Registration Dossier

Administrative data

Description of key information

In an in vitro skin irritation study (OECD 439) test item was not irritating to skin (GLP, Rel. 1)

Based on in vitro eye irritation (OECD 492) and corrosion (OECD 438) studies, the registered substance was considered as irritating to the eyes (GLP, Rel. 1)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20-23 March 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:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted 28 July 2015
Deviations:
no
Qualifier:
according to
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. certificate)
Remarks:
27 April 2017
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: VE16 081-1
- Date received: 21 February 2017
- Manufacturing date: 21 March 2016
- Expiration date: 20 March 2018
- Purity test date: March 2016

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

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Used as supplied
Test system:
human skin model
Source species:
human
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-035) were received on 21 March 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 35 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, as supplied, on a nylon mesh, at the approximate dose of 16 mg, then applied on the epidermal surface of 3 living human skin models during 42 minutes at room temperature.
- 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 slightly brown instead of being whitish as for the coloration of the negative control tissues. Residual test item with brown coloration was noted on all Reconstructed Human epidermis after the rinse. They were incubated for a 42 hours and 05 minutes 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 2 hours 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 and 05 minutes 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:
112.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
PRELIMINARY TESTS
- Test for direct interaction with MTT:
A yellow solution with test item at the bottom of the well was observed after 3 hours of incubation between 36.1°C and 37.0°C, 5% CO2. Therefore, there was no direct interaction between the test item and MTT.
- Spectral analvsis of the test item in isopropanol:
A yellow solution was obtained after 2 hours of incubation at ambient temperature with gentle shaking. The mean of the corrected OD (blank subtracted) was 0.055 which was less than 0.08 (value corresponding to approximately twice the OD of the extracting solvent). 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 112.1% (considered as 100%), versus 1.4% 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

0.731

0.768

0.868

88.5

100.0

11.2

0.784

0.791

2

0.876

0.872

100.5

0.880

0.861

3

0.970

0.963

111.0

0.856

1.063

Positive control

1

0.012

0.012

0.012

1.4

1.4

0.2

0.012

0.012

2

0.012

0.011

1.3

0.011

0.010

3

0.015

0.014

1.6

0.014

0.014

Test item

1

0.877

0.922

0.973

106.3

112.1

5.1

0.959

0.932

2

0.998

0.993

114.4

0.987

0.995

3

0.994

1.003

115.6

0.988

1.027

#: 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 the negative control.

Interpretation of results:
GHS criteria not met
Conclusions:
Under the test conditions and in accordance with Regulation EC No. 1272/2008, the test item was considered as non-irritant to skin. It corresponds to UN GHS No Category. No hazard statement or signal word is 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 item.

As the test item was pasty, it was applied, as supplied, on a nylon mesh then on epidermis, so that to cover the entire surface of the epidermis (corresponding approximatively to 16 mg). The test item was applied on 3 living Reconstructed Human epidermis (SkinEthic RHE® model) during 42 minutes, followed by a rinse with 25 mL of DPBS and a 42-hour and 05-minute 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 item 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 112.1% (considered as 100%), versus 1.4% 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 validated the experiment.

Under the test conditions and in accordance with Regulation EC No. 1272/2008, the test item was considered as non-irritant to skin. It corresponds to UN GHS No Category. No hazard statement or signal word is required.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD Guideline 438 without deviation.
Qualifier:
according to
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:
EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
23 October 2015
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Biolandes / VE16 081-1
- Physical appearance: Brown to dark green sticky solid (moss)
- Manufacturing date: 21 March 2016
- Date received: 21 February 2017
- Expiration date of the lot/batch: 20 March 2018

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
Species:
chicken
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: The eyes collected from chickens obtained from a slaughterhouse (Etablissement Brun, 33820 Etauliers, France) where they are killed for human consumption have been used for this assay.
- Characteristics of donor animals (e.g. age, sex, weight): The age and weight of the chickens used in this test method are that of spring chickens traditionally processed by a poultry slaughterhouse (i.e., approximately 7 weeks old, 1.5 - 2.5 kg).
- 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 12 April 2017 at 8:10 am.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): 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 12 April 2017 at 9:45 am.
- Indication of any existing defects or lesions in ocular tissue samples: None
- Indication of any antibiotics used: None
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): Three manually formed tablets of 0.1609 g, 0.1669 g or 0.1536 g of the test item was applied for 10 seconds to the cornea.
Duration of treatment / exposure:
Test item was applied for 10 seconds to the cornea such that the entire surface of the cornea was evenly covered with the test item.
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 were at a controlled temperature between 31.6 °C and 32.4 °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. 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.
- Once all eyes had been examined and approved (see table in appendix 4), the eyes were incubated between 55 and 70 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
- 1, 3 and 3 eyes for negative & positive control and test item, respectively.

