Lithium iodide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 luciferase KeratinoSens™ test method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase LuSens test method

Test material

Test material information

Constituent 1

Reference substance name:

Lithium iodide

EC Number:

233-822-5

EC Name:

Lithium iodide

Cas Number:

10377-51-2

Molecular formula:

ILi

IUPAC Name:

lithium iodide

Test material form:

solid

Specific details on test material used for the study:

Batch no. 1210621A-0915P

In vitro test system

Details of test system:

Lusens transgenic cell line [442D]

Details on the study design:

PREPARATION
The solubility of the test item was determined in a non-GLP pre-test in dimethyl sulfoxide (DMSO) and medium (Dulbecco´s Modified Eagle Medium, DMEM). The test item is soluble in medium and in DMSO (after sonication) at the required concentration (200 mM). Since DMSO is the preferred solvent according to OECD 442D, DMSO was used as solvent.
The highest test item concentration in the Cytotoxicity Range Finder Test (CRFT) is 2000 µM. Since the final concentration of the solvent during treatment is limited to 1 %, a stock solution containing 200 mM (CRFT) and 200 mM (experiment) test item in DMSO was prepared and sonicated for 17 min. Subsequent dilution to 1 % finally yielded a maximum concentration of 2000 µM in the CRFT and 2000 µM in the experiment.
For that, the stock solution was first used to prepare a geometric series of solutions (CRFT: factor 2; main experiment: factor 1.2) on a master plate. Afterwards all concentrations were further diluted (1:25) in medium no. 3 on a dilution plate. Another 1:4 dilution was achieved by adding 50 µL of each concentration of the dilution plate to the corresponding wells of the test plate containing the cells as well as 150 µL medium no. 3. In the end, the total dilution factor was 1:100. The stock solution as well as the dilutions were freshly prepared on the day of treatment.

Test System:
Reasons for the Choice of the LuSens Cell Line

The LuSens cell line was specially designed for this test system by the BASF SE (Ludwigshafen, Germany). It employs the use of a luciferase reporter gene placed under the control of the antioxidant response element (ARE) and hence monitors Nrf-2 transcription factor activity. For designing this cell line, a human keratinocyte cell line (provided by RWTH, Aachen, Germany) was transfected with the pGL4.20 [luc2/Puro] vector (Promega, Germany) carrying the regulatory antioxidant response element (ARE) upstream of the luciferase gene (Luc2, Promega, Germany) at the Institute of Anatomy and Cell Biology of the RWTH, Aachen (laboratory of PD Dr. Wruck).
Cell Cultures

The LuSens cell line was obtained from the BASF SE (Ludwigshafen, Germany). For mycoplasma contamination screened stocks of LuSens cells are stored in liquid nitrogen in the cell bank of LAUS GmbH to allow a continuous stock of cells (mycoplasma contamination free), which guarantees similar parameters of the experiment and reproducible characteristics of the cells.

For the Cytotoxicity Range Finder Assay cells of passage 8 were used. For the valid repetitions, cells of passage 14 and 12 were used. After thawing the cells were cultivated in DMEM (9 % FBS (Fetal bovine serum)) in cell culture flasks at 37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2.

Vehicle / solvent control:

DMSO

Negative control:

DL-Lactic acid

Positive control:

EGDMA (120 M) [442D]

Results and discussion

In vitro / in chemico

Results

Results 1

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

other: Induction of Luciferase

Cell viability:

82.5 - 90% for the test item

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

In all tested concentrations of the test item no increase ≥ 1.5 fold in luciferase induction in comparison to the solvent control was measured. Therefore, both repetitions are clearly negative.

Results 2

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

other: Induction of Luciferase

Cell viability:

111.7 - 126.1 % for the test item

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

In all tested concentrations of the test item no increase ≥ 1.5 fold in luciferase induction in comparison to the solvent control was measured. Therefore, both repetitions are clearly negative.

Outcome of the prediction model:

negative [in vitro/in chemico]

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Therefore, the test item Lithium Iodide Anhydrous is considered not to have the potential to activate the Nrf2 transcription factor under the conditions of the LuSens test.

Executive summary:

This in vitro study was performed to investigate the potential of Lithium Iodide Anhydrous to activate the Nrf2 transcription factor, by using the LuSens cell line.(OECD442D)

In total five repetitions were performed of which three were invalid (repetition I, III and IV) and had to be repeated. These repetitions are not reported, all documentation is kept with the raw data and will be archived at the GLP test facility. In the end two valid repetitions were performed and evaluated (repetition II and V). Therefore, two independent repetitions were performed.

The exposure time was 48 h. The following nominal concentrations of the test item were investigated in repetition II and V:

269 µM, 323 µM, 388 µM, 465 µM, 558 µM, 670 µM, 804 µM, 965 µM, 1157 µM, 1389 µM, 1667 µM, 2000 µM

None of the real treatment concentrations in all repetitions deviated more than 10 % from the nominal concentration. Precipitation of the test item was not visible up to the highest concentration in the repetitions.

EGDMA (120 µM) was used as positive control. The viability was above 70 % and a distinct increase in luciferase induction above 2.5 fold in comparison to the solvent control was detected. This luciferase induction is well within the historical data range of the positive control.

DL-lactic acid (5000 µM) was used as negative control. The viability was above 70 % and the induction of the luciferase was < 1.5 fold in comparison to the solvent control and well within the historical data range of the negative control.

The induction of the luciferase of the growth control (Medium no. 3) was < 1.5 fold.

Since all acceptability criteria of the assay were met, the study is valid.

No cytotoxic effect was observed in all tested test item concentrations. Therefore, all tested concentrations could be evaluated for luciferase induction.

In all tested concentrations of the test item no increase ≥ 1.5 fold in luciferase induction in comparison to the solvent control was measured.

Therefore, both repetitions are clearly negative.

In conclusion, it can be stated that under the experimental conditions of this study, the test item, Lithium Iodide Anhydrous, was negative in the LuSens assay and is therefore considered not to have the potential to activate the Nrf2 transcription factor.