Magnesium fluoride

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 10th, 2016 - November 8th, 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:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

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: The required amount of the test item (according to the concentration) was mixed in vehicle shortly before the administration (i.e. 100% concentration was obtained by mixing of 1g of test item with 1mL of vehicle).
The preparations were made freshly before each dosing occasion.

FORM AS APPLIED IN THE TEST (if different from that of starting material)
- Suspension. The vehicle (DMSO) was selected from the recommended vehicles according to OECD 429. The test item was insoluble in the vehicle, therefore a homogeneous suspension was obtained.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: AnLab Prague, Czech Republic
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8-11 weeks
- Weight at study initiation: 19.17 - 24.94g
- Housing: The animals were housed individually in polypropylene cages suspended on stainless steel racks, in a room equipped with central air-conditioning. The sanitation was performed according to standard operation procedures.
- Diet (e.g. ad libitum): A laboratory food ssniff (ssniff Spezialdiäten GmbH, Germany) was served ad libitum, each day approximately at the same time. The certificate of analysis is included in the raw data.
- Water (e.g. ad libitum): The animals received tap water for human consumption. Supply of drinking water was unlimited. The quality of drinking water is periodical monitored (including microbiological control) and recorded; certificate of analysis is included in raw data.
- Acclimation period: The animals were acclimated in identical conditions as during the experiment for 6 days prior to the start of treatment. The acclimation was according to standard operation procedures.
- Indication of any skin lesions:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 - 60 %.
- Photoperiod (hrs dark / hrs light): 12-hour light / 12-hour dark cycle
- IN-LIFE DATES: From: 05 October 2016 To: 08 November 2016

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Remarks:

Vehicle – pos. control: Acetone/Olive Oil, 4:1, v/v

Concentration:

The Pre-screen test was performed using a dose of 100 % (v/v).

In the main test the test item at concentrations of 25%, 50% and 100% were administered.

No. of animals per dose:

Pre-test: 2 animals

Main test: 5 animals / 100 % test item
5 animals / 50 % test item
5 animals / 25 % test item
5 animals / control (vehicle)
5 animals / positive control

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: The test item was insoluble in the vehicle (DMSO) , therefore a homogeneous suspension was obtained.
- Irritation: local irritation at the application site were recorded daily
- Systemic toxicity: observed daily for any clinical signs of toxicity
- Ear thickness measurements: Ear thickness was measured by a calliper on Day 1 (pre-dose), Day 3 and Day 6.
- Erythema scores: Observation Score
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beet
redness) to eschar formation preventing grading of erythema 4

MAIN STUDY
Day 1:
Each animal was identified and the body weight was recorded. To the dorsum of each ear 25µL of the appropriate dilution of the test item, or the vehicle alone was applied.
Days 2 and 3:
The application procedure carried out on day 1 was repeated.
Days 4 and 5: No treatment.
Day 6:
The body weight of each animal were recorded. 250µL of phosphate-buffered saline (PBS) containing 2 µCi (7.4 x 104 Bq) of 125I-iododeoxyuridine and 10-5M fluorodeoxyuridine was injected into all test and control mice via the tail vein.
Five hours later, the animals were sacrificed. The draining auricular lymph nodes from each ear were excised and pooled in PBS for each experimental group (pooled treatment group approach) and weighted.

Cell suspension of lymph node cells from pooled treatment groups were prepared by gentle mechanical disaggregation by glass homogenizer. Lymph node cells were centrifuged (SOPA-00156- BIO) by 600g at 4C for 10 min. Suspension of cells were precipitated with 5% trichloroacetic acid (TCA) at 4 degC for 18-20h. Pellets were centrifuged by 2000g at 4C for 5 min., re-suspended in 1 ml TCA and transferred to gamma counting tubes for 125I-counting.

