Ethyl ethyl(phenyl)carbamate

Endpoint summary

Description of key information

Skin irritation in vitro

Key study: OECD guideline 439 and GLP study. The mean corrected percent viability of the treated tissues was 1.5%, therefore test item has to be considered as irritant to skin (Category 2).

Skin corrosion in vitro

Key study: OECD guideline 431 and GLP study. Under the tested conditions, the mean viability of two replicates were 82.18% and 59.15% and based on these results, test item does not have to be considered as corrosive to skin.

Eye irritation in vitro

Isolated Chicken Eye Test Method

Weight of evidence: OECD TG 438 and GLP study. The combination of the three endpoints for the test item was 1 x III, 2 x I, therefore the test item is not predicted as causing serious eye damage or not classified for eye irritation/serious eye damage.

Bovine Corneal Opacity and permeability

Weight of evidence: OECD TG 437 and GLP study. Under the experimental conditions of this study, the IVIS value was evaluated as 2 ±0.1 and therefore, the test item has been identified as not irritant to eye, not requiring classification for eye irritation or serious eye damage (UN GHS No Category).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Remarks:

Reconstructed Human Epidermis Test Method

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 3, 2016 - November 24, 2016

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions:yes
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:NO

Test system:

human skin model

Remarks:

SkinEthic RHE® model,Episkin SA, RHE/S/17

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Source strain:

not specified

Justification for test system used:

The SkinEthic™ RHE model can be use in this study according the OECD guideline for irritation testing (OECD No. 439).

Vehicle:

unchanged (no vehicle)

Remarks:

Test item was applied as supplied.

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:Episkin SA, RHE/S/17
- Tissue batch number(s):16-RHE-122
- Delivery date: 22.11.2016
- Date of initiation of testing:22.11.2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: ROOM TEMPERATURE
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps:25 x 1 mL of DPBS
-Observable damage in the tissue due to washing:NO

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration:300 μL of MTT solution at 1 mg/mL
- Incubation time:3 HOURS at temperaturwe between 36.4-37.8ºC, 5% CO2
- Spectrophotometer: ELx800 absorbance microplate reader
- Wavelength:570nm
- Linear OD range of spectrophotometer: 0- 2.0

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability:Specification :O.D>0.7, Result: O.D.=1.2 (CV=4.6%)
- Barrier function:Specification: 4.0h<=ET50<=10.0h, Result:5.2h
- Morphology:Specification: Number of cell layers >=4, Result:6 cell layers
- Contamination:no

NUMBER OF REPLICATE TISSUES:3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE- no interference

PREDICTION MODEL / DECISION CRITERIA
The test item is considered to be non irritant to skin: if the viability after 42 minutes of exposure and 42 hours of post-treatment incubationis > 50%.
The test item is considered to be irritant to skin: if the tissue viability after 42 minutes of exposure and 42 hours of post-treatment incubation is ≤ 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):16 μL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight):16 μL

POSITIVE CONTROL
- Amount(s) applied (volume or weight):16 μL
- Concentration : 0.5 g of SDS in a 10 mL volumetric flask qsp 10 mL of distilled water

Duration of treatment / exposure:

42 minutes

Duration of post-treatment incubation (if applicable):

41 hours and 15 minutes

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

MEAN viability

Value:

1.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

1.0%

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: NO

- Colour interference with MTT:The direct interaction of MTT with the test item was checked by adding 16 μL of the test item to 300 μL of solution of MTT at 1 mg/mL. After 3 hours of incubation was observed yellow solution - no direct interference between test item and MTT.

In water: The coloration potential of the test item in water was checked by adding 16 μL of the test item to 300 μL of distilled water. A colorless solution was obtained after 3 hours of incubation between 36.4°C and 37.8°C, 5% CO2.

In isopropanol: The coloration potential of the test item in isopropanol was checked by adding 16 μL of the test item to 1.5 mL of isopropanol. A colorless solution was obtained after 2 hours of incubation at room temperature. No significant coloration appeared. Therefore the test item will not interfere with the MTT assay.

DEMONSTRATION OF TECHNICAL PROFICIENCY:YES

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: SD value of the % viability ≤ 21.3%,instead of ≤ 18% as initially scheduled. This is due to a mean optical density (OD) value of the replicate No.3 treated with the negative control, which is slightly above the maximal OD value of 1.5.
- Acceptance criteria met for positive control: SD value of the % viability ≤ 18%.
- Acceptance criteria met for variability between replicate measurements:SD value of the % viability ≤ 18%.

