Ethanol, 2-[(3-nitropyrazolo[1,5-a]pyridin-2-yl)oxy]-

Endpoint summary

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation / corrosion

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Details on test animals or test system and environmental conditions:

human reconstructed epidermis (tissues).The EpiskinTM model consists of an airlifted, living, multilayered epidermal tissue construction (surface 0.38 cm2), reconstructed from normal human epidermal keratinocytes for 13 days and produced in polycarbonate inserts in a serum-free and chemically defined medium. The model features a normal ultra structure and is functionally equivalent to human in vivo epidermis.

Amount / concentration applied:

Test item quantity applied to each tissue: 20 mg ± 2 mg.
The test item was spread over the whole tissue surface without damaging the tissue sample.
As the test item was a solid, 100 µL of 0.9% NaCl were applied over the test item to ensure good contact with the epidermis.
For the negative and positive controls, since they can be sampled using a pipette, a volume of 50 µL was applied evenly to the surface of each tissue, taking care to spread them over the whole tissue surface area without damaging the tissue sample.

Negative control
Name: 0.9% NaCl.
The origin and batch number of the negative control used are recorded in the study files.

Positive control
Name: glacial acetic acid.
The origin and batch number of the positive control used are recorded in the study files.

Details on study design:

1.Preliminary test
1.1Test for direct MTT reduction with the test item
As a test item may directly reduce MTT, thus mimicking mitochondrial succinate dehydrogenase activity, it is necessary to test the ability of the test item to directly reduce MTT before performing the main test. This property of the test item would only be a problem if, at the time of the MTT viability test (after rinsing), remained a sufficient amount of test item present on or in the tissue. In this case, the true metabolic MTT reduction and the false direct MTT reduction should be differentiated and quantified.
To identify any test item interference, the following preliminary test was performed: 20 mg of test item was added to 2 mL of a 0.3 mg/mL freshly prepared MTT solution. The mixture was incubated in darkness at +37°C for 3 hours (± 5 minutes) under continuous stirring. A negative control was tested concurrently by adding 50 µL of water for injection to 2 mL of a 0.3 mg/mL freshly prepared MTT solution.

If the MTT solution color turned blue/purple when compared to the negative control (complete or partial discoloration), the dose formulation was presumed to reduce MTT. In this case additional controls were performed on water killed tissues in parallel to the main test (performed on viable tissues).
1.2Test for the detection of the coloring potential of the test item
The intrinsic color or the ability of the test item to become colored in contact with water (simulating a tissue humid environment) was evaluated by adding 10 mg of test item to 90 µL of water for injection in a transparent recipient. After 1 hour of mixing, the coloration was checked.
If a colored solution was detected, additional controls were performed on viable tissues in parallel to the main test for the evaluation of the non specific OD. Otherwise, the test item was considered as a common chemical.

2 Main test
One 12-well plate was used for each of the three exposure times (3 minutes, 1 hour and 4 hours) for test item-treated tissues.
Positive and negative controls were placed on separate plates.
Test item, and negative and positive controls were applied on duplicate tissues.

2.1 Pre-incubation of the tissues on their day of arrival
A volume of 2 mL maintenance medium, pre-warmed (at 37°C), was added to 2 wells per 12 well plate as follows: duplicate wells for each test item and negative control exposure time and for the 4 hour positive control exposure time.
EpiskinTM kits were opened and one tissue was transferred into each maintenance medium pre-filled well. All 12-well plates were then placed into a cell incubator (37°C, 5% CO2, >95% humidity) for a 1 hour to 48 hours pre-incubation step before treatment.

2.2 Treatment of tissues
A volume of 2 mL assay medium, pre-warmed (at 37°C), was added to 2 wells per 12 well plate as follows: duplicate wells for each test item and negative control exposure time and for the 4 hour positive control exposure time.
The EpiskinTM tissues were removed from the incubator and one tissue was transferred into each assay medium pre-filled well.

The test item, positive control or negative control dose formulations were applied on each designated tissue.
The lids were replaced on each plate before incubation at room temperature as follows: positive control for 4 hours (± 10 minutes); test item and negative control for 3 minutes (± 5 seconds), 1 hour (± 5 minutes) and 4 hours (± 10 minutes).

For MTT reducing test items, duplicate test item-treated water-killed tissues were prepared in a 12-well plate and incubated under the same conditions as test item-treated viable tissues. Duplicate 0.9% NaCl treated water-killed tissues were prepared in a 12-well plate and incubated for 4 hours.

