Reaction mass of 3-[(3aS,7aR)-octahydro-1H-4,7-methanoinden-1-yl]propanal and 3-[(2s,3aR,7aS)-octahydro-1H-4,7-methanoinden-2-yl]propanal and 3-[(2r,3aR,7aS)-octahydro-1H-4,7-methanoinden-2-yl]propanal

Endpoint summary

Description of key information

Skin corrosion (OECD TG 431): Not corrosive
Skin irritation (OECD TG 439): Irritating
Eye severe damage (OECD TG 437): Not irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 January - 26 January, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is considered to be reliability 1 as it has been conducted according to OECD Test Guideline 439 using the EPISKIN™ Reconstructed Human Epidermis Model and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2013)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

(2012)

Deviations:

no

GLP compliance:

yes

Species:

other: EPIKSIN in vitro Reconstructed Human Epidermis (RHE) Model

Strain:

other: Not applicable

Details on test animals or test system and environmental conditions:

EPISKIN™ Reconstructed Human Epidermis Model Kit
Supplier : SkinEthic Laboratories, Lyon, France
EpiSkinTM Tissues (0.38cm2) lot number : 15-EKIN-003
Killed tissues: EPISKIN-SMTM, 0.38 cm2, Lot no.: 14-EKIN-031 and 14-EKIN-035

Type of coverage:

other: Twenty five μl of the undiluted test substance was added into 12-well plates on top of the skin tissues.

Preparation of test site:

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

Vehicle:

unchanged (no vehicle)

Controls:

other: 3 Tissues were treated with 25 μl PBS (negative control) and 3 tissues with 25 μl 5% SDS (positive control) respectively.

Amount / concentration applied:

Test material
- Applied volume: 25 μL

Duration of treatment / exposure:

15-Minute exposure period and 42 hours post-exposure incubation period.

Number of animals:

A total of 9 tissues were used: Triplicate tissues were treated with: test substance, positive control or negative control.

Details on 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 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 thecellular 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.

Test for Direct MTT Reduction:
FRET 09-0536 was checked for possible direct MTT reduction and colour interference in the Skin corrosion test using EpiDerm as a skin model (project 507446):
FRET 09-0536 was checked for possible colour interference before the study was started. Some non-coloured test substances may change into coloured substances in aqueous conditions and thus stain the skin tissues during the 1-hour exposure. To assess the colour interference, 50 μl of FRET 09-0536 50 μl Milli-Q water as a negative control were added to 0.3 ml Milli-Q water. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0°C in the dark.
FRET 09-0536 was checked for possible direct MTT reduction before the study was started. To assess the ability of the test substance to reduce MTT, 50 μl of FRET 09-0536 was added to 1 mg/ml MTT solution in phosphate buffered saline. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0ºC. A negative control, sterile Milli-Q water was tested concurrently.
In case the test substance reacts with the MTT medium in addition to the normal 1-hour procedure, two freeze-killed tissues treated with test substance and two freeze-killed non treated tissues must be used for the cytotoxicity evaluation with MTT.

PRE-INCUBATION:
On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for 25 hours at 37°C. Maintenance medium and Assay medium were supplied by SkinEthic Laboratories, Nice, France.
Living epidermis was transferred to 12 well plates and incubated with 2 ml Milli-Q for 48 ± 1 hours. After incubation, killed epidermis was stored at ≤ -15°C. Killed tissues were thawed by placing them for 1 hour at room temperature in 12 well plates on 2 ml maintenance medium. Further use of killed tissues was similar to living tissues.

APPLICATION/TREATMENT OF TEST SUBSTANCE:
The undiluted test substance was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μl PBS (negative control) and 3 tissues with 25 μl 5% SDS (positive control) respectively. In addition three killed tissues treated with test substance and three killed non treated tissues were used for the cytotoxicity evaluation with MTT. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test substance. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.

CELL VIABILITY MEASUREMENT:
After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-well plate prefilled with 2 ml MTT-medium (0.3 mg/ml). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in prelabeled microtubes and extracted with 500 μl isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for 68.5 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Skin irritation potential of the test substance was classified according to remaining cell viability following exposure of the test substance.

