[1R-(1α,2β,5α)]-2-isopropyl-5-methylcyclohexyl 5-oxo-L-prolinate

Endpoint summary

Description of key information

- Skin irritation/corrosion: irritating (OECD 439 and OECD 431, GLP, Rel. 1)

- Eye irritation: irritating (OECD 437 and OECD 492, GLP, Rel. 1)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 May - 01 June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to OECD Guideline 439 without any deviation.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

dated 23 July 2009

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

27 April 2017

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Used as supplied

Test system:

human skin model

Source species:

other: RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE

Cell type:

non-transformed keratinocytes

Cell source:

other: foreskin

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- 0.50 cm² reconstructed epidermis (Episkin SA, RHE/S/17 Batch No. 17-RHE-063) were received on 30 May 2017.
- On the same day, the insert (filter + epidermis) was gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. The inserts were placed in 6 wells culture plate which had been previously filled with 1 mL of growth medium (Episkin SA) during 2 hours and 25 minutes. Then just before treatment, the inserts were placed in 24 wells culture plate which had been previously filled with 300 µL of maintenance medium (Episkin SA).

TREATMENT
- The test item was applied on a nylon mesh, at the approximate dose of 16 mg, then applied during 42 minutes at room temperature on the epidermal surface of 3 living human skin models previously moistened with 10 µL of distilled water.
- In the same experimental conditions, a positive control (5% SDS) and a negative control (DPBS) were carried out. The 5% SDS solution was prepared by weighing 0.5 g of SDS in a 10 mL volumetric flask qsp 10 mL of distilled water. Then, the preparation was magnetically stirred, just before the treatment. To ensure a good contact with the epidermises, during all the treatment period, the test item was recovered with a nylon mesh provided by Episkin SA.

REMOVAL OF TEST MATERIAL AND CONTROLS
- 42 minutes after the test item application, the nylon mesh was removed and the human epidermises were washed with 25 x 1 mL of DPBS. The rinsed tissues were checked for any coloration and noted to be whitish, comparable to negative control tissues. They were incubated for a 42 hours post-treatment incubation period in fresh medium at 37°C, 5% CO2. Then, the epidermises were put in contact with the MTT solution.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- The cell viability was quantified by the measurement of the cell succinate dehydrogenase activity. This enzyme was responsible for the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS No. 298-93-1)] reduction into blue formazan crystal that is quantitatively measured by Optical Density (OD) after extraction from tissues. The measured OD were proportional to the number of living cells.
- The skin samples were placed in 300 µL of a MTT solution at 1.0 mg/mL for 3 hours at 37°C, 5% CO2. The precipitated blue formazan product was then extracted using isopropanol during 1 hour and 57 minutes under gentle agitation in the dark, and the concentration of formazan was measured by determining the OD (Optical Density) at 570 nm, just after dilution of the extracts (1:2 in isopropanol).
- The OD of MTT extract was measured in triplicate. The measurement of OD was performed using the ELx800 absorbance microplate reader (controlled every year and calibrated if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek.

VIABILITY CALCULATION:
- The results were expressed as a viability percentage compared with the negative control: viability % = (mean OD test item / mean OD negative control) * 100
- Data from individual replicate tissues (OD values and calculated percent tissue viability data for the test item and controls), mean percent tissue viability and standard deviation for each individual test item and control were reported in Table 7.3.1/1.

PREDICTION MODEL / DECISION CRITERIA
The OD values obtained for each test sample were used to calculate a percentage of viability relative to the negative control, which was arbitrarily set at 100%. The cut-off values for the prediction of irritation associated with the RHE models were as follows:
- The test item is considered to be non-irritant to skin if the tissue viability after 42 minutes of exposure and 42 hours of post-treatment incubation is >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% and the result of a skin corrosion test is "non-corrosive". In accordance with Regulation EC No. 1272/2008, the test item has to be classified in Category 2 “Irritant”. The corresponding hazard statement is “H315: Causes skin irritation” with the signal word “Warning”.
- The test item is considered to be irritant or corrosive to skin if the mean percent tissue viability after 42 minutes exposure and 42 hours of post-treatment incubation is ≤50% and in absence of information on a skin corrosion test. In accordance with the Regulation (CE) No.1272/2008 and in absence of information on a skin corrosion test, the item has to be classified in Category 2 "Irritant" or in Category 1 "Corrosive". The corresponding hazard statement is respectively, "H315: Causes skin irritation" with the signal word "Warning" or "H314: Causes severe skin burns and eye damage" with the signal word "Danger".

