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

Endpoint summary

Description of key information

Skin Irritation

In a study according OECD TG 439 the test item is not considered to possess an irritant potential to skin (reference 7.3.1 -1).

Eye Irritation

In a study according to OECD TG 437 the test item did not show an eye hazard potential (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:

July 17, 2017 - October 10, 2017

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:

Commission Regulation (EU) No. 640/2012 amending, for the purpose of its adaption to technical progress, Regulations (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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 28, 2015

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:

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: EpiSkin/SkinEthic Laboratories, Lyon, France
- Tissue batch number: 17-RHE-086

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:
After the end of the treatment interval, the residual test item was removed immediately by gently rinsing with a minimum volume of 25 mL DPBS using a pipette. Excess DPBS was removed by gently shaking the inserts and blotting the bottom with blotting paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: ELx800, BioTek Instruments GmbH, Bad Friedenshall, Germany
- Wavelength: 570 nm
- Linear OD range of spectrophotometer: 0 - 3.0

FUNCTIONAL MODEL CONDITIONS
- Viability: OD 1.4 (CV =4.5%)
- Barrier function: 4.3 h
- Morphology: 6 cell layers, absence of significant histological abnormalities, well differentiated epidermis consisting of basal, spinous, granular layers and a stratum corneum
- Contamination: no

NUMBER OF REPLICATE TISSUES: 3

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

PREDICTION MODEL / DECISION CRITERIA
A test item is considered as non-irritant to skin (UN GHS No Category) if the tissue viability after exposure and post-treatment incubation is > 50%. A test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post-treatment incubation is < 50%. Since the in vitro skin irritation test according to OECD 439 cannot resolve between UN GHS Categories 1 and 2, further information on skin corrosion is required to decide on its final classification.

Acceptability of the Quality Control Data of the Skin Model with Reference to Historical Batch Data:
The negative control OD values for the RHE-model have to be in the range of ≥0.8 and ≤3.0 as given in OECD Guideline 439.

Acceptability of the Positive and Negative Control stated by Episkin/SkinEthic Laboratories:
The negative control data meet the acceptance criteria if the mean OD value of the 3 tissues is ≥ 1.2 at 570 nm.The standard deviation value is considered valid if ≤ 18% of group mean-value.
The positive control data meet the acceptance criteria if the mean viability value, expressed as % of the negative control, is < 40%.The standard deviation value is considered valid if ≤ 18% of group mean-value.

Acceptability of the Positive and Negative Control based on Historical Data of the Testing Laboratory:
The negative control data meet the acceptance criteria if the mean OD value is higher or equal than a historically established boundary at 570 nm. The boundary is two standard deviations below the current historical mean (1.463).
The positive control data meet the acceptance criteria if the mean viability value, expressed as % of the negative control, is lower than or equal to a historically established boundary. The boundary is three standard deviations above the current historical mean (2.98%).

Test Substance Data Acceptance Criteria
The standard deviation of the three tissues treated with the test item should be ≤18%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 16 mg of solid test material

NEGATIVE CONTROL
- Amount(s) applied: 16 µL

POSITIVE CONTROL
- Amount(s) applied: 16 µL
- Concentration: 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

Value:

96.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

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:

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 ≥ 1.455 2.019
Mean viability positive control ≤ 2.97% 1.0%

Test Item Data Acceptance Criteria:

Acceptance Criterion Result
SD of group-mean value ≤ 18% 5.9%

The study met all acceptance criteria.

Table 1:

Group	Time / [min]	Mean OD	Mean Relative viability / [%]
Negative Control	42	2.019	100
Positive Control	42	0.021	1.0
Test Material	42	1.957	96.9

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

Executive summary:

A study 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 96.9% 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).