Treatment of the results

% viability= (OD test Item / OD negative control )* 100%

Table No.1: Results

	SKIN	OD	Mean OD/disc	Mean OD/product	Viability %	Mean viability %	SD	CRITERIA
Negative control	1	0.943	1.019	1.309	77.8	100.00	21.3
		1.039
		1.075
	2	1.308	1.334		101.9
		1.352
		1.343
	3	1.512	1.575		120.3
		1.557
		1.657
Positive control	4	0.012	0.013	0.013	1.0	1.0	0.1	Irritant
		0.013
		0.013
	5	0.011	0.011		0.8
		0.012
		0.011
	6	0.013	0.014		1.1
		0.014
		0.014
Test item PH-16/0550	15	0.018	0.018	0.019	1.4	1.5	0.2	Irritant
		0.019
		0.017
	16	0.026	0.023		1.8
		0.027
		0.016
	17	0.009	0.017		1.3
		0.014
		0.027

OD-optical density

The optical density was measured after a 1:2 dilution of the formazan extracts in isopropanol

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Remarks:

Positive control:-irritant, SD value ≤ 18%, test item-SD value ≤ 18%, Negative control:non irritant, OD value in the range: ≥ 0.8 and ≤ 3%, SD value 21.3% / (acceptable)

Conclusions:

The mean corrected percent viability of the treated tissues was 1.5%, therefore test item has to be considered as irritant to skin.

Executive summary:

In accordance with GLP study and OECD TG 439, this study was to evaluate the possible irritating effects of the test item after topical application on in vitro human reconstructed epidermis (SkinEthic RHE® model). The test item was applied at the dose of 16 μL, to 3 living Reconstructed Human epidermis (SkinEthic RHE® model) during 42 minutes, followed by a rinse with 25 mL of DPBS and a 41 hours and 15 minutes post-incubation period at 37°C, 5% CO2. Then the epidermis were put in contact with MTT solution. In the same condition a positive and negative control were carried out. To ensure a good contact with epidermis, all items were recovered with a nylon mesh. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. The measured OD values were used to calculate a percentage of viability. The mean corrected percent viability of the treated tissues was 1.5%, therefore test item has to be considered as irritant to skin.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 17, 2017 - January 18, 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: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:Room temperature
- Stability under test conditions:yes

Test system:

human skin model

Remarks:

epiCS, Cellsystems

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Source strain:

not specified

Justification for test system used:

The human skin model, epidermis has been validated for corrosion testing and it can be use for skin corrosion test accordiing the OECD guideline 431.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:epiCS, cell systems
- Delivery date:17.01.2017
- Date of initiation of testing:17.01.2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: ROOM TEMPERATURE
- Temperature of post-treatment incubation: 37+-2°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps:20 x 1 mL of DPBS
-Observable damage in the tissue due to washing:NO

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration:300 μL of MTT solution at 1 mg/mL
- Incubation time:3 HOURS at temperaturwe between 36.8-37.8ºC, 5% CO2
- Spectrophotometer: ELx800 absorbance microplate reader
- Wavelength:570nm
- Linear OD range of spectrophotometer: 0- 2.0

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- HIV1,HBV,HCV: Specification:negative, Results:pass
- Bacteria, Yeast, Mycoplasma:Specification: negative, Results:pass
- Barrier function:Specification: >50%, Result: 56.91%
- Morphology: Number of cornified layers 5, Result:pass
Number of vital cell layers 4, Result:pass
- Contamination:no

NUMBER OF REPLICATE TISSUES:2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE- no interference

PREDICTION MODEL / DECISION CRITERIA
If the viability after 3 minutes exposure is strictly less than 50% or greater or equal to 50% and the viability after 1 hour exposure is strictly less than 15 %, test item has to be classified as CORROSIVE.

If the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15 %, test item has to be classified as NON-CORROSIVE.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 uL as supplied

NEGATIVE CONTROL
- Amount(s) applied (volume or weight):50 uL


POSITIVE CONTROL
- Amount(s) applied (volume or weight):50 uL

Duration of treatment / exposure:

2 exposure periods: 3 minutes and 1 hour

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean 1

Value:

ca. 82.18

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean 2

Value:

ca. 59.15

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No.
It was checked by adding 16 ul of test item as supplied to 0.3 mL of the solution of MTT at 1 mg/mL. The solution was the same color (yellow) as at the beginning of incubation.

- Colour interference with MTT:
In water: The coloration potential of the test item in water was checked by adding 16 μL of the test item to 1uLof distilled water. A colorless solution was obtained after 3 hours of incubation between 36.4°C and 37.8°C, 5% CO2.

In isopropanol: The coloration potential of the test item in isopropanol was checked by adding 16 μL of the test item to 1.5 mL of isopropanol. A colorless solution was obtained after 2 hours of incubation at room temperature. No significant coloration appeared. Therefore the test item will not interfere with the MTT assay.

