Lithium iodide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05/11/2021

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 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

Suspensions of bacterial cells are exposed to the test substance in the presence and in the absence of an exogenous metabolic activation system. In the plate incorporation method, these suspensions are mixed with an overlay agar and plated immediately onto minimal medium. In the pre-incubation method, the treatment mixture is incubated and then mixed with an overlay agar before plating onto minimal medi-um. For both techniques, after two or three days of incubation, revertant colonies are counted and com-pared to the number of spontaneous revertant colonies on negative control plates.
This study was performed in order to evaluate the mutagenic potential of Lithium Iodide Anhydrous in the Bacterial Reverse Mutation Test using five strains of Salmonella typhimurium.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

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

The test item was stored in the test facility in a closed vessel at room temperature (20 ± 5 °C), kept under inert gas.

Method

Target gene:

hisC3076
hisD3052
hisG46
hisG428
uvrB
rfa
pKM101
pAQ1

Species / strain

Species / strain / cell type:

S. typhimurium TA 98

Remarks:

Species Salmonella typhimurium LT2 Strains TA98, TA100, TA102, TA1535 and TA1537

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Test concentrations with justification for top dose:

In a non-GLP pre-test, the solubility of the test item was determined in demineralized (demin.) water, dimethyl sulfoxide (DMSO), acetone and ethanol. The test item is soluble in a concentration of 50 g/L in demin. water, ethanol and acetone.

Based on the non-GLP pre-test, demin. water was chosen as vehicle, because the test item was suffi-ciently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.
On the day of the start of each experiment, a stock solution containing 50 g/L of the test item in DMSO was prepared.
The stock solution was used to prepare the geometric series of the concentrations to be tested.

The following nominal test item concentrations were prepared for experiment 1:
5000, 1500, 500, 150 and 50 µg/plate.

The following nominal test item concentrations were prepared for experiment 2:
5000, 2500, 1250, 625, 313 and 156 µg/plate.

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

sodium azide

benzo(a)pyrene

other: 2-Amino-anthracene; Mitomycin C

Details on test system and experimental conditions:

CULTURE OF BACTERIE
On the day before the start of each experiment, a nutrient broth (Oxoid nutrient broth no. 2) was inocu-lated with one lyophilizate per strain (for details see chapter 6.2.2, page 11).

=> Conduct of Experiment

A) Preparations
Different media and solutions were prepared preliminary (exact production dates are documented in the raw data).
On the day of the test, the bacteria cultures were checked for growth visually. The incubation chambers were heated to 37 ± 1 °C. The water bath was turned to 43 ± 1 °C. The table surface was disinfected.
The S9-mix was freshly prepared and stored at 0 °C.

Experiment 1
Date of treatment: 05. Oct. 2021
Tested strains: TA98, TA100, TA102, TA1535, TA1537
Test item concentrations: 5000, 1500, 500, 150, 50 µg/plate
(real concentrations see chapter 17, page 41)
Incubation time: 48 h
Incubation temperature: 37 ± 1 °C
Method: plate incorporation method
Positive controls: see chapter 6.2.4, page 13

Experiment 2
Date of treatment: 03. Nov. 2021
Tested strains: TA98, TA100, TA102, TA1535, TA1537
Test item concentrations: 5000, 2500, 1250, 625, 313, 156, 78 µg/plate
(real concentrations see chapter 17, page 41)
Incubation time: 48 h
Incubation temperature: 37 ± 1 °C

Method: pre-incubation method
Positive controls: see chapter 6.2.4, page 13
 
B) Description of the Method
Per bacteria strain and concentration, three plates with (+S9) and three plates without metabolic activa-tion (-S9) were used.
The test item solutions were prepared according to chapter 6.1.3, page 10.
For the top agar 100 mL agar basis was melted in a microwave oven, 10 mL of the histidine-biotin-solution 0.5 mM was added, then the mixture was placed in the water bath at 43 ± 1 °C.