Tissues were processed (application and rinsing) in the same order and at regular time-intervals so the tissue exposure times were equal.

2.3 Rinsing of tissues
For all treated tissues [test item-treated, positive and negative controls, and treated water killed tissues (if applicable)], at the end of the designated incubation period each tissue insert was removed from the well of the treatment plate and rinsed with D-PBS.
Rinsing was achieved by gently filling and emptying each tissue insert 12 times with 2 mL D-PBS and the surface of each tissue was swept with a cotton-bud to gently remove any residual dose formulation. The inserts were then placed in assay medium pre-filled well (2 mL).

2.4 MTT viability assay
Two empty wells of each 12-well plate were filled with 2 mL MTT solution (0.3 mg/mL), and the corresponding tissues were placed in these wells. Each plate was protected from light and incubated for 3 hours (± 15 minutes) at +37°C, 5% CO2 in a humidified incubator.

At the end of the MTT incubation period, the underside of each tissue culture insert was blotted. Then the tissues were removed from its plastic insert using a biopsy punch. Any tissue discoloration was evaluated with the naked eye.

Thereafter, for each tissue, the epidermis was separated from the collagen matrix. Both parts (epidermis and collagen matrix) were put into a stoppered plastic tube and 0.5 mL acidified isopropanol were then added to each tube. After vortexing, each tube was protected from light and left at room temperature overnight to extract the formazan (reduced MTT) from the MTT-loaded tissues.

2.5 Optical density measurements
At the end of the extraction period, each tube was vortexed and briefly centrifuged avoiding the interference of cell fragments with the absorbance readings.

Each tube was used to fill 2 consecutive wells of a 96-well plate with 200 µL of extract per well. One 96 well plate was used for the negative and positive controls (placed at opposite ends of the plate), and a separate 96-well plate was used for the test item dose formulation (the extracts obtained after all exposure times were placed on the same plate) and, if applicable, the extracts from the treated and 0.9% NaCl treated water-killed tissues were each placed on an additional 96-well plates.

For each 96-well plate, the average Optical Density value (OD) of 4 wells containing 200 µL of acidified isopropanol only was used as the blank.

The OD was measured at 570 nm using a plate reader.
The test complies when:
 the mean cOD of the negative control is between 0.600 and 1.500,
 relative mean viability of the positive control is ≤ 20% of the relative mean viability of the negative control,
 in the range 20-100% viability and for ODs ≥ 0.3, difference of viability between the two tissue replicates should not exceed 30%.

Irritant / corrosive response data:

non corrosive

1      PRELIMINARY TESTS

1.1      Test for direct MTT reduction with the test item

During the preliminary test, the MTT solution color turned blue/purple when compared to the negative control, the dose formulation is presumed to reduce MTT. As a result, additional controls were performed on water-killed tissues in parallel to the main test (performed on viable tissues). The MTT‑reducing test item and negative control were applied to two water-killed tissues for each exposure time.

 

1.2      Test for the detection of the coloring potential of the test item

During the preliminary test, the solution did not become colored, the test item is presumed not to stain the tissue. As a result, no additional controls were performed on viable tissues in parallel to the main test.

 

 

2      MAIN TEST

 

2.1   Evaluation of the coloration of tissues at the end of the MTT incubation period

The qualitative evaluation of the MTT staining was performed with the naked eye and all treated tissues appeared blue which was considered indicative for viable tissues.

 

1.2.3      Evaluation of the MTT results

All of the acceptance criteria for the negative and positive controls were fulfilled, therefore the study was considered to be valid.

 

The mean blank OD values (mean OD_blank) are calculated from the four replicates on each plate.

Then, for each tissue, the mean OD_blankis subtracted from individual tissues OD and the background corrected mean OD values (mean cOD) are calculated.

For each exposure duration, the corrected mean OD of the two negative controls (mean cOD_NC) corresponds to 100% viability and is used as a reference.

For the tissues treated with the test item or positive control, the relative mean viabilities are expressed as percentages of the reference viability and are calculated as follows:

             Relative mean viability = (mean cOD_{TI or PC}/ mean cOD_NC) x 100.

 

with        mean cOD_TI: corrected mean OD of the three tissues treated with test item,

             mean cOD_PC: corrected mean OD of the three positive controls.

 

The relative mean viabilities of the test item-treated tissues were:

.            3 minutes exposure: 105%,

.            1 hour exposure: 100%,

.            4 hours exposure: 103%.

