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

Endpoint summary

Description of key information

Skin corrosion (OECD TG 431): Non-corrosive

Skin irritation (OECD TG 439): Non-irritant

Eye irritation (OECD TG 437): No prediction

Eye irritation (OECD TG 492): Potential eye irritant

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:

THe study was conducted between 16 May 2018 and 28 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 439 using the EPISKIN™ Reconstructed Human Epidermis Model and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

These deviations were considered to have not affected the integrity or validity of the study.

GLP compliance:

yes (incl. QA statement)

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 system:

human skin model

Source species:

human

Cell type:

other: human-derived epidermal keratinocytes

Cell source:

other: no information

Source strain:

other: not applicable

Details on animal used as source of test system:

EPISKIN™ Reconstructed Human Epidermis Model Kit
Supplier: EpiSkin Laboratories, Lyon, France
Date received: 23 May 2018
EpiSkinTM Tissues (0.38cm2) lot number: 18-EKIN-021
Maintenance Medium lot number: 18-MAIN3-026
Assay Medium lot number: 18-ESSC-022

Justification for test system used:

The principle of the assay is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue/purple formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42 Hour post exposure incubation period may also be determined for test items which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complementary end point can be used to either confirm a non-irritant result or will be used to override the non irritant result.

Vehicle:

unchanged (no vehicle)

Details on test system:

Pre-Test Procedure
Assessment of Direct Test Item Reduction of MTT
MTT Salt Metabolism, Cell Viability Assay
The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue/purple formazan salt by mitochondrial succinate dehydrogenase in viable cells.
One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified by using killed tissues to act as controls.

Test for Direct MTT Reduction
As specified, a test item may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, the test item is checked for the ability to directly reduce MTT according to the following procedure:
10 µL of the test item was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control.
If the MTT solution containing the test item turns blue/purple, the test item is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and water killed tissues for quantitative correction of the results.

Assessment of Color Interference with the MTT endpoint
A test item may interfere with the MTT endpoint if it is colored. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed.
10 µL of test item was added to 90 µL of sterile water. After mixing for 15 minutes on a plate shaker a visual assessment of the color was made.

Pre-incubation (Day 0: Tissue Arrival)
Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert:
Tissues Satisfactory: Yes
Temperature Indicator Color Satisfactory: Yes
Agar Medium Color Satisfactory: Yes
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre labeled 12 well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37 °C, 5% CO2 in air overnight.

Main Test
Application of Test Item and Rinsing (Day 1)
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the second column of 3 wells of the 12 well plate.
Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. 10 µL (26.3 µL/cm2) of the test item was applied to the epidermis surface. Triplicate tissues treated with 10 µL of DPBS served as the negative controls and triplicate tissues treated with 10 µL of SDS 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the center). After a 7 Minute contact time the SDS solution was re spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period (re-spreading is not required for the negative control or test item). The plates were kept in the biological safety cabinet at room temperature for 15 minutes.
At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 C, 5% CO2 in air for 42 hours.

MTT Loading/Formazan Extraction (Day 3)
Following the 42 Hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre labeled micro tubes and stored in a freezer at 14 to 30 ºC for possible inflammatory mediator determination.
2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates. The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C, 5% CO2 in air. At the end of the 3 Hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKINTM biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labeled 1.5 mL micro tubes containing 500 µL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.
3.4.2.3 Absorbance/Optical Density Measurements (Day 6)
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous colored solution.
For each tissue, duplicate 200 µL samples were transferred to the appropriate wells of a pre labeled 96 well plate. 200 µL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density (OD570) was measured (quantitative viability analysis) at 570 nm (without a reference filter) using the Labtech LT 4500 microplate reader.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

10 µL of the test item
10 µL of DPBS (negative control)
10 µL of SDS 5% w/v (positive controls)

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

Triplicate

Irritation / corrosion parameter:

other: relative mean viability (%)

Run / experiment:

Mean

Value:

105.2

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The test was repeated due to a failure to meet the assay acceptance criteria. The relative mean tissue viability for the positive control treated tissues was 10.5% relative to the negative control treated tissues and the standard deviation value of the viability was 7.7%. The positive control acceptance criteria were therefore satisfied.
The mean OD570 for the negative control treated tissues was 1.065 and the standard deviation value of the viability was 10.5%. The negative control acceptance criteria were therefore satisfied.
The standard deviation calculated from individual tissue viabilities of the three identical test item treated tissues was 8.8%. The test item acceptance criterion was therefore satisfied.