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 mg
- Concentration (if solution): Undiluted

Duration of treatment / exposure:

42 minutes at room temperature

Duration of post-treatment incubation (if applicable):

42 hours post-incubation period at 37°C, 5% CO2

Number of replicates:

3 living human skin models

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Run / experiment:

main test (duration of exposure: 42 minutes)

Value:

1.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

PRELIMINARY TESTS
- Test for direct interaction with MTT:
A yellow solution was observed after 3 hours of incubation between 36.9°C and 37.6°C, 5% CO2. Therefore, there was no direct interaction between the test item and MTT.
- Coloration potential and spectral analysis of the test item:
A colorless solution was obtained in water after 1 hour of incubation between 36.9°C and 37.6°C, 5% CO2. A colorless solution was obtained in isopropanol after 3 hours of incubation at room temperature.Therefore the test item was considered not to interfere with the MTT assay and there was no need to add non-specific coloration controls to the study.


MTT VIABILITY ASSAY RESULTS
- The mean percent viability of the treated tissues was 1.1%, versus1.3% in the positive control (5% Sodium Dodecyl Sulfate).
- The mean percent tissue viabilities obtained with the negative controls and positive controls were within the range of historical data and therefore validated the experiment.

Table 7.3.1/1: Main test - Individual and mean OD values and tissue viabilities for the test item, the negative and positive controls

 

	Skin	OD	Mean OD / disc (#)	Mean OD / product	Viability %	Mean viability %	Standard deviation (SD)
Negative control	1	0.965	0.966	0.903	106.9	100.0	6.2
		0.980
		0.954
	2	0.887	0.887		98.2
		0.873
		0.901
	3	0.860	0.857		94.9
		0.852
		0.859
Positive control	1	0.013	0.012	0.011	1.3	1.3	0.1
		0.011
		0.013
	2	0.011	0.010		1.1
		0.009
		0.010
	3	0.013	0.012		1.3
		0.012
		0.012
Test item	1	0.011	0.012	0.010	1.3	1.1	0.2
		0.013
		0.012
	2	0.011	0.010		1.1
		0.009
		0.010
	3	0.009	0.008		0.9
		0.008
		0.009

#: mean of 3 values (triplicate of the same extract)

OD: optical density

 

Acceptability criteria:

- SD ≤ 18%;

- Negative control: OD value of the 3 replicates in the range ≥0.8 and ≤3.0.

OD was measured after a 1:2 dilution of the formazan extracts in isopropanol; the acceptability criteria should be in the range ≥0.4 and ≤1.5 for negative control.

Interpretation of results:

other: Category 2 (irritating to skin) or Category 1 (corrosive) based on GHS criteria

Conclusions:

Under the test conditions and in accordance with Regulation EC No. 1272/2008 and in absence of information on skin corrosion, the test item has to be classified in Category 2 "Irritating to skin" or in Category 1 "Corrosive". The hazard statement "H315: Causes skin irritation" with the signal word "Warning" or "H314: Causes severe skin burns and eye damage" with the signal word "Danger" are required.

Executive summary:

An in vitro skin irritation test using the Reconstructed Human Epidermis (SkinEthic RHE® model) was performed according to the OECD Guideline 439 and in compliance with GLP to predict the acute skin irritation potential of the test substance.

The test substance was applied on a nylon mesh at an approximate dose of 16 mg in order to cover the entire surface of the epidermis. The nylon mesh was subsequently applied during 42 minutes to

3 living Reconstructed Human epidermis (SkinEthic RHE® model) previously moistened with 10 µL of distilled water. The treatment of the epidermis was followed by a rinse with 25 mL of DPBS and a 42 hours post-incubation period at 37°C, 5% CO2. 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.

In the preliminary tests, the test substance was found not to have direct MTT reducing properties or colouring potential. In the main test, the mean percent viability of the treated tissues was 1.1%, versus 1.3% in the positive control (5% Sodium Dodecyl Sulfate).

The mean percent tissue viabilities obtained with the positive control and negative controls were within the range of historical data and therefore validate the experiment.

