4,6-dimethyl-2-(1-phenylethyl)-3,6-dihydro-2H-pyran

Endpoint summary

Description of key information

Skin corrosion (OECD TG 431): Not corrosive to skin

Skin irritation (OECD TG 439): Irritating to skin

Eye irritation and severe damage (OECD TG 405): Not an occular irritant

Eye irritation (OECD TG 492): Not eye irritating

Eye severe damage (OECD TG 437): Not categorized

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 26 April 2017 and 08 June 2017

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 12-0492
CAS No.: 1945993-03-2
Appearance: Yellow, liquid
Storage Conditions: In the refrigerator

Test system:

human skin model

Source species:

human

Cell type:

other: normal, human-derived epidermal keratinocytes (NHEK)

Cell source:

other: derived from human keratinocytes

Source strain:

other: not applicable

Details on animal used as source of test system:

The In vitro Skin Corrosion: Human Skin Model Test is based on the observation that skin corrosion (necrotic damage of viable skin cells) shows a high correlation with skin cell cytotoxicity, occurring rapidly after brief exposure of the skin barrier (stratum corneum) to a corrosive chemical. It is designed to predict and classify the skin corrosivity potential of a chemical by using a three-dimensional human epidermis model. The epidermis model (e.g. EpiDermTM) is derived from human keratinocytes and consists of normal, human-derived epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK, which are cultured on specially prepared cell culture inserts using serum free medium, attain levels of differentiation at the cutting edge of in vitro skin technology. Ultrastructurally, the skin models closely parallel human skin. The In vitro Skin Corrosion: Human Skin Model Test consists of topical application of the test material to the tissue for 3 minutes and 1 hour, followed by immediate determination of the cytotoxic effect. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the exposure period.

EpiDerm™ Kit, Lot No.: 25817

Justification for test system used:

Validation studies have shown that tests employing human skin models are able to discriminate between known skin corrosives (Optional sub-category 1A, optional sub-category 1B and 1C) and non-corrosives. The test protocol may also enable the distinction between severe and less severe skin corrosives.

Vehicle:

unchanged (no vehicle)

Details on test system:

Epi-200 kits and MTT-100 assays were purchased from MatTek Corporation (Bratislava, Slovakia). The EpiDerm™ tissue consisted of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consisted of organised basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm™ tissues (surface 0.63 cm²) are cultured on specially prepared cell culture inserts (MILLICELL, 10 mm ).
EpiDerm™ tissues were shipped on cool packs and on medium-supplemented agarose gels in a 24-well plate and reached Envigo CRS GmbH on 06 June 2017. On day of receipt the pre-incubation phase of the EpiDerm™ tissues started.

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

NEGATIVE CONTROL, Deionised water
- Amount(s) applied (volume or weight): 50 µl

POSITIVE CONTROL, Potassium Hydroxide
- Amount(s) applied (volume or weight): 50 µl
- Concentration (if solution): 8.0 N

Duration of treatment / exposure:

3 and 60 minutes

Number of replicates:

duplicate

Type of coverage:

other: dispensed directly onto duplicate EpiDermTM tissue surface.

Preparation of test site:

other: not applicable

Vehicle:

unchanged (no vehicle)

Details on study design:

Test for Direct MTT Reduction and Colour Interference
A test item may interfere with the MTT endpoint if: a) it is coloured and/or b) able to directly reduce MTT. The MTT assay is affected only if the test item is present in the tissues when the MTT viability test is performed.
Some non-coloured test items may change into coloured test items in wet or aqueous conditions and thus stain tissues during the 60 min exposure. Therefore, before exposure, a functional check for this possibility should be performed (step 1).
Step 1
50 µL of the test item were added to 0.3 ml of deionised water (transparent glass test-tube). The mixture was incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5 % CO2) for 60 min. At the end of the exposure time, the mixture was shaken and the presence and intensity of the staining (if any) was evaluated. If the solution changed colour significantly, the test item is presumed to have the potential to stain the tissue. An additional test on viable tissues (without MTT addition) should be performed (step 2).
Since the test item did not dye water when mixed with it, step 2 did not have to be performed.
Step 3
All test items (including those already evaluated in step 1 and step 2) should be further evaluated for their potential to interfere with MTT. To test if an item directly reduces MTT, 50 µL of the test item were added to 1 ml of a MTT/DMEM solution (1 mg/mL) and were incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5 CO2) for 60 minutes. Untreated MTT/DMEM solution (1 mg/mL) medium was used as control. If the MTT/DMEM solution (1 mg/mL) turns blue/purple, the test item reduces MTT and an additional test on freeze-killed tissues (step 4) must be performed.
Since the test item did not prove to be a MTT reducer, step 4 did not have to be performed.


