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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:
From March 27th to 30th, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

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:
July 28th, 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Tris(ethyl acetoacetato-O1',O3)aluminium
EC Number:
239-343-8
EC Name:
Tris(ethyl acetoacetato-O1',O3)aluminium
Cas Number:
15306-17-9
Molecular formula:
C18H27AlO9
IUPAC Name:
tris(ethyl acetoacetato-O1',O3)aluminium
Test material form:
solid
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Test animals / tissue source

Species:
human
Details on test animals or tissues and environmental conditions:
The EpiOcularTM tissue consists of normal, human-derived keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells. These cells are not transformed or transfected with genes to induce an extended life span. The EpiOcularTM tissues are cultured in specially prepared cell culture inserts with a porous membrane through which nutrients can pass to the cells. The tissue surface is 0.6 cm^2.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
other: negative Control (sterile demineralised water) and positive control (methyl acetate)
Amount / concentration applied:
51.3 mg tissue 1, 50.5 mg tissue 2.
Duration of treatment / exposure:
6 hours
Duration of post- treatment incubation (in vitro):
18 hours
Number of animals or in vitro replicates:
Two tissue replicates.
Details on study design:
- Details of the test procedure used:
GENERAL PRINCIPLE OF THE STUDY: eye irritant materials are identified by their ability to produce a decrease in cell viability as determined. The cell viability is measured by dehydrogenase conversion of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide), present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percentage reduction of cell viability in comparison of untreated negative controls is used to predict the eye irritation potential.

PRE-TESTS:
-ASSESSMENT OF DIRECT REDUCTION of MTT
The test item Tris(ethyl acetoacetato-O1`,O3)aluminium was tested for the ability of direct formazan reduction. To test for this ability, 47.5 mg of the solid test item were added to 1 ml of MTT reagent in a 6-well plate and the mixture was incubated in the dark at 37 ± 1 °C, 5.0 ± 1 % CO2 and 80 – 100 % relative humidity for 3 hours. 1 ml of MTT reagent plus 50 µl of H2O demin. was used as negative control. The MTT reagent did not change its colour; therefore, direct MTT reduction had not taken place, and no data correction was necessary.
- ASSESSMENT OF COLOURED OR STAINING TEST ITEM
The test item is colourless. To assess, whether the test item will become coloured after contact with water or isopropanol, 49.2 mg of the solid test item were added to 1 ml of sterile H2O demin. in a 6-well plate and incubated in the dark at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 1 hour Moreover, 49.7 mg of test item were added to 2 ml isopropanol and incubated in a 6-well plate for 2 hours at room temperature.
After incubation, no colour development was visible. Therefore, the main test was per-formed without colourant controls.

MAIN TEST:
On the day of the start of the experiment, the MTT concentrate was thawed. The concentrate was diluted with the MTT solvent directly before use. The assay medium was warmed in the water bath to 37 ± 1°C.
The test item was ground. 6-well-plates were labelled with test item, negative control and positive control and filled with 1 ml assay medium in the appropriate wells. All inserts were inspected for viability and the presence of air bubbles between agarose gel and insert. Viable tissues were transferred in the prepared 6-well-plate and incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 1 hour.
After the pre-incubation, the medium was replaced and the wells were filled with 1 ml fresh assay medium. All 6-well-plates were incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 16 hours.
After overnight incubation, the tissues were pre-wetted with 20 µl DPBS buffer and the tissues were incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 30 minutes. After that, 50 µl of the controls and a defined amount of the test item were applied in duplicate in 1- minute- intervals. At the beginning of each experiment (application of negative controls), a stop watch was started. After dosing the last tissue, all plates were transferred into the incubator for 6 hours at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity. At the end of exposure time, the inserts were removed from the plates in 1-minute-intervals using sterile forceps and rinsed immediately. The inserts were thoroughly rinsed with DPBS. Then, the tissues were immediately transferred into 5 ml of assay medium in pre-labelled 12-well plate for 25 minutes post soak at room temperature.
After that, each insert was blotted on absorbent material and transferred into a pre-labelled 6-well plate, containing 1 ml assay medium. For post-treatment incubation, the tissues were incubated for 18 hours at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity.
After the post-treatment incubation, the MTT Assay was performed.

MTT ASSAY AND EXTRACTION
A 24-well-plate was prepared with 300 µl freshly prepared MTT-reagent in each well. The tissue inserts were blotted on absorbent material and then transferred into the MTT solution. The plate was incubated for 180 at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity.
At last, each insert was thoroughly dried and set into a pre-labelled 6-well-plate, containing 2 ml isopropanol, taking care that no isopropanol is flowing into the tissue insert. The plate was firmly sealed to avoid evaporation of the solvent and then shaken for 2 hours at room temperature, protected from light.

MEASURAMENT
The inserts were removed from the 6-well plate and discarded. The content of each well was thoroughly mixed in order to achieve homogenisation.
From each well, two replicates with 200 µl solution (each) were pipetted into a 96-well-plate. Eight wells with 200 µl isopropanol were also pipetted. The plate was read in a plate spectrophotometer at 570 nm.

EVALUATION
The values of the 96-plate-reader were transferred into a validated spreadsheet (Microsoft Excel®).
Note: All calculations are performed with unrounded values. Therefore, re-calculation with rounded values may lead to slightly different results.