Under the test conditions and in accordance with Regulation EC No. 1272/2008 and in absence of information on skin corrosion, the test item has to be classified in Category 2 "Irritating to skin" or in Category 1 "Corrosive". The hazard statement "H315: Causes skin irritation" with the signal word "Warning" or "H314: Causes severe skin burns and eye damage" with the signal word "Danger" are required.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July November 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

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

other: reconstituted epidermis

Cell type:

other: constituted epidermis (epiCS®, CellSystems®)

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: 0.60 cm2 reconstituted epidermis (epiCS®)

EXPOSURE
- The test item has been applied to the epidermal surface of 2 human skin model, during 3 minutes and during 1 hour.

REMOVAL OF TEST MATERIAL AND CONTROLS
- 3 minutes and 1 hour after the test item application, the human epidermis was washed 20 times with 20 mL of DPBS.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
The cell viability is quantified by measurement of the cellular mitochondrial dehydrogenases activity. These enzymes are responsible for the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; EINECS number 206-069-5, CAS number 298-93-1)] reduction into blue formazan in the viable cells. The skin sample is placed in MTT solution of appropriate concentration (e.g. 0.3 or 1 mg/mL) for 3 hours between 36.3 and 37.8 °C. The precipitated blue formazan product is then extracted using a solvent (e.g. isopropanol), and the concentration of formazan is measured by determining the Optical Density (OD) at a wavelength between 540 and 600 nm (preferably 570 nm). The measured absorbances are proportional to the number of living cells.
The measurement of OD was performed using the ELx800 absorbance microplate reader supplied by BioTek and the validated software Gens ELISA V1.05.11 supplied by BioTek.

NUMBER OF REPLICATE TISSUES:
Duplicate skin tissues for test item, negative and positive controls

VIABILITY
Viability = (OD test item / OD negative control) x 100
For each tissue, OD values and calculated percentage cell viability data for the test item, positive and negative controls, should be reported in tabular form, including data from replicate repeat experiments as appropriate, mean and individual values.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg
- Concentration (if solution): Undiluted
Test item was applied to the epidermal surface of the 2 living human skin models previously moistened with 25 µL of DPBS

Duration of treatment / exposure:

3 minutes and 1 hour

Number of replicates:

Duplicate skin tissues for test item, negative and positive controls

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean at 3 minutes

Value:

70.15

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean at 1 hour

Value:

19.71

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item were 70.15% and 19.71% versus 6.81% and 1.04%, respectively, with the positive control item (potassium hydroxide 8N).

Table 7.3.1/1: Skin corrosion assay: Results

 

	Skin	OD	Mean OD / disc (#)	Mean OD / product	Viability %	Mean viability %	Viability difference between replicates %
Treatment: 3 min
Negative control	1	0.978	0.942	0.896	105.13	100	10.3
		0.919
		0.928
	2	0.844	0.850		94.87
		0.848
		0.856
Positive control	3	0.058	0.064	0.061	7.14	6.81	0.7
		0.070
		0.064
	4	0.058	0.058		6.47
		0.059
		0.056
Test item	5	0.704	0.665	0.629	74.22	70.15	8.1
		0.639
		0.651
	6	0.671	0.592		66.07
		0.558
		0.547
Treatment: 1 hour
Negative control	9	0.960	0.922	0.916	100.66	100	1.3
		0.850
		0.955
	10	0.944	0.910		99.14
		0.922
		0.862
Positive control	11	0.008	0.009	0.010	0.98	1.04	0.1
		0.008
		0.010
	12	0.011	0.010		1.09
		0.010
		0.008
Test item	13	0.180	0.182	0.181	19.71	19.71	0.3
		0.187
		0.178
	14	0.158	0.179		19.54
		0.193
		0.185

#: mean of 3 values

OD: optical density

Note:

30 minutes exposure: If the viability obtained for the test substance is greater than 50%, then it is non-corrosive.

1 hour exposure: If the viability obtained for the test substance is greater than15%, then it is non-corrosive.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Under the test conditions, in accordance with the Regulation (EC) No. 1272/2008, the results obtained enable to conclude that the test item does not have to be classified in Category 1 “Corrosive”. No hazard statement or signal word are required.

Executive summary:

An in vitro skin corrosion study was performed according to OECD Guideline 431 and in compliance with GLP to evaluate the possible corrosive effects of the test item after topical administration on in vitro human reconstituted epidermis (epiCS®, CellSystems®).

