4-ethoxy-2,3-difluoro-4'-propyl-1,1'-biphenyl

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2013-02-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

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:

2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2009-09-01

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-ethoxy-2,3-difluoro-4'-propyl-1,1'-biphenyl

EC Number:

638-734-4

Cas Number:

157248-24-3

Molecular formula:

C₁₇H₁₈F₂O

IUPAC Name:

4-ethoxy-2,3-difluoro-4'-propyl-1,1'-biphenyl

Test material form:

solid

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: from slaughterhouse (Odenwaldschlachthof Brensbach, Brensbach, Germany)
- Number of animals: not specified
- Characteristics of donor animals: not specified
- Storage, temperature and transport conditions of ocular tissue: The eyes were kept and transported in transport medium (HBSS, Hanks’ Balanced Salt Solution) with added Streptomycin and Penicillin (5 mL/ 500 mL HBSS). No further details provided.
- Time interval prior to initiating testing: Eyes were freshly isolated from donor cattle and were transported and immediately after delivery corneas were prepared.
- Indication of any existing defects or lesions in ocular tissue samples: Eyes were carefully examined macroscopically for defects (like vascularization, pigmentation, opacity or scratches). Those with defects were discarded. Furthermore, corneas were macroscopically checked after preparation and tissues with damages (e.g. scratches, pigmentation, neovarcularization) or opacity > 7 opacity units were discarded.
- Indication of any antibiotics used: Streptomycin and Penicillin were added in transport medium.
- 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 °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 with opacity values close to the median value for all corneas were selected as negative control corneas. The remaining corneas were distributed into treatment and positive control groups.

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 750 µL
- Concentration: 20 %

VEHICLE
- Amount applied: 750 µL
- Concentration: 0.9 %
- Lot No: 12084014

POSITIVE CONTROL
- Amount applied: 750 µL
- Concentration: 20 %

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: at least 3 times (or until no visual evidence of the test substance was observed)

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.
- 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

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

-0.8

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.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative (vehicle) control: Yes, the calculated IVIS was 1.2 and, thus, within two standard deviations of the historical mean (IVIS: -0.6 to 3.9).
- Acceptance criteria met for positive control: Yes, the calculated IVIS was 81.6 and, thus, within two standard deviations of the historical mean (IVIS:76.6 to 142.7).

Any other information on results incl. tables

		Opacity	Permeability	IVIS
				per cornea	per group (mean value)	SD
Negative Control	0.9 % NaCl Solution	0.258	0.002	0.293	1.2	1.5
		2.907	-0.001	2.897
		0.364	0.000	0.369
Positive Control	20 % Imidazole solution	52.926	1.679	78.116	81.6	12.3
		49.082	1.494	74.492
		63.143	2.144	95.298
Test material	Test item	-1.962	0.000	-1.957	-0.8	1.0
		-0.182	-0.001	-0.192
		-0.178	-0.002	-0.208

Applicant's summary and conclusion

Interpretation of results:

other: not corrosive, not severe irritant

Conclusions:

Under the conditions of the present study, the test item did not show an ocular corrosive or severe irritant potential in this in vitro assay.

Executive summary:

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 and was conducted according to OECD 437. 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 1.2 and, thus, within two standard deviations of the historical mean (IVIS: -0.6 to 3.9). Treatment with the positive control (20 % Imidazole) revealed an IVIS of 81.6 and was thus, within two standard deviations of the historical mean (IVIS:76.6 to 142.7). Therefore, the study fulfilled the validity criteria. The IVIS obtained after treatment with test item was -0.8 and, thus, lower than 55. Under the conditions of the present study, the test item did not show an ocular corrosive or severe irritant potential in this in vitro assay.