Lithium hydroxide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-01-20 to 2010-07-27

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Lithium Hydroxide monohydrate

EC Number:

603-454-3

Cas Number:

1310-66-3

Molecular formula:

H3LiO2

IUPAC Name:

Lithium Hydroxide monohydrate

Test material form:

solid

Method

Target gene:

Thymidine kinase (TK)

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The indicator cell used for this study was the L5178Y mouse lymphoma cell line that is heterozygous at the TK locus (+/-). The particular clone (3.7.2C) used in this assay is isolated by Dr. Donald Clive (Burroughs Wellcome Company, Research Triangle Park, NC).

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

12.5, 25, 50 100 and 200 ug/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Aqua ad iniectabilia

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

positive control for non-activation mutation studies.

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

positive control for assays performed with S9 metabolic activation.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

ASSAY WITHOUT METABOLIC ACTIVATION
The cells for the experiments were obtained from logarithmically growing laboratory stock cultures and were seeded into a series of tubes at 1 x 107 cells per tube. The cells were pelleted by centrifugation, the culture medium was removed, and the cells were resuspended in a final volume of 20.0 mL of treatment medium that contained 5 % heat inactivated fetal bovine serum. The dosed tubes were closed, vortexed and placed on a roller drum at approx. 37 °C at 10 - 15 rpm for an exposure period of 3 hours. The cells were washed and resuspended in growth medium.
Cell densities were adjusted to 2 x 105/mL and the cells were plated for survival and incubated for the expression period in parallel, i.e. an aliquot of the cells was diluted to 8 cells/mL and 0.2 mL of each culture were placed in two 96 well microtiter plates (192 wells, averaging 1.6 cells/well) and incubated for 1 week at 37 ± 0.4 °C whereas the rest of the cells was incubated for 2 days at 37 ± 0.4 °C for the expression period.
The cells for the plating of survival were counted after 1 week and the number of viable clones was recorded. The cells in the expression period were maintained below 106 cells per mL and a minimum of 4 concentration levels plus positive and negative control was selected for 5-trifluoro-thymidine (TFT) resistance.
At the end of the expression period, the selected cultures were diluted to 1 x 104 cells/mL and plated for survival and TFT resistance in parallel (plating efficiency step 2). The plating for survival was similar to the above described method. For the plating for TFT resistance, 3 μg/mL TFT (final concentration) were added to the cultures and 0.2 mL of each suspension was placed into four 96-well microtiter plates (384 wells, averaging 2 x 103 cells/well). The plates were incubated for 12 days at 37 ± 0.4 °C and wells containing clones were identified microscopically and counted.
In addition, the number of large and small colonies was recorded with an automated colony counter that can detect colony diameters equal or greater than 0.2 to 0.3 mm. Large colonies are defined as >= 1/3 and small colonies < 1/3 of the well diameter of 6 mm.

ASSAY WITH METABOLIC ACTIVATION
The activation assay is often run concurrently with the non-activation assay; however, it was an independent assay performed with its own set of solvent and positive controls. In this assay, the above-described activation system was added to the cells together with test item.

Evaluation criteria:

The minimum criterion considered necessary to demonstrate mutagenesis for any given treatment was a mutant frequency that was >= 2 times the concurrent background mutant frequency. The observation of a mutant frequency that meets the minimum criterion for a single treated culture within a range of assayed concentrations was not sufficient evidence to evaluate the test item as a mutagen.
A concentration-related or toxicity-related increase in mutant frequency should be observed.
The ratio of small : large colonies will be calculated from the results of the determination of small to large colonies.
If the test item is positive, the ratio of small to large colonies for the test item will be compared with the corresponding ratios of the positive and negative controls. Based on this comparison, the type of the mutagenic properties (i.e. basepair substitutions, deletions or large genetic changes frequently visible as chromosomal aberrations) of the test item will be discussed.
A test item is evaluated as non-mutagenic in a single assay only if the minimum increase in mutant frequency is not observed for a range of applied concentrations that extends to toxicity causing 10% to 20% relative growth or a range of applied concentrations extending to at least twice the solubility limit in culture media.

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the main study, cytotoxicity (decreased survival) was noted immediately after treatment (plating efficiency step 1) and in the following plating for 5-trifluoro-thymidine (TFT) resistance (plating efficiency step 2) in the presence and absence of metabolic activation at the top concentration of 200 µg/mL.
Cytotoxicity is defined as a reduction in the number of colonies by more than 50 % compared with the negative control. Exposure to the test item at the concentration of 200 µg/mL in the absence of metabolic activation resulted in relative survival of 28 % and 34 % (plating efficiency step 1) and 20 % and 33 % (plating efficiency step 2), and in the presence of metabolic activation of 28 % and 26 % (plating efficiency step 1) and 23 % and 17 % (plating efficiency step 2). Therefore, the test item was considered cytotoxic at the top concentration of 200 µg/mL.