EXPERIMENTAL PERFORMANCE
Pre-warming of EpiDerm™ Tissues
18 to 19 hours before dosing, EpiDerm™ tissues were unpacked, and the inserts were transferred into 6-well plates containing the pre-warmed assay medium under sterile conditions using sterile forceps. A 24-well plate was prepared as holding plate containing 300 µL assay medium. The holding plate was pre warmed in an incubator (37 ± 1.5 °C, 5 ± 0.5 % CO2) until use.

Treatment
Duplicate EpiDermTM tissues were treated with the test item, positive control or negative control for the following exposure times:
• Test Item: 3 ± 0.5 minutes, 60 ± 5 minutes
• Negative Control: 3 ± 0.5 minutes, 60 ± 5 minutes
• Positive Control: 3 ± 0.5 minutes, 60 ± 5 minutes

After the pre-incubation of the EpiDermTM tissues was completed the DMEM-based medium in each well was replaced with 0.9 mL fresh assay medium. The 6-well plates for the 3 ± 0.5 minutes exposure periods stayed at room temperature in the sterile bench, the 6-well plates for the 60 ± 5 minutes exposure period were placed into an incubator (37 ± 1.5 °C, 5 ± 0.5% CO2).
At the end of the exposure period the tissues were removed from the 6-well plate and gently rinsed with DPBS to remove any residual test material (20 times). Excess DPBS was removed by gently shaking the tissue insert and blotting the lower surface with blotting paper. The tissues were placed in the prepared holding plate until all tissues were rinsed.

MTT Assay
Two 24-well plates were prepared prior to the end of the tissue pre-warming period. MTT solution (300 µL) was added to each well and the plates were kept in an incubator (37 ± 1.5 °C, 5 ± 0.5% CO2) until required.
Following rinsing, the tissues were transferred from the holding plates to the MTT-plates. After a 3 hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO2) the tissues were rinsed three times with DPBS and carefully dried with blotting paper. The inserts were transferred into new 24-well plates. The tissues were each immersed in 2 mL of extractant solution (isopropanol) pipetted in each well ensuring that the tissues were completely covered. The 24-well plates were sealed to minimise isopropanol evaporation. The formazan salt was extracted for nearly 20 hours without shaking at room temperature.
After the extraction period the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken, and the insert was discarded. 24-well plates were then placed on a shaker for 15 minutes until the solution was homogeneous in colour.
3 x 200 µL aliquots of the blue formazan solution were transferred from each tissue into a 96-well flat bottom microtiter plate. The OD was determined in a microplate reader (Versamax®, Molecular Devices, SoftMax Pro Enterprise (version 4.7.1)) at 570 nm (OD570) without reference filter. The mean values were calculated for each set of 3 wells per tissue.

Irritation / corrosion parameter:

other: Relative absorbance (percentage of negative control)

Run / experiment:

3 minute treatment

Value:

108.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

other: Relative absorbance (percentage of negative control)

Run / experiment:

60 minute treatment

Value:

108.3

Vehicle controls validity:

not applicable

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 did not lead to a relevant change in colour.

Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

The test item is considered to be non-corrosive to skin:
• since the viability after 3 minutes exposure is greater than 50% and
• the viability after 1 hour exposure is greater than 15%.

The acceptance criteria are met:
• the mean OD of the tissue replicates treated with the negative control is ≥ 0.8 and ≤ 2.8 for every exposure time (range: 1.455 to 1.575)
• the mean viability of the tissue replicates treated with the positive control for 1 hour, is <15% compared to the negative control (3.2%)
• the Coefficient of Variation (CV) in the range 20 – 100% viability between tissue replicates is ≤ 30% (range: 0.2% to 4.6%)

Results after treatment with FRET 12-0492 and the controls

Dose Group	Ex-posure Inter-val	Absor-bance Well 1 (Tissue 1/2)	Absor-bance Well 2 (Tissue 1/2)	Absor-bance Well 3 (Tissue 1/2)	Mean Absor-bance (Tissue 1/2)	Mean Ab-sorbance (OD) of 3 Wells minus Blank	Mean Ab-sorbance (OD) of 2 Tissues	Absor-bance [% of Negative Control]*	CV [%]	Rel. Absor-bance [% of Negative Control]*
Blank		0.037	0.037	0.037	0.037	0.000
Negative Control	3 minutes	1.562	1.522	1.526	1.537	1.500	1.502	99.8	0.2	100.0
		1.536	1.546	1.543	1.542	1.505		100.2
Positive Control		0.392	0.377	0.382	0.384	0.347	0.339	23.1	3.3	22.6
		0.368	0.368	0.368	0.368	0.331		22.0
Test Item		1.694	1.623	1.644	1.654	1.617	1.626	107.6	0.8	108.2
		1.664	1.677	1.672	1.671	1.634		108.8
Blank		0.036	0.036	0.041	0.038	0.000
Negative Control	1 hour	1.575	1.528	1.524	1.542	1.505	1.469	102.5	3.5	100.0
		1.481	1.476	1.455	1.470	1.433		97.5
Positive Control		0.082	0.082	0.082	0.082	0.044	0.047	3.0	8.1	3.2
		0.088	0.087	0.088	0.087	0.049		3.4
Test Item		1.647	1.610	1.595	1.617	1.579	1.590	107.5	1.0	108.3
		1.649	1.633	1.634	1.639	1.601		109.0

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

This in vitro study was performed to assess the corrosive potential of FRET 12-0492 by means of the Human Skin Model Test with EpiDerm™ tissues models.

