Magnesium fluoride

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 7, 2016 - October 28, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Magnesium fluoride

EC Number:

231-995-1

EC Name:

Magnesium fluoride

Cas Number:

7783-40-6

Molecular formula:

F2Mg

IUPAC Name:

magnesium difluoride

Test material form:

solid: crystalline

Details on test material:

- median particle size 873 um

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Materion / lot 662599-1
- Expiration date of the lot/batch: 31-Jul 2017
- Purity test date: 18- Jan-2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: RT, away from moisture
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: low solubility
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not expected

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: 25 mg of test item were applied topically onto the tissue surface (after wetting by 25 uL sterile DPBS) by spoon, for 60 minutes.

FORM AS APPLIED IN THE TEST (if different from that of starting material) liquid or suspension

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ (EPI-200-SIT)
- Tissue batch number(s): Lot No. 23367
- Delivery date:October 27, 2016
- Date of initiation of testing: October 28, 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: incubator 37±1°C
- Temperature of post-treatment incubation (if applicable): incubator 37±1°C, 5±1% CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 15 times with DPBS
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1mg/ml
- Incubation time: 3 h
- Spectrophotometer: spectrophotometer MRX II (Dynex) Sr. No 1CXD3229
- Wavelength: 540 nm

NUMBER OF REPLICATE TISSUES: three

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 ul

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 ul

Duration of treatment / exposure:

60 ± 1 minutes

Duration of post-treatment incubation (if applicable):

42 ± 2 hours

Number of replicates:

3

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): 25 mg of test item were applied topically onto the tissue surface (after wetting by 25 uL sterile DPBS) by spoon, for 60 minutes.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 ul Dulbecco's Phosphate-Buffered Saline (DPBS)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 ul 5% SDS

Duration of treatment / exposure:

60 ± 1 minutes

Observation period:

42 ± 2 hours

Number of animals:

not used, in vitro study

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): All tissue inserts were rinsed with DPBS (15 times) to remove any residual test material, than was submerged 3 times in 150 mL of DPBS. Finally, each insert was blotted on sterile blotting paper and transferred to new 6-well plates pre-filled with 0.9 mL of fresh medium.
- Time after start of exposure: 60 min

OBSERVATION TIME POINTS
Tissue constructs were exposed to test item for 60 minutes and were then incubated at 37±1°C in a humidified atmosphere of 5±1% CO2 for 42 -hours.

SCORING SYSTEM:
- Method of calculation:
An ability of test item by to produce a decrease in cell viability was determined by using the MTT reduction assay. The celll cultures were transferred to 24-wells plate containing 0.3 mL/well of MTT reagent (1 mg/mL) and incubated at 37±1°C in a humidified atmosphere of 5±1% CO2 in air for 3 hr ± 5 min. After incubation, the cultures were rinsed with DPBS twice and transferred to new 24-well plate and extracted in 2 mL of isopropanol for 2 hours with shaking at room temperature.
2 x 200 μL of each extraction solution were transferred to a 96-well plate and the absorbances were recorded using a wavelength 540 nm.

For each individual tissue treated with test item, PC and NC, the individual relative tissue viability was calculated. For each test item, PC and NC, the mean relative viability of the three individual tissues were calculated and used for classification according to the Prediction Model. The results are shown as % of relative viability ± SD.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

94.8

Negative controls validity:

valid

Remarks:

viability % 100.0

Positive controls validity:

valid

Remarks:

viability % 6.9

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: 25 mg of the test item was added to 1 mL of the MTT medium and incubated in the incubator (37±1°C in a humidified atmosphere of 5±1% CO2 in air) for 60 min. Untreated MTT medium was used as control. The colour of treated MTT remained unchanged and it was concluded that the test item did not reduce MTT directly.
- Colour interference with MTT: The test item in a volume of 30 µL was added into 0.3 mL of purified water. The mixture was incubated in glass test tube in the incubator at 37±1°C in a humidified atmosphere of 5±1% CO2 in air for 60 min. At the end of the exposure time the presence of the staining was evaluated. The colour of mixture was unchanged and the test item has not the potential to stain tissue.


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD of negative control was 1.544. Tissue viability is meeting the acceptance criterion if the mean OD of the NC tissues is ≥ 1.0 and ≤ 2.5.
- Acceptance criteria met for positive control: The viability of culture treated by positive control 5% SDS was 6.9%. The positive control met the acceptance criterion: mean tissue viability less than 20%
- Acceptance criteria met for variability between replicate measurements: The assay met the acceptance criterion as the SD calculated from individual % tissue viabilities of three identically treated replicates was < 18%.

Any other information on results incl. tables

Skin irritation potential of Magnesium Fluoride after 60 minutes exposure in reconstructed human epidermal model EpiDerm^TM

Test item	OD	SD	Viability	SD	in vivo
	mean	of OD	mean (%)	of viabilities	prediction
Negative control^a	1.544	0.081	100.0	5.24	NI
Positive control^b	0.106	0.020	6.9	1.27	I
Magnesium Fluoride	1.464	0.075	94.8	4.83	NI

a  DPBS

b  5% SDS

Evaluation Criteria

In vitro result	In vivo prediction
mean tissue viability ≤ 50%	Irritant (I), (R38 or UN GHS category 2)
mean tissue viability > 50%	Non-irritant (NI), (UN GHS No Category)

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item Magnesium Fluoride was examined for skin irritation in reconstructed human epidermal model EpiDermTM. Based on the results of the study, the test item is considered to be Non-irritant (NI) to the skin.

Executive summary:

Magnesium Fluoride was examined for in vitro skin irritation in reconstructed human epidermal model EpiDerm^TM. The magnitude of viability was quantified by using MTT test. Validity of the test method was ascertained by positive control 5% SDS. Three tissue replicates were used for each treatment (exposure time 60 minutes), including negative and positive control.  

The tissue viability met the acceptance criterion (mean OD of negative control was 1.544). The viability of culture treated by positive control 5% SDS was 6.9%. The positive control met the acceptance criterion: mean tissue viability less than 20%.

 Determined viability of culture treated by Magnesium Fluoride (94.8%) fulfilled the criteria for non-irritancy. Therefore, Magnesium Fluoride is considered to be non-irritant to the skin.