DEMONSTRATION OF TECHNICAL PROFICIENCY:Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The values of mean OD of negative control for treatment time 3 minutes and 1 hour were 1.316 and 1.217 respectively. These values remained in the range of historical negative control.
- Acceptance criteria met for positive control:Mean viability of tissue replicates exposed for 1 hour, expressed as % of the negative control is 0.15 %
- Acceptance criteria met for variability between replicate measurements: Viability in the range 20-100%, OD values ≥0.3. Difference of viability between the two tissue replicates are: 26.5% for 3 minutes exposure time and 8.8% for 1 hour exposure time.
- Range of historical values if different from the ones specified in the test guideline:

Historical control value of MEAN OD for negative control DPBS:
2 replicates, 3 minutes exposure time
17/02/2016, MEAN OD = 1.256
24/02/2016, MEAN OD = 1.074
06/04/2016, MEAN OD = 1.149
13/04/2016, MEAN OD = 1.260
04/05/2016, MEAN OD = 1.324
19/05/2016, MEAN OD = 1.194
08/06/2016, MEAN OD = 1.342
20/07/2016, MEAN OD = 1.049
10/08/2016, MEAN OD = 0.917
07/09/2016, MEAN OD = 1.081
28/09/2016, MEAN OD = 1.379
19/10/2016, MEAN OD = 1.337
23/11/2016, MEAN OD = 0.990
21/12/2016, MEAN OD = 0.934

2 replicates,1 hour exposure time
17/02/2016, MEAN OD = 1.266
24/02/2016, MEAN OD = 1.074
06/04/2016, MEAN OD = 1.149
13/04/2016, MEAN OD = 1.260
04/05/2016, MEAN OD = 1.324
19/05/2016, MEAN OD = 1.194
08/06/2016, MEAN OD = 1.342
20/07/2016, MEAN OD = 1.049
10/08/2016, MEAN OD = 0.917
07/09/2016, MEAN OD = 1.081
28/09/2016, MEAN OD = 1.379
19/10/2016, MEAN OD = 1.337
23/11/2016, MEAN OD = 0.990
21/12/2016, MEAN OD = 0.934

Historical control value of MEAN VIABILITY % for positive control KOH 8N:
2 replicates, 1 hour exposure time
17/02/2016, MEAN VIABILITY % = 1.30
24/02/2016, MEAN VIABILITY % = 1.63
06/04/2016, MEAN VIABILITY % = 0.39
13/04/2016, MEAN VIABILITY % = 0.71
04/05/2016, MEAN VIABILITY % = -0.42
19/05/2016, MEAN VIABILITY % = 0.50
08/06/2016, MEAN VIABILITY % = 2.20
20/07/2016, MEAN VIABILITY % = 1.00
10/08/2016, MEAN VIABILITY % = 7.42
07/09/2016, MEAN VIABILITY % = 0.74
28/09/2016, MEAN VIABILITY % = 1.09
19/10/2016, MEAN VIABILITY % = 0.75
23/11/2016, MEAN VIABILITY % = 0.76
21/12/2016, MEAN VIABILITY % = 0.59

Table No.1 Results of mean viability % of the test item.

	MEAN VIABILITY %
	3 min exposure	1 hour exposure	Conclusion
POSITIVE CONTROL	58.81	0.74	Corrosive subcategory 1B/1C
TEST ITEM PH-16/0550	82.18	59.15	Non corrosive

Table No.2: INDIVIDUAL AND AVERAGE VALUES AFTER 3 MINUTES EXPOSURE

	Skin	OD	Mean OD/disc	Mean OD/product	Viability %	Mean viability %	Viability difference between replicates %
Negative control	1	1.745	1.575	1.316	119.68	100	39.4
		1.417
		1.565
	2	1.086	1.057		80.32
		1.032
		1.055
Positive control	3	0.705	0.773	0.774	58.74	58.81	0.15
		0.750
		0.866
	4	0.890	0.775		58.89
		0.734
		0.703
Test ítem PH-15-0550	9	1.099	1.256	1.082	95.44	82.18	26.5
		1.329
		1.340
	10	0.871	0.907		68.92
		1.012
		0.840

INDIVIDUAL AND AVERAGE VALUES AFTER 1 HOUR EXPOSURE

	Skin	OD	Mean OD/disc	Mean OD/product	Viability %	Mean viability %	Viability difference between replicates %
Negative control	11	0.921	1.028	1.217	84.50	100	30
		1.149
		1.014
	12	1.642	1.405		115.50
		1.469
		1.105
Positive control	13	0.007	0.007	0.009	0.58	0.74	0.3
		0.007
		0.007
	14	0.013	0.011		0.90
		0.011
		0.009
Test ítem PH-15-0550	19	0.765	0.773	0.720	63.54	59.15	8.8
		0.820
		0.736
	20	0.678	0.666		54.75
		0.680
		0.641

Interpretation of results:

GHS criteria not met

Conclusions:

Under the tested conditions, the mean viability of two replicates were 82.18% and 59.15% and based on these results, test item does not have to be considered as corrosive to the skin..

Executive summary:

In accordance with OECD 431 and GLP study, test item was tested for the skin corrosion test. Test substance was applied as supplied, in liquid form, at the dose level of 50 uL, to human skin model surfaces epiCS, Cell Systems at two exposure times - 3 minutes and 1 hour at room temperature. At the same experimental conditions a positive control KOH 8N and a negative control DPBS have been carried out. After the treatment time, the test substance was rinsed with 20 mL of DPBS and tissues were checked for any coloration and noted to be whitish, comparable con negative control. Then cell viability was measured by enzymatic conversion of the vital dye MTT into a blue formazan salt. The concentration of blue formazan was measured by determinating the Optical density at 570 nm. The OD values were used to calculate the viability% and used for results expression. The mean percent viability of the epidermis skins treated with the test item were 82.18 % (3 minutes) and 59.15% (1 hour). Based on these results and the clasification criteria, test item does not have to be considered as corrosive to the skin.

