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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:
04 - 06 Apr 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Version / remarks:
adopted in 2015
Deviations:
yes
Remarks:
No information on historical data given.
GLP compliance:
yes (incl. QA statement)
Remarks:
Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test material

Constituent 1
Chemical structure
Reference substance name:
4-benzoylbenzene-1,2,3-triyl tris(6-diazo-5,6-dihydro-5-oxonaphthalene-1-sulphonate)
EC Number:
227-030-9
EC Name:
4-benzoylbenzene-1,2,3-triyl tris(6-diazo-5,6-dihydro-5-oxonaphthalene-1-sulphonate)
Cas Number:
5610-94-6
Molecular formula:
C₄₃H₂₂N₆O₁₃S₃
IUPAC Name:
4-benzoylbenzene-1,2,3-triyl tris(6-diazo-5,6-dihydro-5-oxonaphthalene-1-sulphonate)
Test material form:
solid

Test animals / tissue source

Species:
human
Strain:
other: not applicable
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability:
The reconstructed human cornea-like epithelium (RhCE) model is an accepted in vitro method to replace in vivo animal testing. The human eye EpiOcular™-model closely mimics the biochemical and physiological properties of the human eye, i.e. the cornea.

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live:
The EpiOcularTM human cell construct (OCL-200, OCL-212, MatTek Corporation) is used in this assay. It is a nonkeratinized epithelium prepared from normal human keratinocytes. All biological components of the EpiOcularTM tissue and the kit culture medium have been tested and are free of contamination. An analysis for tissue functionality and quality was performed: the barrier function, cell viability were meeting the acceptance criteria as well as the histological appearance of the cells.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 50 mg
- Concentration: undiluted
Duration of treatment / exposure:
6 h ± 15 min
Observation period (in vivo):
not applicable
Duration of post- treatment incubation (in vitro):
25 ± 2 min (Post-exposure immersion incubation)
18 h ± 15 min (Post-treatment incubation)
Number of animals or in vitro replicates:
2 replicates
Details on study design:
- Details of the test procedure used
The cytotoxicity of the test item (and thus the ocular irritation potential) is evaluated by measuring the relative viability of the treated RhCE tissues in comparison to the negative control-treated tissues. Viability is determined by the NAD(P)H-dependent microsomal enzyme reduction of MTT in control and test item-treated cultures.

- RhCE tissue construct used, including batch number
The EpiOcularTM human cell construct (OCL-200 and OCL-212, MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia, Lot number: 23769)

- Doses of test chemical and control substances used
50 mg test item; 50 μL positive control (methyl acetate) and 50 μL negative control (sterile deionized water)

- Wavelength used for quantifying MTT formazan
570 nm

- Description of the method used to quantify MTT formazan
Inserts were removed from the 24-well plate after the incubation time in MTT solution (3 h ± 10 min), the bottom of the insert was blotted on absorbent material and transferred to a 6-well plate containing 2 mL isopropanol per well in a manner avoiding the isopropanol to flow into the insert. The plate was sealed with standard plate sealer. To extract the MTT, the plates were placed on an orbital plate shaker and shaken for 2-3 h at room temperature. 200 μL samples from each tube were placed into the wells of a 96-well plate and the absorbance/optical density was measured in a 96-well plate spectrophotometer to determine cell viability.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
The test item requires classification and labelling according to UN GHS (Category 2 or Category 1), if the mean percent tissue viability after exposure and post-exposure incubation is ≤ 60% of the negative control.
The test item requires no classification and labelling according to UN GHS (No Category), if the mean percent tissue viability after exposure and post-exposure incubation is > 60%.

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria
No information is given on historical data, but the results obtained for the negative and positive control meet the acceptance criteria given in the OECD guideline 492.

- Complete supporting information for the specific RhCE tissue construct used
The barrier function integrity test resulted in an ET50 of 4.6, cell viability was determined to have an OD of 1.3, the RHE model was tested to be a well differentiated epidermis with 5.5 cell layers without any significant histological abnormality, thus meeting the acceptance criteria.

- Reference to historical data of the RhCE tissue construct
no further information available in the study report

- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals
no information provided in the study report

- Positive and negative control means and acceptance ranges based on TG OECD 492
negative control mean OD: 1.381 (acceptance range: 0.8 - 2.5)
positive control mean viablity: 11.2% (acceptance criteria: < 50% of the negative control)

- Acceptable variability between tissue replicates for positive and negative controls
The variability between tissue replicates for positive and negative controls meets the acceptance criteria of < 20%.

- Acceptable variability between tissue replicates for the test chemical
The variability between tissue replicates for the test chemical meets the acceptance criteria of < 20%.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
other: mean viability (%)
Run / experiment:
negative control
Value:
100
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: mean viability (%)
Run / experiment:
positive control
Value:
11.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: mean viability (%)
Run / experiment:
test item
Value:
67.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, as the mean OD value (1.381) of the two negative control tissues lies between 0.8 and 2.5.
- Acceptance criteria met for positive control: Yes, as the mean percentage viability for the positive control (11.2%) lies below 50% of the control viability.
- The variability between tissue replicates for positive, negative controls and test chemical were 1.9%, 17.4% and 12.1% respectively, thus meeting the acceptance criteria of < 20%.

The pre-test for direct MTT-reducing capacity of the test item did not result in blue color, i.e. the test item is not a direct MTT reducer. In the pre-test medium coloration by the test item was observed, but no tissues were stained during the study. Therefore, no additional tissues for color control were treated.

Any other information on results incl. tables

Table 1: MTT results of eye irritation study

Controls

 

Optical Density (OD)

Viability (%)

Δ%

Negative Control: Sterile deionized water

1

1.261

91.3

17.4

2

1.501

108.7

mean

1.381

100.0

-

standard deviation (SD)

12.30

 

Positive Control:
Methyl acetate

1

0.141

10.2

1.9

2

0.167

12.1

mean

0.154

11.2

-

standard deviation (SD)

1.34

 

Test item

1

1.015

73.5

12.1

2

0.848

61.4

mean

0.932

67.5

-

standard deviation (SD)

8.56

-

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the present study, the test item did not show an eye hazard potential.
Executive summary:

The objective of the present study was to investigate the potential of the test item to induce eye irritation in an in vitro human cornea model. 

The test item was applied topically to a reconstructed human cornea-like epithelium model (EpiOcular) followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. 

Duplicates of the EpiOcular-model were treated with the test item, the negative or the positive control for 6 hours (+/- 15 min). 50 mg of the test item and 50 µL of either the negative control (sterile deionized water) or the positive control (methyl acetate) were applied to the tissues. 

After treatment with the negative control (sterile deionized water) the mean OD was 1.381 (study acceptance criterion: > 0.8 and < 2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 11.2% (study acceptance criterion: < 50%). Thus, the acceptance criterion were met. 

Following treatment with the test item, the tissue viability was 67.5% and, thus, higher than 60%, i.e. according to OECD 492 the test item did not show an eye hazard potential.