Direct MTT Reduction

The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT. 

Assessment of Color Interference with the MTT endpoint

The solution containing the test item was colorless. It was therefore unnecessary to run color correction tissues.

Test Item, Positive Control Item and Negative Control Item

It was considered unnecessary to perform IL-1α analysis as the results of the MTT test were unequivocal.

Mean OD₅₇₀Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD570 of tissues	Mean OD570 of triplicate tissues	±SD of OD570	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	1.168	1.065	0.112	109.7	100*	10.5
	0.946			88.8
	1.081			101.5
Positive Control Item	0.206	0.112	0.082	19.3	10.5	7.7
	0.052			4.9
	0.078			7.3
Test Item	1.220	1.120	0.093	114.6	105.2	8.8
	1.105			103.8
	1.035			97.2

OD = Optical Density

SD = Standard deviation

* = The mean viability of the negative control tissues is set at 100%

Interpretation of results:

other:

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

The relative mean viability of FRET 15-0735 treated tissues was 105.2% after the 15 Minute exposure period and 42 Hours post exposure incubation period. Since the mean relative tissue viability for the substance was above 50% after 15 minutes treatment, the substance is considered to be non- irritant.

Executive summary:

The possible skin irritation potential of the substance was tested through topical application for 15 minutes. The study procedures described in this report were based on the OECD TG 439. Skin tissue was treated by topical application of 10 µL undiluted test substance. After 42 hours incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) MTT at the end of treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean viability of FRET 15-0735 treated tissues was 105.2% after the 15 Minute exposure period and 42 Hours post exposure incubation period. Since the mean relative tissue viability for the substance was above 50% after 15 minutes treatment, the substance is considered to be non- irritant. The relative mean tissue viability for the positive control treated tissues was 10.5% relative to the negative control treated tissues and the standard deviation value of the viability was 7.7%. The positive control acceptance criteria were therefore satisfied.

The mean OD570 for the negative control treated tissues was 1.065 and the standard deviation value of the viability was 10.5%. The negative control acceptance criteria were therefore satisfied.

The standard deviation calculated from individual tissue viabilities of the three identical test item treated tissues was 8.8%. The test item acceptance criterion was therefore satisfied.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

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.

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)

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

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.

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.

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.

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

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.

Reference 2

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 27 June 2018 and 05 July 2018.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other:

Remarks:

The study is considered to be a reliability 1. The study is considered reliable and according to the SkinEthic Reconstructed Human Corneal Epithelial Model and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Deviations:

yes

Remarks:

This deviation was considered to have not affected the integrity or validity of the study.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: FRET 15-0735
Appearance: Colourless to yellowish*, liquid
Storage Conditions: At room temperature, protected from light

Species:

other: Reconstructed Human Corneal Epithelial Model

Strain:

other: Not applicable

Details on test animals or tissues and environmental conditions:

EpiOcular Kit Components Needed for the Assay
Lot No.: 27056
Sealed 24-well plate Contains 12/24 inserts with EpiOcular™ tissues on agarose
Serum-free test medium DMEM-Medium
Positive control Methyl Acetate (CAS#79-20-9)
12-well plate Holding plate
24-well plates For MTT viability assay
6-well plates For storing inserts, or for topically applying test agents
Ca++Mg++-Free DPBS Dulbecco´s Phosphate Buffered Saline

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Test and control items: 50 µL

Duration of treatment / exposure:

30 minutes

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

Duplicate

Details on study design:

Test Item Preparation
50 µL (83.3 µL/cm² according to guideline) of the test item were dispensed directly atop duplicate EpiOcular™ tissue for 30 minutes.