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:

November 13, 2017 - January 10, 2018

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 i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

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) 2017/735 of 14 February 2017 amending, for the purpose of its adaption to technical progress, the Annex to 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
- Amount(s) applied: 750 µL (150 mg/750 µL)
- Concentration: The test item was prepared as a 20% (w/v) suspension in a 0.9% sodium chloride solution. The stability in the vehicle was not investigated. The test item preparation was administered within <1 hour after preparation.

VEHICLE
- Amount(s) applied: 750 µL
- Concentration: 0.9% sodium chloride solution

Duration of treatment / exposure:

240 minutes

Number of animals or in vitro replicates:

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

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Freshly isolated bovine eyes of cattle were collected from the slaughterhouse. Excess tissue was removed from the eyes. The eyes were kept and transported in transport medium cooled on ice. The corneas were prepared immediately after delivery of the eyes to the laboratory. All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity or scratches were discarded. The corneas were carefully removed from the eyes using scalpel and rounded scissors. A rim of about 2 to 3 mm of tissue (sclera) was left for stability and handling of the isolated cornea. All corneas used in the study were collected in incubation medium (pre-warmed at 32 ± 1°C) and the corneal diameter of each cornea was measured and recorded. Each cornea was mounted in a cornea holder (CiToxLAB, Veszprém, Hungary) with the endothelial side against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring without stretching the cornea. Afterwards, the anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex form.

QUALITY CHECK OF THE ISOLATED CORNEAS
For equilibration, the corneas in the holder were incubated (BSS 160, Grumbach Brutgeräte GmbH, Asslar, Germany) in a vertical position at 32 ± 1°C for about one hour. At the end of the incubation period, the incubation medium was replaced by fresh pre-warmed (32 ± 1°C) incubation medium in both compartments. The baseline opacity was determined with a calibrated opacitometer (BASF-OP2.0, BASF SE, Ludwigshafen, Germany). The light transmission through the corneas, given as lux value, was recorded in a table and thereafter converted into an opacity value (baseline opacity values). Any corneas that showed macroscopic tissue damage (e.g.scratches, pigmentation, neovascularization) or an opacity >7 opacity units were discarded. Three corneas were selected as negative control corneas. The remaining corneas were distributed into treatment and positive control group.

NUMBER OF REPLICATES
Three corneas were used per group (negative control, positive control or test item group). Therefore, a total number of 9 corneas was used in this study.

NEGATIVE CONTROL USED
Negative/Vehicle Control: 0.9% sodium chloride solution, B. Braun Melsungen AG, Batch: 16455011,

POSITIVE CONTROL USED
Imidazole was dissolved with 0.9% sodium chloride solution to a concentration of 20% (w/v). Imidazole, Merck KGaA, Batch S6746923, Purity (GC): 99.8% (a/a)

APPLICATION DOSE AND EXPOSURE TIME
Fresh incubation medium was filled into the posterior compartment, while the surface of the cornea in the anterior compartment was treated with 750 µL of either the test item preparation, negative or positive control. The negative and positive control preparations were introduced with the closed-chamber method through the dosing holes of the chamber, whereas the test item was applied to the anterior chamber with the open-chamber method. For this, the window locking-ring and the glass window were removed from the anterior chamber prior to treatment. After application, the corneas were incubated in an incubator in a horizontal position at 32 ± 1°C for 240 minutes.

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: none

REMOVAL OF TEST SUBSTANCE
After the incubation period, the negative and positive control preparations were removed from the anterior chamber without opening the chamber. The corneal surface was washed three times with wash medium. Incubation medium was used as final rinse to ensure removal of the phenol red from the anterior chamber prior to the opacity measurement. Fresh incubation medium was replaced in both compartments prior to reading the opacity value after treatment. To remove the test item from the epithelium, the window locking-ring and the glass window from the anterior chamber were removed. The corneas were gently rinsed with wash medium using a syringe. Before measurement of the opacity value after treatment, fresh incubation medium was replaced in both compartments.

