Lithium fluoride

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2015-07-08 to 2015-07-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2013

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Lithium fluoride

EC Number:

232-152-0

EC Name:

Lithium fluoride

Cas Number:

7789-24-4

Molecular formula:

FLi

IUPAC Name:

lithium(1+) fluoride

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot No.of test material: 1812
- Expiration date of the lot/batch: May 2025

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature (15-30 °C)

In vitro test system

Test system:

isolated skin discs

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Adult donors

Justification for test system used:

The method with reconstructed human skin is approved by international regulatory agencies as a replacement for the identification of irritants / corrosives in the in vivo rabbit skin assay (OECD 404). The test is designed to predict and classify the skin irritant potential of chemicals according to chemical safety regulations, using the reconstructed human epidermis model EpiSkinTM Small Model (EpiSkinTMSM) and parameters related to skin irritation.

Vehicle:

physiological saline

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN
- Tissue batch number: 15-EKIN-027
- Expiry date: July 13, 2015
- Date of initiation of testing: July 8, 2015

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 20 °C
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: once with 1x PBS solution
- 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: 0.3 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Thermo Scientific; Multiscan FC
- Wavelength: 570 nm
- Filter: without reference filter

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- killed tissues
- Procedure used to prepare the killed tissues: Living epidermis is subjected to -20 °C (or -80 °C) for at least 48 hours; Before use killed tissue is defrozen at room temperature
- N. of replicates : 3
- Method of calculation used: The optical density is evaluated und subtracted before the % viability is calculated.

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be non-corrosive (irritant) to skin if the viability after 15 minutes exposure is greater than or equal to 50 % of the negative control.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 10 mg

NEGATIVE CONTROL
- Amount(s) applied: 10 µL

POSITIVE CONTROL
- Amount(s) applied: 10 µL
- Concentration (if solution): 5 %

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean (test substance)

Value:

91

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

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

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Any other information on results incl. tables

Table 1 Irritation results

		Tissue 1	Tissue 2	Tissue 3	Mean	SD
NC	Mean OD570	0.751	0.680	0.648	0.693
	Viability [% of NC]	108	98	93	100	7.65
PC	Mean OD570	0.056	0.070	0.034	0.053
	Viability [% of NC]	8	10	5	8	2.59
Test substance	Mean OD570	0.569	0.671	0.642	0.628
	Viability [% of NC]	82	97	93	91	7.6

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

According to the available in vitro test, lithium fluoride is considered as non-irritant to skin.

Executive summary:

An in vitro skin irritation test was conducted according to OECD guideline 439 and EU method B.46. Disks of epidermal units (three units / chemical) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test material was terminated by rinsing the epidermal units with 1x PBS solution. Epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5 % CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in 5 % CO2 and protected from light. The resulting formazan chrystals were extracted with acidified isopropanol and quantified with the optical densities (OD) recorded spectrophotometrically. SDS 5 % aq. and 1 x PBS treated epidermis units were used as positive and negative controls, respectively. For each treated tissue, viability was expressed as a percentage relative to negative control. The test item is considered to be a skin irritant, if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less than or equal to (≤) 50 % when compared to the viability values obtained from the negative control. In this in vitro skin irritation test using the EPISKIN model, the test item lithium fluoride did not show significantly reduced cell viability in comparison to the negative control (mean relative viability: 90 %). All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid. The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilized testing conditions. According to the current OECD Guideline No. 439, lithium fluoride is considered as non-irritant to skin and is therefore not classified.