Reaction mass of Rel-(3aR,4S,7R,7aS)-1-(methoxymethylene)octahydro-1H-4,7-methanoindene and Rel-(3aR,4S,7R,7aS)-2-(methoxymethylene)octahydro-1H-4,7-methanoindene

Endpoint summary

Description of key information

Skin corrosion (OECD TG 431): FRET 14 -0383 was considered to be non-corrosive to the skin.

Skin irritation (OECD TG 439): FRET 14 -0383 was considered to be non-irritant to the skin.

Skin irritation (OECD TG 404): FRET 14 -0383 was considered to be non-irritant to the skin

Eye irritation (OECD TG 437): FRET 14 -0383 was considered to be non-irritant to the eye.

Eye irritation (OECD TG 405): FRET 14 -0383 was considered as a mild irritant.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

The study was conducted between 21 February 2018 and 23 February 2018.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: FRET 14-0383
Envigo Description: Colorless liquid
Sponsor Description: Clear yellow liquid
Storage Conditions: Cold at ≈ 4°C in the dark

Test system:

human skin model

Source species:

human

Cell type:

other: epithelial

Cell source:

other: derived from human

Source strain:

other: derived from human

Details on animal used as source of test system:

EpiDerm™ Reconstructed Human Epidermis Model Kit
Supplier: MatTek
Date received: 20 February 2018
EpiDermTM Tissues (0.63cm2) lot number: 25882
Assay Medium lot number: 021518TMA
Upon receipt of the EpidermTM tissues, the sealed 24 well plate was stored in a refrigerator until use.

Justification for test system used:

Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.
This model incorporates several features, which make it advantageous in the study of potential dermal corrosivity. The target cells are epithelial, derived from human skin, and formed into a stratified, cornified epithelium. Test items are applied to the culture surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly.

Vehicle:

unchanged (no vehicle)

Details on test system:

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C, 5% CO2
- Temperature of post-treatment incubation (if applicable): 37 °C, 5% CO2

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: Labtech LT 4500 microplate reader
- Wavelength: 570 nm (OD570)

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

PREDICTION MODEL / DECISION CRITERIA
-The test substance is considered to be corrosive to skin if:
- the relative mean tissue viability (% of negative control) is < 50 after a 3 minute exposure
- the relative mean tissue viability (% of negative control) is ≥ 50 after a 3 minute exposure to the test substance AND < 15 after a 1 hour exposure to the test substance
- The test substance is considered to be non-corrosive to skin if:
- the relative mean tissue viability (% of negative control) is ≥ 50 after a 3 minute exposure to the test substance AND ≥ 15 after a 1 hour exposure to the test substance

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL of the test item

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL of sterile distilled water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL of 8.0 N Potassium Hydroxide

Duration of treatment / exposure:

3-min exposure period and 60-min exposure period

Number of replicates:

2

Species:

other: EpiDerm™ Reconstructed Human Epidermis Model Kit

Strain:

other: Not applicable

Details on test animals or test system and environmental conditions:

EpiDerm™ Reconstructed Human Epidermis Model Kit
Supplier: MatTek
Date received: 20 February 2018
EpiDermTM Tissues (0.63cm2) lot number: 25882
Assay Medium lot number: 021518TMA
Upon receipt of the EpidermTM tissues, the sealed 24 well plate was stored in a refrigerator until use.

Type of coverage:

other: The substance was applied directly to the tissues.

Preparation of test site:

other: The test item was applied topically to the corresponding tissues ensuring uniform covering.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL

Duration of treatment / exposure:

3 and 60 minutes

Number of animals:

A total of 12 tissues were used: Duplicate tissues were treated with: test substance, positive control or negative control for each exposure time.

Details on study design:

Pre-Test Procedure
Assessment of Direct Test Item Reduction of MTT
MTT Dye 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 is still a sufficient amount 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.