- POST-EXPOSURE INCUBATION:
none

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacitometer
- Corneal permeability: An increased permeability of the cornea is indicative for an impairment of the integrity of the corneals’ epithelial cell layers. To determine the permeability of the treated corneas, fresh incubation medium was added to the posterior compartments and 1 mL of a fluorescein solution was administered to the anterior compartments. The corneas were incubated again in an incubator in a horizontal position at 32 ± 1°C for 90 minutes. The amount of fluorescein that crossed the cornea was measured spectrophotometrically in the medium from the posterior chamber.

- Others: Each cornea was observed visually and pertinent observations were recorded (e.g.tissue peeling, residual test chemical, non-uniform opacity patterns).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The In Vitro Irritancy Score (IVIS) was calculated for each individual treatment and positive control cornea. The mean In Vitro Irritancy Score (IVIS) value of each treated group was calculated from the individual In Vitro Irritancy Score (IVIS) values.

DECISION CRITERIA: Decision criteria as indicated in the TG was used.
IVIS ≤ 3 results in no classification, IVIS >55 results in GHS-classification as “inducing serious eye damage, category 1”, IVIS between >3 and ≤55 results in ‘no prediction can be made’

Definition of Study Acceptance Criteria
A test is considered acceptable if the positive control gives an IVIS that falls within two standard deviations of the current historical mean (IVIS positive control: 81.7 – 132.9).
The negative control responses should result in an IVIS that falls within three standard deviations of the current historical mean (IVIS negative control: -1.4 – 3.2).
A single test run with three corneas should be sufficient for a test item when the resulting classification is unequivocal. In cases of the following borderline results in the first testing run, a second test run should be considered.
- 2 of the 3 corneas give discordant predictions from the mean of all 3 corneas or
- 1 of the 3 corneas give discordant predictions from the mean of all 3 corneas, and the discordant result is >10 IVIS units from the cut-off threshold of 55

Irritation parameter:

in vitro irritation score

Run / experiment:

Experiment 1

Value:

0.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

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: yes
After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was 0.7 and, thus, within three standard deviations of the current historical mean of the negative control (IVIS: -1.4 – 3.2).
- Acceptance criteria met for positive control: yes
After treatment with the positive control (20% Imidazole) the calculated IVIS was 101.0 and, thus, also within two standard deviations of the current historical mean of the positive control (IVIS: 81.7 – 132.9).
Therefore, the study fulfilled the acceptance criteria.

		Opacity	Permeability	IVIS
				per cornea	per group (mean value)	SD
Negative control	0.9% NaCl Solution	0.9	-0.002	0.870	0.7	0.2
		0.7	-0.004	0.640
		0.5	-0.003	0.455
Positive control	20% Imidazole solution	59.0	2.671	99.065	101.0	9.3
		63.9	3.150	111.150
		64.4	1.897	92.855
Test item	Test item	-0.2	0.003	-0.155	0.6	1.0
		1.8	-0.001	1.785
		0.2	-0.001	0.185

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.

Executive summary:

A study according OECD TG 437 was conducted to determine the eye hazard potential of the test item. 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). After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was 0.7 (study acceptance criteria range: -1.4 – 3.2). Treatment with the positive control (20% Imidazole) revealed an IVIS of 101.0 (study acceptance criteria range: 81.7 – 132.9). Therefore, the study fulfilled the acceptance criteria. The IVIS obtained after treatment was 0.6 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

A study 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 96.9% 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).

Eye irritation

A study according OECD TG 437 was conducted to determine the eye hazard potential of the test item. 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). After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was 0.7 (study acceptance criteria range: -1.4 – 3.2). Treatment with the positive control (20% Imidazole) revealed an IVIS of 101.0 (study acceptance criteria range: 81.7 – 132.9). Therefore, the study fulfilled the acceptance criteria. The IVIS obtained after treatment with the substance was 0.6 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.

Justification for classification or non-classification

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

The available data is reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified for skin or eye irritation under Regulation (EC) No 1272/2008, as amended for the fourteenth time in Regulation (EU) No 2020/217.