Irritation / corrosion parameter:

other: other: relative mean viability

Value:

36

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 15 minutes expoure. Reversibility: other: Not applicable. Remarks: The relative mean tissue viability compared to the negative control tissues. The substance is not corrosive in the in vitro skin corrosion test.. (migrated information)

Irritant / corrosive response data:

The relative mean tissue viability obtained after 15 minutes treatment with FRET 09-0536 compared to the negative control tissues was 36%. Since the mean relative tissue viability for FRET 09-0536 was below 50% it is considered to be irritant.

Direct MTT Reduction

FRET 09-0536 was checked for possible direct MTT reduction and colour interference in the Skin corrosion test using EpiDerm as a skin model (project 507446), and because solutions did turn blue / purple and/or a blue / purple precipitate was observed it was concluded that FRET 09-0536 did interfere with the MTT endpoint.

In addition to the normal procedure, three killed tissues treated with test substance and three killed non-treated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by FRET 09-0536 was <0% of the negative control tissues. Therefore no correction on the OD for non-specific reduction of MTT was made.

 

Test Item, Positive Control Item and Negative Control Item

The relative mean viability of the test item treated tissues was 36 % after a 15-Minute exposure period and 42 hours post-exposure incubation period.

 

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 9% relative to the negative control treated tissues. The positive control acceptance criterion was therefore satisfied.

The mean OD570 for the negative control treated tissues was 1.073 and the standard deviation was 0.047. The negative control acceptance criterion was therefore satisfied. The standard deviation value of the percentage viability of three tissues treated identically was less than 5%, indicating that the test system functioned properly.

Mean OD570 Values and Percentage 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 (%)
Negative Control Item	1.033	1.073	0.047		100
	1.061
	1.124
Positive Control Item	0.087	0.097	0.010	8	9
	0.108			10
	0.098			9
Test Item	0.366	0.386	0.021	35	36
	0.385			36
	0.407			36

SD = Standard deviation

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

Interpretation of results:

irritating

Remarks:

Migrated information 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 36%. Since the mean relative tissue viability for the substance was below 50% after 15 minutes treatment, the substance is considered to be irritant.

Executive summary:

The possible skin irritation potential of FRET 09 -0536 was tested through topical application for 15 minutes. The study procedures described in this report were based on the OECD TG 439 guideline and GLP principles. Skin tissue was treated by topical application of 25 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 36%.

Since the mean relative tissue viability for FRET 09 -0536 was below 50% after 15 minutes treatment the substance is considered to be irritant. This results in skin irritation, Category 2, H315: Causes skin irritation.

The positive control had a mean cell viability of 9% after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was less than 5%, indicating that the test system functioned properly.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 December 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2013)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

(2010)

Deviations:

no

Principles of method if other than guideline:

- The Ocular Toxicity Working Group (OTWG) of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Interagency Centre for the Evaluation of Alternative Toxicological Methods (NICEATM), Background Review Document (BRD): current status of in vitro test methods for identifying ocular corrosives and severe irritants: The Bovine Corneal Opacity and Permeability (BCOP) Test Method, March 2006.
- In Vitro Techniques in Toxicology Database (INVITTOX) protocol 127. Bovine Opacity and Permeability (BCOP) Assay, 2006.
- Gautheron P., Dukic M., Alix D. and Sina J.F., Bovine corneal opacity and permeability test: An in vitro assay of ocular irritancy. Fundam Appl Toxicol 18:442-449, 1992.

GLP compliance:

yes

Species:

other: Bovine eyes from young cattle

Strain:

other: Not applicable

Details on test animals or tissues and environmental conditions:

Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter. Eyes were collected and transported in physiological saline in a suitable container under cooled conditions. The corneas were prepared immediately on arrival.

Vehicle:

unchanged (no vehicle)

Controls:

other: positive control: 10% (w/v) Benzalkonium Chloride

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:

Three corneas / substance or control.

Details on study design:

Preparation of corneas
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.

The isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Invitrogen Corporation, Breda, The Netherlands) containing 1% (v/v) L-glutamine (Invitrogen Corporation) and 1% (v/v) Foetal Bovine Serum (Invitrogen Corporation)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of MC2 (Clermont-Ferrand, France) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.