- Criteria used to consider a positive response:
Results are expressed as the Stimulation Index (SI). The SI was obtained by dividing the pooled radioactive incorporation for each treatment group by the radioactive incorporation of the pooled vehicle control group; this yields a mean SI. A substance is regarded as sensitizer in the LLNA test when SI≥3.

TREATMENT PREPARATION AND ADMINISTRATION:
The required amount of the test item (according to the concentration) was mixed in vehicle shortly before the administration (i.e. 100% concentration was obtained by mixing of 1g of test item with 1mL of vehicle).
The vehicle (DMSO) was selected from the recommended vehicles according to OECD 429. The test item was insoluble in the vehicle, therefore a homogeneous suspension was obtained.
The test item was administrated in volume of 25 uL to the dorsum of each ear. The alfa-Hexyl cinnamaldehyde (25%) as positive control and vehicle as a negative control were administrated in the same volume.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:

Lymph node Number of
weight (g) lymph nodes DPM SI

Positive Control 0.0838 10 7037 12.79

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

0.96

Test group / Remarks:

Magnesium Fluoride 25%

Results 2

Key result

Parameter:

SI

Value:

1.07

Test group / Remarks:

Magnesium Fluoride 50%

Results 3

Key result

Parameter:

SI

Value:

0.98

Test group / Remarks:

Magnesium Fluoride 100%

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION: The pooled lymph node weights of treated groups were 0.0407g for 25% concentration, 0.0442g for 50% concentration and 0.0415g for 100% concentration of tested item. The lymph node weight of control group and positive control group were 0.0414g and 0.0838g, respectively. The DPM values for the three treated groups were 873 (25%), 976 (50%) and 889 (100%), respectively. The SI values for the three treated groups were 0.96 (25%), 1.07 (50%) and 0.98 (100%), respectively.

EC3 CALCULATION: The EC3 value could not be calculated, as all measured points were below the Stimulation Index of three.

CLINICAL OBSERVATIONS: No clinical signs of toxicity observed

BODY WEIGHTS No marked changes of mean body weight were observed during the study

Any other information on results incl. tables

Lymph node weight, DPM, SI, EC3 values.

	Lymph node	Number of
	weight (g)	lymph nodes	DPM	SI	EC3
Control	0.0414	10	911	-	-
Positive Control	0.0838	10	7037	12.79
Magnesium Fluoride25%	0.0407	10	873	0.96
Magnesium Fluoride50%	0.0442	10	976	1.07
Magnesium Fluoride100%	0.0415	10	889	0.98

Historical Control Data for Positive Control

Date	Study No.	DPM - negative control (vehicle)	DPM - positive control	Stimulation Index
09/2016	600349510	777	4355	5.60
07/2016	600342910	1014	7601	7.49
04/2016	600333730	1093	6811	6.23
08/2015	600292090	313	3925	12.54*
03/2014	6500000060250	1184.99	5055.63	4.27
01/2013	600170570	476.85	1887.81	3.96
08/2010	600079020	772.02	3915.76	5.07
06/2010	600071910	1067.21	4760.68	4.46

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The skin sensitizing potential of Magnesium Fluoride was assessed using the murine local lymph node assay. Based on the results of this study, Magnesium Fluoride is not considered a skin sensitizer under the condition of this study.

Executive summary:

The skin sensitization potential of Magnesium Fluoride was evaluated by LLNA method, which was developed on the basis of scientific understanding of the immunological mechanism of skin sensitization.

In our experiments, the test item was applied over three consecutive days, at three concentrations. All animals survived throughout the test period without showing any clinical signs of toxicity or local irritation. In comparison with control group, the increase in lymph node weight was observed in groups treated at 50 and 100% concentration. The increase of lymph node weight was not dose dependent. A similar trend was registered in the evaluation of DPM of the lymph nodes. Calculated SI values in treated groups remained under the value of 3, which is the threshold to consider the substance a sensitizer. Therefore, it was not possible to calculate an EC3 value.

The study is considered reliable without restrictions since it is conducted according the current guidelines and in compliance with GLP.