The test item passed the MTT- and the colour interference pre-tests.

Independent duplicate tissues of EpiDerm^TMwere exposed to the test item, the negative control (deionised water) or the positive control (8.0 N KOH) for 3 minutes and 1 hour, respectively.

Afterwards, the test and the control items were rinsed off the tissues, and a 3 hour incubation period (37 ± 1 °C, 5 ± 0.5 % CO₂) with MTT solution followed. MTT solution was then aspirated from the wells and the wells were rinsed with DPBS. Inserts were transferred into new 24 well plates. The formazan salt was extracted for nearly 20 hours at room temperature.

The required acceptability criteria were met.

Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 minutes exposure period (22.6%) and for the 1 hour exposure period (3.2%) thus confirming the validity of the test system and the specific batch of tissue models.

After exposure to the test item FRET 12-0492 the relative absorbance value did not decrease after 3 minutes as well as after 1 hour exposure. The viability values were 108.2% and 108.3%. Both values did not fall below the threshold for corrosivity which is defined to be 50% after the 3 minutes exposure and 15% after the 1 hour exposure. Therefore, the test item is not considered to be corrosive.

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

The test item, FRET 12-0492, was classified as non-corrosive to the skin. The following classification criteria apply:
EU CLP (1272/2008/EC)/UN GHS: 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™ Reconstructed Human Epidermis Model. The relative absorbance value after 3 and 60 minutes exposure were above 50% and 15% respectively 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 26 April 2017 and 09 June 2017

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:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: FRET 12-0492
Chemical Name: 4,6-dimethyl-2-(1-phenylethyl)-3,6-dihydro-2H-pyran
Appearance: Yellow, liquid
Storage Conditions: In the refrigerator
Stability in Solvent: Not indicated by the Sponsor
Purpose of Use: Industrial chemical

Test system:

human skin model

Source species:

human

Cell type:

other: Normal Human-Derived Epidermal Keratinocytes

Cell source:

other: no information

Source strain:

other: not applicable

Details on animal used as source of test system:

EpiSkin™ Kit
Supplier : SkinEthic Laboratories (69007 Lyon, France).
Date received : 07 June 2017
EpiSkinTM Tissues (0.38cm2) lot number : 17-EKIN-023

Justification for test system used:

In an international validation study performed by ECVAM, the in vitro skin irritation test using the human skin models EpiSkin™ and EpiDerm™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for skin irritancy potential .

Vehicle:

unchanged (no vehicle)

Details on test system:

Cell Culture
EpiSkin™ kits are purchased from SkinEthic Laboratories (69007 Lyon, France). The EpiSkin™ tissue consists of NHEK, which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiSkin™ tissues (surface 0.38 cm²) are cultured on specially prepared cell culture inserts.
EpiSkin™ tissues were shipped at ambient temperature on medium-supplemented agarose gels in a 12-well plate and reached Envigo CRS GmbH on 07 June 2017. On the day of experiment the pre-incubation phase of the EpiSkin™ tissues started.

Test for Direct MTT Reduction and Colour Interference
Prior to the start of the test, the colour interference potential of the test item had to be evaluated. For this purpose 10 µL of the test item was mixed with 90 µL of deionised water in a pre-experiment. The mixture was gently shaken for 15 minutes at room temperature.
The colour of the mixture did not change during the incubation period compared with the colour of the pure test item. Therefore, the measurement of the OD of the test item in water at 570 nm was not required and consequently not performed.
For correct interpretation of results it is necessary to assess the ability of the test item to directly reduce MTT. To test for this ability 10 µL of the test item was added to 2 mL of MTT solution (0.3 mg/mL) and the mixture was incubated in the dark at
37 ± 1.5 °C (5 ± 0.5% CO2) for 3 hours. MTT solution was used as the control. If the MTT solution colour turns blue/purple, the test item is presumed to have reduced the MTT.
Since the colour did not turn blue/purple, the test item was not considered to be a MTT reducer.

EXPERIMENTAL PERFORMANCE

Pre-warming of EpiSkin™ Tissues
Under sterile conditions using sterile forceps, the inserts were transferred into 12-well plates containing the pre-warmed (37 ± 1.5 °C) maintenance medium. The EpiSkin™ tissues were incubated for approximately 3 hours.

