Registration Dossier

Administrative data

Description of key information

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

- Eye irritation: irritating (OECD TG 492, GLP, Rel. 1 and OECD TG 438, 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:
04-17 February 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
10 January 2013
Test material information:
Composition 1
Test system:
human skin model
Remarks:
human reconstructed epidermis (tissues)
Source species:
other: human reconstructed epidermis (tissues)
Cell type:
other: human reconstructed epidermis (tissues)
Vehicle:
unchanged (no vehicle)
Details on test system:
Supplier: SkinEthic Laboratories, Lyon, France.
Selection: At receipt, the pH (color of the agar medium) and temperature indicators were checked to ensure the good quality of the tissues before use.
Storage conditions: At receipt, the living EpiskinTM tissues were kept at room temperature in their packaging until required.
Description: The EpiskinTM model consists of an airlifted, living, multilayered epidermal tissue construction (surface 0.38 cm2), reconstructed from normal human epidermal keratinocytes for 13 days and produced in polycarbonate inserts in a serum-free and chemically defined medium. The model features a normal ultra-structure and is functionally equivalent to human in vivo epidermis.
Type of coverage:
other: not applicable
Preparation of test site:
other: not applicable
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): 10 µL ± 2 μL of the test item was applied to the epidermis surface.
- Concentration (if solution): undiluted

CONTROLS
- Negative control: Dulbecco’s Phosphate-Buffered Saline (D-PBS)
- Positive control: 5 % (w/v) aqueous solution of Sodium Dodecyl Sulfate (SDS)
Duration of treatment / exposure:
Triplicate tissues were treated with the test item for an exposure period of 15 minutes (± 1 minute).
At the end of the exposure period, tissues were rinsed and incubated at 37 °C, 5 % CO2 in a humidified incubator for 42 h.
Number of animals:
Triplicate tissues for test item, negative and positive controls
Details on study design:
A new 12-well plate was used for each test and control items: one plate for the test item, one plate for the negative control and one plate for the positive control. Each item was applied onto triplicate tissues.

PRE-INCUBATION OF THE TISSUES ON THEIR DAY OF ARRIVAL (DAY 0)
A volume of 2 mL of pre-warmed maintenance medium was added to the first column of 3 wells of 12-well plates (one plate per item). Then, each EPISKIN™ tissue was transferred into the maintenance medium pre-filled wells (three tissues per plate). The plates were incubated at 37 °C, 5 % CO2 in a humidified incubator for at least 24 h (± 2 h).

TREATMENT OF TISSUES (DAY 1)
A volume of 2 mL of pre-warmed maintenance medium was pipetted into the second column of 3 wells of the 12-well plates for each item, respectively. Then, each test or control item was applied on three tissues for an exposure period of 15 minutes. The items were applied topically to the corresponding tissues and gently spread over the epidermis surfaces to ensure uniform covering of the tissues. The tissues were processed (treatment and rinsing) in the same order and at regular time-intervals to ensure each tissue received an equal exposure period.
The exposure of the tissues to the test and control items was performed at room temperature for 15 minutes (± 1 minute).

RINSING OF TISSUES AND INCUBATION FOR 42 HOURS (DAY 1)
At the end of the treatment period, each tissue was rinsed with D-PBS to remove any residual test or control items and then incubated for 42 h at 37 °C, 5 % CO2 in a humidified incubator.

MTT VIABILITY ASSAY (DAY 3)
On Day 3, following the 42 h post-exposure incubation period: MTT test (MTT Loading/Formazan Extraction) was performed and tissues were incubated for 3 h (± 5 minutes) at 37 °C, 5 % CO2 in a humidified incubator.

OPTICAL DENSITY MEASUREMENTS (DAY 6)
On Day 6, at the end of the formazan extraction period: The optical density was measured at 570 nm using a plate reader.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
7
Negative controls valid:
yes
Positive controls valid:
yes
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
6
Negative controls valid:
yes
Positive controls valid:
yes
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3
Value:
7
Negative controls valid:
yes
Positive controls valid:
yes
Other effects / acceptance of results:
Evaluation of the coloration of tissues at the end of the MTT incubation period: All treated tissues appeared white which was considered to be indicative of dead tissues.
Following a 15 minutes exposure and 42 h of recovery period, the relative mean viability of the tissues treated with the test item was 7 % with a Standard Deviation of 1 % as assessed by the MTT assay. As the mean viability was < 50 % the results met the criteria for an irritant response.

