Lithium chloride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The in vitro tests performed according to the respective OECD TG and GLP (bacteria reverse mutation test, chromosome aberration test, mouse lymphoma cell mutation test) did not indicate a genotoxic potential for lithium chloride. However, positive results were reported in some of the published non-GLP studies, like a dose-related increase in micronuclei in CHO cells, most likely due to aneugenicity and possibly influenced by cytotoxicity (Pastor et al., 2009).

As a concern for genotoxicity cannot be fully ruled out and there are no acceptable in vivo tests a combined in vivo micronucleus test (OECD TG 474) combined with the in vivo comet assay (OECD TG 489) is proposed.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

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

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:

TA 1535 without S9 Migrated to IUCLID6: NaN3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA 1537 without S9 Migrated to IUCLID6: 9AA

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: daunomycine (DA)

Remarks:

TA 98 without S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

TA 100 without S9 Migrated to IUCLID6: MMS

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

WP2uvrA without S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2-AA)

Remarks:

TA 1537, TA 1535, TA 98, TA 100, E. coli WP2uvrA with S9

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:

S. typhimurium TA 100

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 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

Positive controls validity:

valid

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

Positive controls validity:

valid

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

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.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

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:

Interpretation of results (migrated information):
negative with and without metabolic activation

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:

A Bacteria Reverse Mutation test with Lithium chloride is not available. Consequently, read across was applied using study results obtained from Lithium hydroxide.

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 tryptophane-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 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000-04-06 to 2000-08-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

July 1999

Principles of method if other than guideline:

NA

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

mammalian cell line, other: human lymphocytes

Details on mammalian cell type (if applicable):

Stimulated cultured human lymphocytes were used because they are sensitive indicators of clastogenic activity of a broad range of chemical classes.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix of Aroclor 1254 induced rat liver

Test concentrations with justification for top dose:

Dose range finding test:
10, 33, 100, 133, 1000 µg/mL with and without S9 mix;

Chromosome aberrations:
Without S9 mix: 275, 300, and 530 µg Lithium Hydroxide/mL culture medium (24 h treatment, 24 h fixation time);
350, 375 and 400 µg Lithium Hydroxide/mL culture medium (48 h treatment, 48 h fixation time),
With S9 mix: 400, 425 and 450 µg lithium Hydroxide/mL culture medium (3 h treatment, 48 h fixation time);

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Remarks:

MMC-C

Positive control substance:

mitomycin C

Remarks:

without S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Remarks:

CP

Positive control substance:

cyclophosphamide

Remarks:

with S9 mix

Details on test system and experimental conditions:

Cytogenetic assay:
Lithium Hydroxide was tested in the absence and presence of 1.8 % (v/v) S9-fraction in duplicate in two independent experiments.

Experiment 1:
Lymphocyte cultures (0.4 mL blood of a healthy male donor was added to 5 mL or 4.8 mL culture medium, without and with metabolic activation respectively and 0.1 mL (9 mg/mL) Phytohaemagglutinin) were cultured for 48 h and thereafter exposed in duplicate to selected doses of Lithium Hydroxide for 3 h in the absence and presence of S9-mix.
After 3 h treatment, the cells exposed to Lithium Hydroxide were rinsed once with 5 mL of HBSS and incubated in 5 mL of culture medium for another 20-22 h (24 h fixation time).
Based on the mitotic index of the dose range finding test and the first cytogenetic assay appropriate dose levels were selected for the second cytogenetic assay The independent repeat was performed with the following modification of experimental conditions.

Experiment 2:
Lymphocyte cultures (0.4 mL blood of a healthy male donor was added to 5 mL or 4.8 mL culture medium, without and with metabolic activation respectively and 0.1 mL (9 mg/mL) Phytohaemagglutinin) were cultured for 48 h and thereafter exposed in duplicate to selected doses of Lithium Hydroxide for 3 h in the absence and presence of S9-mix.
After 3 h treatment, the cells exposed to Lithium Hydroxide in the presence of S9-mix were rinsed once with 5 mL of HBSS and incubated in 5 mL of culture medium for another 44-46 h (48 h fixation time).
The cells which were treated for 24 and 48 h in the absence of S9-mix were not rinsed after treatment but were worked up immediately after 24 h and 48 h (24 h and 48 h fixation time).

Chromosome preparation:
During the last 3 h of the culture period, cell division was arrested by the addition of the spindle inhibitor colchicine (0.5 µg/mL medium). Thereafter the cell cultures were centrifuged for 5 min at 1300 rpm (150 g) and the supernatant was removed. Cells in the remaining cell pellet were swollen by a 5 min treatment with hypotonic 0.56 % (w/v) potassium chloride solution at 37 °C. After hypotonic treatment, cells were fixed with 3 changes of methanol:acetic acid fixative (3:1 v/v).