Test for Direct MTT Reduction
As specified, a test item may interfere with the MTT endpoint, if it was able to directly reduce MTT and at the same time was present on or in the tissues when the MTT viability test was performed. To identify this possible interference, the test item was checked for the ability to directly reduce MTT according to the procedure below:
50 µL of the test item was added to 1 mL of a freshly prepared 1.0 mg/mL MTT solution. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 60 minutes. Untreated MTT solution was tested concurrently to act as a control.
If the MTT solution containing the test item turns blue/purple relative to the control, the test item was presumed to have reduced the MTT.

Assessment of Color Interference with the MTT Endpoint
A test item may interfere with the MTT endpoint if it is colored or if it becomes colored when in wet or aqueous conditions. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed.
50 µL of test item was added to 300 µL of sterile water. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 60 minutes. A visual assessment of the color was then made.

Main Test
Pre-Incubation
The assay medium was brought to room temperature before use. 0.9 mL of this assay medium was pipetted into the appropriate wells of two pre-labeled 6-well plates for both the 3 Minute and 60 Minute exposure periods. EpiDerm™ tissues were transferred into the 6 well plates containing the assay medium. The 6 well plates containing the EpiDerm™ samples were pre-incubated (37 °C, 5% CO2) for approximately 1 hour before dosing.

Application of Test Item and Rinsing
Before pre-incubation was complete, a 24 well plate was prepared for use as a “holding plate” for both the 3 Minute and 60 Minute exposure periods. This plate was used to maintain the viability of the tissue inserts between rinsing following chemical exposure and MTT loading. Another 24 well plate was prepared for the MTT loading. 300 µL of either pre warmed assay medium (holding plate) or MTT medium (MTT loading plate) was dispensed into each well. The two plates were placed into the incubator until required.

After pre incubation of the EpiDerm™ tissues, the medium was aspirated and replaced with 0.9 mL of fresh assay medium. The 6-well plate for the 3 Minute exposure period was returned to the incubator, while the other was being dosed for the 60 Minute exposure. For the 60 Minute exposure period, 50 µL of sterile distilled water (negative control) was added to the first two tissues. The tissues were dosed at regular intervals to allow for the time taken to rinse each tissue following exposure and to ensure that each tissue gets an equal exposure time. 50 µL of the test item and 50 µL of 8.0 N Potassium Hydroxide (positive control) were also applied to the corresponding tissues in turn. The plate was returned to the incubator (37 °C, 5% CO2) for the 60 Minute exposure period.

When dosing for the 60 Minute exposure period was complete, the same procedure was repeated for the 3 Minute exposure period. Because the exposure time was so short, the tissues were dosed at regular intervals to ensure that each tissue received an equal exposure time and to allow for the time taken to rinse each tissue following exposure. Rinsing was achieved by filling and emptying each tissue under a constant soft stream of Dulbecco’s Phosphate Buffered Saline (DPBS) (without Ca++ Mg++) for approximately 40 seconds, to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper. Each tissue was placed into the prepared holding plate until all tissues were rinsed. They were then blotted and transferred to the 24 well plate prepared for MTT loading. The plate was incubated (37 °C, 5% CO2) for 3 hours. Once the 60 Minute exposure period was complete, the same rinsing and MTT loading procedure was repeated.

After the 3 Hour MTT incubation was complete, the inserts were blotted and transferred to labeled 24 well plates for MTT extraction. 2 mL of MTT extractant (isopropanol) was used to completely immerse each insert and the plate was covered with plate sealer to prevent Isopropanol evaporation. The plates stood overnight at room temperature, to allow extraction to proceed.

Absorbance/Optical Density Measurements
After extraction, each tissue was pierced with a pipette fitted with a 1000 µL tip and the extraction solution was forced vigorously up and down to form a homogenous solution. 3 x 200 µL aliquots of the extract were transferred to the appropriate wells of a pre labeled 96 well plate. 200 µL of isopropanol alone was added to the three wells designated as blanks. Absorbency at 570 nm (OD570) of each well was measured using the Labtech LT 4500 microplate reader.