 

As the mean viability was≥35% at each exposure time,the results met the criteria for a non corrosive response.

Interpretation of results:

other: non corrosive

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the experimental conditions of this study, the test item, R0056895A, tested in its original form, is considered to be non corrosive to the reconstructed skin model.

According to these results, the classification of the test item should be the followingconsidered as: non corrosive (Directive 67/548/EEC, UN 2009 and Regulation 1272/2008/EEC.

Executive summary:

The objective of this study was to evaluate the corrosive potential of the test item, R0056895A, using the

EpiskinTM reconstructed skin model.

This study was conducted in compliance with CiToxLAB France’s standard operating procedures and the

principles of Good Laboratory Practices.

Preliminary tests were performed to detect the ability of the test item to interfere with cell viability

measurements by directly reducing MTT.

Following the preliminary test, the corrosive potential of the test item was tested in the main assay.

Test item, and negative and positive controls were applied on duplicate tissues and incubated at room

temperature as follows: positive control for 4 hours (± 10 minutes); test item and negative control for 3 minutes (± 5 seconds), 1 hour (± 5 minutes) and 4 hours (± 10 minutes).

At the end of the designated incubation period, each tissue insert was rinsed with D-PBS and put into 2 mL assay medium pre-filled well. Then, two empty wells of each 12-well plate were filled with 2 mL MTT solution (0.3 mg/mL), and the corresponding tissues were placed in these wells. Each plate was protected from light and incubated for 3 hours (± 15 minutes) at +37°C, 5% CO2 in humidified atmosphere.

At the end of the MTT incubation period, the tissues were removed from its plastic insert. Any tissue discoloration was evaluated with the naked eye (discolored surface area and intensity).

Then, for each tissue, the epidermis was separated from the collagen matrix and both parts were put into acidified isopropanol overnight to extract the formazan (reduced MTT) out of the MTT-loaded tissues.

At the end of the extraction period, the optical density of each extract was measured at 570 nm.

Relative viability values were calculated for each tissue and expressed as percentages of the negative control tissues viability which was set at 100% (reference viability).

In the preliminary tests, the test item was found not to have direct MTT reducing properties or coloring potential.

In the main test, all acceptance criteria for the negative and positive controls were fulfilled. The study was therefore

considered to be valid.

At the end of the MTT incubation period, any tissue discoloration was evaluated with the naked eye.

The blue discoloration of the test item-treated tissues following the 3 minutes, 1 hour and 4 hours exposure periods was representative of viable tissue.

The relative mean viabilities of the test item-treated tissues were:

. 3 minutes exposure: 98%,

. 1 hour exposure: 98%,

. 4 hours exposure: 100%.

As all the mean viabilities were ≥ 35%, the results met the criteria for a non corrosive response.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not 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:

2014

Reliability:

1 (reliable without restriction)

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

Species:

other: Bovine cornea

Details on test animals or tissues and environmental conditions:

Species: bovine cattle.
Origin: bovine eyes were obtained from freshly slaughtered cattle at the abattoir EVA, Saint Pierre sur Dives, France.
Age: bovine cattle were up to 12 months old.
Reason for choice: bovine corneas are recommended by regulatory authorities for this type of study. They are adapted for the evaluation of potential ocular irritants since they are part of the target organ.
Transport from supplier to CiToxLAB France: the eyes were transported to CiToxLAB France at ambient temperature, immerged in buffered Hanks medium containing an antibiotic [Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin (100 units/100 µg/mL final)]. A container with smooth internal surfaces was used for the transport to avoid damage to the corneas.

Preparation of the corneas:
Upon arrival at CiToxLAB France, the selection and preparation of corneas was performed as soon as possible. At each step of the preparation procedure, care was taken to avoid touching the corneas in order not to damage them.

Selection: a careful macroscopic examination was performed on all eyes to detect the presence of any defects (opacity, scratches, pigmentation, etc). Any eyes with defects were discarded. The examination was performed under a lamp, using HBSS in order to keep the eyes moistened and shiny. Particular attention was paid to the corneas and the eyes were swiveled in order to observe the fringe areas and any scratches directly under the light.
Preparation of the selected corneas: the tissues surrounding the eyeball were carefully pulled away and the cornea, surrounded by approximately 2 to 3 mm of sclera, was dissected out. The isolated corneas were stored in HBSS until all corneas had been prepared.
Washing of the corneas: the corneas were washed for 15 minutes, three times, in HBSS plus penicillin/streptomycin (100 units/100 µg/mL final) at room temperature. The corneas were used within a maximum of 24 hours.
Storage of the corneas: as the corneas were not used immediately, they were stored after washing. Each cornea was stored individually in 12 mL of M199 medium containing 5% dextran, plus penicillin/streptomycin, at +4°C, for a maximum of 24 hours before use.