Preparation of slides:
Fixed cells were dropped onto cleaned slides which were immersed for 24 h in a 1:1 mixture of 96 % (v/v) ethanol/ether and cleaned with a tissue. The slides were marked with the study identification number and group number. Two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 5 % (v/v) Giemsa solution in tap water.
Thereafter the slides were rinsed in tap-water and allowed to dry. The dry slides were cleared by dipping them in xylene before they were embedded in MicroMount and mounted with a coverslip.

Mitotic index/dose selection for scoring the cytogenetic assay:
The mitotic index of each culture was determined by counting the number of metaphases per 1000 cells. At least three analysable concentrations were used. Chromosomes of metaphase spreads were analysed of those cultures with an inhibition of the mitotic index of about 50 % or greater whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control. Also cultures treated with an intermediate dose were examined for chromosome aberrations.

Analysis of slides for chromosome aberrations:
To prevent bias, all slides were randomly coded before examination of chromosome aberrations and scored. An adhesive label with study identification number and code was stuck over the marked slide. At least 100 metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations. In case the number of aberrant cells, gaps excluded, was >= 25 in 50 metaphases no more metaphases were examined. Only metaphases containing 46 chromosomes were analysed. The number of cells with aberrations and the number of aberrations were calculated.

Evaluation criteria:

A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations.
b) a statistically significant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, P < 0.05) in crease in the number of cells with chromosome aberrations.

The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Statistics:

The incidence of aberrant cells (cells with one or more chromosome aberrations, inclusive or exclusive gaps) for each treatment group was compared to that of the solvent control using Chi-square statistics:

X*2 = (N-1)x(ad-bc)*2/(a+b)(c+d)(a+c)(b+d)

where b = the total number of aberrant cells in the control cultures,
d = the total number of non aberrant cells in the control cultures,
n0 = the total number of cells scored in the control cultures,
a = the total number of aberrant cells in treated cultures to be compared with the control,
c = the total number of non aberrant cells in treated cultures to be compared with the control,
n1 = the total number of cells scored in the treated cultures,
N = sum of n= and n1

If P [ X*2 > (N-)x(ad-bc)*2/(a+b)(c+d)(a+c)(b+d)] (two-tailed) is small (P < 0.05) the hypothesis that the incidence of cells with chromosome aberrations is the same for both the treated and the solvent group is rejected and the number of aberrant cells in the test group is considered to be significantly different from the control group at the 95 % confidence level.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1000 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Dose range finding test:
Lithium Hydroxide precipitated in the culture medium at a concentration of 1000 µg/mL, therefore a concentration of 1000 µg/mL was used as the highest concentration of Lithium Hydroxide.
In the dose range finding test, blood cultures were treated with 10, 33, 100, 333 and 1000 µg Lithium Hydroxide per mL culture medium with and without S9-mix.
The pH of a concentration of 1000 mg Lithium Hydroxide/mL was 11.83 (compared to 8.15 in the solvent control).

Cytogenetic assay:
Based on the results of the dose range finding test the following dose levels were selected for the cytogenetic assay:

Experiment 1A:
Without S9-mix: 100, 180, 333, 420 and 560 µg Lithium Hydroxide/mL culture medium (3 h treatment time, 24 h fixation time);
With S9-mix: 100, 333, 420 and 560 µg Lithium Hydroxide/mL culture medium (3h treatment, 24 h fixation time);
Lithium Hydroxide precipitated in the culture medium at a concentration of 560 µg/mL, therefore a concentration of 560 µg/mL was used as the highest concentration of Lithium Hydroxide in the first cytogenetic assay.
Since the highest dose level of 560 µg Lithium Hydroxide/mL was too cytotoxic to the cells (mitotic index of 21 % both in the absence and in the presence of S9-mix) and no dose level resulting in a mitotic index of 50 % could be selected in both the absence and presence of S9-mix, an additional experiment was performed with the following dose levels:

Experiment 1B:
With and without S9-mix: 300, 350, 400. 450, 500 and 550 µg Lithium Hydroxide/mL culture medium (3 h treatment, 24 h fixation time);
Because of the high cytotoxicity in cultures treated with 350 µg/mL Lithium Hydroxide and upwards in the presence and absence of S9-mix, the test was not used for evaluation but a third experiment was performed with the following dose levels:

Experiment 1C:
With and without S9-mix: 275, 300, 325, 350, 375, 400, 425, 450, 475 and 500 µg Lithium Hydroxide/mL culture medium (3 h treatment, 24 h fixation time).