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean % tissue viability

Run / experiment:

3 minute exposure period

Value:

92

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean % tissue viability

Run / experiment:

60 minute

Value:

93.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Direct MTT Reduction

The MTT solution containing the test item did not turn blue/purple. This was taken to indicate the test item did not reduce MTT.

Assessment of Color Interference with the MTT endpoint

The solution containing the test item did not become colored. This was taken to indicate the test item did not have the potential to cause color interference.

Test Item, Positive Control Item and Negative Control Item

Mean OD₅₇₀values and viabilities for the negative control, positive control and test item are given inAppendix 1.

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

Exposure Period	Percentage Viability
	Negative Control	Positive Control	Test Item
3 minute	100*	4.5	92.0
60 minute	100*	5.2	93.1

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

Quality Criteria

The mean OD₅₇₀for the negative control treated tissues was 1.959 for the 3‑Minute exposure period and 1.933 for the 60‑Minute exposure period. The negative control acceptance criteria were therefore satisfied.

The relative mean tissue viability for the positive control treated tissues was 0.101% relative to the negative control following the 60‑Minute exposure period. The positive control acceptance criterion was therefore satisfied.

In the range of 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

Interpretation of results:

other: non-corrosive

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

The test item, FRET 14-0383, was classified as non-corrosive to the skin. The following classification criteria apply:
EU CLP (1272/2008/EC)/UN GHS: Not classified for corrosivity.
EU DSD (67/548/EEC): Not classified for corrosivity.
UN Packing Group: Non-Corrosive.

Executive summary:

The skin corrosivity of the test substance was determined according to OECD Guideline 431 using the EpiDerm™ Human Skin Model. The relative mean viability of 30 and 60 were all above 100% in relation to the control. Therefore the substance is not corrosive to skin, according to EU CLP criteria.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 14 March 2018 and 02 April 2018.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

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 14-0383
Envigo Description: Colorless liquid
Sponsor Description: Clear yellow liquid
Storage Conditions: Approximately 4 °C, in the dark

Test system:

human skin model

Source species:

human

Cell type:

other: reconstructed human epidermis

Cell source:

other: not specified

Source strain:

not specified

Details on animal used as source of test system:

EPISKIN™ Reconstructed Human Epidermis Model Kit
Supplier : EpiSkin Laboratories, Lyon, France
Date received : 27 March 2018
EpiSkinTM Tissues (0.38cm^2) lot number : 18-EKIN-013
Maintenance Medium lot number : 18-MAIN3-015
Assay Medium lot number : 18-ESSC-012

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 complimentary end point can be used to either confirm a non-irritant result or will be used to override the non irritant result.
The EPISKINTM model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13 Day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
Following a full validation study the EpiSkinTM reconstructed human epidermis model showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation when the endpoint is measured by MTT reduction and for being used as a replacement for the Draize Skin Irritation Test for the purpose of distinguishing between Irritating and Non-Irritating test items.

Vehicle:

unchanged (no vehicle)

Details on test system:

Reference Items
Negative Control
Information as provided by the Supplier.
Identification: Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca++ and Mg++
Batch: 1754643
Purity: Not applicable
Expiry Date: 01 February 2019
Storage Conditions: Approximately 4 °C in the dark
Supplier: Gibco

Positive Control
Information as provided by the Supplier.
Identification: Sodium dodecyl sulphate
Batch: SLBT3991
Purity: 99.5%
Expiry Date: 01 March 2020
Storage Conditions: Room temperature
Supplier: Sigma-Aldrich

Preparation of Negative and Positive Control Items and MTT
The negative control item, Dulbecco’s Phosphate Buffered Saline (DPBS), was used as supplied.
The positive control item, Sodium dodecyl sulphate (SDS), was prepared as a 5% w/v aqueous solution. The positive control was formulated within 2 hours of being applied to the test system.
A 3 mg/mL MTT stock solution was prepared in DPBS. The stock solution was diluted to 0.3 mg/mL with assay medium when required.
A 0.04 N solution of hydrochloric acid in isopropanol was prepared when required.

Study Design
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/cm^2) 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.