Treatment
The negative control, positive control and the test item were added into the insert atop the concerning EpiSkin™ triplicate tissues for 15 minutes.
After the end of the treatment interval the inserts were immediately removed from the 12-well plate. The tissues were gently rinsed with PBS to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper. The inserts were placed in the plates with 2 mL maintenance medium. The tissues were incubated for approximately 42 hours at 37 ± 1.5 °C, 5 ± 0.5% CO2.

IL-1 α Immunoassay
Samples of all treatment groups were taken from the wells. The plates were shaken for approximately 15 minutes to homogenise the released mediators in the medium before sampling. At least 1.6 mL medium from each well was taken and was stored in the freezer at –20 °C. Since the results derived from the MTT assay were not unclear or borderline, the IL-1 α concentration in the medium was not determined and the taken samples were discarded after report finalization.

MTT Assay
A 12-well plate was filled with 2 mL assay medium containing 0.3 mg/mL MTT per well.
After the 42 ± 1 hour incubation period was completed for all tissues the cell culture inserts were transferred from the holding plates to the MTT-plates. After a 3 hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO2) MTT solution was rinsed three times with PBS and the tissues were plotted. Tissue samples were cut out of the inserts with a biopsy punch and transferred into plastic vials. The tissue samples were immersed into extractant solution by gently pipetting 0.5 mL extractant solution (isopropanol containing 0.04 N HCl) into each vial. The tissue samples were completely covered by isopropanol. The formazan salt was extracted for about 2.5 hours at room temperature with gentle agitation.
Per tissue sample 2 x 200 µL aliquots of the formazan extract were transferred into a 96-well flat bottom microtiter plate. OD was read in a microplate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, version 4.7.1) with 570 nm filter. Mean values were calculated from the 2 wells per tissue sample.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10µl

NEGATIVE CONTROL (PBS)
- Amount(s) applied (volume or weight): 10µl

POSITIVE CONTROL (SLS, Fluka, Sigma-Aldrich)
- Amount(s) applied (volume or weight): 10µl
- Concentration (if solution): 5% solution in deionised water

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hrs

Number of replicates:

3

Vehicle:

unchanged (no vehicle)

Irritation / corrosion parameter:

other: Relative absorbance (% of negative control)

Value:

30.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

The optical pre-experiment (colour interference pre-experiment) to investigate the colour change potential of the test item in water did not lead to a change in colour.
Optical evaluation of the MTT-reducing capacity of the test item after 3 hour incubation with MTT-reagent did not show blue colour.
The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 30.9% (threshold for irritancy: ≤ 50%), consequently the test item was irritant to skin.
Concerning acceptance criteria:
• The mean OD of the three negative control exposed tissues is  0.6 till ≤ 1.5 (range: 1.384 to 1.468).
• The rel. standard deviations between tissues of the same treatment group were ≤ 18% (3.2% (negative control) and 2.3% (test item)). For the positive control, the acceptance criteria were not met (28.7%). Reason: extremely low absorbance values of 0.014, 0.014 and 0.022, where small differences cause high rel. standard deviations. The OECD guideline 439 and the SkinEthic protocol request only the simple standard deviation being ≤ 18% as acceptance criteria between tissues of the same treatment group. If the simple standard deviation is considered (0.3%), the acceptance criteria are met. This deviation from the acceptance criteria is not considered to have an impact on the outcome of the study.
• The mean relative tissue viability of the positive control was ≤ 40% (1.2%).
• The acceptance limit of the IC50 of the respective EpiSkin™ lot was between 1.5 and 3.0 mg/mL after 18 hours treatment with SLS (1.8 mg/mL).
• Historical Data for the negative control are not available. Reason: Envigo CRS GmbH changed the negative control for the test system “In vitro Skin Irritation Assay using RHE supplied by SkinEthic” from deionized water to PBS. The test is performed at Envigo CRS GmbH a long time prior to a former version of OECD 439 guideline, where deionized water or PBS are suggested as negative control. Originally, SkinEthic requested deionized water to be used as negative control in their protocol. All of Envigo CRS GmbH’s historical data are based on deionized water. In the current SkinEthic protocol, PBS is suggested as negative control. Therefore, Envigo CRS GmbH decided to change the negative control to be in accordance with the SkinEthic protocol, but the number of performed assays using PBS as negative control is still too low to issue historical data.
Historical Data for the positive control are available. But the determined viability value of 1.2% is not within the historical data (viability range: 3.90% - 32.73%). Since the historical data at Envigo CRS GmbH are updated at least once a year, after next update the present positive control viability of 1.2% will be within the historical range. Since neither the OECD guideline nor SkinEthic requests historical data for the positive control, the outcome of the study is not impacted by this.