Despite the narrow concentration range used, the mitotic index of cultures treated with 375 and 400 µg/mL Lithium Hydroxide (without S9-mix) drastically decreased from 128 % to 0 %. In the presence of S9-mix, cytotoxicity was observed at a concentration of 375 µg/mL Lithium Hydroxide and upwards.
The pH of the concentrations 275, 300, 325, 350, 375, 400 and 425 µg/mL was 9.61, 9.69, 9.66, 9.68, 9.66, 9.80 and 10.19, respectively. Possibly these high pH values also play a role in the cytotoxicity of Lithium Hydroxide.

Since it was not possible to determine a concentration which caused the appropriate 50 % inhibition of the mitotic index, the following doses were selected for scoring of chromosome aberrations:
From experiment 1A:
With and without S9-mix: 333, 420 and 560 µg Lithium Hydroxide/mL (3 h treatment, 24 h fixation time);

From experiment 1C:
Without S9-mix: 325, 350, and 375 µg lithium Hydroxide/mL culture medium (3 h treatment time, 24 h fixation time);
With S9-mix: 325, 350, 375, and 400 µg Lithium Hydroxide/mL culture medium (3 h treatment, 24 h fixation time);
For cultures with S9-mix four doses were selected, since only one of the duplicate cultures contained scorable metaphases at concentrations of 375 and 400 µg/mL Lithium Hydroxide.

Based on the results of the dose range finding test and experiments 1A, 1B and 1C the following dose levels were selected to perform an independent repeat:

Experiment 2:
Without S9-mix: 275, 300, 325, 350, 375, 400 and 425 µg Lithium Hydroxide/mL culture medium (24 and 48 h treatment time, 24 and 48 h fixation time);
With S9-mix: 350, 375, 400, 425, 450, 475, 500 and 525 µg Lithium Hydroxide/mL culture medium (3 h treatment time, 48 h fixation time).
Based on these observations the following doses were selected for scoring of chromosome aberrations:
Without S9-mix: 275, 300 and 350 µg Lithium Hydroxide/mL culture medium (24 h treatment time, 24 h fixation time);
350, 375 and 400 µg Lithium Hydroxide/mL culture medium (48 h treatment time, 48 h fixation time),
With S9-mix: 400, 425 and 450 µg Lithium Hydroxide/mL culture medium (3 h treatment time, 48 h fixation time).

Evaluation of the results:
The ability of Lithium Hydroxide to introduce chromosome aberrations in human peripheral lymphocytes was investigated. The test was carried out in duplicate in three independent experiments.
The number of cells with chromosome aberrations found in the solvent control cultures were within the laboratory historical control data range {min = 0, max = 5 (mean = 0.8, standard deviation = 1.0) aberrant cells per 100 metaphases in the absence of S9-mix; gaps excluded and min = 0 max = 5 (mean = 0.8, standard deviation = 0.9)aberrant cells per 100 metaphases in the presence of S9-mix, gaps excluded}.
The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Experiments 1A and 1C:
Due to the steepness of the dose response curve for cytotoxicity of Lithium Hydroxide it was not possible to determine the number of chromosomal aberrations at a mitotic index of 50 %. Therefore, chromosome aberrations were scored from two independent experiments (experiment 1A and 1C) at different concentrations. As a result of extreme cytotoxicity, only 102 and 103 metaphases could be scored in the absence and presence of S9-mix respectively, in experiment 1A at a concentration of 560 µg/mL Lithium Hydroxide. At the other concentrations tested, 200 metaphases were scored per concentration. In experiment 1C in the presence of S9-mix at the highest concentrations of 375 and 400 µg/mL only one of the two duplicate cultures could be scored due to extreme cytotoxicity.
Both in the absence and presence of S9-mix Lithium Hydroxide did not induce a statistically or biologically significant increase in the number of cells with chromosome aberrations in both experiments 1A and 1C.

Experiment 2:
In the absence of S9-mix, at the 24 hours continuous treatment time, Lithium Hydroxide induced statistically significant increases in the number of cells with chromosome aberrations at the lowest tested concentration of 275 µg/mL (only when gaps were included) and at the highest cytotoxic concentration of 350 µg/mL both when gaps were included and excluded. At the intermediate concentration of 300 µg/mL Lithium Hydroxide did not induce a statistically significant increase in the number of cells with chromosome aberrations.
Since the increase of chromosome aberrations at 275 µg/mL was observed only when gaps were included and furthermore the increase was within the historical control data range the increase was not considered biologically relevant.

