Magnesium fluoride

Endpoint summary

Description of key information

Skin irritation:

Not Irritating (in vitro skin irritation in reconstructed human epidermal model EpiDerm^TM). Key study; Klimisch score 1.

 

Eye irritation:

Not Irritating (in vitro eye irritation in human model EpiOcular^TM). Key study; Klimisch score 1.

 

Respiratory irritation:

Irritating. No data available of the substance. Inorganic fluorides generally are considered highly irritating substances.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

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

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

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.

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%.

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)

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

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

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

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

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

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

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:

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)

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Additional information

Skin irritation

 

A good quality guideline compliant skin irritation study (OECD 439 -in vitro skin irritation in reconstructed human epidermal model EpiDerm^TM) was conducted for magnesium fluoride (Bednáriková, M. 2016a).The purpose of the study was to evaluate the skin irritation potential of the test item Magnesium Fluoride by EpiDerm^TM in vitro model by measuring test item induced decrease in cell viability (as determined by using the MTT reduction assay) below defined threshold levels. The method involved a 60- minute exposure of tissues to the test item.

In the study three tissue replicates were used for each treatment, including negative and positive control. After exposure each tissue was washed and post-incubated for 42 -hours post-incubated. The cultures were transferred to 24-wells plate containing MTT reagent (1 mg/mL) and incubated in a humidified atmosphere 3 h. After incubation, the cultures were rinsed twice and transferred to new 24-well plate and extracted in isopropanol with shaking at room temperature.

Each extraction solution was transferred to a 96-well plate and the absorbances were recorded.

 

Validity of the test method was ascertained by positive control (5% SDS) and by negative control (sterile DPBS). The tissue viability of negative control and viability of culture treated by positive control determined in this study were compared with the historical data values. The tissue viability met the acceptance criterion (mean OD of negative control was 1.544). The positive control met the acceptance criterion: mean tissue viability was 6.9% (less than 20%). The viability of culture treated by Magnesium Fluoride was 94.8%. Therefore, magnesium fluoride is considered to be non-irritant to the skin.

Eye irritation

A good quality guideline compliant eye irritation study (OECD 492 –in vitro eye irritation in Reconstructed Human Cornea-like Epithelium (RhCE)) was conducted for magnesium fluoride (Bednáriková, M. 2016b). Aim of the study was to examine the acute eye irritation potential of the test item by measurement of its cytotoxic effect on the EpiOcular™ cornea epithelial model. The EpiOcular tissue construct is a nonkeratinized epithelium prepared from normal human keratinocytes (MatTek).

In this assay, the test article was applied to the surface of the cornea epithelial construct for 6 hours. After exposure period, each tissue was extensively rinsed, and the tissue was allowed to express the resulting damage.

Two construct tissues are used for the treatment with test item and for negative and positive controls. The MTT assay was performed to determine tissue viability. The tissues were transferred in the 24-wells plate containing MTT medium (1 mg/mL) and incubated in a humidified atmosphere for 3 hours. After incubation the blue formazan salt formed was extracted in isopropanol and the optical density of extracted formazan was determined using a spectrophotometer at 540 nm.

Validity of the test method was ascertained by positive control (methyl acetate) and negative control (aqua pro injection). The tissue viability met the acceptance criterion as the mean OD of negative control was 1.172 (acceptance criteria OD > 1.0 and < 2.6). The positive control met the acceptance criterion as the viability of culture treated by methyl acetate was 17.3% (less than 60%).The viability of culture treated by magnesium fluoride was 109.1%. Therefore, the test item magnesium fluoride is considered to be non-irritant to the eye.

 

Respiratory irritation

Potential respiratory irritation properties of magnesium fluoride have not been thoroughly investigated and recorded. However, it is generally known that the exposure of humans to airborne contaminants and particulates, may cause adverse effects on the respiratory system. The particle size distribution of the magnesium fluoride (Smeykal, H. 2016) showed median particle size D50 = 873 um and only approximately 0.3 Vol.-% is of inhalable size i.e. smaller than 100 um.

However, inorganic fluorides are considered highly irritating substances. Epidemiological investigations of workers in aluminium smelters (i.e. occupationally exposed to airborne fluorides) have reported e.g., reduced lung capacity, irritation of the respiratory tract, asthma, cough and/or bronchitis)(WHO, 2002). However, the effects observed on these studies may not be possible to attribute solely to fluoride per se owing to the exposure of these workers to other airborne contaminants and particulates.

Justification for classification or non-classification

The result of the key study (Bednáriková, M. 2016a) does not indicate Magnesium Fluoride to be classified for skin irritant according to CLP Regulation 1272/2008.

The result of the key study (Bednáriková, M. 2016b) does not indicate Magnesium Fluoride to be classified for eye irritant according to CLP Regulation 1272/2008.

Evidence on respiratory tract irritation of inorganic fluorides (WHO, 2002) suggests that the substance has to be classified to hazard class STOT SE 3 H335 according to CLP Regulation 1272/2008.