Specific equipment used for the study:
 water bath (32°C),
 identified bovine cornea holders: OPKIT (polypropylene), diameter 18 mm, ref. ED 4004 SB (MC2, Clermont-Ferrand, France),
 electric screwdriver,
 vacuum pump with metal gavage tube,
 OPKIT opacitometer and calibrators (MC2, Clermont-Ferrand, France).

Vehicle:

other: As the test item is a non-surfactant solid, it was tested in a vehicle. The vehicle was selected according to the results of solubility assays : 0.9% NaCl

Controls:

yes

Amount / concentration applied:

Treatment of corneas
The medium of the anterior chamber was removed and the dose formulation was applied onto the epithelium of the cornea:
The test item was first of all grounded to a fine powder using a mortar and a pestle and then suspended in the required quantity of vehicle. The dose formulation was a yellow suspension. it was prepared at the concentration of 20% (w/v) in the vehicle.
The test item dose formulation was prepared within 4 hours before use and kept at room temperature.As the test item was tested using a 4-hour treatment, the positive control was 20% imidazole solution in 0.9% NaCl (w/v).
As the dose formulation could be applied using a micropipette, a volume of 750 µL (± 8 µL) was used using the closed-chamber method. The dose formulation was introduced into the anterior chamber of the corneal holder, through the dosing holes to cover the epithelial side of the cornea. The dosing holes were then sealed.

For the positive and vehicle controls, a volume of 750 µL ± 8 µL was used for each cornea using the closed-chamber method.


The treatment time of each series of three corneas was carefully measured with a chronometer, starting from the beginning of treatment of the first cornea of each series. Then each further operation (rinsing, measurement, etc.) was carried out in the same order for the three corneas of each series.

After application of the dose formulation, the holders were incubated vertically (cornea positioned horizontally with the treated side uppermost) in a water bath at +32°C (± 1°C), for4h.

Duration of treatment / exposure:

As the test item is a non-surfactant solid, a treatment time of 4 hours (± 5 minutes) was used in the study.

Details on study design:

Assembly of the corneas and the holders
The corneas were carefully examined macroscopically before their assembly in the holders, in order to detect the presence of any defects. Any corneas with defects were discarded. The corneas were then mounted in the corneal holders with the endothelial side against the O-ring of the posterior chamber. Each cornea was identified with the corresponding holder number.

Pre-incubation
For pre-incubation, both chambers of the corneal holder were filled to overflowing with MEM culture media supplemented with 1% fetal bovine serum plus penicillin/streptomycin (cMEM) at room temperature. The posterior chamber was always filled first to maintain the natural concave shape of the cornea.
After making sure that no air bubbles were present within the holder, it was immersed in a water bath, horizontally (cornea positioned vertically), up to approximately three quarters of its height. The holders were pre incubated for 1 hour and 5 minutes (± 5 minutes) at +32°C (± 1°C).

At the end of the pre-incubation period, the medium was removed from both chambers of the holder using a metal gavage tube attached to a vacuum pump to ensure complete evacuation. They were refilled with fresh cMEM (previously heated to +32°C), starting with the posterior chamber and taking care that no air bubbles were present. The chambers were re-sealed and the corneas were examined macroscopically through the holder to detect the presence of any defects. Then, the opacity of the cornea was measured to obtain OPT0.
Corneas that showed any macroscopic defect or an OPT0 value over 7 were discarded.

The test item, the vehicle control and the positive control were tested on three corneas each.

The corneas were distributed as follows:
 the median value of the OPT0 values of all pre-incubated corneas (with OPT0 ≤ 7) was calculated,
 three corneas with opacity values close to the median value were selected as vehicle control corneas,
 the remaining corneas were shared out between test item and positive control treated series using a manual distribution procedure.

To rigorously respect the treatment and any other incubation times, the three corneas from the same series were always processed in the same order at each step.
As the test item was tested using a 4-hour treatment, the positive control was 20% imidazole solution in 0.9% NaCl (w/v).