NEGATIVE CONTROL USED: Physiological saline

POSITIVE CONTROL USED: Sodium hydroxide

APPLICATION DOSE AND EXPOSURE TIME
- Immediately following the zero reference measurements, the eye (in its holder) was removed from the superfusion apparatus and placed in a horizontal position. Three manually formed tablets of 0.1609 g, 0.1669 g or 0.1536 g of the test item was applied for 10 seconds to the cornea such that the entire surface of the cornea was evenly covered with the test item.

REMOVAL OF TEST SUBSTANCE
- After exposure, the eyes were rinsed twice with 10 mL of physiological saline at ambient temperature. The eye (in its holder) was subsequently returned to the superfusion apparatus in the original upright position.

OBSERVATION PERIOD
- Treated corneas were evaluated before the pre-treatment and at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse.

METHODS FOR MEASURED ENDPOINTS:
- All observations of the cornea and measurement of corneal thickness were performed using a Haag-Streit BP900 slit-lamp microscope with depth-measuring device no. I. For the measurement of corneal thickness, the slit-width was set at 9½, equalling 0.095 mm.
- The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects (e.g., pitting or loosening of the epithelium). All of the endpoints, with the exception of fluorescein retention (which was determined only at pretreatment and 30 minutes after exposure to the test item) were determined at each of the above time points.

SCORING SYSTEM:
- Mean corneal swelling (%): Corneal swelling was determined from corneal thickness measurements made with an optical pachymeter on a slit-lamp microscope.
Corneal swelling (%) = ((corneal thickness at time t - corneal thickness at time = 0) / (corneal thickness at time = 0)) x 100
The mean percentage of corneal swelling for all tested 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 the test item.
- Mean maximum opacity score: Corneal opacity was calculated by using the area of the cornea that was most densely opacified for scoring. The mean corneal opacity value for all tested eyes was calculated for all observation time points. Based on the highest mean score for corneal opacity, as observed at any time point, an overall category score was then given for each test or control item.
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 iris 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: The mean fluorescein retention value for all tested 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.
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:
Results from corneal opacity, swelling, and fluorescein retention were evaluated separately to generate an ICE class for each endpoint. The ICE classes for each endpoint were then combined to generate an Irritancy Classification for the test item.
Once each endpoint was evaluated, ICE classes were assigned based on a predetermined range. Interpretation of corneal thickness, opacity, and fluorescein retention using four ICE classes was done according to the table 7.3.2/1, 7.3.2/2, 7.3.2/3.
Irritation parameter:
cornea opacity score
Run / experiment:
Mean of three runs with two replicates each /10 seconds
Value:
0.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
fluorescein retention score
Run / experiment:
Mean of three runs with two replicates each /10 seconds
Value:
1.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
percent corneal swelling
Run / experiment:
Mean of three runs with two replicates each /10 seconds
Value:
4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OCULAR REACTIONS:
- maximal mean score of corneal opacity: 0.3, corresponding to ICE class I;
- mean score of fluorescein retention: 1.2, corresponding to ICE class II;
- maximal mean corneal swelling: 4%, corresponding to ICE class I.
The combination of the three endpoints for test item was 2 x I, 1 x II.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The combination of the three endpoints for the negative control, physiological saline, was 2 x I, 1 x II. Therefore, the negative control is classified as “No Category”, as expected.
- Acceptance criteria met for positive control: The combination of the three endpoints for the positive control, sodium hydroxide was 3 x IV. Therefore, the positive control is classified as “Corrosive/Severe Irritant”, as expected.

Table 7.3.2/5: Individual and average values for evaluation of corneal lesions after treatment

 

Endpoint measured

Eye No.