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

 

Group

Tissue No.

OD measurements

Mean

ODblank

cOD

Mean cOD

Viability (%)

1st

2nd

1st

2nd

 

 

Negative control

1

0.993

1.027

0.037

 

 

0.956

0.990

0.973

101

2

0.975

0.988

0.938

0.951

0.945

98

3

1.019

1.028

0.982

0.991

0.987

102

Positive control

1

0.091

0.090

0.037

 

 

0.054

0.053

0.054

6

2

0.136

0.130

0.099

0.093

0.096

10

3

0.096

0.088

0.059

0.051

0.055

6

Test item

1

0.100

0.105

0.037

 

 

0.063

0.068

0.066

7

2

0.097

0.092

0.060

0.055

0.058

6

3

0.102

0.105

0.065

0.068

0.067

7

OD = optical density

cOD = blank corrected optical density

 

Table 7.3.1/2: Mean tissue viability and Standard Deviations for the test item, the negative and positive controls

 

Group

cOD

Viability (%)

Mean

SD

Mean

SD

Negative control

0.968

0.021

100

2

Positive control

0.068

0.024

7

2

Test item

0.063

0.005

7

1

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
Nopol is classified in category 2 (H315) according to Regulation (EC) No. 1272/2008 (CLP) and GHS.
Executive summary:

An in vitro skin irritation study was performed according to Guideline OECD 439 and in compliance with GLP, using the EPISKINTM reconstructed human epidermis model.

The test item, Nopol, was applied topically on triplicate tissues and incubated at room temperature for 15 minutes. At the end of the treatment period, each tissue was rinsed with D-PBS and incubated for 42 h at 37°C, 5% CO2 in a humidified incubator. The cell viability was then assessed by means of the colorimetric MTT reduction assay. Relative viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (reference viability). In the preliminary tests, the test item was found not to have direct MTT reducing properties or coloring potential.

 

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

 

All treated tissues appeared white, which was considered to be indicative of dead tissues. Following a 15 minute exposure and 42 h of recovery period, the relative mean viability of the tissues treated with the test item was 7% with a Standard Deviation of 1% as assessed by the MTT assay. As the mean viability was < 50% the results met the criteria for an irritant response.

 

Therefore, Nopol is classified in category 2 (H315) according to Regulation (EC) No. 1272/2008 (CLP) and GHS.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (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:
09-12 May 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study performed according to OECD Guideline 492 without any deviation
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Reference:
Composition 0
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
Test material information:
Composition 1
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. 23709] were received on 10 May 2016.
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 µL
- Concentration (if solution): Undiluted
Duration of treatment / exposure:
30 minutes at 37°C, 5% CO2, 95% humidity (standard culture conditions)
Duration of post- treatment incubation (in vitro):
- Post-exposure immersion period: 12 minutes at room temperature
- Post-exposure incubation period: 1 hour and 56 minutes 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 50 µL of the test item to 1 mL of MTT solution 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 50 µL 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 17 hours and 51 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, as supplied, at the dose of 50 µL, to the entire surface of 2 living RhCE tissue replicates during 30 minutes 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 30 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. The rinsed tissues were checked for any colouration and noted for comparable colour with the negative control. This rinsing step was followed by a 12-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 1-hour and 56-minute 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 300 µL of a MTT solution at 1.0 mg/mL for 3 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 17 hours and 05 minutes at 6±3°C 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 was performed at 570 nm 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:
13.21
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
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 37.3°C and 38.0°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 colourless solution was obtained in water after 1 hour of incubation between 37.3°C and 38.0°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
- MTT assay results: The mean percent tissue viability of the RhCE replicates treated with the test substance was 13.21%, versus 31.99% in the positive control (Methyl acetate). Refer Tables 7.3.2/1 for more details.