CALCULATION
% VIABILITY= [OD corrected test item/OD corrected mean negative control]*100%

- RhCE tissue construct used, including batch number: the EpiOcularTM tissue consists of normal, human-derived keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells. These cells are not transformed or transfected with genes to induce an extended life span. The EpiOcularTM tissues are cultured in specially prepared cell culture inserts with a porous membrane through which nutrients can pass to the cells. The tissue surface is 0.6 cm^2. EpiOcularTM tissues were procured from MatTek In Vitro Life Science Laboratories, Myln-ské Nivy 73, 82105 Bratislava, Slovakia (Batch number 23773)

- Doses of test chemical used: 51.3 mg (Tissue 1) and 50.5 mg (Tissue 2)

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable)
Exposure: 6 hours at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity.
Post-exposure incubation: 18 hours at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity.

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable): not applicable: the pre-tests regarding direct reduction of MTT and regarding colouring potential of the test item were negative.

- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable): two replicates

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): quantification of MTT formazan performed at 570 nm.

- Description of the method used to quantify MTT formazan: spectrophotometer.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model:
% Viability > 60 % : Non eye irritant: No GHS category for eye irritation
% Viability: ≤ 60 %: Eye irritant: GHS category 1 or 2

- ACCEPTANCE CRITERIA:
OD of negative control: > 0.8 and < 2.5
Tissue viability of positive control: < 50 % (relative to negative control)
Variation within two tissue replicates of test item and controls: < 20 %



Results and discussion

In vitro

Results
Irritation parameter:
other: Viability %
Run / experiment:
Mean of 2 tissue replicates
Value:
ca. 29.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
RESULTS OF MAIN TEST:
After 6h exposure followed by product washing and a post incubation of 18h a residual cell viability (mean of two replicates) of 29.9 % has been quantified for test item.

RESULTS OF PRELIMINARY TESTS:
In the preliminary test to assess the direct reduction of MTT, MTT reagent did not change its colour after incubation with test item; therefore, direct MTT reduction had not taken place, and no data correction was necessary.
In the preliminary test to assess coulored test item, no colour development was visible; therefore, the main test was performed without colourant controls.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The mean viability for negative and positive controls was in the range of historical data.

ACCEPTANCE OF RESULTS:
OD of negative control: > 0.8 and < 2.5: fulfilled (1.5)
Tissue viability of positive control: < 50% (relative to negative control): fulfilled (42.1%)
Variation within two tissue replicates of test item and controls: < 20%: fulfilled (1.5 % for negative control; 4.0 % for positive control and 0.1 % for test item)

Any other information on results incl. tables

Absorbance Values Negative Control, Positive Control and Test Item (OD at 570 nm)

Designation

Measurement

Negative Control

Positive Control

Tris(ethyl acetoacetato-O1`,O3)aluminium-

Tissue 1 

1

1.605

0.656

0.503

2

1.572

0.653

0.491

Tissue 2 

1

1.573

0.720

0.503

2

1.559

0.711

0.495

Mean Absorbance Negative Control, Positive Control and Test Item (Note Mean Blank = 0.037)

Designation

Negative Control

Positive Control

Tris(ethyl acetoacetato-O1`,O3)aluminium

Mean – blank

(Tissue 1)

1.552

0.618

0.460

Mean – blank

(Tissue 2)

1.529

0.679

0.462

% Viability Positive Control and Test Item

Designation

Positive Control

Tris(ethyl acetoacetato-O1`,O3)aluminium-

% Viability (Tissue 1)

40.1 %

29.9 %

% Viability (Tissue 2)

44.1 %

30.0 %

% Viability Mean

42.1 %

29.9 %

Historical data

Parameter

Optical Density

Negative Control

Relative Viability %

Positive Control

 

Demineralised H2O

Methyl acetate

Exposition time

6 hours

Mean

1.526

32.4 %

Standard deviation

0.249

8.3 %

Range

1.047 – 2.080

21.1 – 53.9 %

Study17021405G891

1.540

42.1%

Applicant's summary and conclusion

Interpretation of results:
other: eye irritant according to the CLP Regulation (EC n.1272/2008)
Conclusions:
Tris(ethyl acetoacetato-O1`,O3)aluminium is eye irritant according to the CLP Regulation (EC n.1272/2008).
Executive summary:

The potential eye irritation of Tris(ethyl acetoacetato-O1`,O3)aluminium was examined according to OECD TG 492 (EpiOcularTMEye Irritation test).

The test item was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 6 hours. The solid test item was applied to each tissue. After treatment, the respective substance was rinsed from the tissue; then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to formazan. The formazan production was evaluated by measuring the optical density (OD) of the resulting solution.

Demineralised water was used as negative control, methyl acetate was used as positive control.

The controls showed the following results: after treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD > 0.8 and < 2.5, OD was 1.5. The positive control showed clear eye irritating effects, the relative absorbance value was reduced to 42.1 % (< 50 %). Variation within tissue replicates was acceptable (< 20 %).

 

After treatment with the test item, the mean value of tissue viability was 29.9 %.

This value is below the threshold for eye irritation potential (≤ 60 %).

According to the OECD Guideline TG 492, the EpiOcularTMEye Irritation Test does not allow discrimination between eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible effects on the eye (Category 1). Under the conditions of the test, Tris(ethyl acetoacetato-O1`,O3)aluminium-CHA109 is considered as eye irritant or inducing serious eye damage in the EpiOcularTMEye Irritation Test.

Considering the results of in vitro tests for skin and eye irritation, the structure of the molecule and its pH in water (6.2), Tris (ethyl acetoacetato-O1’,O3) aluminium is classified H319 (eye irritant category 2) according to the CLP Regulation (EC.n.1272/2008).

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