Time (minutes)

0

30

75

120

180

240

Corneal opacity

7

0

0

0

0

0

0

8

0

0

0

0

0.5

0.5

9

0

0

0

0

0.5

0.5

Mean

0.0

0.0

0.0

0.0

0.3

0.3

ICE class

I

Fluorescein retention

7

0.5

0.5

-

-

-

-

8

0.5

2

-

-

-

-

9

0.5

1

-

-

-

-

Mean

0.5

1.2

 

-

-

-

ICE class

II

Corneal thickness

7

0.63

0.65

0.65

0.65

0.65

0.65

8

0.64

0.66

0.66

0.67

0.69

0.69

9

0.59

0.59

0.60

0.60

0.60

0.60

Corneal swelling (%)

7

-

3

3

3

3

3

8

-

3

3

5

8

8

9

-

0

2

2

2

2

Mean

-

2

3

3

4

4

ICE class

I

Combination of the three endpoints

2 x I, 1 x II

Classification

No category

Note:No morphological effects were noted, whatever the examination time

Interpretation of results:
GHS criteria not met
Conclusions:
Under the test conditions, the test item is not classified for eye irritation and serious eye damage as defined by the UN GHS (No Category) according to the Regulation (EC) No. 1272/2008.
Executive summary:

An ex vivo eye irritation study was performed according to the OECD Guideline 438 and in compliance with GLP to evaluate the possible ocular corrosive or severe irritating effects of the test item after administration on enucleated chicken eyes.

 

In order to cover the entire surface of the cornea, three manually formed tablets made of test item were shaped at an approximately amount of 0.1605 g each. The tablets were subsequently applied to 3 enucleated chicken eyes during 10 seconds. The eyes were then rinsed twice with 10 mL of physiological saline. 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.

 

The ocular reactions observed in eyes treated with the test item were:

- maximal mean score of corneal opacity: 0.3, corresponding to ICE class I;

- mean score of fluorescein retention: 1.2, corresponding to ICE class II;

- maximal mean corneal swelling: 4%, corresponding to ICE class I.

 

The combination of the three endpoints for test item was 2 x I, 1 x II.

 

The combination of the three endpoints for the positive control, sodium hydroxide was 3 x IV. Therefore, the positive control is classified as “Corrosive/Severe Irritant”, as expected. The combination of the three endpoints for the negative control, physiological saline, was 2 x I, 1 x II. Therefore, the negative control is classified as “No Category”, as expected.

 

Under the test conditions, the test item is not classified for eye irritation and serious eye damage as defined by the UN GHS (No Category) according to the Regulation (EC) No. 1272/2008.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21-23 March 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study performed according to OECD Guideline 492 without any deviation
Qualifier:
according to
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:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
23 October 2015
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: VE16 081-1
- Date received: 21 February 2017
- Manufacturing date: 21 March 2016
- Expiry date: 20 March 2018
- Purity test date: March 2016

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

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

FORM AS APPLIED IN THE TEST (if different from that of starting material): Test item was applied on a nylon mesh in order to cover the entire surface of the epidermis (corresponding to approximatively 50 mg).
Species:
human
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. 23772] were received on 21 March 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): ca. 50 mg
- Concentration (if solution): Undiluted
Duration of treatment / exposure:
5 hours and 50 minutes 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 MTT reduction with the test item:
The direct interaction of MTT with the test item was checked by adding approximatively 16 mg of the test item, previously applied on a nylon mesh, to 300 µL of a solution of MTT at 1 mg/mL.
- Test for the detection of the colouring potential of the test item:
The spectral properties at 570 nm of test item in isopropanol were checked by adding approximatively 16 mg of the test item, previously applied on a nylon mesh, to 1.5 mL of isopropanol.