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
10 µL of SDS 5% w/v

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:

57.1

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 9.9% relative to the negative control treated tissues and the standard deviation value of the viability was 2.8%. The positive control acceptance criteria were therefore satisfied.
The mean OD570 for the negative control treated tissues was 0.685 and the standard deviation value of the viability was 3.5%. The negative control acceptance criteria were therefore satisfied.
The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 2.7%. 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

The relative mean viability of the test item treated tissues was 57.1% after a 15‑Minute exposure period and 42‑Hour post‑exposure incubation period.

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

Mean OD₅₇₀ Values 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	0.671	0.685	0.024	98.0	100*	3.5
	0.671			98.0
	0.712			104.0
Positive Control Item	0.061	0.068	0.019	8.9	9.9	2.8
	0.053			7.7
	0.090			13.1
Test Item	0.412	0.391	0.019	60.1	57.1	2.7
	0.385			56.2
	0.376			54.9

OD = Optical Density

SD = Standard deviation

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

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

The relative mean viability of FRET 14-0383 treated tissues was 57.1% 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 FRET 14 -0383 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 14-0383 treated tissues was 57.1% 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 positive control had a mean cell viability of ≤40% relative to the negative control treated tissues after 15 minutes treatment. The standard deviation value of the percentage viability of three tissues treated identically was less than 18%, indicating that the test system functioned properly.

Reference 3

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 8 January 2019 and 14 January 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2500 (Acute Dermal Irritation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, Skin Irritation (2-1-4), 12 Nousan No. 8147, Agricultural Production Bureau, November 24, 2000.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test item FRET 14-0383
Appearance Clear yellow liquid.
Storage conditions Refrigerated (2 to 8°C), protected from light.
Supplier Sponsor.
Batch number RDGM435-97
Expiry date October 2019
Purity 95.1%
Supplier’s responsibilities Characterization of the test item and the documentation of the methods of synthesis, fabrication or derivation and stability.

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

3Animal Information
Animals for this study were selected from a stock supply of healthy adult rabbits of the New Zealand White strain. They were in the weight range of 3.73 to 4.59 kg and were 56 or 57 weeks of age, prior to treatment (Day 1). All rabbits were acclimatized to the experimental environment for a period of 41 weeks prior to the start of the study.

Animal Care and Husbandry
Each animal was housed individually in a plastic cage with perforated floors and was offered 150 g of a standard laboratory rabbit diet per day; drinking water was provided ad libitum. The batch of diet used for the study is analyzed for nutrients, possible contaminants and micro-organisms likely to be present in the diet and which, if in excess of specified amounts, might have an undesirable effect on the test system. A dietary supplement of hay was offered.

During the acclimatization and study period the animals were given small soft white untreated wood blocks for environmental enrichment.

Results of routine physical and chemical examination of drinking water, as conducted by the supplier are made available to Covance.

Animal room environmental controls were set to maintain temperature within the range 15 to 21°C, and relative humidity within 45 to 70%. These environmental parameters were recorded and the permanent record archived with other departmental raw data. Lighting was controlled by means of a time switch to give 12 hours of artificial light (06:00 to 18:00 GMT) in each 24 hour period.

Each animal was identified by a numbered tag placed through the edge of one ear. Each cage was identified by a label displaying the study number and animal number.

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0.5mL of test item was applied to the treatment site

Duration of treatment / exposure:

A single animal (number 7) received three exposures of three minutes, one or four hours duration in a step-wise manner and acted as a preliminary screen.

Observation period:

Examination of the treated skin was made immediately before administration and approximately 1, 24, 48 and 72 hours after the removal of the dressings.
In the absence of a severe effect on removal of the dressings the next exposure was initiated. In the absence of a severe effect in this animal two further animals were committed to the study.