The test item was applied as supplied, at the dose of 25 mg, to 2 living Human skin model surfaces (epiCS®, CellSystems®) during 3 minutes and 1 hour, followed by a rinse with 20 mL of DPBS. 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.

3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item were 70.15% and 19.71% versus 6.81% and 1.04%, respectively, with the positive control item (potassium hydroxide 8N).

Under the test conditions, in accordance with the Regulation (EC) No. 1272/2008, the results obtained enable to conclude that the test item does not have to be classified in Category 1 “Corrosive”. No hazard statement or signal word are required.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02-05 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to OECD Guideline 492 with a minor deviation: difference of viability between negative control tissues replicates was greater than 20%. Considering the results obtained, this deviation was considered as without impact on the conclusion of the study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted 28 July 2015

Deviations:

yes

Remarks:

difference of viability between negative control tissues replicates was greater than 20%

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

27 April 2017

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Used as supplied

Species:

other: Reconstructed human Cornea-like Epithelia

Details on test animals or tissues and environmental conditions:

- Description of the cell system used: 0.60 cm² Reconstructed human Cornea-like Epithelia [EpiOcular(TM) OCL-212-ver2.0, supplied by MatTek Corporation, batch No. 23778] were received on 03 May 2017.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 mg
- Concentration (if solution): Undiluted

Duration of treatment / exposure:

6 hours at 37°C, 5% CO2, 95% humidity (standard culture conditions)

Duration of post- treatment incubation (in vitro):

- Post-exposure immersion period: 25 minutes at room temperature
- Post-exposure incubation period: 18 hours at standard culture conditions

Number of animals or in vitro replicates:

Test item, negative and positive controls were applied on duplicate tissues.

Details on study design:

PRELIMINARY TESTS
- Test for direct interaction with MTT:
The direct interaction of MTT with the test item was checked by adding approximatively 100 mg of the test item, previously applied on a nylon mesh, to 1 mL of solution of MTT at 1 mg/mL.
- Test for the detection of the colouring potential of the test item:
The colouration potential of the test item in water or isopropanol was checked by adding approximatively 100 mg of the test item to 1 mL of distilled water or 2 mL of isopropanol, respectively.


MAIN TEST
- Pre-incubation of the tissues:
On the day of receipt, the 12 tissues in their 24-well shipping container were equilibrated at room temperature for 15 minutes. Then the inserts (filter + epithelium) were gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. They were placed in 6 wells culture plate which had been previously filled with 1 mL of 37°C pre-warmed assay medium and incubated during 19 hours and 50 minutes at standard culture conditions.
After the overnight incubation, the tissues were pre-wetted with 20 μL of Ca2+Mg2+Free-DPBS. The tissues were incubated at standard culture conditions for 30 minutes.
- Treatment and post-treatment incubation of the tissues:
The test item was applied on a nylon mesh at an approximate amount of 50 mg, and then applied to the entire surface of 2 living RhCE tissue replicates during 6 hours at standard culture conditions. In the same experimental conditions, a positive control (Methyl acetate), and a negative control (distilled water) were used. The controls were applied, as supplied, at the dose of 50 µL, to the surface of 2 viable RhCE tissue replicates during 6 hours at standard culture conditions.
After the treatment, the test item and control substances were carefully washed from the RhCE tissues by extensive rinsing with Ca2+Mg2+Free-DPBS. The rinsed tissues were checked for any colouration and noted for comparable colour with the negative control. This rinsing step was followed by a 25-minute post-exposure immersion period at room temperature in 5 mL of fresh medium to remove any test item absorbed into the tissue. The RhCE constructs were then incubated for a 18-hours post-exposure incubation at standard culture conditions in 1 mL of fresh medium at 37°C, 5% CO2.
- MTT viability assay:
Following the exposure to the test item, viability measurements are performed immediately after the post-exposure incubation period of the rinsed tissues in fresh medium. This period allows both for recovery from weak cytotoxic effects and for appearance of clear cytotoxic effects.
The RhCE tissue viability was measured by enzymatic conversion of the vital dye MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1] by the viable cells of the tissue into a blue MTT formazan salt that is quantitatively measured by Optical Density (OD) after extraction from tissues. The measured OD was proportional to the number of living cells.
The RhCE constructs were placed in 2 mL of a MTT solution at 1.0 mg/mL for 2 hours at standard culture conditions. The precipitated blue formazan product was then extracted from the tissues by placing each insert in 2 mL of isopropanol during 2 hours at room temperature in the dark. The concentration of formazan was measured by determining the OD at 570 nm, just after dilution of the extractions in isopropanol (1:2). The OD measurement of formazan extracts at 570 nm was measured in triplicate samples using the ELx800 absorbance microplate reader (controlled every year and calibrated if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek.