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

1.035

1.006

1.079

93.28

100.00

13.44

1.023

0.961

2

1.158

1.151

106.72

1.128

1.167

Positive control

1

0.367

0.358

0.345

33.19

31.99

2.41

0.351

0.357

2

0.335

0.332

30.78

0.333

0.328

Test item

1

0.139

0.135

0.143

12.52

13.21

1.39

0.134

0.133

2

0.152

0.150

13.91

0.149

0.149

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

OD: optical density

Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
In accordance with Regulation EC No. 1272/2008 (CLP) and GHS, the test substance should be classified in Category 2.
Executive summary:

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

The test substance was applied, as supplied, at the dose of 50 µL to two DPBS pre-treated RhCE (EpiOcular™ tissue model) during 30 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 12-minute post-exposure immersion period at room temperature and a 1-hour and 56-minute post-exposure incubation at standard culture conditions. The tissue viability was measured by performing a 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 13.21%, versus 31.99% in the positive control (Methyl acetate).

In accordance with Regulation EC No. 1272/2008 (CLP) and GHS, the test substance should be classified in Category 2.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12-13 February 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD test Guideline No. 437 without any deviation.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
10 January 2013
Test material information:
Composition 1
Species:
other: Bovine eye
Details on test animals or tissues and environmental conditions:
Origin: Bovine eyes were obtained from EVA, Saint-Pierre-sur-Dives, France.
Age: Bovine cattle were up to 12 months old.
Transport from supplier to testing laboratory: The eyes were transported to testing laboratory 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.
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) of test item was applied on each cornea
- Concentration (if solution): undiluted

CONTROLS:
Negative control: 0.9 % sodium chloride solution
Positive control: 10 % sodium hydroxide solution
Duration of treatment / exposure:
The test item was evaluated by using a treatment time of 10 minutes ± 30 seconds.
Duration of post- treatment incubation (in vitro):
2 hours ± 10 minutes in a water bath at 32 ± 1°C.
Number of animals or in vitro replicates:
Total: 9 corneas - 3 corneas/group for test item, negative and positive controls
Details on study design:
Dose formulation application:
As the test item was a non-viscous liquid, the closed-chamber method was used as follows: the test and control items were introduced into the anterior chamber of the corneal holder through the dosing holes, to cover the epithelial side of the cornea. Then the dosing holes were sealed.

Treatment of corneas:
Corneas obtained from freshly slaughtered cattle (from abattoir) were mounted in corneal holders with the endothelial side against the O-ring of the posterior chamber. For pre-incubation, both chambers of the corneal holder were filled with MEM culture media supplemented with 1% fetal bovine serum plus penicillin/streptomycin (cMEM) and pre-incubated for 1 h and 5 minutes ± 5 minutes at 32 ± 1 °C. Then MEM was removed & then refilled with fresh cMEM and corneas were examined macroscopically for any defects. Then, the opacity of the cornea was measured to obtain OPT0. The medium was removed from anterior chamber and the test item was applied onto the epithelium of the cornea. After application of the dose formulation, the holders were incubated, vertically (cornea positioned horizontally with the treated side uppermost) in a water bath at 32 ± 1 °C, for 10 minutes. At the completion of the treatment period, the test and control items were removed from the front opening of the anterior chamber and the corneas were rinsed. The corneas were then incubated for 2 h ± 10 minutes in a water bath at 32 ± 1 °C and the second opacity measurement (OPT2) was performed.

Permeability determination
- Application of sodium fluorescein: After the second opacity measurement, the medium of the anterior chamber was removed and filled with a fluorescein solution (4 mg/mL). The holders were then incubated vertically (cornea positioned horizontally with the fluorescein-treated side uppermost) in a water bath at 32 ± 1 °C for 90 ± 5 minutes. At the end of the incubation, the optical density at 490 nm (OD490) of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea.

OTHERS:
- 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.
Irritation parameter:
in vitro irritation score
Run / experiment:
1
Value:
11
Negative controls valid:
yes
Positive controls valid:
yes
Irritation parameter:
in vitro irritation score
Run / experiment:
2
Value:
6
Negative controls valid:
yes
Positive controls valid:
yes
Irritation parameter:
in vitro irritation score
Run / experiment:
3
Value:
12
Negative controls valid:
yes
Positive controls valid:
yes
Other effects / acceptance of results:
Macroscopic examinations: No notable opaque spots or irregularities were observed on negative control corneas. Fluorescein fixation was observed on all test item-treated corneas. Opacity, fluorescein fixation and thickening of the corneas were observed on those treated with the positive control.
- In Vitro Irritancy Score (IVIS) for test item and positive control were 10 and 187, respectively.
Other effects:
None