Number of animals:

Three

Details on study design:

Approximately 0.5mL of test item was applied to the treatment site under a 2-ply 25 mm x 25 mm porous gauze pad secured with a single strip of surgical adhesive tape. An additional site was similarly treated with the exception of the test substance and acted as a control. The treatment site was covered with cotton wool and "Tubigrip" elasticated bandage dressing (the posterior end was secured by strip of surgical tape) for the duration of the exposure of one hour or more. The animals were not restrained during the exposure period and returned to their cages immediately after treatment.
At the end of the exposure period the semi-occlusive dressing and gauze pad were removed and the treatment site was washed with warm tap water (30 - 40°C) to remove any residual test substance. The treated area was blotted dry with absorbent paper.

Irritation parameter:

erythema score

Basis:

animal: 7F, 8F, 9F

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: No effects observed

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

animal: 7F, 8F, 9F

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: No effects observed

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

No dermal reaction was observed in any animal throughout the duration of the study.

Other effects:

Clinical Signs
There was no sign of toxicity or ill health in any rabbit during the observation period.

Interpretation of results:

GHS criteria not met

Conclusions:

The mean Primary Irritation Index was calculated to be 0.0; FRET 14-0383 was classified as a ‘Non-irritant’ and did not require labeling in accordance with Commission Regulation 1272/2008.

Executive summary:

Summary

The purpose of this study was to assess the skin irritation potential of FRET 14-0383 to the rabbit. The method followed was that described in:

B4 Acute Toxicity (Skin Irritation) of Commission Regulation (EC) No. 440/2008.

Three rabbits received a single four hour, semi-occlusive, dermal administration of approximately 0.5 mL of the test item as supplied and were observed for three days.

No dermal reaction was observed in any animal throughout the duration of the study.

The means of scores for these reactions at approximately 24, 48 and 72 hours after administration, calculated separately for each animal, are summarized below:

Means of scores at approximately 24, 48 and 72 hours
Animal number	Erythema	Oedema
7	0.0	0.0
8	0.0	0.0
9	0.0	0.0
EC trigger values*	³2.3	³2.3
*Classification according to Commission regulation 1272/2008 is triggered if means of scores for either effect are³2.3 for two or three animals (or if effects persist to Day 14 in at least two animals).

The Primary Irritation Index was calculated to be 0.0; FRET 14-0383 was classified as a ‘Non‑irritant’ and did not require labeling in accordance with Commission Regulation 1272/2008.

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:

key study

Study period:

The study was conducted between 07 March 2018 and 08 March 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 Ocular Corrosives and Severe Irritants)

Deviations:

yes

Remarks:

The deviation was 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 14-0383
Physical state/Appearance: Clear colorless liquid
Storage Conditions: Approximately 4 °C in the dark

Species:

other: Eyes from adult cattle (typically 12 to 60 months old)

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

Observation period (in vivo):

120 minutes

Number of animals or in vitro replicates:

3 cornea per group

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. The average opacity for all corneas was calculated.
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 in vitro irritation score

Value:

2

Vehicle controls validity:

not applicable

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.

 In Vitro Irritancy Score

The In Vitro irritancy scores are summarized as follows:

Treatment	In Vitro Irritancy Score
Test Item	2.0
Negative Control	1.0
Positive Control	46.7

Individual and Mean Corneal Opacity and Permeability Measurements

Treatment	Cornea Number	Opacity					Permeability (OD492)		In VitroIrritancy Score
		Pre-Treatment	Post-Treatment	Post Incubation	Post-Incubation - Pre‑Treatment	Corrected Value		Corrected Value
Negative Control	2	4	3	5	1		0.003
	3	4	4	5	1		0.002
	6	2	2	3	1		0.000
					1.0*		0.002¨		1.0
Positive Control	7	3	29	28	25	24.0	0.989	0.987
	10	4	36	36	32	31.0	1.008	1.006
	11	2	28	30	28	27.0	1.885	1.883
						27.3·		1.292·	46.7
Test Item	12	2	6	6	4	3.0	0.054	0.052
	13	3	7	6	3	2.0	0.006	0.004
	14	7	6	7	0	0.0	0.003	0.001
						1.7·		0.019·	2.0

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	Clear
	13	Clear	Clear
	14	Clear	Clear

Interpretation of results:

other: Not classified for irritation

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

The test item FRET 14-0383 was considered not to be an ocular corrosive or severe irritant.