MTT-100 Assay Kit Components
1 vial, 2 mL MTT concentrate
1 vial, 8 mL MTT diluent (supplemented DMEM) For diluting MTT concentrate prior to use in the MTT assay
1 bottle, 60 mL Extractant solution (Isopropanol) For extraction of formazan crystals

Cell Culture
EpiOcular™ kits and MTT-100 kits were purchased from MatTek Corporation (82105 Bratislava, Slovakia). The EpiOcular™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. The EpiOcular™ tissues (surface 0.6 cm²) were cultured on specially prepared cell culture inserts (MILLICELL®, 10 mm Ø).
EpiOcular™ tissues were received at 2 - 8 °C on medium-supplemented agarose gels in a 24-well plate. On day of receipt (03 July 2018) of the EpiOcular™ tissues, the equilibration step (15 minutes at room temperature in the 24-well shipping container) started. 1.0 mL of the medium was aliquoted into the appropriate wells of pre-labelled 6-well plates.
Each 24-well shipping container was removed from its plastic bag under sterile conditions and its surface disinfected by wiping with 70% isopropanol- or ethanol-soaked tissue paper. The sterile gauze was removed and each tissue was inspected for air bubbles between the agarose gel and insert. Cultures with air bubbles under the insert covering greater than 50% of the insert area were not used. The tissues were carefully removed from the 24-well shipping containers using sterile forceps. Any agarose adhering to the inserts was removed by gentle blotting on sterile filter paper or gauze. The insert was then transferred aseptically into the 6-well plates and pre-incubated at standard culture conditions for one hour in the Assay Medium. After one hour, the Assay Medium was replaced by 1 mL fresh Assay Medium at 37 °C and the EpiOcular™ tissues were incubated at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5% CO2) overnight (about 18 hours).

MTT-Solution
On the day of the experiment a MTT solution 1.0 mg/mL in DMEM was prepared.

Assessment of Direct MTT Reduction by the Test Item
Test items may have the ability to directly reduce MTT and to form a blue/purple reaction product which could have an impact on the quantitative MTT measurement. Therefore, it was necessary to assess this ability for the test item prior to conducting any assays with viable tissues. For this purpose approximately 50 µl of the test item were added to a 1 mL of a 1.0 mg/mL MTT solution (in DMEM) in a glass tube and the mixture was incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air for three hours. A control (50 µL of deionised water in 1 mL of 1.0 mg/mL MTT solution) was performed concurrently. If the MTT solution colour turned blue/purple, the test item will be presumed to have reduced the MTT.
Since the MTT solution colour did not turn blue/purple, the test item is not presumed to be a MTT reducer. An additional test with freeze-killed tissues did not have to be performed.
 
Assessment of Coloured or Staining Materials
Coloured test items or test items which become coloured after application to the tissues could interfere with the quantitative photometric MTT measurement if the colorant bound to the tissue and would be extracted together with MTT. Therefore, each test item had to be checked for its colouring properties.
Since the test item was coloured (yellowish), it had to be tested for its ability to absorb significantly light at the wavelength used for the MTT determination. 50 µL of the test item were added to 2 mL of isopropanol, incubated in a glass tube, and placed on an orbital plate shaker for 2 to 3 hours at room temperature. Two 200 µL aliquots of isopropanol solutions and of pure isopropanol were transferred to a 96-well plate and the absorbance was measured with a plate reader at the MTT measurement wavelength.
Since the test item did not dye water or isopropanol and the OD of the test item solution was not > 0.08, additional tests with viable tissues did not have to be performed.
 
EXPERIMENTAL DESIGN AND STUDY CONDUCT
Experimental Performance
After the overnight incubation, the tissues were pre-wetted with 20 µL of Ca++Mg++free-DPBS. The tissues were incubated at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5% CO2) for 30 minutes.
After the 30 minute Ca++Mg++free-DPBS pre-treatment, the test and control items were tested by applying 50 µL topically on the EpiOcular™ tissues. The tissues were incubated at standard culture conditions for 30 minutes.
At the end of the 30 minutes treatment time, the test item was removed by extensively rinsing the tissues with Ca++Mg++-free DPBS (brought to room temperature). Three clean beakers containing a minimum of 100 mL each of Ca++Mg++-free DPBS were used per test item. Each test item utilized a different set of three beakers. The inserts containing the tissues were lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. To assure throughput, the tissues were rinsed two at a time by holding replicate inserts together by their collars using forceps. The test or control items were decanted from the tissue surface onto a clean absorbent material and the cultures dipped into the first beaker of DPBS, swirled in a circular motion in the liquid for approximately 2 seconds, lifted out so that the inserts were mostly filled with DPBS, and the liquid was decanted back into the container. This process was performed two additional times in the first beaker. The culture was then rinsed in the second and third beaker of DPBS three times each in the same fashion. Finally, any remaining liquid was decanted onto the absorbent material by rotating the insert to approximately a 45° angle (open end down) and touching the upper lip to the absorbent material (to break the surface tension).
After rinsing, the tissues were immediately transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a pre-labelled 12-well plate for a 12 minute immersion incubation (post-soak) at room temperature. This incubation in Assay Medium was intended to remove any test item absorbed into the tissue.
At the end of the post-soak immersion, each insert was removed from the Assay Medium, the medium was decanted off the tissue, and the inserts were blotted on absorbent material, and transferred to the appropriate well of the pre-labelled microtiter plate containing 1 mL of warm Assay Medium. The tissues were incubated for 120 minutes at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 (post-treatment incubation).