Irritation parameter:

other: % mean viability of the tissues

Run / experiment:

Main test

Value:

3.11

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

PRELIMINARY TESTS
- Test for direct interaction with MTT:
A yellow solution was observed after 3 hours of incubation between 36.9°C and 37.6°C, 5% CO2. Therefore, there was no direct interaction between the test item and MTT.
- Test for the detection of the colouring potential of the test item:
A colourless solution was obtained in water after 1 hour of incubation between 36.9°C and 37.6°C, 5% CO2. A colourless solution was obtained in isopropanol after 3 hours of incubation at room temperature. No significant colouration appeared, therefore the test item was considered to be not interfering with the MTT assay.

MAIN TEST
- The mean percent tissue viability of the RhCE replicates treated with the test substance was 3.11%, versus 24.05% in the positive control (Methyl acetate). Refer Tables 7.3.2/1 for more details.
- The mean percent tissue viabilities obtained with the positive control and negative controls were within the range of historical data and therefore validated the experiment.

Table 7.3.2/1: Main test - Individual and mean corrected OD values and tissue viabilities for the test item, the negative and positive controls

 

	Tissue	OD	Mean OD/disc (#)	Mean OD/product	Viability %	Mean viability %	Difference of viability %
Negative control	1	0.475	0.471	0.564	83.58	100.00	32.83
		0.477
		0.461
	2	0.668	0.656		116.42
		0.638
		0.663
Positive control	1	0.120	0.130	0.136	23.07	24.05	1.95
		0.134
		0.138
	2	PH	0.141		25.02
		0.142
		0.139
Test item	1	0.018	0.017	0.018	3.02	3.11	0.18
		0.016
		0.017
	2	0.018	0.018		3.19
		0.018
		0.020

#: mean of 3 values (triplicate of the same extract)

OD: optical density

 

Acceptability criteria:

- The difference of viability between the two replicates of the negative control group was 32.83% instead of <20%. Considering the results obtained, this deviation was considered as without impact on the conclusion and the validity of the study.

Interpretation of results:

other: Category 2 (irritating to eyes) or Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

Under the test conditions and in accordance with Regulation EC No. 1272/2008, the test item was identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1).

Executive summary:

An in vitro eye irritation test using the Reconstructed Human Cornea-like Epithelium (RhCE) (EpiOcular™ tissue) model was performed according to the OECD Guideline 492 and in compliance with GLP to predict the acute eye irritation potential of the test item.

The test item was applied on a nylon mesh, at an approximate amount of 50 mg in order to cover the entire surface of the epidermis and it was subsequently applied on epidermis. The test item was applied to 2 DPBS pre-treated RhCE (EpiOcular™ tissue model) during 6 hours at 37°C, 5% CO2, 95% humidity (standard culture conditions). The exposure period was followed by extensive rinsing with DPBS at room temperature, a 25 minutes post-exposure immersion period at room temperature and an 18 hours post-exposure incubation at standard culture conditions. The tissue viability was measured by performing an MTT assay.

In the preliminary tests, the test item was found not to have direct MTT reducing properties or colouring potential. In the main test, the mean percent tissue viability of the RhCE replicates treated with the test item was 3.11%, versus 24.05% in the positive control (Methyl acetate). The mean percent tissue viabilities obtained with the positive control and negative controls were within the range of historical data and therefore validate the experiment.

Under the test conditions and in accordance with Regulation EC No. 1272/2008, the test item was identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1).

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

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

Species:

cattle

Strain:

other: bovine cattle (Bos taurus)

Details on test animals or tissues and environmental conditions:

bovine eyes were obtained from freshly slaughtered cattle at the abattoir EVA, Saint Pierre sur Dives, France

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 mg (± 75 mg)

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

2 hours

Number of animals or in vitro replicates:

Three corneas for each treated series (test item formulation, positive control and vehicle control) were used.