Executive summary:

The eye irritancy potential of the test substance FRET 14-0383 was assessed according to OECD Test Guideline 437 using the Bovine Corneal Opacity and Permeability Assay method and is not classified as an eye irritant , according to EU CLP criteria.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 18 June 2018 and 29 June 2018.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

2017

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 14-0383
Physical state/Appearance: clear colorless liquid
Storage Conditions: approximately 4 °C in the dark

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Animal Information
Two New Zealand White (Hsdlf:NZW) strain rabbits were supplied by Envigo RMS (UK) Limited, Leicestershire, UK. At the start of the study the animals weighed 2.95 or 3.11 kg and were 12 to 52 weeks old. After an acclimatization period of at least 5 days each animal was given a number unique within the study which was written with a black indelible marker pen on the inner surface of the ear and on the cage label.

Animal Care and Husbandry
The animals were individually housed in suspended cages. Free access to mains drinking water and food (2930C Teklad Global Rabbit diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study. The diet and drinking water were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
The temperature and relative humidity were set to achieve limits of 17 to 23 °C and 30 to 70% respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Vehicle:

unchanged (no vehicle)

Controls:

other: One eye of each animal remained untreated and served as the reference control.

Amount / concentration applied:

Amount applied (volume or weight with unit): 0.1 mL

Duration of treatment / exposure:

Single instillation on Day 1

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

2 females

Details on study design:

Immediately before the start of the test, both eyes of the provisionally selected test rabbits were examined for evidence of ocular irritation or defect with the aid of a light source from a standard ophthalmoscope. Only animals free of ocular damage were used.
Initially, a single rabbit was treated. A subcutaneous injection of buprenorphine 0.01 mg/kg was administered 60 minutes prior to test item application to provide a therapeutic level of systemic analgesia. Five minutes prior to test item application, a pre dose anesthesia of ocular anesthetic (two drops of 0.5% proxymetacaine hydrochloride) was applied to each eye.
A volume of 0.1 mL of the test item was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test item, and then released. The left eye remained untreated and was used for control purposes. Immediately after administration of the test item, an assessment of the initial pain reaction was made according to the six point scale.
Eight hours after test item application, a subcutaneous injection of post dose analgesia, buprenorphine 0.01 mg/kg and meloxicam 0.5 mg/kg, was administered to provide a continued therapeutic level of systemic analgesia. The treated animal was checked for signs of pain and suffering approximately 12 hours later. No further analgesia was required.
After consideration of the ocular responses produced in the first treated animal, a second animal was similarly treated.
Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment, according to the numerical evaluation (Draize, J.H, 1977).
Any other ocular effects were also noted. Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.
Any clinical signs of toxicity, if present, were also recorded.
Individual body weights were recorded on Day 0 (the day of dosing) and at the end of the observation period.

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

conjunctivae score

Remarks:

Redness score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.33

Max. score:

6

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

conjunctivae score

Remarks:

Redness score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.33

Max. score:

1

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.33

Max. score:

1

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.67

Max. score:

1

Reversibility:

fully reversible within: 72 hours

Irritant / corrosive response data:

Ocular Reactions
No corneal effects were noted.
Iridial inflammation was noted in both treated eyes one hour after treatment.
Moderate conjunctival irritation was noted in both treated eyes one hour after treatment. Minimal conjunctival irritation was noted in both treated eyes at the 24-Hour observation and persisted in one treated eye at the 48-Hour observation.
Treated eyes appeared normal at the 48 or 72-Hour observation.

Body Weight
Both animals showed a slight loss in body weight during the study.

