Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Skin irritation/corrosion

The test item was demonstrated to be non-irritant in an in vitro study performed according to OECD guideline 439 (Reconstructed Human Epidermis Test method, EPISKIN) and in agreement with GLP requirements ( Balázs Orovecz, 2017). This study was considered reliable without restrictions (Klimisch 1) and is considered as the key study for endpoint coverage.

Furthermore, the pH value and the acidic/alkaline reserve was determinated by the registrant on the test substance. The pH value of a 10% solution of Lutetium oxalate was determined to be 5.04 and the alkaline reserve 0.05. Based on these data, the calculation for corrosivity was 5.04 (out of the tresholds for corrosivity <= -0.5 for acid and >=14.5 for alkaline). Therefore the substance was considered not to be corrosive.

 

Eye irritation

The test item was demonstrated to be non-irritant to eyes in an in vitro study performed according to OECD guideline 492 (Reconstructed Human Cornea-like Epithelium test method, EpiOcular study) and in agreement with GLP requirements (Richez, 2017). This study was considered reliable without restrictions (Klimisch 1) and is considered as the key study for endpoint coverage.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation / corrosion, other
Remarks:
Determination of pH-value and acidic reserve
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Report date: 20 July 2016
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
Electrochemic determination of pH
Determination of the acidic reserve via titration
GLP compliance:
no
Remarks:
Internal data
Details on study design:
Purpose:
The higher the buffer capacity of a mixture (solution or slurry) is, the stronger the irritating and corrosive potential is, respectively. The physiological effects of acidic or alkaline solutions are not only determined by the pH value but also by the buffering capacity. That is why it is necessary to determine the alkaline or acidic reserve of identified products.

Method:
First, the pH-value of a 10% solution (or slurry) is determined electrochemically at 20°C with a calibrated pH meter (MA 235 Mettler Toledo). This value is recorded.

Determination of the acidic reserve titration:
- For the determination of the alkaline reserve: the titration volume (in mL) of a 0.5 mol/L H2SO4 solution, which is required to achieve a pH of 10 of a 10% solution (or slurry) at 20°C, is recorded.
- For the determination of the acid reserve: the titration volume (in mL) of a 1 mol/L NaOH solution, which is required to achieve a pH of 4 of a 10% solution (or slurry) at 20°C, is recorded.

Calculations:
Titration of the 10% solution/slurry
Alkaline or acid reserve = titration volume [mL] x 0.4

A product is classified as corrosive if:
pH + 1/12 alkaline reserve >= 14.5
pH - 1/12 acid reserve <= -0.5
Irritation parameter:
other: acid/alcaline reserve
Basis:
other: acid reserve
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
Not corrosive (based on an alkaline reserve of 0.05 and pH of 5.04, the calculation for corrosivity was 5.04 which is higher than -0.5).
Interpretation of results:
other: not corrosive
Conclusions:
The pH value of a 10% solution of Lutetium oxalate was determined to be 5.04 and the alkaline reserve 0.05. Based on these data, the calculation for corrosivity was 5.04 (> -0.5). Therefore the substance was considered not to be corrosive.
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 20 March 2017 to 19 July 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP compliance
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
22 September 2015
Specific details on test material used for the study:
No correction factor is applied for this type of study
Test system:
human skin model
Source species:
human
Cell type:
other: Adult human-derived epidermal keratinocytes
Cell source:
other: three-dimensional human epidermis model
Justification for test system used:
The EPISKINTM (SM) model has been validated for irritation testing in an international validation study and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: three-dimensional human epidermis model
- Tissue batch number(s): 17-EKIN-012
- Expiration date: 27 March 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature of pre-incubation: 37°C
- Temperature used during treatment / exposure: 15 minutes at room temperature (26.0 - 27.1°C)
- Temperature of post-treatment incubation: 37°C

APPLICATION
- Test item: As the test item was solid, first an appropriate amount (10 µL) distilled water was applied to the epidermal surface in order to improve further contact between test item and epidermis and then 20 mg of the test item was applied evenly to the epidermal surface. If necessary, the test item was spread gently on the skin surface with a pipette tip (or other appropriate tool) without damaging the epidermis. The amount was sufficient to cover the epidermal surface.
- Negative and positive controls: 50 µL of negative control (PBS) or positive control (5% (w/v) SDS solution) were added to each skin unit by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all
the epidermal surface if necessary (without damaging the epidermis).
- Additional control for test item having colouring potential: For potentially colouring test item, in addition to the normal procedure, two additional viable chemical-treated tissues were used for the non specific OD evaluation. These tissues followed the same treatment steps as the other tissues except for the MTT step: MTT incubation step was replaced by incubation with fresh Assay Medium. OD reading were made following the same conditions as the other tissues.

