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

Endpoint summary

Description of key information

The test item is not considered to be irritating to skin (reference 7.3.1-1).

The test item is not considered to be irritation to eye (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:

2017-08-16 to 2017-08-18

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:

2012

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

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

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”.

 

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.

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.

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:

2017-11-10

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:

2017-10-09

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:

2017-02-14

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Odenwaldschlachthof Brensbach, 64395 Brensbach, Germany
- Number of animals:
- Characteristics of donor animals: age: 15 - 29 months, cornea diameter: 24 – 26 mm
- Storage, temperature and transport conditions of ocular tissue: 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.
- Time interval prior to initiating testing: Corneas were prepared immediately after delivery of the eyes to the laboratory, testing started on the day of collection
- Indication of any existing defects or lesions in ocular tissue samples: Any corneas that showed macroscopic tissue damage (e.g. scratches, pigmentation, neovascularization) or an opacity >7 opacity units were discarded.
- Indication of any antibiotics used: Streptomycin and Penicillin were added in transport medium (5 mL/ 500 mL HBBS).
- Selection and preparation of corneas: The corneas were carefully removed from the eyes but a rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. All corneas used in the experiment were collected in incubation medium (EMEM, pre-warmed at 32 +/- 1 °C). Each cornea was mounted in a cornea holder with the endothelial side against the sealing ring (O -ring) of the posterior part of the holder. The anterior part of the holder was positioned on top of the cornea. Both compartments of the holder were filled with incubation medium (EMEM). The posterior compartment was filled first to return the cornea to its natural convex form. For equilibration, the corneas in the holder were incubated (incubator: Grumbach BSS 160) in a vertical position at 32 ± 1 °C for about one hour. At the end of the incubation period, the incubation medium (EMEM) was removed from both compartments and replaced by fresh incubation medium (EMEM).
- Quality check of the isolated corneas: The baseline opacity was determined with an opacitometer (BASF-OP2.0). The opacitometer measured the light transmission passing through the corneas and displayed a lux value. This value is converted into an opacity value. Three corneas were selected as negative control corneas. The remaining corneas were distributed into treatment and positive control groups.

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

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

NEGATIVE / VEHICLE CONTROL: 750 µL, 0.9 % sodium chloride solution

POSITIVE CONTROL: 750 µL, Imidazole was dissolved with 0.9 % sodium chloride solution to a concentration of 20 % (w/v).

Duration of treatment / exposure:

240 minutes

Duration of post- treatment incubation (in vitro):

Incubation with fluorescein solution: 90 min

Number of animals or in vitro replicates:

3 replicates in all groups

Details on study design:

TREATMENT METHOD: closed chamber / open chamber, not specified

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 3

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity was determined with an opacitometer (BASF-OP2.0). The change in opacity was calculated by subtracting the initial baseline opacity from the post treatment opacity. In addition, the opacity values of treatment and positive control groups were corrected for the man negative control opacity value.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of spectrophotometry (BioTek ELx800) at 490 nm (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: according to TG

Irritation parameter:

in vitro irritation score

Run / experiment:

Run 1 / Experiment 1

Value:

0.1

Vehicle controls validity:

valid

Negative controls validity:

not applicable

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 0.7 and, thus, within three standard deviations of the current historical mean of the negative control (IVIS: -1.5 – 3.2).
- Acceptance criteria met for positive control: After treatment with the positive control (20 % Imidazole) the calculated IVIS was 114.6 and, thus, within two standard deviations of the current historical mean of the positive control (IVIS: 81.5 – 132.9).

		Opacity	Permeability	IVIS
				per cornea	per group (mean value)	SD
Negative control	0.9 % NaCl Solution	1.5	-0.003	1.455	0.7	0.7
		0.2	-0.003	0.155
		0.6	-0.003	0.555
Positive control	20 % Imidazole solution	81.5	2.182	114.230	114.6	3.7
		67.8	2.884	111.060
		66.5	3.549	118.385
Test item	Test item	1.7	0.002	1.730	0.1	1.5
		-1.0	0.042	-0.370
		-1.2	0.004	-1.140

 

Interpretation of results:

GHS criteria not met

Conclusions:

The test item did not show an eye hazard potential.

Executive summary:

A study according to OECD TG 437 was conducted 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. 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). After treatment with the negative control the calculated IVIS was 0.7 (study acceptance criteria range: -1.5 – 3.2). Treatment with the positive control revealed an IVIS of 114.6 (study acceptance criteria range: 81.5 – 132.9). Therefore, the study fulfilled the acceptance criteria. The IVIS obtained after treatment with the test item was 0.1 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). Under the conditions of the present study, the test item did not show an eye hazard potential.

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 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.

 

Eye irritation

A study according to OECD TG 437 was conducted 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. 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). After treatment with the negative control the calculated IVIS was 0.7 (study acceptance criteria range: -1.5 – 3.2). Treatment with the positive control revealed an IVIS of 114.6 (study acceptance criteria range: 81.5 – 132.9). Therefore, the study fulfilled the acceptance criteria. The IVIS obtained after treatment with the test item was 0.1 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). Under the conditions of the present study, the test item did not show an eye hazard potential.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008.

Based on available data on skin irritation/corrosion, the test item does not require classification for causing skin irritation or corrosion according to Regulation (EC) No 1272/2008 (CLP).

Based on available data on eye irritation/corrosion, the test item does not require classification according to Regulation (EC) No 1272/2008 (CLP).