Details on study design:

SELECTION AND PREPARATION OF CORNEAS:
- Bovine eyes were obtained from freshly slaughtered cattle (age: typically 5-8 months old) at the abattoir EVA, Saint-Pierre-sur-Dives, 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.
- Upon arrival at CiToxLAB France, the tissues surrounding the eyeball of selected corneas 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.
- In both experiments, corneas obtained from freshly slaughtered calves were mounted in corneal holders [OPKIT (polypropylene), diameter 18 mm, ref. ED 4004 SB (MC2, Clermont-Ferrand, France)]. 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 (± 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.

QUALITY CHECK OF THE ISOLATED CORNEAS: 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.

NUMBER OF REPLICATES: Three corneas for each treated series (test item formulation, positive control and vehicle control) were used.

APPLICATION DOSE AND EXPOSURE TIME: The test item, was evaluated in these two experiments using a treatment time of 10 minutes and using the open-chamber method.

TREATMENT METHOD: The medium of the anterior chamber was removed and each item was applied onto the epithelium of the cornea. The test item [750 mg (± 75 mg)], was applied for a treatment time of 10 minutes (± 30 secondes) using the open-chamber method. Vehicle and positive controls (750 µL ±8 µL) were applied using the same treatment time and the closed-chamber treatment 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 items, the holders were incubated vertically (cornea positioned horizontally with the treated side uppermost) in a water bath at +32°C (± 1°C), for the selected treatment time.

RINSING OF THE CORNEAS: On completion of the treatment period, the test item formulation was removed from the front opening of the anterior chamber (open-chamber method) and the epithelium was rinsed as follows:
- any test item formulation adhering to the walls of the anterior chamber was removed using a pipette of heated cMEM (+32°C),
- the corneas were rinsed four times with pre-warmed cMEM containing phenol red (i.e. until the test item formulation had been completely removed from the chamber or until the phenol red was not discoloured). 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 colour changing of the rinsing medium (containing phenol red). Despite the rinsing performed,residual test item was noted over the three test item-treated corneas.
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 the rinsing step, 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 was used to measure light transmission (i.e. the level of opacity) through the center of each mounted cornea. 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.
- Corneal permeability: After the second opacity measurement, the medium of the anterior chamber was removed and the anterior 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 each 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. For both experiments, as the values obtained were between 0.850 and 0.940, the fluorescein solution was validated.
For each series of three corneas, a chronometer started from the fluorescein solution 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. 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).
- 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.

SCORING SYSTEM: The In Vitro Irritancy Score (IVIS) was determined from the opacity and permeability measurements, as described below.
- Opacity: The change in opacity value of each individual cornea treated with test item, negative control or positive control was calculated by subtracting the initial base-line opacity measurement (OPT0) from the post-treatment opacity reading (OPT2). The average change in opacity for the corneas treated with the vehicle control was calculated and this value was subtracted from the change in opacity for each cornea treated with test item or positive control to obtain a corrected opacity value (cOPT). When the average change in opacity for the corneas treated with the negative/vehicle control was negative, it is considered equal to 0. The mean cOPT value of each series of three corneas was calculated from the individual corrected opacity values.
- Permeability: The corrected OD490 nm (cOD490 nm) value (i.e. permeability) for each cornea treated by test item or positive control was calculated by subtracting the average vehicle control cornea OD490 nm value from the original OD490 nm value of each cornea. When the average vehicle control cornea OD490 nm value was negative, it is considered equal to 0. The mean cOD490 nm value of each series of three corneas was calculated from the individual cOD490 nm values.
- In Vitro Irritancy Score calculation: 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.

ACCEPTANCE CRITERIA:For the validation of an experiment, the following criteria had to be fulfilled:
- the mean In Vitro Irritancy Score (IVIS) of the positive control corneas should fall within two standard deviations of the historical mean,
- the mean opacity and mean OD490 nm of the vehicle control corneas should be less than the established upper limit of historical mean

DECISION CRITERIA: The IVIS cut-off values for identifying the test item as inducing serious eye damage (UN GHS Category 1) or as not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are the following:
- 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
Two experiments composed of three corneas were performed to unequivocally classify the test item.

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

49

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

MACROSCOPIC EXAMINATION:

No notable opaque spots or irregularities were observed on negative control-treated corneas.
Opacity, residual test item on corneas and fluorescein fixation were observed on the three corneas treated with the test item.
Opacity, fluorescein fixation and thickening of the corneas were observed on those treated with the positive control.