Table 7.3.2/1: Eye irritation – results

GROUP

OPACITY

PERMEABILITY

SCORE

 

Holder

OPT0

OPT2

OPT2-OPT0

cOPT

OD490 nm

cOD490 nm

Negative

control

22

3

2

-1.0

-

0.002

-

-

35

3

2

-1.0

-

0.004

-

-

8

3

2

-1.0

-

0.021

-

-

Mean

-

-

-1.0

-

0.009

-

-

SD

-

-

0.0

-

0.010

-

-

Test item

7

2

5

3.0

4.0

0.472

0.463

11

46

3

7

4.0

5.0

0.092

0.083

6

39

3

13

10.0

11.0

0.077

0.068

12

Mean

-

-

-

6.7

-

0.205

10

SD

-

-

-

3.8

-

0.224

3.1

Positive

control

42

3

129

126.0

127.0

5.248

5.239

206

18

3

111

108.0

109.0

5.312

5.303

189

16

4

115

111.0

112.0

3.736

3.727

168

Mean

-

-

-

116.0

-

4.756

187

SD

-

-

-

9.6

-

0.892

18.9

 

OD: optical density

cOD: corrected optical density

cOPT: corrected corneal opacity

SD: standard deviation

OPT0: corneal opacity before treatment

OPT2: corneal opacity after the 2 h recovery period

Interpretation of results:
other: further testing should be conducted
Conclusions:
The ocular corrosive or severe irritant potential of Nopol could not be predicted. The test item could not be identified as inducing serious eye damage (UN GHS Category 1) or as a test chemical not requiring classification for eye irritation or serious eye damage (UN GHS No Category).
According to Guideline OECD 437, further testing should be conducted for classification and labeling purposes.
Executive summary:

In an in vitro eye irritation study performed according to Guideline OECD 437 and in compliance with GLP, 750 μL (± 8 μL) of undiluted test item, Nopol, was applied to isolated bovine corneas for 10 minutes followed by an incubation period of 2 h at 32°C. After the opacity measurements, the permeability of the cornea was determined by filling the anterior chamber with a fluorescein solution and then holders were incubated vertically for 90 minutes at 32°C. At the end of the incubation, the optical density of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea.Three corneas were used for each treated series (undiluted test item; negative control; positive control). The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

No notable opaque spots or irregularities were observed on negative control corneas. Fluorescein fixation was observed on all test item-treated corneas. Opacity, fluorescein fixation and thickening of the corneas were observed on those treated with the positive control. In Vitro Irritancy Score (IVIS) for the test item and positive control were 10 and 187, respectively. 

 

Therefore, the ocular corrosive or severe irritant potential of test item Nopol could not be predicted. The test item could not be identified as inducing serious eye damage (UN GHS Category 1) or as a test chemical not requiring classification for eye irritation or serious eye damage (No Category in UN GHS).

According to OECD Guideline 437, further 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 study was performed according to Guideline OECD 439 and in compliance with GLP, using the EPISKINTM reconstructed human epidermis model.

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

The relative mean viability of the tissues treated with the test item was 7% . As the mean viability was < 50% the results met the criteria for an irritant response.

Therefore, the test item, Nopol is classified in category 2 (H315) according to Regulation (EC) No. 1272/2008 (CLP).

Eye irritation:

An in vitro eye irritation test using the Reconstructed human Cornea-like Epithelium (RhCE) (EpiOcular™ tissue) model was performed according to Guideline OECD 492 in compliance with GLP to predict the acute eye irritation potential of the test substance.The mean percent tissue viability of the RhCE replicates treated with the test substance was 13.21%, versus 31.99% in the positive control (Methyl acetate).

Also, in an in vitro eye irritation study performed according to Guideline OECD 437 in compliance with GLP, In Vitro Irritancy Score (IVIS) for test item and positive control were 10 and 187, respectively. 

According to the results of those studies, Nopol should be classified in category 2 (H319) according to Regulation EC N° 1272/2008 (CLP) and GHS.

Justification for classification or non-classification

Based on the results of a study conducted according to Guideline OECD 439, Nopol is classified in category 2 (H315) according to Regulation EC No. 1272/2008 (CLP) and GHS.

Based on the results of studies conducted according to Guidelines OECD 438 and 492, Nopol should be classified in category 2 (H319) according to Regulation EC No. 1272/2008 (CLP) and GHS.