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:

weight of evidence

Study period:

November 7, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:room temperature
- Stability under test conditions:yes

Species:

chicken

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source:Slaughterhouse (Etablissement Brun, 33820 Etauliers, France)
- Characteristics of donor animals (e.g. age, sex, weight): 7 weeks old, 1.5-2.5 kg.
Animals were killed for human consumption.

- Storage, temperature and transport conditions of ocular tissue:
Heads have been removed immediately after sedation of the chickens by electric shock, and incision of the neck for bleeding. The heads have been collected on 07 November 2016 at 8:20 am.Because eyes were dissected in the laboratory, the intact heads were transported from the slaughterhouse at ambient temperature in plastic boxes humidified with towels moistened with physiological saline. The eyes were enucleated at Phycher on 07 November 2016 at 9:45 am.
- Time interval prior to initiating testing: November 7,2016 (8:20-9:45)
- indication of any existing defects or lesions in ocular tissue samples: NO
- Indication of any antibiotics used: NO

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 μL of the test item was applied, as supplied.

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

240 minutes (± 5 minutes)

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES:
The eyelids were carefully excised, taking care not to damage the cornea. Then, the eye was further dissected from the skull, taking care not to damage the cornea. The eyeball was pulled from the orbit by holding the nictitating membrane firmly with surgical forceps, and the eye muscles were cut with a bent, blunt-tipped scissor. When the eye is removed from the orbit, a visible portion of the optic nerve should be left attached. Once removed from the orbit, the eye was placed on an absorbent pad and the nictitating membrane and other connective tissue were cut away. The enucleated eye was mounted in a stainless steel clamp with the cornea positioned vertically. The clamp was then transferred to a chamber of the superfusion apparatus. The clamps were positioned in the superfusion apparatus such that the entire cornea was supplied with the physiological saline drip (in the range 0.1 to 0.15 mL/min). The chambers of the superfusion apparatus was temperature controlled between 32.3°C and 32.6°C. After being placed in the superfusion apparatus, the eyes were examined with a slit-lamp microscope to ensure that they have not been damaged during the dissection procedure using sodium fluorescein. Corneal thickness was also measured at this time at the corneal apex using the depth measuring device on the slit-lamp microscope.Eyes with; (i), a fluorescein retention score of > 0.5; (ii) corneal opacity > 0.5; or, (iii), any additional signs of damage were replaced. For eyes that are not rejected based on any of these criteria, individual eyes with a corneal thickness deviating more than 10% from the mean value for all eyes are to be rejected.

EQUILIBRATION AND BASELINE RECORDINGS
Once all eyes had been examined and approved the eyes were incubated between 45 and 65 minutes to equilibrate them to the test system prior to dosing. Following the equilibration period, a zero reference measurement was recorded for corneal thickness and opacity to serve as a baseline (i.e., time = 0). The fluorescein score determined at dissection was used as the baseline measurement for that endpoint.

NUMBER OF REPLICATES:3

NEGATIVE CONTROL USED: physiological saline 30 μL (one eye)

SOLVENT CONTROL USED: not applicable

POSITIVE CONTROL USED: 5% Benzalkonium chloride 30 μL (three eyes)

APPLICATION DOSE AND EXPOSURE TIME: The test item was applied for 10 seconds

OBSERVATION PERIOD: Pretreatment and starting at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse.


REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period:20 mL of physiological saline at ambient temperature
- Indicate any deviation from test procedure in the Guideline: No

METHODS FOR MEASURED ENDPOINTS:
- CORNEAL OPACITY: It was calculated by using the area of the cornea that was most densely opacified for scoring. The mean corneal opacity value for all test eyes was calculated for all observation time points. Based on the highest mean score for corneal opacity, as observed at any time

- SWELLING: measured with Haag-Streit BP900 slit-lamp microscope with depth-measuring device no. I and slit-width setting at 9½, equaling 0.095 mm: was determined from corneal thickness measurements made with an optical pachymeter on a slit-lamp microscope. It was expressed as a percentage and was calculated from corneal thickness measurements according to the following formula:
((corneal thickness at time t - corneal thickness at gtime=0 )/ corneal thicnkness at time=0 )*100. The mean percentage of corneal swelling for all test eyes was calculated for all observation time points. Based on the highest mean score for corneal swelling, as observed at any time point, an overall category score was then given for each test item.

- Damage to epithelium based on fluorescein retention: The mean fluorescein retention value for all test eyes was calculated for the 30-minute observation time point only, which was used for the overall category score given for each test or control item.