Results after treatment with the test item and controls

Test Group	Absor-bance 570 nm Tissue Well 1	Absor-bance 570 nm Tissue Well 2	Mean Absor-bance 570 nm	Mean Absor-bance 570 nm*	Mean Absor-bance of 3 Tissues	Standard Deviation of Tissue Absorbance	Relative Absor-bance [%] Tissue 1, 2, 3**	Standard Deviation [%]	Rel. Standard Deviation [%]****	Rel. Absorbance [% of Negative Control]***
Blank	0.050	0.048	0.049	0.000
Negative Control	1.480	1.457	1.468	1.420	1.387	0.045	102.4	3.2	3.2	100.0
	1.469	1.438	1.453	1.405			101.3
	1.432	1.337	1.384	1.336			96.3
Positive Control	0.063	0.062	0.062	0.014	0.017	0.005	1.0	0.3	28.7	1.2
	0.061	0.065	0.063	0.014			1.0
	0.076	0.066	0.071	0.022			1.6
Test Item	0.491	0.485	0.488	0.439	0.428	0.010	31.7	0.7	2.3	30.9
	0.475	0.470	0.472	0.424			30.6
	0.466	0.475	0.470	0.422			30.4

*         Mean of two replicate wells after blank correction

**       relative absorbance per tissue [rounded values]: (100 x (absorbance _tissue)) / (mean absorbance _{negative control})

***     relative absorbance per treatment group [rounded values]: (100 x (absorbance_{test item})) / (mean absorbance_{negative control})

 

****      relative standard deviation per treatment group [rounded values]: (100 x standard deviation _{tissue absorbance}) / mean absorbance _tissue)

Discussion

This in vitro study was performed to assess the irritation potential of FRET 12-0492 by means of the Human Skin Model Test.

The test item passed the MTT and colour interference pre-tests.

Each three tissues of the human skin model EpiSkin™ were treated with the test item, the negative or the positive control for 15 minutes.

The test item and the positive and negative controls were washed off the skin tissues treatment. After further incubation for approximately 42 hours the tissues were treated with the MTT solution for 3 hours following about 2.5 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.

The acceptance criteria were met. Exceptions:

·        The rel. standard deviation between tissues exposed to the positive control was > 18% due to very low absorbance values.

·        Due to recent change of negative control at Envigo CRS GmbH for being in accordance with the tissue supplier’s test protocol, historical data for the negative control are not yet available.

The exceptions from the acceptance criteria did not have an impact on the outcome of the study.

After treatment with the test item the mean relative absorbance value was reduced to 30.9%. This value is below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

The relative mean tissue viability after 15 minutes treatment with the substance compared to the negative control tissue was 30.9%. Since the mean relative tissue viability for the substance was below 50% after 15 minutes treatment, the substance is considered to be a potential 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 tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissue was 30.9%.

Since the mean relative tissue viability for FRET 12 -0492 was below 50% after 15 minutes treatment the substance is considered to be irritant. The positive control had a mean cell viability of 1.2% after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was less than 18% (with the exception of the positive control because extremely low absorbance values were recorded), indicating that the test system functioned properly.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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 26 April 2017 and 12 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: FRET 12-0492
Chemical Name: 4,6-dimethyl-2-(1-phenylethyl)-3,6-dihydro-2H-pyran
Appearance: Yellow, liquid
Storage Conditions: In the refrigerator

Species:

other: Reconstructed Human Corneal Epithelial Model

Strain:

other: Not applicable

Details on test animals or tissues and environmental conditions:

EpiOcular™ kits and MTT-100 kits are 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).
EpiOcular™ tissues were shipped at 2 - 8 °C on medium-supplemented agarose gels in a 24-well plate. On day of receipt (10 May 2017) 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 of fresh Assay Medium at 37 °C and the EpiOcular™ tissues was incubated at standard culture conditions overnight (nearly 17 hours).

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount applied (volume with unit): 50 µL.

Negative control: Deionised water
Positive control: Methyl acetate

Duration of treatment / exposure:

30 minutes

Observation period (in vivo):

No data

Duration of post- treatment incubation (in vitro):

12 minute post-soak to remove any absorbed test material followed by 120 minute post-treatment incubation

Number of animals or in vitro replicates:

tested in duplicate for all test groups

Details on study design:

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, 95% RH) for 30 minutes.
Test item exposure: 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 beakers 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 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 glass tube containing 1 mL of warm assay medium. The tissues are incubated for 120 minutes at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 (post-treatment incubation).

MTT Assay
After post-treatment incubation of 120 minutes the MTT assay was performed.
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 are 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 (17 hours) and then shaken for 2.5 hours at room temperature. 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 absorbance at 570 nm (OD570) of each well was measured with a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro, version 4.7.1). No reference wavelength measurement was used.

Irritation parameter:

other: Mean relative absorbance

Value:

83.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

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 colour.
The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 83.4% (threshold for irritancy: ≤ 60%), consequently the test item was not irritant to eye.
Concerning acceptance criteria:
• The negative control OD is > 0.8 and < 2.5 (1.813 and 1.937).
• The mean relative viability of the positive control is below 50% of the negative control viability (20.6%).
• The difference of viability between the two relating tissues of a single item is < 20% (values between 1.0% and 2.3%) in the same run (for positive and negative control tissues and tissues of single test items).