The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min).

REMOVAL OF TEST MATERIAL
After the 15 minutes incubation time, the EPISKINTM(SM) units were removed and rinsed thoroughly with PBS to remove any remaining material from the epidermal surface as much as possible. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis).
After rinsing the units were placed into the plate wells with fresh pre-warmed Maintenance Medium (2 mL/well) below them and then incubated for 42 hours (± 1h) at 37°C in an incubator with 5% CO2, in a > 95% humidifed atmosphere.

MTT TEST
After the 42 hours incubation, all EPISKINTM(SM) units (except of two colour control units) were transferred into the MTT working solution filled wells (2 mL of 0.3 mg/mL MTT per well). Then, all transferred EPISKINTM(SM) units were incubated for 3 hours (± 5 min) at 37°C in an incubator with 5% CO2, in a > 95% humidified atmosphere, protected from light.

FORMAZAN EXTRACTION
After the incubation with MTT, a formazan extraction was undertaken. A disk of epidermis was cut from each skin unit (this involved the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube containing 500 μL acidified isopropanol (one tube corresponded to one well of the assay plate).
The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material and the acidified isopropanol, and then incubated for about two hours at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.
A blank sample containing 2 mL of acidified isopropanol was processed in parallel.

CELL VIABILITY MEASUREMENTS
Following the formazan extraction, 2×200 μL sample from each tube were placed into the wells of a 96-well plate (labelled appropriately). The OD (optical density or absorbance) of the samples was measured using a plate reader at 570 nm. The mean of 6 wells of acidified isopropanol solution (200 μL/well) was used as blank.

NUMBER OF REPLICATE TISSUES: 3

PREDICTION MODEL / DECISION CRITERIA
In the present study, the irritancy potential of test substances is predicted by the mean tissue viability of tissues exposed to the test item. The test item considered to be irritant to skin (Category 2), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control.
Mean tissue viability % is ≤ 50%: Category 2 or Category 1
Mean tissue viability % is > 50%: Non-Irritant

VALIDITY OF THE TEST
The mean OD value of the three negative control tissues should be between 0.6 and 1.5, and the stand ard deviation value (SD) of the % viability values should be ≤ 18.
The acceptable mean percentage viability range for positive controls is 0-40% and the standard deviation value (SD) of the % viability values should be ≤ 18.
The SD calculated from individual % tissue viability values of the three test item treated replicates should be <18.
The mean OD value of the blank samples (acidified isopropanol) should be <0.1.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST ITEM
first an appropriate amount (10 μL) and then ~64.8 mg of the test item
NEGATIVE AND POSITIVE CONTROLS
50 μL of negative control (PBS) or positive control (5% (w/v) SDS solution)
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3 tissues
Irritation / corrosion parameter:
% tissue viability
Value:
107.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
ADDITIONAL CONTROLS
As no colour change (yellow colour) was observed after three hours of incubation of the test item in MTT working solution, the test materials did not interact with MTT and additional controls and data calculations were not necessary. The false estimation of viability can be excluded.

As the test item was coloured, two additional test item-treated tissues were used for the non-specific OD evaluation. The optical density (measured at 570 nm) of tissues were 0.014, Non-specific Colour % was calculated as 2.2%. This value was below 5%, therefore additional data calculation was not necessary.

VIABILITY RESULTS
The results of the optical density (OD) measured at 570 nm of each sample and the calculated relative viability % values are presented in Table 1 below (Any other information on results incl. tables). The OD values for the test item treated skin samples showed 107.7% relative viability.

VALIDITY OF THE TEST
After receipt, the two indicators of the delivered kit were checked. Based on the observed colours, the epidermis units were in proper conditions.

The mean OD value of the three negative control tissues was in the recommended range (0.668). Standard deviation of the viability results for negative control samples was 3.0.
The positive control treated tissues showed 7.0% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 1.6.
The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 4.9.
The mean OD value of the blank samples (acidified isopropanol) was 0.046.
All these parameters met the acceptability criteria, therefore the study was considered to be valid.