- Macroscopic morphological damage to the surface: pitting of corneal epithelial cells, loosening of epithelium, rougheningof the corneal surface and sticking of the test item to the cornea. This findings can vary in severity and may occur simultaneously.

SCORING SYSTEM:
- Mean maximum opacity score:
0 - No opacity,
0.5 -Very faint opacity
1- Scattered or diffuse areas; details of the iris clearly visible
2- Easily discernible translucent area; details of the ris are slightly obscured,
3-Severe corneal opacity; no specific details of the iris are visible; size of the pupil is barely discernible
4-Complete corneal opacity; iris invisible

- Mean fluorescein retention score at 30 minutes post-treatment :
0-No fluorescein retention,
0.5-Very minor single cell staining,
1-Single cell staining scattered throughout the treated area of the cornea,
2-Focal or confluent dense single cell staining,
3-Confluent large areas of the cornea retaining fluorescein

DECISION CRITERIA:YES as indicated in the TG was used.

Irritation parameter:

cornea opacity score

Run / experiment:

MEAN

Value:

ca. 0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class I

Irritation parameter:

fluorescein retention score

Run / experiment:

MEAN

Value:

ca. 1.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class III

Irritation parameter:

percent corneal swelling

Run / experiment:

MEAN

Value:

ca. 0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class I

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No, no morphological effects were noted, whatever the examination time.

Three separated tests were conducted with this substance (performed on 05 January 2015, 02 March 2015 and 15 Apri l 2015), and all tests lead to the conclusion "No prediction can be made". This result is considered as not critical since all test items that come out "No prediction can be made" would be subsequently tested with other adequately validated in vitro test(s), or as a last option in rabbits, depending on regulatory requirements, using a sequential testing strategy.

DEMONSTRATION OF TECHNICAL PROFICIENCY: YES

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, 3xI. The negative control is classified as “No Category”.
- Acceptance criteria met for positive control: Yes, 3xIV,The positive control is classified as “Corrosive/Severe Irritant.

Other effects:

- Lesions and clinical observations: NO
- Ophthalmoscopic findings:NO
- Effects of rinsing or washing: NO
- Other observations: NO

Table No.1: Selected eyes for the performance of the ICE test

Acceptability criteria ensuring eyes quality:

Eyes with (i) a fluorescein retention score > 0.5; (ii) corneal opacity > 0.5; or, (iii) any additional signs

of damage were replaced.

For eyes that were not rejected based on any of these criteria, individual eyes with a corneal thickness

deviating more than 10% from the mean value for all eyes were rejected.

Chamber	Fluorescein retention	Corneal opacity	Morphological effects	Corneal thickness (e)
1	0.5	0	N.t.R.	0.54
2	0.5	0	N.t.R.	0.60
3	0.5	0	N.t.R.	0.56
4	0.5	0	N.t.R.	0.57
5	0.5	0	N.t.R.	0.54
6	0.5	0	N.t.R.	0.57
7	0.5	0	N.t.R.	0.54
8	0.5	0	N.t.R.	0.62
9	0.5	0	N.t.R.	0.60
10	0.5	0	N.t.R.	0.61
11	0.5	0	N.t.R.	0.62
12	0.5	0	N.t.R.	0.60
13	0.5	0	N.t.R.	0.62
14	0.5	0	N.t.R.	0.59
15	0.5	0	N.t.R.	0.60
16	0.5	0	N.t.R.	0.54
Mean corneal thickness value = Range of accepted thickness :				0.58 0.52≤e≤0.64

N.t.R.: Nothing to Report

Table No.2: Results for negative control, 30 μL of negative control, 07 November 2016

Endpoint measured	Eye No.	0	30	75	120	180	240
Corneal opacity	16	0.0	0.0	0.0	0.0	0.0	0.0
ICE class					I
Fluorescein retention	16	0.5	0.5	-	-	-	-
ICE class			I	-	-	-	-
Corneal thickness	16	0.54	0.54	0.54	0.55	0.55	0.55
Corneal swelling (%)	16	-	0	0	2	2	2
ICE class					I
Combination of the 3 Endpoints	3 x I
CLASSIFICATION	No Category

No morphological effects were noted, whatever the examination time.

Table No.3:Results of positive control, 30 μL of positive control, 07 November 2016

Endpoint measured	Eye No.	0	30	75	120	180	240
Corneal opacity	1 2 3	0 0 0	3 3 3	3 3 3	3 3 3	3 3 3	3 3 3
Mean		0.0	3.0	3.0	3.0	3.0	3.0
ICE class					IV
Fluorescein retention	1 2 3	0.5 0.5 0.5	3 3 3	- - -	- - -	- - -	- - -
Mean		0.5	3.0	-	-	-	-
ICE class			IV	-	-	-	-
Corneal thickness	1 2 3	0.54 0.6 0.56	0.63 0.78 0.72	0.98 0.89 0.72	1.06 0.99 0.88	1.20 0.99 0.88	1.20 0.99 0.90
Corneal swelling (%)	1 2 3	- - -	17 30 29	81 48 29	96 65 57	122 65 57	122 65 61
Mean		-	25	53	73	81	83
ICE class					IV
Combination of the 3 Endpoints	3 x IV
CLASSIFICATION	Category 1 : Corrosive/Severe irritant

Severe loosening of the corneal epithelium noted from 30 minutes post-dose in eyes No. 1, 2 & 3.

