Lithium Citrate Tetrahydrate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In the MAK Value Documentation (2014) lithium citrate is reported to be not mutagenic with and without metabolic activation in the strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 of S. typhimurium up to a dose of 7137 µg/plate and in E. coli K12/343/113 up to a concentration of 2099 µg/mL.

Read-across with Lithium hydroxide (monohydrate)

OECD 471: Lithium hydroxide is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

OECD 476: Lithium hydroxide monohydrate was negative with respect to the mutant frequency in the L5178Y TK +/- mammalian cell mutagenicity test in the absence and  presence of metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2010-01-20 to 2010-07-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

NA

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase (TK)

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).

Additional strain / cell type characteristics:

not specified

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9-mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

with S9-mix

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 degree 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 degree C whereas the rest of the cells was incubated for 2 days at 37 ± 0.4 degree 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 suspensions 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 degree 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 is 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 a 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.

Statistics:

No data

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 ug/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 ug/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 ug/mL.

No relevant change in pH and osmolality was noted.

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 ug/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 ug/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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Please refer to attached "Read-across justification" in section 13.

Reason / purpose for cross-reference:

read-across source

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

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1999-11-25 to 2000-01-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 1997

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- lot/batch No.of test material: 27.05.97
- Expiration date of the lot/batch: 19 November 2000

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under test conditions: Stability of aqueous solution: at least 96 hrs

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- -> his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA 1535, TA 100) and frameshift (TA 1537, TA 98) mutations. The Escherichia coli WP2 uvrA (trp) reversion system measures trp– -> trp+ reversions. The Escherichia coli WP2 uvrA detect mutagens that cause other base-pair substitutions (AT to GC).

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

Lithium hydroxide was tested in concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate with and without S9 mix.

Vehicle / solvent:

None

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without S9: TA 1535

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without S9: TA 1537

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: daunimycine

Remarks:

Without S9: TA 98

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without S9: TA 100

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without S9: WP2uvrA

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With S9: TA 1537, TA 1535, TA 98, TA 100, E. coli WP2uvrA

Details on test system and experimental conditions:

The test substance was dissolved in Milli-Q-water. The test substance was ground and the stock solution was filter (0.22 µm)-sterilized. Test substance concentrations were prepared directly prior to use.

Range finding study:
Lithium Hydroxide was tested in the tester strains TA 100 and WP2uvrA with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and in the presence of S9 mix.

Mutation assay:
Based on the results of the dose range finding study, Lithium Hydroxide was tested up to concentrations of 5000 µg/plate in the absence and in the presence of S9-mix in two mutation experiments. The first mutation experiment was performed with the strains TA 1535, TA 1537 and TA 98 and the second mutation experiment was performed with the strains TA 1535, TA 1537, TA 98 TA 100 and WP2uvrA.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) It induces a number of revertant colonies, dose related, greater than two-times the number of revertants induced by the solvent control in any tester strains, either with or without metabolic activation.
However, any mean plate count of less than 20 is considered to be not significant.
b) The positive response should be reproducible in at least one independently repeated experiment.

Statistics:

Not indicated

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98 and 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

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

GENOTOXICITY:
Please refer to tables 1 and 2, which are presented under Sect. "Remarks on results including tables and figures"
- without metabolic activation: No increase in the number of revertants/plate observed
- with metabolic activation: No increase in the number of revertants/plate observed

CYTOTOXICITY:
No reduction of the bacterial background lawn was observed in all dose levels tested.

Experiment 1

Mutagenic response of lithium hydroxide in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay:

Dose (μg/plate)	Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain
	TA 1535	TA 1537	TA 98	TA 100	WP2uvrA
Without S9-mix
positive control	219±33	435±100	371±39	466±22	172±32
solvent control	12±6	7±3	16±3	65±2	9±2
3				77±13	12±3
10				72±7	11±3
33				79±8	8±2
100	12±1	7±5	18±3	74±8	12±3
333	15±1	7±1	16±4	65±7	7±3
1000	14±3	4±2	19±3	80±8	9±2
3330	11±3	8±3	12±4	77±13	5±2
5000	12±1	6±2	12±5	77±11	4±1
With S9-mix[1]
positive control	296±23	703±22	1346±230	1199±176	237±37
solvent control	14±6	6±2	26±3	90±10	11±3
3				96±4	12±3
10				99±5	12±4
33				95±9	9±3
100	11±3	6±3	31±4	78±11	12±2
333	15±1	4±1	27±9	101±5	10±1
1000	18±7	9±1	26±6	83±12	11±3
3330	15±6	5±1	25±3	86±14	7±4
5000	14±2	4±1	22±3	65±1	4±1

Solvent control: 0.1 mL Milli-Q water

[1]The S9-mix contained 5% (v/v) S9 fraction

Experiment 2

Mutagenic response of lithium hydroxide in the Salmonella typhimurium reverse mutation assay and in the escherichia coli reverse mutation assay:

Dose (μg/plate)	Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain
	TA 1535	TA 1537	TA 98	TA 100	WP2uvrA
Without S9-mix
positive control	195±2	287±105	642±125	608±24	696±16
solvent control	10±1	4±2	15±4	69±10	8±1
100	12±4	4±2	13±2	79±13	10±1
333	8±3	4±3	16±7	68±10	8±3
1000	12±5	5±2	15±2	70±7	11±2
3330	11±4	4±2	10±4	60±11	6±1
5000	7±1	5±2	11±3	64±8	7±3
With S9-mix[1]
positive control	203±14	367±45	551±25	709±131	70±8
solvent control	9±1	3±2	23±3	67±6	11±5
100	10±4	3±1	23±3	84±14	12±3
333	8±2	3±3	25±4	70±8	12±2
1000	9±4	6±3	22±6	68±11	7±2
3330	11±5	3±2	14±4	49±6	7±2
5000	5±3	3±2	13±2	54±8	3±1

 

Solvent control: 0.1 mL Milli-Q water

[1]The S9-mix contained 5% (v/v) S9 fraction

Conclusions:

Based on the results of this study it is concluded that lithium hydroxide is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

Lithium hydroxide was tested in the Salmonella typhimurium reverse mutation assay according to OECD Guideline 471. The test was performed with four histidine-requiring strains of Salmonella typhimurium (TA 1535, TA 1537, TA 100 and TA 98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli WP2uvrA in two independent experiments. Lithium hydroxide was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix. Lithium hydroxide did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at all concentrations tested. Reduction in the number of revertants was observed in the tester strain TA 1535, TA 98, TA 100 and WP2uvrA. Lithium hydroxide did not induce a dose-related, two-fold, increase in the number of revertant (His+) colonies in each of the four tester strains (TA 1535, TA 1537, TA 98 and TA 100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. Based on the results of this study it is concluded that lithium hydroxide is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

Please refer to attached "Read-across justification" in section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98 and 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

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537, TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

E. coli, other: K12/343/113

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

other: OECD 471; 279067, 2000

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537, TA 1538

Species / strain / cell type:

E. coli, other: K12/343/113

Metabolic activation:

with and without

Test concentrations with justification for top dose:

S. typhimurium TA, 98, TA 100, TA 1535, TA 1537, TA 1538: up to 7137 µg/plate (34 µmol/plate)
E. coli K12/343/113: up to 2099 µg/mL (10 mM)

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Key result

Species / strain:

S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537, TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

E. coli, other: K12/343/113

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Conclusions:

In the MAK Value Documentation (2014) lithium citrate is reported to be not mutagenic with and without metabolic activation in the strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 of S. typhimurium up to a dose of 7137 µg/plate and in E. coli K12/343/113 up to a concentration of 2099 µg/mL.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A genetic toxicity test with lithium citrate in bacteria is available. Further, read-across was applied using the structural analogue substance, lithium hydroxide.

 

In vitro assay with bacteria

Read-across with lithium hydroxide (NOTOX B.V, 279067, 1999)

Lithium hydroxide was tested in the Salmonella typhimurium reverse mutation assay according to OECD Guideline 471. The test was performed with four histidine-requiring strains of Salmonella typhimurium (TA 1535, TA 1537, TA 100 and TA 98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli WP2 uvr A in two independent experiments. Lithium hydroxide was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix. Lithium hydroxide did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at all concentrations tested. Reduction in the number of revertants was observed in the tester strain TA 1535, TA 98, TA 100 and WP2 uvr A at the limit concentration of 5000 µg/plate, indicating some bacterial toxicity. Lithium hydroxide did not induce a dose-related, two-fold, increase in the number of revertant (His+) colonies in each of the four tester strains (TA 1535, TA 1537, TA 98 and TA 100) and in the number of revertant (Trp+) colonies in the tester strain WP2 uvr A both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. Based on the results of this study it is concluded that lithium hydroxide is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

 

Additionally, a study with lithium citrate is available, which is cited in the MAK Value Documentation (2014) for lithium compounds. The test substance was exposed to Salmonella typhimurium strains TA 98, Ta 100, TA 1535, TA 1537, TA 1538 and Escherichia coli K12/343/1113 in concentrations up to 7137 µg/plate and 2099 µg/mL, respectively. Based on the results, the test substances is not mutagenic in the S. typhimurium reverse mutation assay and in the E. coli reverse mutation assay with and without metabolic activation.

 

In vitro assay with mammalian cells

According to ECHA Guidance Document Chapter R.7a (July 2017) bacterial mutagenesis assays of inorganic metal compounds are frequently negative due to limited capacity for uptake of metal ions and/or the induction of large DNA deletions by metals in bacteria potentially leading to an increased death rate in mutants. The high prevalence of false negatives for metal compounds might suggest that mutagenesis assays with mammalian cells, as opposed to bacterial cells, would be the preferred starting point for testing for this class of Annex VII substances. Thus, an in vitro mammalian cell assay with the read-cross substance lithium hydroxide monohydrate was included.

Read-across with lithium hydroxide monohydrate (FMC, Albemarle, 25361, 2010) 

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. 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. 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 10^6 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.

Methylmethanesulfonate was employed as positive control in the absence of exogenous metabolic activation and 3-Methylcholanthrene in the presence of exogenous metabolic activation and indicated that the test conditions were adequate and that the metabolic activation system functioned properly.

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. 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.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008:

The available experimental test data with the substance lithium citrate and read-across data with lithium hydroxide (monohydrate) are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genetic toxicity, lithium citrate tetrahydrate is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.