IN VITRO IRRITANCY SCORE:

The mean In Vitro Irritancy Score (IVIS) of the test item-treated corneas was: 49
However, one of the three test item-treated corneas gave a discordant prediction from the mean of all three corneas and this discordant results was more than 10 IVIS units above the cut-off threshold of 55 (i.e. cornea No. 54, IVIS = 69). The results are therefore considered borderline.
It was also noted that despite successive rinsing, the test item stuck to the corneas and probably tended to increase opacity values. Based on these results, it can therefore not be clarified which part of the opacity signal is due to residual test item and which part is due to potential test article irritant properties. It was also noted that permeability values were greater in the test item series than in the positive control group, suggesting an irritant effect of the test item.

Table 7.3.2/1: Results

Calibration of the opacitometer	Before OPT0	After OPT0	Before OPT2	After OPT2
Calibrator No. 1	75	75	75	76
Calibrator No. 2	145	np	np	np
Calibrator No. 3	214	np	np	np

Group	Holder	Opacity					Permeability			IVIS
		OPT0	OPT2	OPT2-OPT0	Neg correction	cOPT	OD490 nm	Neg correction	cOD 490 nm
Vehicle control	52	0	1	1	1	-	0.005	0.005	-	-
	58	0	0	0	0	-	0.013	0.013	-	-
	69	0	1	1	1	-	0.010	0.010	-	-
	Mean	-	-	-	0.0	-	-	0.009	-	-
	SD	-	-	-	0.0	-	-	0.004	-	-
Test item	54	0	19	19	19	18	3.376	3.376	3.367	69
	68	0	8	8	8	7	1.804	1.804	1.795	34
	59	0	8	8	8	7	2.544	2.544	2.535	45
	Mean	-	-	-	-	11.0	-	-	2.565	49
	SD	-	-	-	-	6.4	-	-	0.786	17.7
Positive control	63	0	22	22	22	21	2.420	2.420	2.411	57
	64	0	19	19	19	18	1.644	1.644	1.635	43
	80	0	17	17	17	16	0.984	00984	0.975	31
	Mean	-	-	-	-	18.7	-	-	1.673	44
	SD	-	-	-	-	2.5	-	-	0.719	13.3

np: not performed

OD: optical density

cOD: optical density corrected by mean OD value of vehicle control

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

OPT0: corneal opacity before treatment

OPT2: corneal opacity after treatment

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

SD: standard deviation

IVIS: In Vitro Irritation Score

Negative control:0.9% sodium chloride

Positive control: 20% imidazole solution in 0.9% NaCl

Interpretation of results:

other: no prediction can be made

Conclusions:

Under the experimental conditions of this study, the ocular corrosive or severe irritant potential of the test item, could not be predicted and further testing should be conducted for classification and labeling purposes.

Executive summary:

An ex vivo eye irritation study was performed according to the OECD Guideline 437 and in compliance with GLP to evaluate the possible ocular corrosive or severe irritating effects of the test item after administration on bovine corneas.

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.

A single experiment was performed using three corneas for each treated series (test item, positive control and negative control).

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

The test item was applied undiluted, in a single experiment using a treatment time of 10 minutes and using the open-chamber method. Negative and positive controls were applied using the same treatment time and using the closed-chamber method. At the completion of the treatment period, all items were removed from the front opening of the anterior chamber and the epithelia were rinsed.

The corneas were then incubated for 2 hours (± 10 minutes)at +before a second opacity measurement was performed.

After the second opacity measurement, the medium of the anterior chamber was removed and filled with a fluorescein solution. The holders were then incubated vertically for 90 minutes(± 5 minutes)at +.

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.

Opacity, residual test item on corneas and fluorescein fixation were observed on each cornea treated with the test item.

All acceptance criteria were fulfilled. The study was therefore considered as valid.

The mean In Vitro Irritancy Score (IVIS) of the test item-treated corneas was: 49.

Under the experimental conditions of this study,the ocular corrosive or severe irritant potential of the test item,could not be predicted andfurther testing should be conducted for classification and labeling purposes.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

An in vitro skin irritation test using the Reconstructed Human Epidermis (SkinEthic RHE® model) was performed according to the OECD Guideline 439 and in compliance with GLP to predict the acute skin irritation potential of the test substance.

