Neodymium(3+) acetate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 January 2017 to 10 February 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU Method B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test"

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Neodymium(3+) acetate

EC Number:

228-244-5

EC Name:

Neodymium(3+) acetate

Cas Number:

6192-13-8

Molecular formula:

C2H4O2.1/3Nd

IUPAC Name:

neodymium(3+) triacetate

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: pink powder

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark, over silica gel

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: the test material was used as supplied

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

This model incorporates several features, which make it advantageous in the study of potential dermal corrosivity. The target cells are epithelial, derived from human skin, and formed into a stratified, cornified epithelium. Test materials are applied to the culture surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ (MatTek)
- Tissue lot number(s): 23392
- Delivery date: 08 February 2017, stored refrigerated until use.
- Medium lot number: 020217TMA

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: A constant soft stream of DPBS was used to remove the test material, excess DPBS was removed by blotting the insert with tissue paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL

TEST FOR DIRECT MTT REDUCTION
A test material may interfere with the MTT endpoint if it was able to directly reduce MTT and at the same time was present on or in the tissues when the MTT viability test was performed. To identify this possible interference, the test material was checked for the ability to directly reduce MTT according to the procedure below:
25 mg of the test material was added to 1 mL of a freshly prepared 1.0 mg/mL MTT solution. The solution was incubated in the dark at 37°C, 5% CO2 in air for 60 minutes. Untreated MTT solution was tested concurrently to act as a control. If the MTT solution containing the test material turns blue/purple relative to the control, the test material was presumed to have reduced the MTT.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
A test material may interfere with the MTT endpoint if it is coloured. The MTT assay is affected only if the test material is present in the tissues when the MTT viability assay is performed.
25 mg of test material was added to 300 μL of sterile water. The solution was incubated in the dark at 37°C, 5% CO2 in air for 60 minutes. A visual assessment of the colour was then made.
The test material produced a coloured solution. There was a possibility that if the test material could not be totally rinsed off the tissues, any residual test material present on or in the tissue may interfere with the MTT endpoint and could have given rise to a false negative result. Therefore, the determination of skin corrosion potential was performed in parallel on colour correction tissue groups.
In addition to the normal test procedure, the coloured test material was applied to two tissues per exposure period. In addition, two tissues per exposure period were treated with the negative control. These colour correction tissues were handled identically to the procedures of the main test with the exception of the MTT incubation being replaced by an incubation in assay medium under the same incubation conditions.

PRE-INCUBATION
The assay medium was pre-warmed before use, 0.9 mL was pipetted into the appropriate wells of two pre-labeled 6-well plates for both the 3-Minute and 60-minute exposure periods. EpiDerm™ tissues were transferred into the 6-well plates containing the assay medium. The 6-well plates containing the EpiDerm™ samples were pre-incubated (37°C, 5% CO2) for approximately 1 hour before dosing.

APPLICATION OF THE TEST MATERIAL AND RINSING
Before pre-incubation was complete, a 24-well plate was prepared for use as a “holding plate” for both the 3-minute and 60-minute exposure periods. This plate was used to maintain the viability of the tissue inserts between rinsing following chemical exposure and MTT loading. Another 24-well plate was prepared for the MTT loading. 300 μL of either pre-warmed assay medium (holding plate) or MTT medium (MTT loading plate) was dispensed into each well. The two plates were placed into the incubator until required.
After pre-incubation of the EpiDerm™ tissues, the medium was aspirated and replaced with 0.9 mL of fresh assay medium. The 6-well plate for the 3-minute exposure period was returned to the incubator, while the other was being dosed for the 60-Minute exposure. For the 60-Minute exposure period, 50 μL of sterile distilled water (negative control) was added to the first two tissues. The tissues were dosed at regular intervals to allow for the time taken to rinse each tissue following exposure and to ensure that each tissue gets an equal exposure time. 25 mg of the test material and 50 μL of 8.0 N Potassium Hydroxide (positive control) were also applied to the corresponding tissues in turn. 25 μL of sterile water was added for wetting of the test material to increase tissue surface contact. The plate was returned to the incubator (37°C, 5% CO2) for the 60-minute exposure period.
When dosing for the 60-minute exposure period was complete, the same procedure was repeated for the 3-minute exposure period. Because the exposure time was so short, the tissues were dosed at regular intervals to ensure that each tissue received an equal exposure time and to allow for the time taken to rinse each tissue following exposure. Rinsing was achieved by filling and emptying each tissue under a constant soft stream of DPBS to gently remove any residual test material. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper. Each tissue was placed into the prepared holding plate until all tissues were rinsed. They were then blotted and transferred to the 24-well plate prepared for MTT loading. The plate was incubated (37°C, 5% CO2) for 3 hours. Once the 60-minute exposure period was complete, the same rinsing and MTT loading procedure was repeated.
After the 3-hour MTT incubation was complete, the inserts were blotted and transferred to labelled 24-well plates for MTT extraction. 2 mL of MTT extractant (isopropanol) was used to completely immerse each insert and the plate was covered with plate sealer to prevent Isopropanol evaporation. The plates stood overnight at room temperature, to allow extraction to proceed.