Selection of corneas and opacity readings
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (OP-KIT, MC2, Clermont-Ferrand, France). The opacity of each cornea was read against an air filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

Treatment of corneas
The medium from the anterior compartment was removed and 750 μl of either the negative control, positive control (10% (w/v) Benzalkonium Chloride) or test substance was introduced onto the epithelium of the cornea. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or the test substance over the entire cornea. Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32 ± 1°C. After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Eagle’s Minimum Essential Medium, Invitrogen Corporation) and thereafter with cMEM. Possible pH effects of the test substance on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120 ± 10 minutes at 32 ± 1°C. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.

Application of sodium fluorescein
Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Merck, Darmstadt, Germany) was evaluated.
The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 ml of 4 mg Na-fluorescein/ml cMEM solution. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 ± 5 minutes at 32 ± 1°C.

Permeability determinations
After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 μl of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.

Histopathology
Not applicable.

Evaluation of results
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.

Opacity measurement
The opacitometer determined the difference in the light transmission between each control or treated cornea and an air filled chamber. The numerical opacity value (arbitrary unit) was displayed and recorded. The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each positive control or test substance treated cornea was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each positive control or test substance treated cornea.
The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.

Permeability measurement
The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution was performed, the OD490 of each reading was corrected for the mean negative control OD490 before the dilution factor was applied to the readings.

In vitro irritancy score
The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:
In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)
Additionally the opacity and permeability values were evaluated independently to determine whether the test substance induced irritation through only one of the two endpoints.

Visual observation
After the 10 minutes exposure, possible pH effects of the test substance on the corneas were recorded. After the 120 minutes incubation period, Each cornea was inspected visually for dissimilar opacity patterns.

Irritation parameter:

overall irritation score

Remarks:

(In vitro irritancy score (IVIS))

Basis:

mean

Time point:

other: 10 minutes

Score:

0.5

Reversibility:

other: Not applicable

Irritant / corrosive response data:

Corneal epithelium condition
The corneas were clear after the 10 minutes of treatment with FRET 09-0536 and the negative control. The corneas treated with the positive control substance were turbid after the 10 minutes of treatment.
In vitro irritancy scores:
The in vitro irritancy score for the test item was 0.5
The in vitro irritancy score for the negative control was 0.0
The in vitro irritancy score for the positive control was 125.3

Other effects:

No other effects were observed

Criterion for an acceptable test

The positive control in vitro irritancy score was within the range of 81 to 264. The positive control acceptance criterion was therefore satisfied.

The corneas treated with FRET 09 -0536 showed opacity values ranging from 0 to 1 and permeability values ranging from 0.005 to 0.023. The corneas were clear after the 10 minutes of treatment with FRET 09 -0536. No pH effect of the test substance was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -0.3 to 0.6 after 10 minutes of treatment with FRET 09 -0536.

Opacity score

Eye	Opacity before treatment	Opacity after treatment		Final Opacity^1		Negative control corrected Final Opacity^2		Mean Opacity
Negative control
1	2	2		0	-0.3			0.0
2	1	2		1	0.7
3	1	1		0	-0.3
			Mean final opacity: 0.3
Positive control
4	1	80		79	78.7			78.7
5	3	85		82	81.7
6	3	79		76	75.7
FRET 09-0536
10	1	1		0	-0.3			0.3
11	1	2		1	0.7
12	2	3		1	0.7

¹ Final Opacity = Opacity after treatment – Opacity before treatment.

² Negative control corrected Final Opacity = Final opacity – Mean final opacity negative control. Permeability score individual values (uncorrected)

Eye	Dilution factor	OD490 1	OD490 2		OD490 3		Average OD		Final OD		Mean final negative control
Negative control
1	1	0.004		0.005		0.004		0.004		0.004		0.005
2	1	0.004		0.004		0.004		0.004		0.004
3	1	0.007		0.007		0.007		0.007		0.007
Positive control(Benzalkonium Chloride)
4	6	0.400		0.401		0.402		0.401		2.406
5	6	0.334		0.330		0.331		0.332		1.990
6	6	0.835		0.826		0.850		0.837		5.022
FRET 09-0536
10	1	0.029		0.026		0.030		0.028		0.028
11	1	0.015		0.014		0.015		0.015		0.015
12	1	0.010		0.010		0.010		0.010		0.010

