Tris[oxalato(2-)]dilutetium

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 17 February 2017 to 28 April 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP compliance

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

2 November 2016

Test material

Test material information

Constituent 1

Reference substance name:

Tris[oxalato(2-)]dilutetium

EC Number:

222-328-5

EC Name:

Tris[oxalato(2-)]dilutetium

Cas Number:

3426-45-7

Molecular formula:

C6Lu2O12

IUPAC Name:

4,4'',5,5''-tetraoxo-1',4'-dihydro-1λ³,1''λ³,3λ³,3''λ³-dispiro[1λ³,3λ³-dioxa-2-lutetacyclopentane-2,5'-1λ³-oxa-3λ³-oxa-2-lutetacyclopenta[4,5-d]1λ³-oxa-3λ³-oxa-2-lutetacyclopentane-2',2''-[1λ³,3λ³]dioxa-[2]lutetacyclopentane]-2',2',2',2,2,2-hexakis(ylium)-1',4',1,1'',3,3''-hexaide

Constituent 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Water

Test material form:

solid: particulate/powder

Details on test material:

Name: Tris[oxalate(2-)]dilutetium

Specific details on test material used for the study:

No correction factor is applied for this type of study

Test animals / tissue source

Species:

human

Strain:

other: Reconstructed human Cornea-like Epithelium (tissues)

Details on test animals or tissues and environmental conditions:

TEST SYSTEM
- Source: MatTek, Bratislava, Slovak Republic
- Expiry date: The EpiOcularTM tissues were used within 72 hours of their production.
- Selection: At receipt, the tissues were inspected for obvious defects as they could have been rejected based on blistering, excess fluid or air bubbles below the tissue insert. Cultures with air bubbles under the insert covering greater than 50% of the insert area were not used.
- Storage conditions: At receipt, the living EpiOcularTM tissues were stored at room temperature on their day of arrival.
- Description: The EpiOcularTM model constists of an airlifted, living, multilayered ocular tissue construction (surface 0.60 cm2), reconstructed from normal (non-transformed) human-derived keratinocytes. This is a non-keratinized epithelium which models the corneal epithelium with progressively stratified, but not cornified cells. The cells are cultured in proprietary serum-free culture media, which induces corneal differentiation and the formation of the organotype 3D cornea-like model. The 3D tissue consists of highly organized cell layers similar to that found in the cornea. The model features a normal ultra-structure and is functionally equivalent to human in vivo tissue.

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): 51 mg
- Preparation: Since the test item was solid (which could not be sampled with a pipette), the required quantity was weighed before treatment and was stored at room temperature until application.
- Administration: A quantity of 51 mg +/- 1 mg was applied evenly to the surface of each tissue using a spoon, taking care to spread it over the whole tissue surface area without damaging the tissue sample.

For the negative and positive controls, since they could be sampled using a pipette, a volume of 50 μL was evenly applied to the surface of each tissue, taking care to spread them over the whole tissue surface area without damaging the tissue sample.

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

18 hours

Number of animals or in vitro replicates:

2 replicates

Details on study design:

- Doses of test chemical and control substances used: 51 mg of test item, 50 μL of sterile deinonized water for negative control, 50 μL methyl acetate for positive control.
- Main test:
One 6-well plate was used for the test item-treated tissues.
Positive and negative controls were placed on separate 6-well plates (one plate for each).
- Pre-incubation of the tissues:
The tissues were equilibrated (in the 24-well shipping container) to room temperature for at least 15 minutes. The underside of each tissue was inspected for air bubbles between the agarose gel and the insert. Tissues with air bubbles under the insert covering greater than 50% of the insert area were not used. Any unusual observation was noted separately. The plastic bag containing the 24-well plate shipping container was disinfected by wiping with 70% ethanol-soaked tissue paper. Each 24-well shipping container was then removed from its plastic bag under sterile conditions. A volume of 1 mL assay medium pre-warmed (+37°C) was added to 2 wells per pre-labeled 6-well plate. The tissues were carefully removed from the 24-well shipping containers using sterile forceps. Any agarose adhering to the inserts was removed by gentle blotting on sterile filter paper or gauze.
The insert was then transferred aseptically into the 6-well plate and pre-incubated at +37°C, 5% CO2 in a humidified incubator for 1 hour. After the pre-incubation period, the assay medium was removed and replaced by 1 mL of fresh assay medium before incubation overnight (16-24h) at +37°C, 5% CO2 in a humidified incubator.
- Treatment of tissues:
Following the pre-incubation period, the tissues were pre-wetted with 20 μL of D-PBS. If the D-PBS do not spread across the tissue, the plate was tapped to ensure that the entire tissue surface was wetted.
The tissues were then incubated at +37°C, 5% CO2 in a humidified incubator for 30 (± 2) minutes.
As the test item was solid, the insert was removed from the medium and placed onto a sterile surface (e.g. the lid of a microtiter plate) to avoid that test item was spilled into the assay medium under the tissue insert. The test item was then applied by pouring it onto the tissue surface so that the surface was completely covered by the test item. The test item, negative and positive controls were applied topically on each designated tissue, and gently spread onto the epithelium surfaces to ensure uniform covering of the tissues. Inserts were then tapped on the wall of the plate to ensure that the items were applied evenly to the surface of each tissue.
The tissues were placed back into the assay medium after treatment with the test item.
All tissues (test item, negative and positive controls) were incubated at +37°C, 5% CO2 in a humidified incubator for 6 hours (± 15 minutes).
- Rinsing of tissues:
At the end of the treatment period, each tissue was removed from the well of the treatment plate and rinsed to gently remove any residual test or control items. A set of three clean beakers containing a minimum of 100 mL each of D-PBS was used per test or control item. The tissues were rinsed two at time by holding replicate inserts together. The test or control items were firstly decanted from the tissue surface onto a clean absorbent paper. The tissues were then dipped into the first beaker of D-PBS, swirled in a circular motion during approximately 2 seconds, lifted out and decanted back into the beaker. This process was performed three times per beaker. Any remaining liquid was decanted onto an absorbent paper.
- Post-soak and post-incubation:
The rinsed tissues were transferred to new wells of a pre-labeled 12-well plate containing 5 mL of assay medium pre-warmed at room temperature. This incubation in assay medium was intended to remove any test article from the tissue.
Each tissue was incubated for 25 minutes (± 2 minutes) at room temperature to remove any solid test item or negative and positive controls from the tissue.
At the end of the post-soak immersion, each insert was blotted on absorbent material and transferred to appropriate well of the pre-labeled 6-well plate containing 1 mL of assay medium. The tissues were then incubated for 18 hours (± 15 minutes) at +37°C, 5% CO2 in a humidified incubator for test item, negative and positive controls.
- MTT viability assay:
Following the post-treatment incubation, a volume of 0.3 mL of a freshly prepared MTT solution (1.0 mg/mL) was added into new wells of pre-labeled 24-well plates.
At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent paper. The tissues were then transferred to the MTT pre-filled wells and incubated for 3 hours (± 10 minutes) at +37°C, 5% CO2 in a humidified incubator.
At the end of the 3-hour incubation period, the underside of each tissue was blotted on absorbent paper to dry. Each tissue was examined with the naked eye and the degree of MTT staining was evaluated.
For the negative and positive control-treated tissues, the inserts were transferred to new wells of the 24-well plate containing 2 mL of isopropanol per well so that isopropanol was flowing into the insert on the tissue surface.
As the test item was a non-colorant solid, the inserts were transferred to a 6-well plate containing 2 mL of isopropanol in each well so that no isopropanol was flowing into the insert. This avoided any potential contamination of the isopropanol solution with any test item that may have remained on the tissue.
Plates were surrounded with parafilm to prevent evaporation. Formazan extraction was performed overnight at +2-8°C and protected from light.
- Optical density measurements:
At the end of the formazan extraction period, plates were placed under orbital shaking at room temperature for 16 minutes prior using them. Then tissues (test item, negative and positive control-treated tissues) were not pierced.
The extract solution was mixed using a pipette and two 200 μL aliquots were transferred to the appropriate wells of a pre-labeled 96-well plate.
One 96-well plate was used for the negative and positive controls (placed at opposite end of the plate), and a separate 96-well plate was used for all test item-treated tissues For each 96-well plate, the average Optical Density value (OD) of 4 wells containing 200 μL of isopropanol only was used as the blank.
The OD was measured at 570 nm using a plate reader.
- Acceptance criteria:
The results of the study were considered acceptable if the following criteria are fully met:
* the mean cOD of the negative controls is between 0.8 and 2.5,
* relative mean viability of the positive control is < 50% of the relative mean viability of the negative control,
* the difference of viability between the two tissue replicate is < 20%.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

other: Optical density measurement

Run / experiment:

Mean of duplicate tissues

Value:

1.591

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

other: Relative viability in %

Run / experiment:

Mean of duplicate tissues

Value:

101

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

PRELIMINARY TESTS
- Test for direct MTT reduction with the test item:
The MTT solution containing the test item did not turn blue/purple when compared with the negative control. The test item was therefore considered not to have direct MTT reducing properties. As a result, no additional controls were performed on freeze-dead tissues in parallel to the main test.
- Test for the detection of the coloring potential of the test item:
During this test, as both water and isopropanol solutions containing the test item did not change color, the test item was found not to have a coloring potential. As a result, no additional controls were used in parallel to the main test.