Table No.4:Results of test item, 30 μL of pure test item, 07 November 2016

Endpoint measured	Eye No.	0	30	75	120	180	240
Corneal opacity	10 11 12	0 0 0	0 0 0	0 0 0	0 0 0	0 0 0	0 0 0
Mean		0.0	0.0	0.0	0.0	0.0	0.0
ICE class					I
Fluorescein retention	10 11 12	0.5 0.5 0.5	1 2 2	- - -	- - -	- - -	- - -
Mean		0.5	1.7	-	-	-	-
ICE class			III
Corneal thickness	10 11 12	0.6 0.62 0.60	0.61 0.62 0.60	0.61 0.62 0.60	0.61 0.62 0.60	0.61 0.62 0.60	0.61 0.62 0.60
Corneal swelling (%)	10 11 12	0 0 0	0 0 0	0 0 0	0 0 0	0 0 0	0 0 0
Mean		-	0	0	0	0	0
ICE class					I
Combination of the 3 Endpoints	1 x III, 2 x I
CLASSIFICATION	No prediction can be made

No morphological effects were noted, whatever the examination time.

Interpretation of results:

study cannot be used for classification

Conclusions:

The combination of the three endpoints for the test item was 1 x III, 2 x I, therefore the test item is not predicted as causing serious eye damage or not classified for eye irritation/serious eye damage.

Executive summary:

In accordance with OECD TG 438 and GLP study, the test item was tested for evaluation the possible ocular corrosive or severe irritating effects. Eyes were collected from the chicken killed for human consumption and transported in plastic boxes humified with towels moistened with physiological saline at ambient temperature in 1 hour 25 minutes to the laboratory at the same day. Thes test item was applied as supplied in amount 30 μL to 3 enucleated chicken eyes during 10 seconds. Then the eyes were rinsed twice with 20 mL of physiological saline at ambient temperature. Three eyes were treated in the same manner with a positive control and one eye with a negative control. Damages by the test item were assessed by determination of corneal swelling, opacity, and fluorescein retention at 30, 75, 120, 180 and 240 minutes post-dose. Then the results of each endpoint were assigned to ICE classes according the OECD guideline 438. Their combination - 1xIII and 2xI leads to the category : no prediction can be made.Therefore, the test item is not predicted as causing serious eye damage (Category 1) or as not classified for eye irritation/serious eye damage (No category) with ICE method.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

June 22, 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 i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: yes

Species:

cattle

Strain:

other: Bos taurus (bovine cattle)

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: freshly slaughtered cattle at the abattoir EVA, Saint Pierre sur Dives, France.
- Characteristics of donor animals: up to 12 months old, typically, 5 to 8 months old (French Authorities avoid the use of any organs from the head of bovines aged more than 12 months).
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Transported 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.
- Time interval prior to initiating testing: Upon arrival to the laboratory, preparation and selection was performed immediately. The corneas were used within a maximum of 24 hours.
- indication of any existing defects or lesions in ocular tissue samples: No - A macroscopic examination was performed on all eyes to detect the presence of any defects.
- Indication of any antibiotics used: Antibiotic used during transport in buffered Hanks medium [Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin (100 units/100 µg/mL final)].

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):750 µL (± 8 µL) applied on each cornea.

Duration of treatment / exposure:

10 minutes (± 30 seconds)

Duration of post- treatment incubation (in vitro):

Yes, post-exposure incubation: 2 hours (± 10 minutes).

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Every eye with some defects was discared.The examination of defects was performed under a lamp, using HBSS. 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. 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. 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.

QUALITY CHECK OF THE ISOLATED CORNEAS: A pre-incubation was performed before treatment aplication. Both chambers of the corneal holder were filled with MEM culture media supplemented with 1% fetal bovine serum plus penicillin/streptomycin (cMEM) at room temperature. 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 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.

NUMBER OF REPLICATES:3

NEGATIVE CONTROL USED: Yes, absolute (100%) ethanol.

SOLVENT CONTROL USED (if applicable): Not applicable

POSITIVE CONTROL USED: Yes, 0.9% NaCl

APPLICATION DOSE AND EXPOSURE TIME: 750 µL (± 8 µL), 10minutes.

TREATMENT METHOD: The closed-chamber method was used for the test item, positive and negative control.
The medium of the anterior chamber was removed and the each item was applied onto the epithelium of the cornea. 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 items, the holders were incubated vertically (cornea positioned horizontally with the treated side uppermost) in a water bath at +32°C (± 1°C), for 10 minutes (± 30 seconds).

