Magnesium fluoride

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 08, 2016 - September 22, 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 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

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

Test animals / tissue source

Species:

other: in vitro study

Details on test animals or tissues and environmental conditions:

TEST SYSTEM
- Protocol: In vitro EpiOcularTM eye irritation test (OCL-200-EIT)
- Source: MatTek In Vitro Life Science Laboratories, SR
- Cell line: The EpiOcular™ human cell construct
- Lot: 23735

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 37±1°C
- Cell Culture Conditions: a humidified atmosphere of 5±1% CO2 in air

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL AND CONTROLS
- Amount(s) applied (volume or weight with unit): 50 mg
- Concentration (if solution): as such

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

18 hours

Number of animals or in vitro replicates:

2

Details on study design:

- Details of the test procedure used
The EpiOcularTM cultures were pre-incubated at 37±1°C in a humidified atmosphere of 5±1% CO2 for 60 min. At the end of the first pre-incubation period, the medium was replaced by 1 mL of fresh assay medium. Further, the tissues were pre-incubated in incubator overnight prior to dosing for release of transport stress related compounds and debris. After pre-incubation tissues were pre-wetted with 20 µL of Dulbecco's Phosphate Buffered Saline (DPBS), then 50 µL of control items and 50 mg of the test item were applied topically onto the tissue surface for 6 hours. Two tissues were used per treatment, negative and positive controls. After exposure period, each tissue was rinsed gently with DPBS to remove any residual test item.

- RhCE tissue construct used, including batch number
The EpiOcular™ human cell construct Lot: 23735 was delivered one day before the pre-incubation of tissues and was stored in original package at 2-8°C. At day 1, each culture was removed from the agarose gel using a sterile forceps, inspected and transferred to a pre-labeled 6-well plates containing 1 mL of assay medium per well.

- Doses of test chemical and control substances used
50 mg and 50 µl, respectively

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable)
After post-soak immersion at room temperature, tissues than were transferred to fresh medium and incubated for 18 hours.

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable)
Before testing, test item was checked for its ability to reduce MTT directly. For this purpose, a 1.0 mg/mL MTT solution (in DMEM) was prepared. 50 mg of the test item was added to 1 mL of the MTT solution in a 6-well plate and the mixture was incubated in the dark at 37 +/- 1°C in a humidified atmosphere of 5 +/- 1% CO2 in air (standard culture conditions) for three hours. A negative control (50 μL of aqua pro injectione) was run concurrently. At the end of the exposure time the colour of treated MTT remained unchanged and it was concluded that the test item did not reduce MTT directly.

- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable)
Two

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer)
Spectrophotometer MRX II (Dynex), wavelength 540nm

- Description of the method used to quantify MTT formazan
The MTT assay was performed by transferring the tissues to 24-wells plate containing MTT medium (1 mg/mL) and incubated at 37±1°C in a humidified atmosphere of 5±1% CO2 in air for 3 hours. After incubation, the blue formazan salt formed by cellular mitochondria was extracted with 2 mL/tissue of isopropanol for 2 hours with shaking at room temperature. Each extraction solutions in a volume of 200 μL were transferred to a 96-well plate and the optical density of extracted formazan was determined using a spectrophotometer at 540 nm.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
60%, established cut-off value (Draft revised TG492, July 2016)

- Positive and negative control means and acceptance ranges based on historical data
Negative Control (NC):
The negative control OD > 1.0 and < 2.6.
Positive Control (PC):
The mean relative viability of the positive control is at 6 hours exposure below 60% of control viability.

- Acceptable variability between tissue replicates for positive and negative controls and for the test chemical
The difference of viability between the two relating tissues of a single chemical is < 20% in the same run

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

The mean tissue viability (%)

Value:

109.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100.0

Positive controls validity:

valid

Remarks:

17.3

Other effects / acceptance of results:

OTHER EFFECTS:
not observed

DEMONSTRATION OF TECHNICAL PROFICIENCY:
not demonstrated

ACCEPTANCE OF RESULTS:
Concurrent positive and negative controls were used to ensure adequate performance of the experimental model. Validity of the test method was ascertained by positive control methyl acetate and negative control aqua pro injectione. The tissue viability met the acceptance criterion; mean OD of negative control was 1.172.
The viability of culture treated by positive control methyl acetate was 17,3 %. The positive control met the acceptance criterion: mean tissue viability less than 60%.

- Range of historical values if different from the ones specified in the test guideline:

Any other information on results incl. tables

Test item	OD	SD	Viability	SD	in vivo
	mean	of OD	mean (%)	of viabilities	prediction
Negative control^a	1.172	0.033	100.0	2.77	NI
Positive control^b	0.203	0.020	17.3	1.66	I
Magnesium Fluoride	1.279	0.010	109.1	0.81	NI

a  H₂O

b  methyl acetate

Evaluation Criteria

In vitro result                                   In vivo prediction

mean tissue viability ≤ 60%                     Irritant (I)

mean tissue viability > 60%                     Non-irritant (NI)

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item Magnesium Fluoride was examined for eye irritation potential in EpiOcularTM Eye Irritation Test (OCL-200-EIT). Based on the results of the study, the test item is considered to be Non-irritant (NI).

Executive summary:

The test item Magnesium Fluoride was examined for in vitro eye irritation in human model EpiOcular^TM. The magnitude of viability was quantified by using MTT test.

 

Validity of the test method was ascertained by positive control methyl acetate. Two tissue replicates were used for each treatment (exposure time 6 hours), including negative and positive controls. The tissue viability met the acceptance criterion (mean OD of negative control was 1.172). The viability of culture treated by positive control methyl acetate was 17.3%. The positive control met the acceptance criterion: mean tissue viability less than 60%.

 

Determined viability of culture treated by Magnesium Fluoride (109.1%) fulfilled the criteria for irritancy. 

Therefore, the test item Magnesium Fluoride is considered to be non-irritant to the eye.