MAIN TEST
- Evaluation of the coloration of tissues at the end of the MTT incubation period:
The qualitative evaluation of the MTT staining was performed with the naked eye and results are presented in Table 1 (Any other information on results incl. tables).
All test item-treated tissues appeared blue/white which was considered to be indicative of semi-viable tissue.
- Evaluation of the MTT results:
The individual and mean OD values, standard deviations and viabilities for the test item, negative control and positive control tissues are presented in Table 2 (Any other information on results incl. tables).
All of the acceptance criteria for the negative and positive controls were fulfilled (Table 2), therefore the study was considered to be valid.
The relative mean viability of the tissues treated with the test item was 101% with a difference of 2% between duplicate tissues.
As the mean viability was > 60% after the MTT reduction, the results met the criteria for a non-irritant response.

Any other information on results incl. tables

Table 1: Qualitative assessment of tissue viability

 

Treatment	Tissue 1	Tissue 2
Negative control	B	B
Positive control	B/W b	B/W b
Test item	B/W	B/W

B: blue discolouration of the tissue

B/W: blue/white discolouration of the tissue

b: blue colouration at the periphery of tissues and white discolouration at the center of tissues

 

Table 2:

*A: Individual and mean corrected OD values and tissue viabilities for the test item, the negative and positive controls

*B: Mean tissue viability and standard deviations for the test item, the negative and positive controls

*A

Group	Exposure duration	Tissue No.	OD570 nmmeasurements		Mean blank	cOD570 nmmeasurements		Mean cOD570 nm	Viability (%)
			1^st	2^nd		1^st	2^nd
Negative control	6h	1	1.576	1.570	0.041	1.535	1.529	1.532	98
		2	1.651	1.639		1.610	1.598	1.604	102
Positive control	6h	1	0.390	0.388	0.041	0.349	0.347	0.348	22
		2	0.353	0.354		0.312	0.313	0.312	20
Test item	6h	1	1.610	1.615	0.042	1.569	1.574	1.571	100
		2	1.649	1.654		1.608	1.613	1.610	103

OD = optical density

cOD = blank corrected optical density

*B

Group	Exposure duration	cOD570 nm		Viability (%)
		Mean	SD	Mean	SD	Difference (%)
Negative control	6h	1.568	0.051	100	3	5
Positive control	6h	0.330	0.025	21	2	2
Test item	6h	1.591	0.028	101	2	2

cOD = blank corrected optical density

SD = standard deviation

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this study, the test item, Tris[oxalato(2-)]dilutetium, is considered to be non-irritant to Reconstructed human Cornea-like Epithelium.
According to the results of this study, the classification of the test item should be:
. No Category (GHS 2015 and Regulation (EC) No. 1272/2008).

Executive summary:

The purpose of this study was to predict the acute eye irritation potential of the test item, Tris[oxalato(2-)]dilutetium, by measurement of its cytotoxic effect on the EpiOcularTMcornea epithelial model.

The study was conducted in compliance with CiToxLAB France standard operating procedures and the principles of Good Laboratory Practice.

 

Methods

Preliminary tests were performed to detect the ability of the test item to directly reduce MTT as well as its coloring potential.

Following the preliminary tests, the eye irritation potential of the test item was assessed in the main test.

The test item and both negative and positive controls were applied topically on duplicate tissues and incubated at +37°C for 6 hours. At the end of the treatment period, each tissue was rinsed with D-PBS, incubated for 25 minutes at room temperature to remove any remaining test item from the tissue, blotted on absorbent material, and then incubated for another 18 hours at 37°C, 5% CO2in a humidified incubator.

The cell viability was then assessed by means of the colorimetric MTT reduction assay.

Mean viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (reference viability).

Results

Preliminary tests

In the preliminary tests, the test item was found not to have direct MTT reducing properties or coloring potential.

 

Main test

All acceptance criteria for the negative and positive controls were fulfilled. The study was therefore considered to be valid.

 

The relative mean viability of the tissues treated with the test item was 101% with a difference of 2% between duplicate tissues. As the mean viability was > 60% after the MTT reduction, the results met the criteria for a non-irritant response.

 

Conclusion

Under the experimental conditions of this study, the test item, Tris[oxalato(2-)]dilutetium, is considered to be non-irritant to Reconstructed human Cornea-like Epithelium.

According to the results of this study, the classification of the test item should be:

*No Category (GHS 2015 and Regulation (EC) No. 1272/2008).