Registration Dossier

Toxicological information

Eye irritation

Currently viewing:

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 August - 21 September 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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
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:
27 April 2017

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Name (as cited in study report): LABDANUM ABSOLUTE / F
- Storage condition of test material: room temperature, darkness, closed container

Test animals / tissue source

Species:
human
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability: the OECD 492 (adopted in 2015), is validated and has regulatory acceptance. This test guideline is applicable to solid and waxes, so is considered to be applicable to the test item.

RECONSTRUCTED HUMAN CORNEA-LIKE EPITHELIUM (RhCE)
- Model used: EpiOcularTM OCL-212-ver2.0, supplied by MatTek Corporation
- Tissue batch number(s): 27005
- Keratinocyte strain: 4F1188

FUNCTIONAL MODEL CONDITIONS
- Tissue viability: 1.419, within the acceptance criteria (1.1-3.0)
- Barrier function: 15.97 min, within the acceptance criteria (12.2-37.5)
- Sterility: Sterile

Test system

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

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): undiluted

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- 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:
2
Details on study design:
- Details of the test procedure used: method for solids (not pipetteable), as described in the OECD TG 492
- RhCE tissue construct used, including batch number: EpiOcularTM OCL-212-ver2.0, supplied by MatTek Corporation, batch No. 27005
- Doses of test chemical and control substances used: 50 mg / 50 µL
- Duration and temperature of
Exposure: 6 hours at 37°C, 5% CO2, 95% humidity (standard culture conditions)
Post-exposure immersion: 25 minutes at room temperature
Post-exposure incubation periods: 18 hours at standard culture conditions
- Description of any modifications to the test procedure: None
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable): not applicable.
- Number of tissue replicates used per test chemical and controls (positive control, negative control): 2
- Wavelength used for quantifying MTT formazan, measuring device (e.g. spectrophotometer): 570 nm, 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.
- Description of the method used to quantify MTT formazan
The RhCE constructs were placed in 300 µL of a MTT solution at 1.0 mg/mL for 2 hours and 52 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 2 hours at 6±3°C in the dark.
The concentration of formazan was measured by determining the OD (Optical Density) 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.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
The test substance is considered to be corrosive or irritant to eyes if the mean percent tissue viability after exposure and post-exposure incubation is ≤ 60%. When the final mean percent tissue viability is ≤ 60%, further testing with other test methods will be required because the RhCE test method shows a certain number of false positive results and cannot resolve between UN GHS Categories 1 and 2.
The test substance is considered to be non-irritant to eyes if the mean percent tissue viability after exposure and post-exposure incubation is > 60%.
- Complete supporting information for the specific RhCE tissue construct used
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: included in the report, follows the OECD TG 492.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
other: % mean viability of the tissues
Run / experiment:
1
Value:
29.05
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
40.34%
Remarks on result:
positive indication of irritation
Irritation parameter:
other: % mean viability of the tissues
Run / experiment:
2
Value:
8.73
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
14.96 %
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
MAIN TEST
- MTT assay results - First run: The mean percent tissue viability of the RhCE replicates treated with the test substance was 29.05 versus 40.34% in the positive control (Methyl acetate).
- MTT assay results - Second run: The mean percent tissue viability of the RhCE replicates treated with the test substance was 8.73 versus 14.96% in the positive control (Methyl acetate).

OTHER EFFECTS:
- Visible damage on test system: none

ACCEPTANCE OF RESULTS:
FIRST RUN
- Acceptance criteria met for negative control: yes, the negative control OD is > 0.4 and < 1.25 (values between 0.528 and 0.700). [The optical density was measured after a 1:2 dilution of the formazan extracts in isopropanol : the acceptability criteria should be in the range ≥ 0.4 and ≤1.25 for the negative control.]
- Acceptance criteria met for positive control: no, the percentage of viability of 1/2 epidermis of the positive control (Methyl acetate) was higher than 50%
<=> a second run was performed
SECOND RUN:
- Acceptance criteria met for negative control: yes, the negative control OD is > 0.4 and < 1.25 (values between 0.528 and 0.700).
- Acceptance criteria met for positive control: yes, the mean relative viability of the positive control is below 50% of the negative control viability (14.96%).
- Acceptance criteria met for variability between replicate measurements: yes, the difference of viability between the two relating tissues of the test item and the positive control are < 20% (1.83% and 2.66%, respectively). The difference of viability between the two relating tissues is slightly above the threshold of 20% for the negative control (21.11%). However, this deviation is not considered to have affected the integrity of the study or the overall conclusion.
- Range of historical values if different from the ones specified in the test guideline: The positive and negative control OD were within the historical control ranges

Any other information on results incl. tables

Table 7.3.2/1: First run - 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

0.861

0.858

0.992

86.54

100.00

26.93

0.855

0.858

2

1.185

1.125

113.46

1.092

1.098

Positive control

1

0.502

0.497

0.400

50.13

40.34

19.57

0.486

0.502

2

0.305

0.303

30.56

0.308

0.295

Test item

1

0.181

0.182

0.228

18.36

29.05

21.38

0.182

0.182

2

0.395

0.394

39.75

0.393

0.395

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

OD: optical density

Table 7.3.2/2: Second run - 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

0.700

0.665

0.602

110.56

100.00

21.11

0.640

0.657

2

0.550

0.538

89.44

0.537

0.528

Positive control

1

0.111

0.098

0.090

16.29

14.96

2.66

0.094

0.091

2

0.076

0.082

13.62

0.087

0.085

Test item

1

0.047

0.047

0.053

7.81

8.73

1.83

0.048

0.048

2

0.058

0.058

9.64

0.059

0.058

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

OD: optical density

Applicant's summary and conclusion

Interpretation of results:
other: Category 1 (irreversible effects on the eye) or Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
With a percentage of tissue viability < 60%, the test item requires classification as irritating or corrosive to eyes according to the Regulation (EC) No 1272/2008 (CLP) and to the GHS.
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.

As the test item was a paste considered as a solid, it was administered after being directly applied on a nylon mesh in order to cover the entire surface of the epidermis (corresponding to 50 mg).

The test itemLABDANUM ABS SUPER - 953418 was applied, to 2 DPBS pre-treated RhCE (EpiOcularTMtissue 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. The experimental protocol was established in accordance withO.E.C.D. Test Guideline No. 492 adopted 28 July 2015.

 

During the first run, the percentage of viability of 1/2 epidermis of the positive control (Methyl acetate) was higher than 50%. It was not possible to conclude for the test item because the acceptability criteria defined in OECD 439 was not respected. A second run was necessary.

 

In the second run, the mean percent tissue viability of the RhCE replicates treated with the test item LABDANUM ABS SUPER - 953418 was 8.73%, versus 14.96% in the positive control (Methyl acetate).

 

In the second run, the quality criteria required for acceptance of results in the test were satisfied:

- Acceptance criteria met for negative control: yes, the negative control OD is > 0.4 and < 1.25 (values between 0.528 and 0.700).

- Acceptance criteria met for positive control: yes, the mean relative viability of the positive control is below 50% of the negative control viability (14.96%).

- Acceptance criteria met for variability between replicate measurements: yes, the difference of viability between the two relating tissues of the test item and the positive control are < 20% (1.83% and 2.66%, respectively).  The difference of viability between the two relating tissues is slightly above the threshold of 20% for the negative control (21.11%). However, this deviation is not considered to have affected the integrity of the study or the overall conclusion.

 

The positive and negative control OD were within the historical control ranges.

With a percentage of tissue viability < 60%, the test item requires classification as irritating or corrosive to eyes according to the Regulation (EC) No 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for eye irritation endpoint.