POST-INCUBATION PERIOD: No

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: The anterior chamber was emptied using a metal gavage tube attached to a vacuum pump. The test item was removed with a cotton bud.The corneas were rinsed six times with pre-warmed cMEM containing phenol red until the test item 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). Some residual amounts of test item adhered to the walls of the anterior chamber, but were removed at the end of the rinsing step.

- POST-EXPOSURE INCUBATION: Yes,Following the 10-minute treatment and the rinsing of corneas, the holders were incubated horizontally
(corneas placed vertically) for 2 hours (± 10 minutes) in a water bath at +32°C (± 1°C). On completion of the 2-hour incubation period, the medium of both anterior and posterior chambers was renewed with pre-warmed cMEM (+32°C (± 1°C)), the second opacity measurement (OPT2) was then performed.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:An opacitometer (OPKIT opacitometer and calibrators (MC2, Clermont-Ferrand, France) was used to measure light transmission (the level of opacity) through the center of each mounted cornea. It was determinated by reading each holder in the right hand chamber of the calibrated opacitometer versus an empty holder (without cMEC, cornea and glasses) placed in the left hand chamber.
- Corneal permeability:passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry Cary 100.It is determined by the amount of sodium fluorescein (4mg/mL) dye that penetrates all corneal cell layers. Before the use, the fluorescein solution was validated.
- Others: Macroscopic examination: Observation for opaque spots, other irregularities and any separation of epithelium.


SCORING SYSTEM: In Vitro Irritancy Score (IVIS). The following formula was used to determine the In Vitro Irritancy Score (IVIS):
IVIS = cOPT + (15 x cOD490 nm)
The IVIS was calculated for each test item and positive control cornea. The mean IVIS for each series of three corneas was calculated from the individual scores.When the cOPT or cOD490 nm values were negative, they are considered equal to 0.

DECISION CRITERIA: as indicated in the TG.
IVIS UN GHS
If the test item induces an IVIS ≤ 3 No category
If the test item induces an 3 < IVIS ≤ 55 No prediction can be made
If the test item induces an IVIS > 55 Category 1

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

ca. 2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: SD = 0.1

Irritation parameter:

cornea opacity score

Run / experiment:

Mean

Value:

ca. 2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: Permeability

Run / experiment:

Mean

Value:

ca. 0.002

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: SD=0.004

Irritation parameter:

other: Fluorescein fixation

Remarks on result:

other: observed on the three corneas treated with the test item.

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, the mean opacity (0.0) and mean OD490 nm of the negative control corneas (0.011) should be less than the established upper limit of historical mean ( acceptable range: opacity <= 2, mean OD490 <=0.023, Mean+2SD: opacity 1.90 and OD 490 =0.023).

- Acceptance criteria met for positive control: Yes, the mean In Vitro Irritancy Score (IVIS) of the positive control corneas (50) should fall within two standard deviations of the historical mean (mean+2SD=66.22, acceptable range 25-67).

Irritant / corrosive response data:

Since the mean IVIS was < 3, the test item was considered as a test chemical not requiring classification for eye irritation or serious eye damage (UN GHS No Category). A single experiment composed of three corneas was sufficient for the test item formulation since the resulting classification was unequivocal.

Results:

Negative control	Opacity				Permeability
HOLDER	OPT0	OPT2	OPT2-OPT0	NEG. correction	OD490nm	NEG. correction
55	2	1	-1	0	0.009	0.0009
60	2	1	-1	0	0.0010	0.010
77	2	1	-1	0	0.015	0.015
Mean				0.0		0.011
SD				0.0		0.003

Test item	Opacity					Permeability
HOLDER	OPT0	OPT2	OPT2-OPT0	NEG. correction	cOPT	OD490nm	NEG. correction	cOD490nm	IVIS
82	1	3	2	2	2	0.011	0.011	0.000	2
50	2	4	2	2	2	0.018	0.018	0.007	2
62	2	4	2	2	2	0.009	0.009	0.000	2
Mean					2.0			0.002	2
SD					0.0			0.004	0.1

POSITIVE CONTROL	Opacity					Permeability
HOLDER	OPT0	OPT2	OPT2-OPT0	NEG. correction	cOPT	OD490nm	NEG. correction	cOD490nm	IVIS
51	1	31	30	30	30	2.148	2.148	2.137	62
84	1	24	23	23	23	31.384	1.384	1.373	44
88	3	24	21	21	21	1.500	1.500	1.489	43
Mean					24.7			1.666	50
SD					4.7			0.412	10.7

Np- not performed

OD: optical density

cOD-optical density corrected by mean OD value of negative control

cOPT: corneal opacity corrected by mean opacity value of negative control

OPT0: corneal opcity before treatment

OPT2: corneal opacity after 2 hours recovery period

Neg. correction: all individual values below 0 are set at 0

SD: standart deviations

IVIS:in vitro irritation source

Negative control: 0.9% NaCl

Positive control: 100% ethanol

Interpretation of results:

GHS criteria not met

Remarks:

No irritating

Conclusions:

Under the experimental conditions of this study, the test item has been identified as not irritant to eye (IVIS value 2.0±0.1), not requiring classification for eye irritation or serious eye damage (UN GHS No Category).