MTT Assay
At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into the 24-well plate containing 0.3 mL of MTT solution. Once all the tissues were placed into the 24-well plate, the plate was incubated for 180 minutes at standard culture conditions.
Each insert was removed from the 24-well plate after 180 minutes, the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labelled 24-well plate containing 2.0 mL of isopropanol in each designated well so that isopropanol was flowing into the insert on the tissue surface. The plates were sealed with a standard plate sealer, and were stored overnight at 2-8 °C in the dark. At the end of the extraction period, the tissue was pierced and the liquid within each insert was decanted into the well from which it was taken.
The extract solution was mixed and two 200 µL aliquots were transferred to the appropriate wells of a pre-labelled 96-well plate.
The absorbance at 570 nm (OD570) of each well was measured with a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro Enterprise, version 4.7.1). No reference wavelength measurement was used.

Data Recording
The data generated were recorded in the laboratory protocol. The results are presented in tabular form, including experimental groups with the test item and the controls.

Irritation parameter:

other: Mean relative viability (%)

Run / experiment:

Mean

Value:

29.51

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water or isopropanol did not lead to a change in colour.
Optical evaluation of the MTT-reducing capacity of the test item with MTT-reagent did not show blue/purple colour.
The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 29.51% (threshold for irritancy: ≤ 60%), consequently the test item was irritant to eye.
Concerning acceptance criteria:
• The negative control OD is > 0.8 and < 2.5 (2.305 and 2.445).
• The mean relative viability of the positive control is below 50% of the negative control viability (39.09%).
• The difference of viability between the two relating tissues of a single item is < 20% (values between 1.31% and 4.19%) in the same run (for positive and negative control tissues and tissues of single test items).

Results after treatment for 30 minutes withFRET 15-0735and the controls

Treatment Group	Tissue No.	OD 570 nm Well 1	OD 570 nm Well 2	Mean OD of 2 Wells	Mean OD of 2 Wells blank corrected	Mean OD of Treatment Group blank corrected	Rel. Viability [%] Tissue 1, 2 *	Absolute Value of the Difference of Rel. Viability  Tissue 1,2[%]	Mean Rel. Viability [%]**
Blank		0.037	0.036	0.036
Negative Control	1	2.340	2.305	2.322	2.286	2.333	98.0	4.01	100.0
	2	2.387	2.445	2.416	2.380		102.0
Positive Control	1	0.878	0.920	0.899	0.863	0.912	37.0	4.19	39.09
	2	0.978	1.016	0.997	0.961		41.2
Test Item	1	0.746	0.734	0.740	0.704	0.688	30.2	1.31	29.51
	2	0.715	0.704	0.709	0.673		28.9

*                      Relative viability [rounded values]: ((100 x (absorbance _{test item/positive control/negative control}))/mean absorbance _{negative control})

 **                    Mean relative viability [rounded value]:

((100 x (absorbance_{test item/positive control/negative control}))/mean absorbance_{negative control})

Interpretation of results:

other:

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, FRET 15-0735 possesses an eye irritating potential.

Executive summary:

This in vitro study was performed to assess the eye irritation potential of FRET 15-0735 by means of the Human Cornea Model Test.