After treatment of corneas, a rinsing was done to eliminate as much dose formulation as possible, while taking care not to damage the cornea. On completion of the treatment period, the dose formulation was removed from the front opening of the anterior chamber (open-chamber method) and the epithelium was rinsed as follows:
 the chamber was emptied using a metal gavage tube attached to a vacuum pump,
 the corneas were rinsed four times with pre-warmed cMEM containing phenol red (i.e. until the dose formulation had been completely removed from the chamber or until the phenol red was not discolored). Then, the corneas were finally rinsed with pre-warmed cMEM without phenol red.

The rinsing efficiency was visually confirmed by observing the transparency and the color changing of the rinsing medium (containing phenol red). No difficulties were encountered during the rinsing.

The anterior chamber was refilled with fresh pre-warmed cMEM without phenol red. The front cover was replaced. Care was taken to make sure that no air bubbles were present within the holders (by ensuring that each chamber was filled to overflowing with pre-warmed cMEM).
Following the 4-hour treatment, the medium of the posterior chamber was renewed with pre-warmed cMEM (+32°C) and the second opacity measurement (OPT2) was performed directly.

Opacity measurements :
An opacitometer was used to measure light transmission (i.e. the level of opacity) through the center of each mounted cornea. A numerical opacity measurement (arbitrary unit) was displayed and recorded.

Just before the first opacity measurement (i.e. OPT0), the opacitometer was calibrated using specific calibrators. Values obtained for each calibrator were as follows:
 calibrator No. 1: set to 75,
 calibrator No. 2: from 145 to 155,
 calibrator No. 3: from 218 to 232.

Just before the second opacity measurement (i.e. OPT2), the opacitometer was calibrated using the calibrator No. 1 set to 75.

Just after each opacity measurement (OPT0 and OPT2), the calibration of the opacitometer was checked by using the calibrator No. 1. The value obtained was between 73 and 77.

Corneal opacity was determined by reading each holder in the right hand chamber of the calibrated opacitometer, versus an empty holder (without cMEM, cornea and glasses), placed in the left hand chamber.

For opacity measurement, care was taken to make sure that no air bubbles were present within the holders containing corneas (by ensuring that each compartment was filled to overflowing with heated cMEM) and each holder was wiped dry.

Permeability determination:
After the second opacity measurement, the medium of the anterior chamber was removed and the chamber received 1 mL of a fluorescein solution.
As the test item is a non-surfactant solid, the concentration of the fluorescein solution was 5 mg/mL.

Before use, the fluorescein solution was validated. For this purpose, the solution of fluorescein was diluted in cMEM in order to obtain a 5 µg/mL solution and the optical density at a wavelength of 490 nm (OD490 nm) of this dilution was measured. As the value obtained was between 0.850 and 0.940 (see Table 1), the fluorescein solution was validated.

For each series of three corneas, a chronometer started from the fluorescein application time of the first cornea of the series. The holders were incubated vertically (cornea positioned horizontally with the fluorescein-treated side uppermost) in a water bath at +32°C (± 1°C) for 90 minutes (± 5 minutes).

At the end of incubation, the maximum volume of cMEM recoverable from the posterior chamber of each holder was transferred into an identified tube using a single use plastic syringe and a needle. The medium was homogenized prior to determination of OD490 nm, using single-use cuvettes (1 cm path length) and a spectrophotometer (cMEM used as the blank). Any solutions giving an OD490 nm beyond the linear range of the spectrophotometer was diluted in cMEM and measured again. The corresponding OD490 nm was multiplied according to the dilution factor used.

Macroscopic examination:
After permeability determination, the corneas were removed from the holders and observed for opaque spots, other irregularities and any separation of the epithelium.
Then, the corneas were discarded

Irritant / corrosive response data:

No notable opaque spots or irregularities were observed on vehicle control corneas.
Fluoresceine fixation was observed on the corneas treated with the test item.
Opacity, fluoresceine fixation and thickening of the corneas were observed on those treated with the positive control.
IN VITRO IRRITANCY SCORE : All acceptance criteria were fulfilled. The study was therefore considered as valid.
The mean In Vitro Irritancy Score (IVIS) was: 9.

As the test item induced a mean IVIS > 3 and ≤ 55, the eye hazard potential of the test item could not be predicted. The test item could not be identified as inducing serious eye damage (UN GHS Category 1) or as a test chemical not requiring classification for eye irritation or serious eye damage (UN GHS No Category).