Permeability score individual values (corrected)

Eye	Dilution factor	Negative control corrected OD4901^1	Negative control corrected OD4902^1	Negative control corrected OD4903^1	Negative control corrected OD490 Average	Negative control corrected final OD490	Average OD
Negative control
1	1	-0.001	0.000	-0.001	-0.001	-0.001	0.000
2	1	-0.001	-0.001	-0.001	-0.001	-0.001
3	1	0.002	0.002	0.002	0.002	0.002
Positive control(Benzalkonium Chloride)
4	6	0.395	0.396	0.397	0.396	2.375	3.109
5	6	0.329	0.325	0.326	0.327	1.959
6	6	0.830	0.821	0.845	0.832	4.991
FRET 09-0536
10	1	0.024	0.021	0.025	0.023	0.023	0.013
11	1	0.010	0.009	0.010	0.010	0.010
12	1	0.005	0.005	0.005	0.005	0.005

¹ OD₄₉₀values corrected for the mean final negative control permeability (0.005).

In Vitro irritancy score

Eye	Negative control correctedFinal Opacity	Negative control correctedFinal OD490	In vitroIrritancy Score^1
Negative control
1	-0.3	-0.001	-0.3
2	0.7	-0.001	0.7
3	-0.3	0.002	-0.3
Positive control(Benzalkonium Chloride)
4	78.7	2.375	114.3
5	81.7	1.959	111.1
6	75.7	4.991	150.5
FRET 09-0536
10	-0.3	0.023	0.0
11	0.7	0.010	0.8
12	0.7	0.005	0.7

¹ In vitroirritancy score (IVIS) = opacity value + (15 x OD₄₉₀value).

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item was considered not to be an ocular corrosive or severe irritant, as FRET 09-0536 induced an IVIS ≤ 3.

Executive summary:

The eye irritancy potential of FRET 09 -0536 was assessed according to OECD Test Guideline 437 using the Bovine Corneal Opacity and Permeability Assay method and GLP principles.

FRET 09-0536 did not induce ocular irritation through both endpoints, opacity and permeability, resulting in a mean in vitro irritancy score of 0.5 after 10 minutes of treatment.

As FRET 09-0536 induced an IVIS ≤ 3, the substance is considered not to be an ocular corrosive or severe irritant, according to EU CLP criteria.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro tests

Skin corrosion: The skin corrosivity of FRET 09 -0536 was determined according to OECD Guideline 431 and GLP principles using the EpiDerm™ Reconstructed Human Epidermis Model. The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with FRET 09-0536 compared to the negative control tissues was 104% and 95% respectively. Because the mean relative tissue viability for FRET 09-0536 was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment FRET 09-0536 is considered to be not corrosive to skin, according to EU CLP criteria.

Skin irritation:

The possible skin irritation potential of FRET 09 -0536 was tested through topical application for 15 minutes. The study procedures described in this report were based on the OECD TG 439 guideline and GLP principles. Skin tissue was treated by topical application of 25 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 36%.

Since the mean relative tissue viability for FRET 09 -0536 was below 50% after 15 minutes treatment the substance is considered to be irritant. The positive control had a mean cell viability of 9% after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was less than 5%, indicating that the test system functioned properly.

In vitro eye irritation

The eye irritancy potential of FRET 09 -0536 was assessed according to OECD Test Guideline 437 using the Bovine Corneal Opacity and Permeability Assay method and GLP principles.

FRET 09-0536 did not induce ocular irritation through both endpoints, opacity and permeability, resulting in a mean in vitro irritancy score of 0.5 after 10 minutes of treatment.

As FRET 09-0536 induced an IVIS ≤ 3, the substance is considered not to be an ocular corrosive or severe irritant, according to EU CLP criteria.

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

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

Effects on skin irritation/corrosion: irritating

Justification for classification or non-classification

Based on the positive results in the in vitro skin irritation test the substance needs to be classified as a skin irritant. According to EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 this results in skin irritation, Category 2, H315: Causes skin irritation. According to EU Directive 67/548/EEC this results inXi-R38: May cause skin irritation.