Plate incorporation method
The following materials were gently vortexed in a test tube and poured onto the selective agar plates:

• 100 µL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control).
• 500 µL S9-mix (see chapter 6.4.13, page 16 for test with metabolic activation) or phosphate buffer (for test without metabolic activation).
• 100 µL bacteria suspension (see chapter 6.2.2, page 11, test system, culture of the strains)
• 2000 µL overlay agar (top agar)
The plates were closed and left to solidify for a few minutes, then inverted and placed in the dark incuba-tor at 37 ± 1 °C.

Pre-incubation method
The following materials were gently vortexed in a test tube and incubated at 37 ± 1 °C for 20 minutes:

• 100 µL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control).
• 500 µL S9-mix (see chapter 6.4.13, page 16 for test with metabolic activation) or phosphate buffer (for test without metabolic activation).
• 100 µL bacteria suspension (see chapter 6.2.2, page 11, test system, culture of the strains)

After the pre-incubation for 20 minutes, 2000 µL top agar was added and the tube was gently slewed. The mixture was poured onto the selective agar plate.
The plates were closed and left to solidify for a few minutes, then inverted and placed in the incubator at 37 ± 1 °C.
 
=> References and Validity

A) Genotype Confirmation
Confirmation of genotype is performed for each batch of lyophilized bacteria by the supplier Trinova Bio-Chem GmbH. The batches used of lyophilized bacteria met the criteria

B) Spontaneous Revertants
The number of spontaneous revertants was determined for each solvent, used in the test by investigat-ing three replicates with and without metabolic activation, incubation for 48 hours at 37 ± 1 °C for each strain.

C) Determination of Titre
The titre was determined by dilution of the overnight culture using sodium chloride solution and placing 0.1 mL on maximal-soft agar. It should give a density of 109 cells/mL (at the least).
Two replicates with and without metabolic activation, incubation for 48 hours at 37 ± 1 °C.

D) Sterility Control
Performed analogously to the test item but with solvent only and S9 (without adding bacteria) on top agar.
Four replicates, incubation for 48 hours at 37 ± 1 °C.

E) Solubility
Plates were checked for precipitation of test item at the end of the incubation by visual inspection.

F) Positive Controls
Using diagnostic mutagens (see chapter 7.3.6, page 19). The stock solutions of the substances were dilut-ed to achieve an application volume of 0.1 mL/plate.
Three replicates with and without metabolic activation, incubation for 48 hours at 37 ± 1 °C.
 

Evaluation criteria:

Five different analysable concentrations were used for the evaluation of the mutagenic potential of the test item.
The colonies were counted visually and the numbers were recorded. A validated spreadsheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, negative control and positive control.
The mean values and standard deviations of each threefold determination were calculated as well as the increase factor of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (mean re-vertants minus mean spontaneous revertants) is given.
A result is considered as clearly positive if all following criteria are fulfilled:
• A concentration-related increase, in revertants
• a clear biologically relevant increase in at least one concentration compared to the concurrent solvent control
• at least one concentration with an increase above the distribution of historical solvent control data (mean ± 3 SD).

A biologically relevant increase is described as follows:
• if in the bacteria strains TA98, TA100, TA102 the number of revertants is at least twice as high than the reversion rate of the negative controls (increase factor of at least 2.0)
• if in the bacteria strains TA1535 and TA1537 the number of revertants is at least three times higher than the reversion rate of the negative controls (increase factor of at least 3.0).

A test result is considered as clearly negative, if it does not meet the criteria above.
Tables of the values obtained in this test are included in this final report.
 

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The study was performed with the plate incorporation (exp. 1) and pre-incubation method (exp. 2.) in the absence and presence of a metabolic activation system (S9). Under these conditions the influence of the test item on bacterial test strains was evaluated. The test item Lithium Iodide Anhydrous showed no rele-vant or dose-related increase in the number of revertants in the Salmonella typhimurium test strains TA98, TA100, TA102, TA1535 and TA1537 in all evaluated experiments.
Based on the results of this study it is concluded that Lithium Iodide Anhydrous is not mutagenic in the Salmonella typhimurium test strains TA98, TA100, TA102, TA1535 and TA1537 in the absence and presence of metabolic activation under the experimental conditions of the present study.