This in vitro study was performed to assess the eye irritation potential of FRET 12-0492 by means of the Human Cornea Model Test.
Additional tests with viable or freeze-killed tissues were not performed, since the test item was not coloured intensively, did not dye water or isopropanol, and did not prove to be a MTT reducer.
Tissues of the human cornea model EpiOcular™ were treated with the test item, the positive and the negative control for 30 minutes each in duplicate.
Treatment with the positive control induced a decrease in the mean relative absorbance compared with the negative control to 20.6%, thus the validity of the test system is ensured.
Since the viability value of the test item exposed tissues did not decrease below 60%, the test item is not considered to possess an eye irritating potential.

Results after treatment for 30 minutes with FRET 12-0492 and the controls

Dose Group	Ab-sorbance Well 1 (Tissue 1/2)	Ab-sorbance Well 2 (Tissue 1/2)	Mean Absor-bance* (Tissue 1/2)	Mean Absorbance* Tissue 1 and 2	Mean Absorbance of 2 Tissues*	Rel. Absorbance [%] Tissue 1 and 2**	Absolute Value of the Difference of the Rel. Absorbances [%] Tissue 1 and 2	Mean Rel. Absorbance [% of Negative Control]**
Blank	0.038	0.038	0.038	0.000
Negative Control	1.813	1.937	1.875	1.837	1.846	99.5	1.0	100.0
	1.893	1.894	1.894	1.855		100.5
Positive Control	0.424	0.453	0.439	0.401	0.379	21.7	2.3	20.6
	0.395	0.398	0.396	0.358		19.4
Test Item	1.603	1.593	1.598	1.560	1.540	84.5	2.2	83.4
	1.558	1.559	1.558	1.520		82.4

*         Mean of two replicate wells after blank correction

**       Relative absorbance [rounded values]: (100 x (absorbance _{testitem/positivecontrol}) / (meanabsorbance_{negativecontrol})

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

According to the study plan followed the test item, was considered to be non-irritant.

EU CLP (1272/2008/EC)/UN GHS: Not classified for irritation.
UN GHS: Not classified for irritation.

Executive summary:

The eye irritation potential of the test substance FRET 12-0492 was assessed using the Human Cornea Model after a treatment period of 30 minutes. Cell viability is measured by dehydrogenase conversion of MTT [(3-4,5-dimethyl thiazole 2-yl) 2,5-diphenyl-tetrazoliumbromide], present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison of untreated negative controls is used to predict eye irritation potential.

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. The EpiOcular ™ Eye Irritation Test (EIT) was validated by the European Union Reference laboratory for Alternatives to Animal Testing (EURL ECVAM) and cosmetics Europe between 2008 and 2013. The test consists of a topical exposure of the neat test item to a human reconstructed cornea model followed by a cell viability test.

Relative mean tissue viability of ≤ 60 % results in a prediction of ocular irritancy. 

After a 30 minute exposure, the relative mean viability of the test item treated tissues was 83.4% and the test substance is therefore considered to be non-irritant, according to EU CLP criterial. 

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 06, 2017 - July 20, 2017

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:

(2012)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: FRET 12-0492
Physical state/Appearance: very pale yellow liquid
Storage Conditions: approximately 4 °C in the dark

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

- Source: Two New Zealand White (Hsdlf:NZW) strain rabbits were supplied by Envigo RMS (UK) Limited, Leicestershire, UK
- Age at study initiation: 12 to 52 weeks old
- Weight at study initiation: Body weights were 3.63 or 4.12 kg
- Housing: Individually in suspended cages.
- Diet: 2930C Teklad Global Rabbit diet supplied by Envigo RMS (UK) Limited, Oxon, UK
- Water: mains drinking water
- Acclimation period: a period of at least 5 days.

ENVIRONMENTAL CONDITIONS
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): 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:

STUDY DESIGN
The test material was applied at 0.1 mL in 2 animals.

TREATMENT
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.
In accordance with OECD 405 (2012). The test substance is placed in the conjunctival sac of one eye of each animal after gently pulling the lower lid away from the eyeball. The lids are then gently held together for about one second. The other eye serves at control.
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.

REMOVAL OF TEST SUBSTANCE
-Washing: No

OBSERVATIONS
- Irritation:
The eyes of each animal were examined approximately 1, 24, 48 and 72 hours after instillation of the test substance.
The irritation scores and a description of all other (local) effects were recorded. The irritation was assessed according to OECD 405 (2012).

