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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Auguste - November 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:
liquid
Remarks:
slightly brown flowable liquid
Details on test material:
Name LABDANUM ABSOLUTE
Batch no. 0117/1
Appearance slightly brown flowable liquid
CAS No. 89997-74-0
EINECS-No. 289-711-7
Production date Jan. 2017
Expiry date Jan. 2019
Storage Room Temperature (20 ± 5°C)

Test animals / tissue source

Species:
other: Reconstructed human Cornea-like Epithelia
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, liquids, semi solids 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): 27002

FUNCTIONAL MODEL CONDITIONS
- Tissue viability: 1.269, within the acceptance criteria (1.1-3.0)
- Barrier function: 24.36 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 µl undiluted

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
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: 2 hours and 03 minutes at standard culture conditions
Number of animals or in vitro replicates:
2
Details on study design:
MAIN TEST
- Pre-incubation of the tissues:
On the day of receipt, the tissues in their 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 10 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 2 living PBS pre-treated RhCE (EpiOcular™ tissue model) during 30 minutes at 37°C, 5% CO2, 95% humidity (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 2-hour and 03 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 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 5 hours and 25 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 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.

Results and discussion

In vitro

Results
Irritation parameter:
other: % mean viability of the tissues
Run / experiment:
1
Value:
44.79
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
24.44%
Other effects / acceptance of results:
MAIN TEST
- MTT assay results: The mean percent tissue viability of the RhCE replicates treated with the test substance was 44.79 versus 24.44% 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 0.835 and 0.855).
- Acceptance criteria met for positive control: yes, the mean relative viability of the positive control is below 50% of the negative control viability (24.44%).
- Acceptance criteria met for variability between replicate measurements: yes, the difference of viability between the two relating tissues of the negative and positive control are < 20% (1.42% and 1.78%, respectively). The difference of viability between the two relating tissues is above the threshold of 20% for the test item (28.05%). In addition, one epithelium showed a percentage of viability of 58.82% which is considered to be a bordeline value (60%+/- 5%) according to OECD Test Guideline No 492. However, the two replicates showed concordant positive results, therefore a second test was not deemed necessary.
- 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: 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

0.846

0.839

0.845

99.29

100.00

1.42

0.835

0.836

2

0.845

0.851

100.71

0.855

0.854

Positive control

1

0.227

0.214

0.207

25.33

24.44

1.78

0.211

0.205

2

0.202

0.199

23.55

0.198

0.198

Test item

1

0.257

0.260

0.379

30.77

44.79

28.05

0.264

0.258

2

0.494

0.497

58.82

0.503

0.495

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

OD: optical density

Concerning acceptability criteria:

- The negative control OD is > 0.4 and < 1.25 (values between 0.835 and 0.855).

- The mean relative viability of the positive control is below 50% of the negative control viability (24.44%).

- The difference of viability between the two relating tissues of the negative and positive controls are < 20% (1.42% and 1.78%, respectively).The difference of viability between the two relating tissues is above the threshold of 20% for the test item (28.05%). In addition, one epidermis showed a percentage of viability of 58.82%, which is considered to be a borderline value (60% ± 5%) according to O.E.C.D. Test Guideline No. 492. However, the two replicates showed concordant positive results, therefore a second test was not deemed necessary

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.

The test item was applied, as supplied at the dose of 50 µl, to 2 living DPBS pre-treated RhCE (EpiOcularTMtissue 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 minutes post-exposure immersion period at room temperature and an 2 hours and 03 minutes 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.

 

The mean percent tissue viability of the RhCE replicates treated with the test item Labdanum absolute was 44.79%, versus 24.44% in the positive control (Methyl acetate).

 

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 0.835 and 0.855).

- Acceptance criteria met for positive control: yes, the mean relative viability of the positive control is below 50% of the negative control viability (24.44%).

- Acceptance criteria met for variability between replicate measurements: yes, the difference of viability between the two relating tissues of the negative and positive control are < 20% (1.42% and 1.78%, respectively).  The difference of viability between the two relating tissues is above the threshold of 20% for the test item (28.05%). In addition, one epithelium showed a percentage of viability of 58.82%, which is considered to be a borderline value (60% +/- 5%) according to OECD Test Guideline No. 492. However, the two replicates showed concordant positive results, therefore a second test was not deemed necessary.

 

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.