2-ethyl-N-(3-methoxyphenyl)-N-methylbutanamide

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

The study was conducted between 10 May 2018 and 10 May 2018.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is considered to be reliability 1 as it has been conducted according to OECD Test Guideline 437 using the Bovine Corneal Opacity and Permeability Assay method and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

This deviation is thought not to affect the purpose or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-ethyl-N-(3-methoxyphenyl)-N-methylbutanamide

Cas Number:

2101947-22-0

Molecular formula:

C14H21NO2

IUPAC Name:

2-ethyl-N-(3-methoxyphenyl)-N-methylbutanamide

Test material form:

liquid

Specific details on test material used for the study:

Identification: FRET 15-0735
Physical state/Appearance:Clear colourless liquid
Storage Conditions:Room temperature in the dark

Test animals / tissue source

Species:

other: Eyes from adult cattle

Strain:

other: Not applicable

Details on test animals or tissues and environmental conditions:

Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

0.75 mL of the test item or control items were applied to the cornea.

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

Triplicate

Details on study design:

Preparation of Corneas
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

Selection of Corneas and Opacity Reading
The medium from both chambers of each holder was replaced with fresh complete EMEM.
A pre treatment opacity reading was taken for each cornea using a calibrated opacitometer.
Three corneas were randomly allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

Treatment of Corneas
The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 10 minutes.
At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post treatment opacity reading was taken and each cornea was visually observed.
The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes.
After incubation the holders were removed from the incubator, the medium from both chambers was replaced with fresh complete EMEM and a final opacity reading was taken. Each cornea was visually observed.

Application of Sodium Fluorescein
Following the final opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (4 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.

Permeability Determinations
After incubation the medium in the posterior chamber of each holder was decanted and retained.
360 µL of media representing each cornea was dispensed into the appropriate wells of a pre labeled 96 well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.

Histopathology
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.
No histopathology was required for this study.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

10.8

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The positive control In Vitro Irritancy Score was within the range of 31.6 to 58.7. The positive control acceptance criterion was therefore satisfied.
The negative control gave opacity of ≤3.0 and permeability ≤0.077. The negative control acceptance criteria were therefore satisfied.

Any other information on results incl. tables

Corneal Epithelium Condition

The corneas treated with the test item were clear post treatment and slightly cloudy post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation.

 In Vitro Irritancy Score

The In Vitro irritancy scores are summarized as follows:

Treatment	In Vitro Irritancy Score
Test Item	10.8
Negative Control	0.7
Positive Control	53.4

Individual and Mean Corneal Opacity and Permeability Measurements

Treatment	Cornea Number	Opacity					Permeability (OD492)		In Vitro Irritancy Score
		Pre-Treatment	Post-Treatment	Post Incubation	Post-Incubation - Pre‑Treatment	Corrected Value		Corrected Value
Negative Control	2	2	3	2	0		0.008
	3	4	6	6	2		0.001
	6	2	3	2	0		0.003
					0.7*		0.004¨		0.7
Positive Control	7	3	36	36	33	32.3	2.330	2.326
	10	5	36	35	30	29.3	1.288	1.284
	11	5	37	34	29	28.3	1.083	1.079
						30.0·		1.563·	53.4
Test Item	12	2	5	14	12	11.3	0.045	0.041
	14	2	4	10	8	7.3	0.066	0.062
	15	2	4	14	12	11.3	0.052	0.048
						10.0·		0.050·	10.8

OD= Optical density       * = Mean of the post-incubation -pre‑treatment values           ¨= Mean permeability                     ·= Mean corrected value

Corneal Epithelium Condition Post Treatment and Post Incubation

Treatment	Cornea Number	Observation
		Post Treatment	Post Incubation
Negative Control	2	Clear	Clear
	3	Clear	Clear
	6	Clear	Clear
Positive Control	7	Cloudy	Cloudy
	10	Cloudy	Cloudy
	11	Cloudy	Cloudy
Test Item	12	Clear	Slightly Cloudy
	14	Clear	Slightly Cloudy
	15	Clear	Slightly Cloudy

Applicant's summary and conclusion

Interpretation of results:

other:

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

No prediction of eye irritation can be made on FRET 15-0735.

Executive summary:

The eye irritancy potential of FRET 15 -0735 was assessed according to OECD Test Guideline 437 using the Bovine Corneal Opacity and Permeability Assay method. Based on the results, no prediction of eye irritation can be made.