The test item did not prove to be an MTT reducer in the MTT pre-test. Also, its intrinsic colour was not intensive and it did not prove to dye water or isopropanol in the colour interference pre-test. Therefore, additional tests with freeze-killed or viable tissues did not have to be performed.

Each 50 µL of the test item, the negative control (deionised water) or the positive control (methyl acetate) were applied to each of duplicate tissues for 30 minutes.

After treatment with the negative control, the absorbance values were well within the required acceptability criterion of OD > 0.8 and < 2.5, thus showing the quality of the tissues.

Treatment with the positive control induced a decrease below 50% viability compared with the negative control value in the relative absorbance, thus ensuring the validity of the test system.

The difference of viability between the two relating tissues was < 20% in the same run (for test item tissues, positive and negative control tissues).

Irritating effects were observed following incubation with FRET 15-0735. Compared with the value of the negative control, the relative mean absorption value corresponding to the viability of the tissues decreased below 60% (determined value for the test item: 29.51%).

In conclusion, it can be stated that in this study and under the experimental conditions reported, FRET 15-0735 possesses an eye irritating potential.

Additional information

In vitro tests

Skin irritation:

The skin corrosivity of the test substance was determined according to OECD Guideline 431 using the EpiDerm™ Human Skin Model.

The relative mean viabilities for each treatment group were as follows:

Exposure Period	Percentage Viability
	Negative Control	Positive Control	Test Item
3 minute	100*	3.8	92.6
60 minute	100*	3.0	92.7

*The mean viability of the negative control tissues is set at 100%

Quality criteria: The quality criteria required for acceptance of results in the test were satisfied.

Therefore the substance is not corrosive to skin, according to EU CLP criteria.

Skin irritation:

The possible skin irritation potential of the substance was tested through topical application for 15 minutes. The study procedures described in this report were based on the OECD TG 439. Skin tissue was treated by topical application of 10 µL undiluted test substance. After 42 hours incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) MTT at the end of treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean viability of FRET 15-0735 treated tissues was 105.2% after the 15 Minute exposure period and 42 Hours post exposure incubation period. Since the mean relative tissue viability for the substance was above 50% after 15 minutes treatment, the substance is considered to be non- irritant. The relative mean tissue viability for the positive control treated tissues was 10.5% relative to the negative control treated tissues and the standard deviation value of the viability was 7.7%. The positive control acceptance criteria were therefore satisfied.

The mean OD570 for the negative control treated tissues was 1.065 and the standard deviation value of the viability was 10.5%. The negative control acceptance criteria were therefore satisfied.

The standard deviation calculated from individual tissue viabilities of the three identical test item treated tissues was 8.8%. The test item acceptance criterion was therefore satisfied.

Eye irritation (BCOP):

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.

Eye irritation (OECD 492):

This in vitro study was performed to assess the eye irritation potential of FRET 15-0735 by means of the Human Cornea Model Test.

The test item did not prove to be an MTT reducer in the MTT pre-test. Also, its intrinsic colour was not intensive and it did not prove to dye water or isopropanol in the colour interference pre-test. Therefore, additional tests with freeze-killed or viable tissues did not have to be performed.

Each 50 µL of the test item, the negative control (deionised water) or the positive control (methyl acetate) were applied to each of duplicate tissues for 30 minutes.

After treatment with the negative control, the absorbance values were well within the required acceptability criterion of OD > 0.8 and < 2.5, thus showing the quality of the tissues.

Treatment with the positive control induced a decrease below 50% viability compared with the negative control value in the relative absorbance, thus ensuring the validity of the test system.

The difference of viability between the two relating tissues was < 20% in the same run (for test item tissues, positive and negative control tissues).

Irritating effects were observed following incubation with FRET 15-0735. Compared with the value of the negative control, the relative mean absorption value corresponding to the viability of the tissues decreased below 60% (determined value for the test item: 29.51%).

In conclusion, it can be stated that in this study and under the experimental conditions reported, FRET 15-0735 possesses an eye irritating potential.

Justification for selection of skin/eye irritation / corrosion endpoint:
The result of the study is reliable and adequate for covering the endpoint.

Justification for classification or non-classification

Based on the negative results in the skin irritation tests the substance does not need to be classified for this endpoint according to EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 and according to EU Directive 67/548/EEC (DSD).