DISCUSSION
The test item Lithium Iodide Anhydrous was tested in the Bacterial reverse mutation assay with five strains of Salmonella typhimurium (TA98, TA100, TA102, TA1535 and TA1537).
The test was performed in two valid experiments in the presence and absence of metabolic activation, with +S9 standing for the presence of a metabolic activation, and -S9 standing for absence of metabolic activation.
The test item (dissolved in demin. water) was tested up to concentrations of 5000 µg/plate in the absence and presence of S9 mix in the strains TA98, TA100, TA102, TA1535 and TA1537 using the plate incorpora-tion method (exp. 1) and the pre-incubation method (exp. 2).
The test item showed no precipitates on the plates at any of the concentrations and no signs of cytotoxi-city could be observed in the presence and the absence of metabolic activation in both experiments (exp. 1 and 2).
In exp. 1 no relevant or concentration-related increase of the number of revertant colonies in the treat-ments with and without metabolic activation could be observed.
To verify this result, a further experiment with adapted conditions (pre-incubation method) was per-formed (exp. 2) and again 1 no relevant or concentration-related increase of the number of revertant colonies was found.

Based on the results of this study it is concluded that Lithium Iodide Anhydrous is not mutagenic in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in the presence and absence of metabolic activation under the experimental conditions in this study.

No observations arousing doubts concerning the accuracy of the results and the validity of the study were made.

Any other information on results incl. tables

VALIDITY

All negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

All positive controls showed f(I) values > 2 or > 3 (strain specific threshold) which demonstrated the mutagenic potential of the diagnostic mutagens ).

 

The confirmation tests of the genotype performed by Trinova BioChem GmbH did not show any irregularities. The control of the titre was above the demanded value of 10⁹ bacteria/mL. In the sterility control no growth of bacteria could be detected.

Since all criteria for acceptability have been met, the study is considered valid.

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it is concluded that Lithium Iodide Anhydrous is not mutagenic in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in the presence and absence of metabolic activation under the experimental conditions in this study.

Executive summary:

This study was performed in order to evaluate the mutagenic potential of Lithium Iodide Anhydrous in the Bacterial Reverse Mutation Test using five strains of Salmonella typhimurium (TA98, TA100, TA102, TA1535 and TA1537) based on the most recent Guidelines OECD 471 (2020) and EU Method B.13/14 (2008).

 

Findings and Results:

The test was performed in two valid experiments in the presence and absence of metabolic activation, with +S9 standing for the presence of a metabolic activation, and -S9 standing for absence of metabolic activation.

 

Experiment 1:

In the first experiment, the test item (dissolved in demineralized water, demin. water) was tested up to concentrations of 5000 µg/plate in the absence and presence of S9 mix in the strains TA98, TA100, TA102, TA1535 and TA1537 using the plate incorporation method.

The test item showed no precipitates on the plates at any of the concentrations and no signs of cytotoxicity could be observed in the presence and the absence of metabolic activation .

The results of this experiment showed that none of the tested concentrations induced a relevant or concentration-related increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

 

Experiment 2:

Based on the results of the first experiment, the test item was tested up to concentrations of 5000 µg/plate in the presence and absence of S9 mix in all bacteria strains using the pre-incubation method.

The test item showed no precipitates on the plates at any of the concentrations and no signs of cytotoxicity could be observed in the presence and the absence of metabolic activation .

The results of this experiment showed that none of the tested concentrations induced a relevant or concentration-related increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

 

Conclusion:

Based on the results of this study it is concluded that Lithium Iodide Anhydrous is not mutagenic in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in the presence and absence of metabolic activation under the experimental conditions in this study.