The test substance was applied on a nylon mesh at an approximate dose of 16 mg in order to cover the entire surface of the epidermis. The nylon mesh was subsequently applied during 42 minutes to

3 living Reconstructed Human epidermis (SkinEthic RHE® model) previously moistened with 10 µL of distilled water. The treatment of the epidermis was followed by a rinse with 25 mL of DPBS and a 42 hours post-incubation period at 37°C, 5% CO2. 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.

In the preliminary tests, the test substance was found not to have direct MTT reducing properties or colouring potential. In the main test, the mean percent viability of the treated tissues was 1.1%, versus 1.3% in the positive control (5% Sodium Dodecyl Sulfate).

The mean percent tissue viabilities obtained with the positive control and negative controls were within the range of historical data and therefore validate the experiment.

Skin corrosion:

An in vitro skin corrosion study was performed according to OECD Guideline 431 and in compliance with GLP to evaluate the possible corrosive effects of the test item after topical administration on in vitro human reconstituted epidermis (epiCS®, CellSystems®).

The test item was applied as supplied, at the dose of 25 mg, to 2 living Human skin model surfaces (epiCS®, CellSystems®) during 3 minutes and 1 hour, followed by a rinse with 20 mL of DPBS. 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.

3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item were 70.15% and 19.71% versus 6.81% and 1.04%, respectively, with the positive control item (potassium hydroxide 8N).

Eye irritation:

An in vitro eye irritation test using the Reconstructed Human Cornea-like Epithelium (RhCE) (EpiOcular™ tissue) model was performed according to the OECD Guideline 492 and in compliance with GLP to predict the acute eye irritation potential of the test item.

The test item was applied on a nylon mesh, at an approximate amount of 50 mg in order to cover the entire surface of the epidermis and it was subsequently applied on epidermis. The test item was applied to 2 DPBS pre-treated RhCE (EpiOcular™ tissue model) during 6 hours at 37°C, 5% CO2, 95% humidity (standard culture conditions). The exposure period was followed by extensive rinsing with DPBS at room temperature, a 25 minutes post-exposure immersion period at room temperature and an 18 hours post-exposure incubation at standard culture conditions. The tissue viability was measured by performing an MTT assay.

In the preliminary tests, the test item was found not to have direct MTT reducing properties or colouring potential. In the main test, the mean percent tissue viability of the RhCE replicates treated with the test item was 3.11%, versus 24.05% in the positive control (Methyl acetate). The mean percent tissue viabilities obtained with the positive control and negative controls were within the range of historical data and therefore validate the experiment.

Eye corrosion:

An ex vivo eye irritation study was performed according to the OECD Guideline 437 and in compliance with GLP to evaluate the possible ocular corrosive or severe irritating effects of the test item after administration on bovine corneas.

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.

A single experiment was performed using three corneas for each treated series (test item, positive control and negative control).

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

The test item was applied undiluted, in a single experiment using a treatment time of 10 minutes and using the open-chamber method. Negative and positive controls were applied using the same treatment time and using the closed-chamber method. At the completion of the treatment period, all items were removed from the front opening of the anterior chamber and the epithelia were rinsed.

The corneas were then incubated for 2 hours (± 10 minutes)at +before a second opacity measurement was performed.

After the second opacity measurement, the medium of the anterior chamber was removed and filled with a fluorescein solution. The holders were then incubated vertically for 90 minutes(± 5 minutes)at +.

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.

Opacity, residual test item on corneas and fluorescein fixation were observed on each cornea treated with the test item.

All acceptance criteria were fulfilled. The study was therefore considered as valid.

The meanIn VitroIrritancy Score (IVIS) of the test item-treated corneas was: 49.

Under the experimental conditions of this study,the ocular corrosive or severe irritant potential of the test item,could not be predicted andfurther testing should be conducted for classification and labeling purposes, but the substance is not considered as classified as eye corrosive category 1, as the IVIS score is < to the cut off value of 55.

Justification for classification or non-classification

Harmonized classification:

The registered substance has no harmonized classification according to the Regulation (EC) No. 1272/2008.

Self-classification:

According to results from OECD 439 and OECD 431 studies, the registered substance is classified as category 2 for skin irritation (H315) according to Regulation EC N° 1272/2008 (CLP) and GHS.

According to the results of OECD TG 437 and OECD 492, the registered substance is classified as category 2 for eye irritation (H319) according to Regulation EC N° 1272/2008 (CLP) and GHS.