Irritation parameter:

cornea opacity score

Remarks:

75716 Female

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

cornea opacity score

Remarks:

75764 Female

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

iris score

Remarks:

75816 Female

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

iris score

Remarks:

75764 Female

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

conjunctivae score

Remarks:

75716 Female

Basis:

mean

Time point:

24/48/72 h

Score:

2.6

Max. score:

6

Reversibility:

fully reversible

Irritation parameter:

conjunctivae score

Remarks:

75764 Female

Basis:

mean

Remarks:

(mean)

Time point:

24/48/72 h

Score:

2.67

Max. score:

6

Reversibility:

fully reversible

Irritation parameter:

chemosis score

Remarks:

75716 Female

Basis:

mean

Time point:

24/48/72 h

Score:

0.33

Max. score:

1

Reversibility:

fully reversible

Irritation parameter:

chemosis score

Remarks:

75764 Female

Basis:

mean

Time point:

24/48/72 h

Score:

0.33

Max. score:

1

Reversibility:

fully reversible

Irritant / corrosive response data:

See section "Any other information on results incl. tables"

Ocular Irritant response data (for the undiluted test substance)

Rabbit Number and Sex	75716Female				75764Female
	IPR= 0				IPR = 1
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	0	0	0	0	0	0	0	0
Score (D x 5)	0	0	0	0	0	0	0	0
CONJUNCTIVAE
A = Redness	2	1	1	0	2	1	1	0
B = Chemosis	1	1	0	0	1	1	0	0
C = Discharge	2	1	0	0	2	1	0	0
Score (A + B + C) x 2	10	6	2	0	10	6	2	0
Total Score	10	6	2	0	10	6	2	0

IPR=Initial pain reaction

  Ocular Reactions

No corneal or iridial effects were noted during the study.

Moderate conjunctival irritation was noted in both treated eyes 1 hour after treatment with minimal conjunctival irritation noted at the 24 and 48‑Hour observations.

Both treated eyes appeared normal at the 72‑Hour observation.

 Body Weight

Both animals showed expected gain in body weight during the study.

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 (2012), minimal irritation was observed. Based on the results of this study, the substance is not considered an eye irritant.

Executive summary:

The substance, FRET 12 -0492 was tested in an eye irritation test in rabbits according to OECD 405 (2012). Ocular effects include a summary of the grades were observed, which were fully reversible within 72 hours". The results showed that the substance is not an ocular irritant.

Reference 3

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

The study was conducted on the 28 April 2017

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:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: FRET 12-0492
Chemical Name: 4,6-dimethyl-2-(1-phenylethyl)-3,6-dihydro-2H-pyran
Appearance: Yellow, liquid
Storage Conditions: In the refrigerator

Species:

cattle

Strain:

other: Not applicable

Details on test animals or tissues and environmental conditions:

Freshly isolated bovine eyes of at least 9 month old donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were stored in HBSS containing 1% (v/v) Penicillin/Streptomycin (100 units/mL penicillin and 100 μg/mL streptomycin) in the cooled slaughter-house until transportation on the same morning to the laboratory using a Styrofoam box. The corneae were isolated on the same day after delivery of the eyes.

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:

3 cornea for test and contol items

Details on study design:

Preparation of Corneae
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea.
Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OECD guideline 437, which consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure no air bubbles were present within the compartments.
For equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath.
At the end of the incubation period, the basal opacity was determined (t0).
The basal opacity of all corneae was recorded. Each cornea with a value of the basal opacity > 7 was discarded. Sets of three corneae were used for treatment with the test item and the negative and positive controls.

Exposure of the Corneae to the Test Groups
The anterior compartment received the test item or negative or positive control at a volume of 0.75 mL on the surface of the corneae. The corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath.
The incubation time lasted ten minutes.
After the test item or control items, respectively, were rinsed off from the application side with saline, fresh cMEM was added into the anterior compartment. Then the corneae were
incubated at 32 ± 1 °C for further two hours in a vertical position, followed by a second opacity reading (t130).
In the second step of the assay, permeability of the corneae was determined.

Opacity Measurement
The opacitometer determines changes in the light transmission passing through the corneae, and displays a numerical opacity value. This value was recorded in a table. The opacitometer OP_KiT opacitometer (Electro Design, 63-Riom France) was calibrated as described in the manual and the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea.
After exposure of the corneae to the test groups, after rinsing and further incubation of the corneae for two hours, the opacity value was determined again (t130).

Permeability Determination
Following the opacity readings, the permeability endpoint was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the complete medium was removed from the anterior compartment and replaced by 1 mL of a 0.4% (w/v) sodium fluorescein solution in HBSS. Corneae were incubated again in a horizontal position for 90 minutes in a water-bath at 32 ± 1 °C. Complete medium from the posterior compartment was removed, well mixed and transferred into a 96 well plate and the optical density at 490 nm (OD490) was determined with a spectrophotometer.
The optical density was measured with a microplate reader (Versamax® Molecular Devices) at 490 nm (OD490). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

Data Evaluation
Opacity
The change of opacity value of each treated cornea or positive and negative control corneae is calculated by subtracting the initial basal opacity from the post treatment opacity reading (t130 – t0).
The average change in opacity of the negative control corneae is calculated and this value is subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.