ABSORBANCE/ OPTICAL DENSITY MEASUREMENTS
After extraction, each tissue was pierced with a pipette fitted with a 1000 μL tip and the extraction solution was forced vigorously up and down to form a homogenous solution. 3 x 200 μL aliquots of the extract were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 μL of isopropanol alone was added to the three wells designated as blanks. Absorbency at 562nm (OD562) of each well was measured using the Anthos 2001 microplate reader.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 25 mg
- 25 µL of sterile water was added for wetting of the test material to increase tissue surface contact.

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL
- Concentration (if solution): 8.0 N

Duration of treatment / exposure:

3 minutes of exposure and 1 hour of exposure

Duration of post-treatment incubation (if applicable):

Incubated for 3 hours with MTT

Number of replicates:

The test was performed in duplicate

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure

Value:

89.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minute exposure

Value:

84.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

DIRECT MTT REDUCTION
The MTT solution containing the test material did not turn blue/purple. This was taken to indicate the test material did not reduce MTT.

ASSESSMENT OF COLOUR WITH THE MTT ENDPOINT
The solution containing the test material was a very pale blue colour. Therefore, an additional procedure using colour correction tissues was performed during the determination of skin corrosion potential. However, the results obtained showed a negligible degree of colour interference occurred. It was therefore considered unnecessary to use the results of the colour correction tissues for quantitative correction of results or for reporting purposes.

ACCEPTANCE OF RESULTS:
- The mean OD562 for the negative control treated tissues was 1.648 for the 3-minute exposure period and 1.633 for the 60-minute exposure period. The negative control acceptance criteria were therefore satisfied.
- The relative mean tissue viability for the positive control treated tissues was 4.1% relative to the negative control following the 60-minute exposure period. The positive control acceptance criterion was therefore satisfied.
- In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

Any other information on results incl. tables

Table 1: Summary of results

Exposure period	Percentage viability
	Negative control	Positive control	Test material
3 minutes	100*	3.9	89.1
60 minutes	100*	4.1	84.5

*The mean viability of the negative control tissues is set at 100%

Applicant's summary and conclusion

Interpretation of results:

other: Not corrosive in accordance with EU criteria

Conclusions:

Under the conditions of this study, the test material is not corrosive in the in vitro skin corrosion test.

Executive summary:

An in vitro skin corrosion test was carried out with the test material using a human skin model in accordance with the standardised guidelines OECD 431 and EU Method B.40 BIS under GLP conditions.

During the study, duplicate tissues were treated with the test material for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. The test material was found to have the potential to cause colour interference with the MTT endpoint therefore additional tissue groups were incorporated into the testing to correct for this. At the end of the exposure period the test material was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction.

At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density (OD) was measured at 562 nm (OD562).

The quality criteria required for acceptance of results in the test were satisfied.

After treatment with the test material the percentage viabilities of the tissues were 89.1 and 84.5% after 3 and 60 minutes exposure, respectively.

Under the conditions of this study, the test material is not corrosive in the in vitro skin corrosion test.