Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-07-30 to 2013-09-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
well documented study according to GLP; Up to now, no internationally accepted guidelines are available for the EpiOcular eye irritation test. But the EpiOcular Test has been already used in pre-validation studies and validation by ECVAM is in progress.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: EpiOcular (TM) guideline from MatTek in vitro life science laboratories, Bratislava
Deviations:
no
Principles of method if other than guideline:
Guideline Reference: Ocular Irritation Protocol: Neat Method (MTT ET-50) for use with EpiOcular Tissues, Rev:17.Jan.2012 by MatTek in vitro life science laboratories; Bratislava
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report):FC-131
- Physical state: yellow crystalline powder
- Analytical purity: 99.8% (area HPLC)
- Impurities (identity and concentrations): about 0.1% 4-diethylamino-2-methoxy-benzaldehyde
- Isomers composition:No data
- Purity test date:03.07.2013
- Lot/batch No.:30610045
- Expiration date of the lot/batch:12.06.2015
- Homogeneity: yes
- Storage condition of test material: Room temperature (20+-5 °C), no humidity
- Production date: Jun. 2013

Test animals / tissue source

Species:
other: in vitro test
Strain:
other: in vitro test
Details on test animals or tissues and environmental conditions:
- EpiOcular (TM) tissue: normal, human-derived epidermal keratinocytes cultured to form a stratified squamous epithelium similar that of human cornea
- tissue surface: approx. 0.6 square cm
-origin: OCL-200 tissues produced from MatTek in vitro life science laboratories, Bratislava
- day of delivery: 20.08.2013
- Batch: 18627
- Buffer for tissue rinsing: DPBS
- plates: 6-well plates
- temperature: 37+-1°C
- CO2: 5+-1%

Cell viability test:
-measured after exposure of tissues with test substance
-for calculation of the ET50 value (time needed for 50% reduction of cell viability)
- measured by dehydrogenase conversion of MTT, present in cell mitochondria, into blue formazan, which is quantitatively measured after tissue extraction

Test system

Vehicle:
unchanged (no vehicle)
Controls:
other: in vitro test with tissues: positive and negative controls included
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 100+-10mg of FC-131 in duplicate (2 tissues each)


Duration of treatment / exposure:
3,30 and 60 min
positive control: 15 ad 45 min
negative control: 60 min
Observation period (in vivo):
three hours in MTT medium, and thereafter overnight at room temperature (tissues covered by isopropanol) for formazan production
Number of animals or in vitro replicates:
number of tissues: 2 tissues for each time point and two replicates each
Details on study design:
- first step: substance exposure to tissues
- second step: cell viability test based on MTT coversion to formanzan

- Positive control: 0.3% Triton X 100 solution
- Negative control: Deionised H2O

REMOVAL OF TEST SUBSTANCE
- Washing (if done): washing of tissues with DPBS buffer and soaking in medium for 10min


SCORING SYSTEM: Formanzan relative absorption for calculation of ET50 (time needed for 50% reduction of absorption (cell viability)
ET50 > 60: non/minimal irritant
ET50 31-60: mild irritant
ET50 3-30: moderate irritant
ET50 < 3: severe/extreme irritant

TOOL USED TO ASSESS SCORE: plate spectral photometer at 570m

Results and discussion

In vivo

Results
Irritation parameter:
other: ET50
Basis:
mean
Time point:
other: 60min
Score:
> 60
Reversibility:
other: not applicable
Remarks on result:
other: determination of time leading to a reduction of relative formanzan absorption, mean formanzan production in tissues was 96.5% after 60 min compared to controls
Irritant / corrosive response data:
Values for absorption and relative absorption (in % compared to negative controls) are shown in tables 1-3. After 60 min of treatment with the test substance relative absorbance values were marginal reduced to 96.5%. Hence, the ET50 value is stated as "> 60min" and FC-131 according to this test can be considered as not/minimal irritant.
Other effects:
The ET50 for the positive controls was 29.55 (min). Marginal deviations from historical controls of tested negative controls were observed.

Any other information on results incl. tables

Table 1: Absorption values for negative controls, positive controls and test item

Substance

Tissue 1

Tissue 2

Incubation Time

Negative Control

1.969

1.856

60 minutes

1.969

1.833

1.970

1.842

Positive Control

1.513

1.426

15 minutes

1.507

1.438

1.517

1.433

Positive Control

0.436

0.450

45 minutes

0.434

0.449

0.438

0.445

Test Item

1.860

1.780

3 minutes

1.857

1.800

1.843

1.792

Test Item

1.810

1.806

30 minutes

1.829

1.814

1.836

1.813

Test Item

1.894

1.801

60 minutes

1.871

1.813

1.888

1.783

Table 2: Mean absorption values for negative control, positive control and the test item FC-131

Designation

Negative

control

Positive

control

Positive

control

Test item

Test

Item

Test

item

Incubation time

60 min.

15 min

45 min.

3 min.

30 min.

60 min.

Mean – blank (Tissue 1)

1.931

1.474

0.398

1.815

1.787

1.846

Mean – blank (Tissue 2)

1.806

1.394

0.410

1.753

1.773

1.761

Mean of the two Tissues

1.869

1.434

0.404

1.784

1.780

1.804

Relative Standard Deviation of the two tissues

0.047

0.039

0.021

0.025

0.006

0.033

Table 3: Formanazan production in % compared to untreated (negative) controls and cells treated with FC-131

Designation

Positive

control

Positive

control

Test

item

Test

Item

Test

item

Incubation time

15 min

45 min.

3 min.

30 min.

60 min.

% Formazan production (Tissue 1)

78.9%

21.3%

97.1%

95.6%

98.8%

% Formazan production (Tissue 2)

74.6%

21.9%

93.8%

94.9%

94.2%

% Formazan production Mean

76.7%

21.6%

95.5%

95.2%

96.5%

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: other: from EpiOcular guideline
Conclusions:
FC-131 reduced cell viability of EpiOcular tissue only marginal. The estimated ET50 value was > 60min. Hence, FC-131 can be considered as not/minimal irritant in the Human Cornea Model test under the conditions of the test.
Executive summary:

The eye irritation potential of FC-131 was determined using an in vitro Human Cornea Model (EpiOcular Test). Up to now, no internationally accepted guidelines are available for this test but the validation by ECVAM is in progress. About 100 mg of the test substance was tested for 3, 30 ad 60 min. The irritation potential was measured as reduction of cell viability within the tissue by applying a cell viability test after exposure based on formanzan production of the cells. FC-131 lead to 95.5% formanzan production (cell viability) compared to untreated controls after 60 min treatment. Hence, an ET50 value (time needed for a 50% reduction of cell viability) of ">60 min" could be estimated and FC-131 was considered to be not/mild irritant.