Cerium(3+) acetate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 December 2016 to 20 December 2016

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 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Cerium(3+) acetate

EC Number:

208-654-0

EC Name:

Cerium(3+) acetate

Cas Number:

537-00-8

Molecular formula:

C2H4O2.1/3Ce

IUPAC Name:

cerium(3+) triacetate

Test material form:

solid: granular

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: CEACK1/16
- Expiration date of the lot/batch: 17 October 2018

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

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Recommended in international guidelines

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ Reconstructed Human Epidermis Model Kit, supplied by SkinEthic Laboratories, Lyon, France.
- Tissue lot number: 16-EKIN-050
- Delivery date: 14 December 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature
- Temperature of post-treatment incubation: 37°C

TEST FOR DIRECT MTT REDUCTION
A test material may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, the test material is checked for the ability to directly reduce MTT according to the following procedure: 10 mg of the test material was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37°C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control. If the MTT solution containing the test material turns blue or purple, the test material is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and water killed tissues for quantitative correction of the results.

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. 10 mg of test material was added to 90 µL of sterile water. After mixing for 15 minutes on a plate shaker a visual assessment of the colour was made.

MAIN TEST

PRE-INCUBATION: DAY 0
Before removal from the transport plate each tissue was inspected for: any air bubbles between the agarose gel and the insert, if tissues are satisfactory, if the temperature indicator colour is satisfactory and if the agar medium colour is satisfactory. 2 mL of maintenance medium, warmed to approximately 37°C, was pipetted into the first column of 3 wells of a pre labelled 12 well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test material and each control material. The tissues were incubated at 37°C, 5 % CO2 in air overnight.

APPLICATION OF TEST MATERIAL: DAY 1
2 mL of maintenance medium, warmed to approximately 37°C, was pipetted into the second column of 3 wells of the 12 well plate. Triplicate tissues were treated with the test material for an exposure period of 15 minutes. The test material was applied topically to the corresponding tissues ensuring uniform covering. 5 µL of sterile distilled water was topically applied to the epidermal surface in order to improve contact between the test material and the epidermis. Approximately 10 mg (26.3 mg/cm²) of the test material was then applied to the epidermal surface. Triplicate tissues treated with 10 µL of DPBS served as the negative controls and triplicate tissues treated with 10 µL of SDS 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control material the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the center). After a 7 Minute contact time the SDS solution was re spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period (re-spreading is not required for the negative control or test material). The plates were kept in the biological safety cabinet at room temperature for 15 minutes.

REMOVAL OF TEST MATERIAL AND CONTROLS
At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca²+ and Mg²+. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test material. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37°C, 5% CO2 in air for 42 hours.

MTT LOADING/ FORMAZAN EXTRACTION: DAY 3
Following the 42 hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenise the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre labelled micro tubes and stored in a freezer at 14 to 30°C for possible inflammatory mediator determination.
2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates. The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37°C, 5% CO2 in air. At the end of the 3 hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKIN™ biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labelled 1.5 mL micro tubes containing 500 µL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10°C until day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.

ABSORBANCE/ OPTICAL DENSITY MEASUREMENTS: DAY 6
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous coloured solution. For each tissue, duplicate 200 µL samples were transferred to the appropriate wells of a pre labelled 96 well plate. 200 µL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 562 nm (without a reference filter) using the Anthos 2001 microplate reader.

NUMBER OF REPLICATE TISSUES: 3

DATA EVALUATION
Quantitative MTT Assessment (Percentage Tissue Viability)
For the test material the relative mean tissue viabilities obtained after the 15 minute exposure period followed by the 42 hour post exposure incubation period were compared to the mean of the negative control treated tissues (n=3). The relative mean viabilities were calculated in the following way:
Relative mean viability (%) = (mean OD562 of test material / mean OD562 of negative control) x100
Classification criteria for in vitro interpretation:
Relative mean tissue viability is ≤ 50%: Irritant,
Relative mean tissue viability is > 50%: Non-irritant.

QUALITY CRITERIA
- The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues is ≤ 40% relative to the negative control treated tissues, and the standard deviation (SD) value of the percentage viability is ≤ 18 %.
- The assay establishes the acceptance criterion for an acceptable test if the mean OD562 for the negative control treated tissues is ≥ 0.6 and ≤ 1.5, and the SD value of the percentage viability is ≤ 18%.
- The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues is ≤ 18%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 10 mg (26.3 mg/cm²)

NEGATIVE CONTROL
- Amount(s) applied: 10 µL

POSITIVE CONTROL
- Amount applied: 10 µL
- Concentration: 5 % w/v

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

96.5

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:

DIRECT MTT REDUCTION
The MTT solution containing the test material did not turn blue or purple which indicated that the test material did not directly reduce MTT.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
The solution containing the test material was a white colour. This colour was attributed to the intrinsic colour of the test material itself. It was therefore unnecessary to run colour correction tissues.

MAIN STUDY
A summary of the results from the study is given in Table 1.
The relative mean viability of the test material treated tissues was 96.5% after a 15 minute exposure period and 42 hour post exposure incubation period. It was considered unnecessary to perform IL-1α analysis as the results of the MTT test were unequivocal.

QUALITY CRITERIA
- The relative mean tissue viability for the positive control treated tissues was 8.1% relative to the negative control treated tissues and the standard deviation value of the viability was 1.9%. The positive control acceptance criteria were therefore satisfied.
- The mean OD562 for the negative control treated tissues was 0.714 and the standard deviation value of the viability was 1.0%. The negative control acceptance criteria were therefore satisfied.
- The standard deviation calculated from individual tissue viabilities of the three identically test material treated tissues was 11.9%. The test material acceptance criterion was therefore satisfied.

Any other information on results incl. tables

Table 1: Summary of results

Item	OD562of tissues	Mean OD562of triplicate tissues	± SD of OD562	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control	0.706	0.714	0.007	98.9	100*	1.0
	0.720			100.8
	0.715			100.1
Positive Control	0.042	0.058	0.014	5.9	8.1	1.9
	0.065			9.1
	0.067			9.4
Test Material	0.623	0.689	0.085	87.3	96.5	11.9
	0.785			109.9
	0.658			92.2

OD = Optical density

SD = Standard deviation

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

Applicant's summary and conclusion

Interpretation of results:

other: Not classified in accordance with EU criteria

Conclusions:

Under the conditions of this study, the test material is not classified as a skin irritant.

Executive summary:

The potential of the test material to cause skin irritation was investigated in accordance with the standardised guidelines OECD 439 and EU Method B.46, using a human skin model under GLP conditions.

During the study triplicate tissues were treated with the test material for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre labelled 96 well plate. The optical density was measured at 562 nm.

The relative mean viability of the test material treated tissues was 96.5% after the 15 minute exposure period and 42 hours post exposure incubation period. The quality criteria required for acceptance of results in the test were satisfied.

Under the conditions of this study, the test material is not classified as a skin irritant.