Tetrakis(pentane-2,4-dionato-O,O')zirconium

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 Feb 2019 - 08 Feb 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

29 July 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Tetrakis(pentane-2,4-dionato-O,O')zirconium

EC Number:

241-510-5

EC Name:

Tetrakis(pentane-2,4-dionato-O,O')zirconium

Cas Number:

17501-44-9

Molecular formula:

C20H28O8Zr

IUPAC Name:

2,4,8,8,10,10,13,15-octamethyl-1λ³,5λ³,7λ³,7λ³,11λ³,11λ³,12λ³,16λ³-octaoxa-6-zirconadispiro[5.5⁶.5⁶.5⁶]henicosa-1,4,7,7,10,10,12,15-octaene-6,6,6,6-tetrakis(ylium)-3,9,9,14-tetraide

Test material form:

solid: particulate/powder

Details on test material:

- Identification: Zirconium (IV) acetylacetonate (Zr-acac)
- Description: White to off-white powder
- Storage conditions: At room temperature

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Justification for test system used:

Recommended test system in international guidelines (OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model
- Tissue batch number(s): 29944
- Surface: 0.6 cm^2
- On the day of receipt the tissues were kept on agarose and stored in the refrigerator.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 minute exposure: room temperature, 1 hour exposure: 37.2 - 37.5°C.
- Temperature of post-treatment incubation: 37°C.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: the tissues were washed with phosphate buffered saline (one washing step)
- Observable damage in the tissue due to washing: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE: the test substance was checked for possible color interference before the study was started.
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm
- The test item was checked for interference with the MTT endpoint before the start of the study.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Freeze-killed tissues: EPI-200, Lot no.: 29404
- Procedure used to prepare the killed tissues: Living epidermis was transferred to a freezer (≤-15°C), thawed, and then again transferred to (≤-15°C). The freeze-killed epidermis was stored at ≤ -15°C until use. Freeze-killed tissues were thawed by placing them for 1 hour at room temperature in a 6 well plate on 0.9 mL DMEM.
- N. of replicates: 2 treated with test item, 2 treated with the negative control
- Method of calculation used: Nonspecific MTT reduction was calculated as the difference between the mean OD of the untreated killed tissues and test item treated killed tissues expressed as percentage of the mean of the negative control tissues. True tissue viability is calculated as the difference between the living test item treated tissues incubated with MTT medium and the difference between the mean OD of the untreated killed tissues and the mean OD of the negative control tissues.

OD= ODlt_t+MTT – (ODkt_t+MTT-ODkt_u+MTT)
%Viability = [OD/ mean ODlt_u+MTT] * 100

NUMBER OF REPLICATE TISSUES: 4 (2 tissues for the 3 minute exposure period, 2 tissues for the 1 hour exposure period) + 2 tissues for the negative and the positive control for each exposure period

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
A test item is considered corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test item considered non-corrosive (viability ≥ 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test item is decreased below 15%.

A test item is considered non corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.

ACCEPTABILITY CRITERIA:
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8)
b) The mean relative tissue viability following 1-hour exposure to the positive control should be <15 %.
c) In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be ≤ 30%.
d) The non-specific MTT reduction should be ≤30% relative to the negative control OD.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

other: concurrent MTT reduction control

Amount/concentration applied:

Test material: 24.8 to 36.1 mg
Negative control: 50 μL
Positive control: 50 μL

Duration of treatment / exposure:

3 minutes and 1 hour

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

4 in total; 2 per exposure period

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute treatment

Value:

101

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

Mean tissue viability: 29

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour treatment

Value:

95

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

Mean tissue viability: 13

Other effects / acceptance of results:

RESULTS WITH KILLED TISSUES
The non-specific reduction of MTT by the test substance was -10% and -1.2% of the negative control tissues after 3 minutes and 1 hour respectively. Since the %NSMTT ≤ 0.0, there was no need to correct for interference of the test item.

- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: Yes
- Colour interference with MTT: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the absolute mean OD570 was between the acceptance limits (i.e. 1.444 for the 3-minute exposure and 1.428 for the 1-hour exposure).
- Acceptance criteria met for positive control: yes, the mean relative tissue viability following 1 hour exposure was <15% (i.e., 13%).
- Acceptance criteria met for variability between replicate measurements: yes, CV between tissue replicates was ≤30% (i.e., 12% for the 3 minute exposure period and 5.9% for the 1 hour exposure period).
- Acceptance criteria met for non-specific reduction of MTT: yes, the NSMTT was <30% of the negative control tissues

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Not classified according to Regulation (EC) No. 1272/2008

Conclusions:

The results of a skin corrosion test, performed according to OECD guideline 431, showed that Zirconium (IV) acetylacetonate (ZR acac) was not corrosive to the skin. The test substance is therefore not classified according to GHS and according to Regulation (EC) 1272/2008.

Executive summary:

A skin corrosion test was performed, according to OECD guideline 431, to assess the potential of Zirconium (IV) acetylacetonate (ZR acac) to induce skin corrosion on a human three dimensional epidermal model. The test item was applied at an amount of >25 mg directly on top of an EpiDerm model for a 3 -minute and a 1 -hour exposure period. For each exposure period two tissues were treated with the test item, two tissues were treated with a positive control (potassium hydroxide) and two tissues were treated with Milli-Q water as a negative control. After exposure, the tissues were incubated for 3 hours in MTT-medium and subsequently the cell viability was measured at 570 nm using a spectrophotometer. The mean cell viability for the tissues treated with test item compared to the negative control were 101 and 95% for the 3 -minute and the 1 -hour exposure period, respectively. The mean tissue viability for the positive control after 1 -hour exposure was 13%. All acceptability criteria were met and the study was considered to be valid. Since the mean tissue viability was >50% after the 3 -minute exposure and >15% after the 1 -hour exposure, Zirconium (IV) acetylacetonate (ZR acac) is considered not corrosive to the skin. The test substance is therefore not classified for skin corrosion according to GHS and according to Regulation (EC) 1272/2008.