Individual Scores and Individual Total Scores for Ocular Irritation

Rabbit Number and Sex	76076 Female				76038 Female
	IPR= 0				IPR = 0
Time After Treatment	1 Hour	24 Hours	48 Hours	72 Hours	1 Hour	24 Hours	48 Hours	72 Hours
CORNEA
E = Degree of Opacity	0	0	0	0	0	0	0	0
F = Area of Cornea Involved	0	0	0	0	0	0	0	0
Score (E x F) x 5	0	0	0	0	0	0	0	0
IRIS
D	1	0	0	0	1	0	0	0
Score (D x 5)	5	0	0	0	5	0	0	0
CONJUNCTIVAE
A = Redness	2	1	0	0	2	1	0	0
B = Chemosis	2	1	0	0	2	1	1	0
C = Discharge	2	1	0	0	1	1	0	0
Score (A + B + C) x 2	12	6	0	0	10	6	2	0
Total Score	17	6	0	0	15	6	2	0

IPR = Initial pain reaction

Individual Total Scores and Group Mean Scores for Ocular Irritation

Rabbit Number and Sex	Individual Total Scores At:
	1 Hour	24 Hours	48 Hours	72 Hours
76076 Female	17	6	0	0
76038 Female	15	6	2	0
Group Total	32	12	2	0
Group Mean Score	16.0	6.0	1.0	0.0

 

Individual Body Weights and Body Weight Change

Rabbit Number and Sex	Individual Body Weight (kg)		Body Weight Change (kg)
	Day 0	Day 3
76076 Female	2.95	2.92	-0.03
76038 Female	3.11	3.10	-0.01

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

In an eye irritation study with rabbits, performed according to OECD 405 (2017), mild irritation was observed. Based on the results of this study, FRET 14-0383 does not meet the criteria for classification according to the Globally Harmonized System of Classification and Labelling of Chemicals and to Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.

Executive summary:

The study was performed to assess the irritancy potential of FRET 14-0383 to the eye of the New Zealand White rabbit according to OECD 405 (2017). A single application of the test item to the non-irrigated eye of two rabbits produced iridial inflammation and moderate conjunctival irritation. Treated eyes appeared normal at the 48 or 72-Hour observation.

The test item produced a maximum group mean score of 16.0 and was classified as a mild irritant (Class 4 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.

The test item does not meet the criteria for classification according to the Globally Harmonized Systemn of Classification and Labelling of Chemicals and to Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

In vitro tests

Skin corrosion: The skin corrosivity of the test substance, FRET 14 -0383 was determined according to OECD Guideline 431 using the EpiDerm™ Human Skin Model. The relative mean viability of the test item treated tissues was 92.0 % after a 3-Minute exposure period and and 93.1 % after a 60 -Minute exposure period. This result shows that the substance is not a skin corrosive, according to EU CLP criteria.

 

Skin irritation: The possible skin irritation potential of FRET 14 -0383 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 14-0383 treated tissues was 57.1% 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 positive control had a mean cell viability of ≤40% relative to the negative control treated tissues after 15 minutes treatment. The standard deviation value of the percentage viability of three tissues treated identically was less than 18%, indicating that the test system functioned properly.

In vivo test:

Skin irritation

The study was performed to assess the irritancy ppotential of FRET 14 -0383 to the skin of the New Zealand White rsbbit according to OECD 404. Three rabbits received a single four hour, semi-occlusive, dermal administration of approximately 0.5 mL of the test item as supplied and were observed for three days.

No dermal reaction was observed in any animal throughout the duration of the study.

The Primary Irritation Index was calculated to be 0.0; FRET 14-0383 was classified as a ‘Non‑irritant’ and did not require labeling in accordance with Commission Regulation 1272/2008.

Eye irritation:

The study was performed to assess the irritancy potential of FRET 14-0383 to the eye of the New Zealand White rabbit according to OECD 405 (2017). A single application of the test item to the non-irrigated eye of two rabbits produced iridial inflammation and moderate conjunctival irritation. Treated eyes appeared normal at the 48 or 72-Hour observation.

The test item produced a maximum group mean score of16.0and was classified as a mild irritant (Class 4 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.

The test item does not meet the criteria for classification according to the Globally Harmonized Systemn of Classification and Labelling of Chemicals and to Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.

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 corrosion test 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).

Based on the negative results in the skin irritation test 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).

Based on the negative results in the eye irritation test 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).