No relevant change in pH and osmolality was noted.

Applicant's summary and conclusion

Conclusions:

Under the present test conditions, lithium hydroxide monohydrate, tested up to a pronounced cytotoxic concentration in the absence and presence of metabolic activation in two independent experiments, was negative with respect to the mutant frequency in the L5178Y TK +/- mammalian cell mutagenicity test. Under these conditions positive controls exerted potent mutagenic effects.
In addition, no change was noted in the ratio of small to large mutant colonies. Therefore, lithium hydroxide monohydrate also did not exhibit clastogenic potential at the concentration-range investigated.
According to the evaluation criteria for this assay, these findings indicate that lithium hydroxide monohydrate, tested up to a cytotoxic concentration in the absence and presence of metabolic activation did neither induce mutations nor had any chromosomal aberration potential.

Executive summary:

An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK +/- cells to test the potential of lithium hydroxide to cause gene mutation and/or chromosome damage according to OECD Guideline 476 and the EU method B.17. Lithium hydroxide monohydrate was assayed in a gene mutation assay in cultured mammalian cells (L5178Y TK +/-) both in the presence and absence of metabolic activation by a liver post-mitochondrial fraction (S9 mix) from Aroclor 1254-induced rats. The test was carried out employing 2 exposure times without S9 mix: 3 and 24 hours, and one exposure time with S9 mix: 3 hours; this experiment with S9 mix was carried out twice. The test item was dissolved in aqua ad iniectabilia. A correction factor of 1.73 was used. The dose levels and concentrations given in the text and tables refer to lithium hydroxide monohydrate. The limit of solubility was about 34 mg/mL. In the preliminary experiment without and with metabolic activation, concentrations tested were 0.25, 1, 2.5, 10, 25, 100 and 200 µg/mL. Cytotoxicity (decreased survival) was noted at the top concentration of 200 μg/mL. Hence, in the experiments without or with metabolic activation the concentrations of 12.5, 25, 50 100 and 200 µg/mL were used. In the main study, cytotoxicity (decreased survival) was noted immediately after treatment (plating efficiency step 1) and in the following plating for 5-trifluoro-thymidine (TFT) resistance (plating efficiency step 2) in the presence and absence of metabolic activation at the top concentration of 200 μg/mL. Methylmethanesulfonate was employed as positive control in the absence of exogenous metabolic activation and 3 -methylcholanthrene in the presence of exogenous metabolic activation. The mean values of mutation frequencies of the negative controls ranged from 61.61 to 98.34 per 106 clonable cells in the experiments without metabolic activation, and from 68.23 to 82.61 per 106 clonable cells in the experiments with metabolic activation and, hence, were well within the historical data range. The mutation frequencies of the cultures treated with lithium hydroxide monohydrate ranged from 64.74 to 92.63 per 106 clonable cells (3 hours exposure) and 50.42 to 92.34 per 106 clonable cells (24 hours exposure) in the experiments without metabolic activation and 75.88 to 105.59 per 106 clonable cells (3 hours exposure, first assay) and 45.04 to 99.10 per 106 clonable cells (3 hours exposure, second assay) in the experiments with metabolic activation. These results were within the range of the negative control values and, hence, no mutagenicity was observed according to the criteria for assay evaluation.

Under the present test conditions, lithium hydroxide monohydrate, tested up to a pronounced cytotoxic concentration in the absence and presence of metabolic activation in two independent experiments, was negative with respect to the mutant frequency in the L5178Y TK +/- mammalian cell mutagenicity test. Under these conditions positive controls exerted potent mutagenic effects. In addition, no change was noted in the ratio of small to large mutant colonies. Therefore, lithium hydroxide monohydrate also did not exhibit clastogenic potential at the concentration range investigated. According to the evaluation criteria for this assay, these findings indicate that lithium hydroxide monohydrate, tested up to a cytotoxic concentration in the absence and presence of metabolic activation did neither induce mutations nor had any chromosomal aberration potential.

Based on a read-across approach, results of this study are applied to lithium fluoride and it can therefore be concluded that lithium fluoride is negative with regard to mutant frequency in the absence and presence of metabolic activation. (LPT, 2010)