.

 

Conclusion

Under the conditions of the present study, the test item was not considered to possess an irritant potential to skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 25, 2017 - June 12, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

2013

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

Commission Regulation (EU) No. 1152/2010 amending, for the purpose of its adaption to technical progress, Regulation (EC) No. 440/2008 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

GLP compliance:

yes (incl. QA statement)

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL: 750 µL (i.e. 150mg/750µL)

NEGATIVE / VEHICLE CONTROL: 750 µL

Designation: 0.9% sodium chloride solution
Supplier: B. Braun Melsungen AG, Germany
Batch: 16435011
Storage: 2 to 8°C
Released until: September 2019


POSITIVE CONTROL: 750 µL

Designation: Art. 814223
Synonym: Imidazole
Supplier: Merck KGaA, Germany
Batch: S6746923
Purity (GC) 99.8% (a/a)
Storage: At room temperature
Released until: August 31, 2018

Imidazole was dissolved with 0.9% sodium chloride solution to a concentration of 20% (w/v).

Duration of treatment / exposure:

240 minutes

Number of animals or in vitro replicates:

in vitro: triplicate design

Irritation parameter:

in vitro irritation score

Run / experiment:

Run 1 / Experiment 1

Value:

0.4

Vehicle controls validity:

valid

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 observations (e.g. tissue peeling, residual test chemical, non-uniform opacity patterns) were seen in a visually inspection of the corneas after treatment.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was 0.6 and, thus, within three standard deviations of the current historical mean of the negative control (IVIS: -1.4 – 3.3).
- Acceptance criteria met for positive control: After treatment with the positive control (20% Imidazole) the calculated IVIS was 98.8 and, thus, also within two standard deviations of the current historical mean of the positive control (IVIS 80.1 – 133.1). Therefore, the study fulfilled the validity criteria.

		Opacity	Permeability	IVIS
				per cornea	per group (mean value)	SD
Negative control	0.9% NaCl Solution	-0.2	0.000	-0.200	0.6	0.8
		0.5	-0.001	0.485
		1.5	-0.001	1.485
Positive control	20% Imidazole solution	73.7	2.079	104.885	117.1	11.4
		96.3	2.079	127.485
		82.2	2.410	118.950
Test item	Test item	-0.7	0.002	-0.670	0.4	1.1
		0.2	0.007	-0.305
		0.9	0.047	1.605

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the present study, the test item did not show an eye hazard potential. The test item is not requiring classification for eye irritation or serious eye damage (UN GHS: No Category).

Executive summary:

Objective

The objective of the present study was to examine the potential of the test item to induce serious eye damage in the BCOP assay. The BCOP assay with isolated fresh bovine corneas is an accepted in vitro model for ocular hazard assessment.

 

Study Design

To determine the eye hazard potential the induced opacity and increased permeability was investigated in isolated bovine corneas after exposure to the test item as a 20% (w/v) suspension in a 0.9% sodium chloride solution. As negative control 0.9% sodium chloride solution and as positive control 20% (w/v) Imidazole was used.

Three corneas were used per group (negative control, positive control or test item group).

After a first opacity measurement of the untreated bovine corneas, 750 µL of the suspended test item, positive or negative control were applied on the corneas and incubated for 240 minutes. After the incubation phase the test item, the positive, and the negative control were rinsed from the corneas and the opacity was measured again.

After the opacity measurements, the permeability of the corneas was determined by application of a fluorescein solution for 90 minutes. The amount of fluorescein solution that crossed the cornea was measured spectrophotometrically. The opacity and permeability assessments were combined to determine an In Vitro Irritancy Score (IVIS).

 

Results

After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was 0.6 (study acceptance criteria range: -1.4 – 3.3). Treatment with the positive control (20% Imidazole) revealed an IVIS of 117.1 (study acceptance criteria range: 80.1 – 133.1).

 

Therefore, the study fulfilled the acceptance criteria.The IVIS obtained after treatment with the test item was 0.4 and, thus, lower than 3,i.e.according to OECD 437 the test item is not requiring classification for eye irritation or serious eye damage (UN GHS: No Category).

 

Conclusion

Under the conditions of the present study, the test item did not show an eye hazard potential. The test item is not requiring classification for eye irritation or serious eye damage (UN GHS: No Category).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin irritation

Based on the provided information there is no need for classification for skin irritation according to the EU Regulation (EC) No 1272/2008 on Classification Labelling and Packaging of Substances and Mixtures.

 

Eye irritation

Based on the provided information there is no need for classification for eye irritation according to the EU Regulation (EC) No 1272/2008 on Classification Labelling and Packaging of Substances and Mixtures.