Executive summary:

In accordance with OECD TG 437and GLP study an in vitro (ex vivo) Bovine Corneal Opacity and Permeability study was conducted in order to determine the potential severe eye damaging effects of the test item. The corneas were obtained from freshly slaughtered calves. A single experiment was performed using three corneas for each treated series (test item, positive control and negative control). Before treatment, first opacity measurements were conducted. The test item was applied, as supplied, in liquid form using at treatment time of 10 minutes and the closed-chamber method. Negative and positive controls were applied using the same treatment time and method. After exposure, all items were removed from the front opening of the anterior chamber and the epithelia were rinsed. In the post exposure incubation period, the corneas were incubated for 2 hours (± 10 minutes) at 32ºC before a second opacity measurement was performed. After second opacity measurement the medium was removed and filled with fluorescein solution for measuring the permeability and the corneas were examined for irregularities. The mean corneal opacity and the mean IVIS for the test item was 2 (< 3), and the mean permeability was 0.002. All validity criteria were fulfilled. Therefore test item is considered not irritating to eye (no category).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Irritation in vitro

Key study: In accordance with GLP study and OECD TG 439, this study was to evaluate the possible irritating effects of the test item after topical application on in vitro human reconstructed epidermis (SkinEthic RHE® model). The test item was applied at the dose of 16 μL, to 3 living Reconstructed Human epidermis (SkinEthic RHE® model) during 42 minutes, followed by a rinse with 25 mL of DPBS and a 41 hours and 15 minutes post-incubation period at 37°C, 5% CO2. Positive and negative control groups were treated at the same way.Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. The mean corrected percent viability of the treated tissues was 1.5%, therefore test item has to be considered as irritant to skin (Category 2).

However, in the absence of corrosive results, an in vitro test of skin corrosivity should be performed to know if the test item has to be classified as Category “Corrosive“ or Category 2

“Irritant“.

Skin corrosion in vitro

Key study: In accordance with OECD 431 and GLP study, test item was tested for the skin corrosion test. Test substance was applied as supplied, in liquid form, at the dose level of 50 uL, to human skin model surfaces epiCS, Cell Systems at two exposure times - 3 minutes and 1 hour at room temperature. At the same experimental conditions a positive control KOH 8N and a negative control DPBS have been carried out. After the treatment time, the test substance was rinsed with 20 mL of DPBS.Then cell viability was measured by enzymatic conversion of the vital dye MTT into a blue formazan salt and its concentration was measured by determinating the Optical density at 570 nm. The OD values were used to calculate the viability%. The mean percent viability of the epidermis skins treated with the test item were 82.18 % (3 minutes) and 59.15% (1 hour). Based on these results and the clasification criteria, test item does not have to be considered as corrosive to skin.

Eye Irritation in vitro

Isolated Chicken Eye Test Method

Weight of evidence: An in vitro (ex vivo) study was conducted in order to determine the potential severe eye damaging effects of the test item according to the OECD guideline 438 (GLP study). Three eyeballs per group were isolated from chickens and, after appropriate preparation, were exposed to either 30 μL of the test item, 5% Benzalkonium chloride (positive control) and same amount of physiological saline (negative control).According to the overall in vitro classification (UN GHS), no prediction can be made since the combinations of the 3 endpoints for the test item were 2 x I, 1 x III.

Due to that results from this first in vitro study do not allow a conclusive decision on the classification of the substance or the absence of eye irritation potential, an other in vitro study for this endpoint has been considered. The Bovine Corneal Opacity and Permeability study has been conducted.

The Bovine Corneal Opacity and Permeability

Weight of evidence: An in vitro (ex vivo) Bovine Corneal Opacity and Permeability study was conducted in order to determine the potential severe eye damaging effects of the test item according the OECD TG 437 (GLP study).The corneas were obtained from freshly slaughtered calves. A single experiment was performed using three corneas for each treated series (test item, positive control and negative control). Before treatment, first opacity measurements were conducted. The test item was applied, as supplied, in liquid form using at treatment time of 10 minutes and the closed-chamber method. Negative and positive controls were applied using the same treatment time and method. In the following post exposure incubation period, the corneas were incubated for 2 hours (± 10 minutes) at 32ºC before a second opacity measurement was performed. After second opacity measurement the medium was removed and filled with fluorescein solution for measuring the permeability. Since the mean IVIS was < 3, the test item was considered as a test chemical not requiring classification for eye irritation or serious eye damage (UN GHS No Category).

Justification for classification or non-classification

According to the provided data, and specifically a skin irritation test which concluded a mean corrected percent viability of the treated tissues was 1.5%, the item has to be classified as Skin irritant Category 2 (H315) in accordance with the according to CLP Regulation (EC) No. 1272/2008. The corresponding hazard statement is “H315: Causes skin irritation” with the signal word “Warning”.

Based on the available information and specifically an IVIS of 2, the test item is not classified for eye irritation according to CLP Regulation (EC) no.1272/2008.