Optical density of the fluorescein solution diluted to 5µg/mL: 0.891

Calibration of opacitometer	Before OPT0	After OPT0	Before OPT2	After OPT2
calibratorN°1	75	75	75	76
calibratorN°2	153	np	np	np
calibratorN°3	225	np	np	np

 

GROUP		OPACITY				PERMEABILITY		SCORE
Vehicle control	holder	OPT0	OPT2	OPT2-OPT0		OD 490 nm
	41	1	0	-1		0.026
	32	1	0	-1		0.036
	20	1	3	2		0.022
	MeanSD			0 1.7		0.028 0.007
Test item	Holder	OPT0	OPT2	OPT2-OPT0	cOPT	OD 490 nm	cOD 490 nm
	15	0	3	3	3	0.014	-0.014	3
	17	0	2	2	2	0.027	-0.001	2
	44	0	1	1	1	0.031	0.003	1
	Mean SD				2 1 3.8		-0.004 0.009	2 0.9
Positive control	Holder	OPT0	OPT2	OPT2-OPT0	cOPT	OD 490 nm	cOD 490 nm
	27	0	91	91	91	5.408	5.380	172
	23	0	111	111	111	2.336	2.308	146
	37	0	117	117	117	3.176	3.148	164
	Mean SD				106.3 13.6		3.612 1.588	161 13.4

np: not performed

OD: optical density

cOD: corrected optical density

cOPT: corrected corneal opacity

SD: standard deviation

OPT0: corneal opacity before treatment

OPT2: corneal opacity after treatment

Vehicle control: 09% NaCl

Test item: R0056895A

Positive control: 20% imidazole solution in 0.9% NaCl

Interpretation of results:

other: not corrosive

Conclusions:

Under the experimental conditions of this study, the test item, R0056895A, was identified as a test
chemical not requiring classification for eye irritation or serious eye damage (UN GHS No Category).

Executive summary:

The objective of this study was to evaluate the eye hazard potential of the test item, R0056895A. Indeed,

the Bovine Corneal Opacity and Permeability (BCOP) test method can identify chemicals inducing serious

eye damage and chemicals not requiring classification for eye irritation or serious eye damage.

The design of this study was based on the guideline OECD Guideline 437 and the study was performed in

compliance with the OECD Principles of Good Laboratory Practice.

Corneas obtained from freshly slaughtered calves were mounted in corneal holders. Both chambers of the

corneal holder were filled with complemented MEM culture media (cMEM) and pre-incubated for 1 hour

and 5 minutes (± 5 minutes) at +32°C. Three corneas were used for each treated series (test item, positive control and vehicle control).

Before the treatment, a first opacity measurement was performed using an opacitometer.

The test item was applied at the concentrations of 20% (w/v) in the vehicle (0.9% NaCl), in a single experiment using a treatment time of 4 hours and the closed-chamber method. Vehicle and positive controls were applied using the same treatment time and using the closed-chamber method. At the completion of the treatment period, all formulations were removed from the front opening of the anterior chamber and the epithelia were rinsed.

A second opacity measurement was then performed.

After the second opacity measurement, the medium of the anterior chamber was removed and filled with a fluoresceine solution. The holders were then incubated vertically for 90 minutes at +32°C.

At the end of the incubation period, the optical density of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea. Each cornea was then observed for opaque spots and other irregularities.

At macroscopic examinations, fluoresceine fixation was observed on the corneas treated with the test item.

In Vitro Irritancy Score : all acceptance criteria were fulfilled. The study was therefore considered as valid.

The mean In Vitro Irritancy Score (IVIS) was: 2.

As the test item induced a mean IVIS ≤ 3, the test item was considered as a test chemical not requiring

classification for eye irritation or serious eye damage (UN GHS No Category).

Under the experimental conditions of this study, the test item, R0056895A, was identified as a test

chemical not requiring classification for eye irritation or serious eye damage (UN GHS No Category).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Justification for classification or non-classification

Using the Episkin TM reconstructed skin model, to evaluate the corrosive potential of the test item, R0056895A, a non-corrosive response was observed.

Using the EpiskinTM reconstructed human epidermis model, to evaluate the skin irritation potential of the test item, R0056895A, a

non-irritant skin response was observed.

Using theBovine Corneal Opacity and Permeability (BCOP) to evaluate the eye hazard potential of the test item, R0056895 A,

, the mean In Vitro Irritancy Score (IVIS) was: 2. As the test item induced a mean IVIS < 3, the test item was considered as a test chemical not requiring classification for eye irritation or serious eye damage (UN GHS N° Category).