Permeability
The corrected OD490 value of each cornea treated with positive control and test item is calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

The following formula is used to determine the IVIS of the negative control:
IVIS = opacity value + (15 x OD490 value)
The following formula is used to determine the IVIS of the positive control and the test item:
IVIS = (opacity value – corrected opacity value mean negative control) + (15 x corrected OD490 value)
The mean IVIS value of each treated group is calculated from the IVIS values.
Depending on the score obtained, the test item is classified into the appropriate category according to OECD guideline 437.

Irritation parameter:

in vitro irritation score

Value:

2.02

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 0.75).
The positive control (2-Ethoxyethanol) was tested undiluted and showed clear opacity and distinctive permeability of the corneae (mean IVIS = 76.00) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)).
The test item FRET 12-0492 was tested undiluted. Relative to the negative control, the test item FRET 12-0492 did not cause a relevant increase of the corneal opacity or permeability. The calculated mean IVIS was 2.02 (threshold for serious eye damage: IVIS > 55). According to OECD 437, the test item is not categorized.

Results after 10 Minutes Treatment Time

Test Group	Opacity value = Difference (t130-t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS	Proposed in vitro Irritancy Score
		Mean		Mean
Negative Control	0	0	0.05	0.05	0.75	0.75	Not categorized
	0		0.051		0.77
	0		0.048		0.72
Positive Control	67.00*		0.896*		80.45	76	Category 1
	56.00*		1.093*		72.4
	65.00*		0.677*		75.16
FRET 12- 0492	1.00*		0.041*		1.62	2.02	Not categorized
	2.00*		0.046*		2.7
	1.00*		0.049*		1.74

*corrected values

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

The test item, FRET 12-0492 was considered not to be an ocular corrosive or severe irritant.

Executive summary:

The eye irritancy potential of the test substance, FRET 12-0492 was assessed according to OECD Test Guideline 437 using the Bovine Corneal Opacity and Permeability Assay method and has an IVIS of 2.02, according to EU CLP criteria.According to OECD 437 the test item is not categorized.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

In vitro tests

Skin irritation (OECD TG 439):

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 tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissue was 30.9%.

Since the mean relative tissue viability for FRET 12 -0492 was below 50% after 15 minutes treatment the substance is considered to be irritant. The positive control had a mean cell viability of 1.2% after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was less than 18% (with the exception of the positive control because extremely low absorbance values were recorded), indicating that the test system functioned properly.

Skin corrosion (OECD TG 431):

The skin corrosivity of the test substance was determined according to OECD Guideline 431 using the EpiDerm™ Reconstructed Human Epidermis Model. The relative absorbance value after 3 and 60 minutes exposure were above 50% and 15% respectively in relation to the control. Therefore the substance is not corrosive to skin, according to EU CLP criteria.

Eye irritation (OECD TG 492):

The eye irritation potential of the test substance FRET 12-0492 was assessed according to OECD TG 492 using the Human Cornea Model after a treatment period of 30 minutes. Cell viability is measured by dehydrogenase conversion of MTT [(3-4,5-dimethyl thiazole 2-yl) 2,5-diphenyl-tetrazoliumbromide], present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison of untreated negative controls is used to predict eye irritation potential.

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. The EpiOcular ™ Eye Irritation Test (EIT) was validated by the European Union Reference laboratory for Alternatives to Animal Testing (EURL ECVAM) and cosmetics Europe between 2008 and 2013. The test consists of a topical exposure of the neat test item to a human reconstructed cornea model followed by a cell viability test.

Relative mean tissue viability of ≤ 60 % results in a prediction of ocular irritancy. 

After a 30 minute exposure, the relative mean viability of the test item treated tissues was 83.4% and the test substance is therefore considered to be non-irritant, according to EU CLP criterial. 

Eye severe damage (OECD TG 437):

The eye irritancy potential of the test substance, FRET 12-0492 was assessed according to OECD test guideline 437 using Bovine Corneal Opacity and Permeability Assay method and has an IVIS of 2.02, according to EU CLP criteria the test item is not categorized.

In vivo test

Eye irritation and severe damage (OECD TG 405):

The substance, FRET 12-0492 was tested in an eye irritation test in rabbits according to OECD 405 (2012). Ocular effects include a summary of the grades were observed, which were fully reversible within 72 hours. The results showed that the substance is not an occular irritant.

Justification for classification or non-classification

Base on the positive results in the in vitro skin irritation test, FRET 12 -0492 is classified as Category 2 skin irritant according to EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.

Based on the negative results in the eye irritation tests FRET 12 -0492 does not need to be classified for eye irritation and severe damage.