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 20 hours and 12 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 dose of 50 mg, and then applied to the entire surface of 2 living RhCE tissue replicates during 5 hours and 50 minutes at standard culture conditions. In the same experimental conditions, a positive control (Methyl acetate), and a negative control (distilled water) were carried out. The controls were applied, as supplied, at the dose of 50 µL, to the surface of 2 RhCE tissue replicates during 05 hours and 50 minutes 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. Residue of yellow coloration was noted on all RhCE after the rinse. This rinsing step was followed by a 25 minutes 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 an 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 after extraction from tissues.
The RhCE constructs were placed in 300 µL of a MTT solution at 1.0 mg/mL for 2 hours and 55 minutes 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 02 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 at 570 nm was measured in triplicate samples of formazan extracts. The measured OD are proportional to the number of living cells. 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.
Irritation parameter:
other: % mean viability of the tissues
Run / experiment:
Main test
Value:
16.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
PRELIMINARY TESTS
- Test for direct MTT reduction with the test item:
A yellow solution with test item at the bottom of the well was observed after 3 hours of incubation between 36.1°C and 37.0°C, 5% CO2. Therefore, there is no direct interaction between the test item and MTT.
- Test for the detection of the colouring potential of the test item:
A yellow solution was obtained after 2 hours of incubation at ambient temperature with gentle shaking. The mean of the corrected OD (blank subtracted) was 0.055 which is less than 0.08 (value corresponding to approximately twice the OD of the extracting solvent). 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.

MAIN TEST
- MTT assay results: The mean percent tissue viability of the RhCE replicates treated with the test substance was 16.80%, versus 31.07% in the positive control (Methyl acetate). Refer Tables 7.3.2/1 for more details.

ACCEPTANCE OF RESULTS:
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

0.935

0.910

0.971

93.77

100

12.47

0.920

0.875

2

1.023

1.031

106.23

1.030

1.039

Positive control

1

0.287

0.295

0.302

30.40

31.07

1.34

0.294

0.303

2

0.311

0.308

31.74

0.308

0.304

Test item

1

0.168

0.164

0.163

16.90

16.80

0.21

0.162

0.163

2

0.162

0.162

16.69

0.165

0.158

 

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

OD: optical density

 

Note

- As all extracts were diluted at 50% just before the OD measurement, the acceptability criteria for OD values should be in the range > 0.4 and < 1.25 for the negative control.

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 the Regulation (EC) No. 1272/2008, the test substance is identified as potentially requiring classification and labelling according to the 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 substance.

The test substance was applied on a nylon mesh which was applied on the epidermis, in order to cover the entire surface of the epidermis (corresponding to approximatively 50 mg). The test substance was applied to two DPBS pre-treated RhCE (EpiOcular™ tissue model) during 5 hours and 50 minutes 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 a 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 substance 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 substance was 16.80%, versus 31.07% 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 the Regulation (EC) No. 1272/2008, the test substance is identified as potentially requiring classification and labelling according to the UN GHS (Category 2 or Category 1).

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 item.

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 viability of the treated tissues was 112.1% (considered as 100%), versus 1.4% 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 validated the experiment.

Under the test conditions and in accordance with Regulation EC No. 1272/2008, the test item was considered as non-irritant to skin. It corresponds to UN GHS No Category. No hazard statement or signal word is required.

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 substance.

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 tissue viability of the RhCE replicates treated with the test substance was 16.80%, versus 31.07% 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 the Regulation (EC) No. 1272/2008, the test substance is identified as potentially requiring classification and labelling according to the UN GHS (Category 2 or Category 1).

An ex vivo eye irritation study was performed according to the OECD Guideline 438 and in compliance with GLP to evaluate the possible ocular corrosive or severe irritating effects of the test item after administration on enucleated chicken eyes.

 The ocular reactions observed in eyes treated with the test item were:

- maximal mean score of corneal opacity: 0.3, corresponding to ICE class I;

- mean score of fluorescein retention: 1.2, corresponding to ICE class II;

- maximal mean corneal swelling: 4%, corresponding to ICE class I

The combination of the three endpoints for test itemwas 2 x I, 1 x II.

The combination of the three endpoints for the positive control, sodium hydroxide was 3 x IV. Therefore, the positive control is classified as “Corrosive/Severe Irritant”, as expected. The combination of the three endpoints for the negative control, physiological saline,was 2 x I, 1 x II. Therefore, the negative control is classified as “No Category”, as expected.

Under the test conditions, the test item is not classified for eye irritation and serious eye damage as defined by the UN GHS (No Category) according to the Regulation (EC) No. 1272/2008.

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:

Based on the available information, the registered substance is not classified as skin irritant and is classified as eye irritant category 2 H319 according to the criteria of the Regulation (EC) No. 1272/2008 (CLP).