Table 1: Optical Density (OD) and the calculated relative viability % of the samples

 

Substance

Optical Density (OD)

Viability (% RV)

 

Measured

Blank corrected

Negative Control:

1

0.705

0.659

98.6

Phosphate buffered saline

2

0.738

0.691

103.5

 

3

0.701

0.654

97.9

 

mean

-

0.668

100.0

Positive Control:

1

0.100

0.054

8.0

5% (w/v) SDS* solution

2

0.098

0.052

7.7

 

3

0.081

0.034

5.1

 

mean

-

0.047

7.0

Test Item:

1

0.800

0.753

112.7

tris[oxalate(2-)]dilutetium

2

0.735

0.688

103.0

 

3

0.764

0.717

107.4

 

mean

-

0.720

107.7

* SDS: Sodium Dodecyl Sulphate

Notes:

1. Mean blank value was 0.046.

2. Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

 

Interpretation of results:
GHS criteria not met
Conclusions:
Following exposure with tris[oxalate(2-)]dilutetium, the mean cell viability was 107.7% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered to be non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.
In conclusion, in this in vitro EPISKINTM(SM) model test with tris[oxalate(2-)]dilutetium (Batch number: 1534498), the results indicate that the test item is non-irritant to skin.
Executive summary:

An in vitro skin irritation test of tris[oxalate(2-)]dilutetium test item was performed in a reconstructed human epidermis model. EPISKINTM (SM) is designed to predict and classify the irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The irritation potential of the test item was evaluated according to the OECD No. 439 guideline.

 

Disks of EPISKINTM (SM) (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered saline (PBS). The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2, in a >95% humidified atmosphere.

The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2 protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

 

PBS and 5% (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units / control). Two additional disks were used to provide an estimate of colour contribution (NSCliving) from the test item. For each treated tissue, the viability was expressed as a % relative compared to the negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negativecontrol, the test item is considered to be irritant to skin.

 

Following exposure with tris[oxalate(2-)]dilutetium, the mean cell viability was 107.7% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered to be non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

 

In conclusion, in thisin vitroEPISKINTM(SM) model test with tris[oxalate(2-)]dilutetium (Batch number: 1534498), the results indicate that the test item is non-irritant to skin.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
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
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
Specific details on test material used for the study:
No correction factor is applied for this type of study
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.
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%.
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
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.

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 (%)

1st

2nd

1st

2nd

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

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).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation/corrosion

One reliable (Klimisch 1) in vitro study is available (Balázs Orovescz, 2017). This study was performed according to OECD guideline 439 (Reconstructed Human Epidermis Test method, EPISKIN) and in compliance with GLP requirements. The mean % tissue viability in the three tissue replicates exposed to the test item was 107.7%. A test item is considered to be non-irritant to skin if the mean relative viability of three individual tissues after 15 minutes exposure to the test item and 42 hours post-incubation is more than 50% of the mean viability of the negative controls. Since the mean viability in the negative controls was 100%, the test item was concluded to be non-irritant under the conditions of the study. Based on these results, the test item is considered not classified according to the CLP Regulation. The study of Balázs Orovecz (2017) is considered as the key study for endpoint coverage.

Furthermore, the pH value and the acidic/alkaline reserve was determinated by the registrant on the test substance.The pH value of a 10% solution of Lutetium oxalate was determined to be 5.04 and the alkaline reserve 0.05. Based on these data, the calculation for corrosivity was 5.04 (out of the tresholds for corrosivity <= -0.5 for acid and >=14.5 for alkaline). Therefore the substance was considered not to be corrosive.

 

Eye irritation

One reliable (Klimisch 1) in vitro studies is available (Richez, 2017). The test substance was tested for its eye irritation potential in a Reconstructed Human Cornea-like Epithelium assay according to OECD guideline 492 and in agrrement with GLP requirements (Richez, 2017). Since the mean viability in the treated tissues after MTT reduction was 101%, which is higher than the cut-off level of 60%, the test results met the criteria for a non-irritant response. Therefore, the test substance could be concluded not to be classified for eye irritation under the CLP Regulation. The study of Richez (2017) is considered as the key study for endpoint coverage.

Justification for classification or non-classification

Skin irritation/corrosion

The test item was demonstrated not to be irritant to skin in an in vitro study performed according to OECD guideline 439 and is therefore not to be classified according to the CLP regulation.

Eye irritation

The test item was demonstrated not to be irritant to eyes in an in vitro study performed according to OECD guideline 492 and is therefore not to be classified according to the CLP Regulation.