Scoring of the additional 200 metaphases at the concentration of 350 µg/mL Lithium Hydroxide verified the statistically significant increase. However, the observed increase within or just on the border of our historical control data range (min = 0, max = 5 aberrant cells per 100 metaphases, gaps excluded), and is observed at a very toxic concentration. In addition, higher concentrations tested at the prolonged treatment time of 48 hours in the absence of metabolic activation did not induce significant increases in the number of cells with chromosome aberrations. Furthermore, the irregular toxicity profile and the non-physiological test conditions (pH > 9) may be considered cofounding factors. Therefore, the observed increase in the number of aberrant cells at the concentration of 350 µg/mL is considered not biologically relevant.

At the continuous treatment time of 48 hours exposure of cells to 350, 375 or 400 µg/mL Lithium Hydroxide did not induce a significant increase in the number of cells with chromosome aberrations.

In the presence of S9-mix, Lithium Hydroxide did not induce a statistically or biologically significant increase in the number of cells with chromosome aberrations.

Conclusion:
Finally, it is concluded that this test is considered valid and that Lithium Hydroxide is not clastogenic under the experimental conditions of this test.

No other information

Conclusions:

Interpretation of results (migrated information):
negative

The effect of Lithium Hydroxide on the induction of chromosome aberrations in culture peripheral human lymphocytes in the presence and absence of a metabolic activation system (Aroclor-1254 induced rat liver S9-mix) was investigated. It is concluded that this test is considered valid and that Lithium Hydroxide is not clastogenic under the experimental conditions of this test.

Executive summary:

A Chromosome Aberration test with Lithium carbonate is not available. Consequently, read across was applied using study results obtained from Lithium hydroxide.

The effect of Lithium Hydroxide on the induction of chromosome aberrations in culture peripheral human lymphocytes in the presence and absence of a metabolic activation system (Aroclor-1254 induced rat liver S9-mix) was investigated according to OECD Guideline 473 and EU method B.10.  

In the absence of S9-mix Lithium Hydroxide was tested up to 560 µg/mL for a 3 h treatment time with a 24 h fixation time in experiment 1A and up to 375 µg/mL in experiment 1C. In the second experiment Lithium Hydroxide was tested up to 350 µg/mL for a 24 hours continuous treatment time and up to 400 µg/mL for a 48 hours continuous treatment time.  

In the presence of 1.8 % (v/v) S9-fraction Lithium Hydroxide was tested up to 560 ug/mL for a 3 h treatment time with a 24 h fixation time in experiment 1A and up to 400 µg/mL in experiment 1C. In the second experiment Lithium Hydroxide was tested up to 450 ug/mL for a 3 h treatment time with a 48 h fixation time.  

Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.  

Experiment 1A and 1C:

Both in the absence and presence of S9-mix Lithium Hydroxide did not induce a statistically or biologically significant increase in the number of cells with chromosome aberrations in both experiments 1A and 1C.  

Experiment 2:

In the absence of S9-mix, at the 24 hours continuous treatment time, Lithium Hydroxide induced statistically significant increases in the number of cells with chromosome aberrations at the lowest tested concentration of 275 ug/mL (only when gaps were included) and at the highest cytotoxic concentration of 350 µg/mL both when gaps were included and excluded. At the intermediate concentration of 300 ug/mL Lithium Hydroxide did not induce a statistically significant increase in the number of cells with chromosome aberrations.

Since the increase of chromosome aberrations at 275 ug/mL was observed only when gaps were included and furthermore the increase was within the historical control data range and revealed no dose-response-relationship, the increase was not considered biologically relevant.  

Scoring of the additional 200 metaphases at the concentration of 350 ug/mL Lithium Hydroxide verified the statistically significant increase. However, the observed increase within or just on the border of the historical control data range (min = 0, max = 5 aberrant cells per 100 metaphases, gaps excluded), and is observed at a very toxic concentration. In addition, higher concentrations tested at the prolonged treatment time of 48 hours in the absence of metabolic activation did not induce significant increases in the number of cells with chromosome aberrations. Furthermore, the irregular toxicity profile and the non-physiological test conditions (pH > 9) may be considered cofounding factors. Therefore, the observed increase in the number of aberrant cells at the concentration of 350 ug/mL is considered not biologically relevant.

At the continuous treatment time of 48 hours exposure of cells to 350, 375 or 400 ug/mL Lithium Hydroxide did not induce a significant increase in the number of cells with chromosome aberrations.  

In the presence of S9-mix, Lithium Hydroxide did not induce a statistically or biologically significant increase in the number of cells with chromosome aberrations.  

Finally, it is concluded that this test is considered valid and that Lithium Hydroxide is not clastogenic under the experimental conditions of this test.

   

 

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

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:

positive control for non-activation mutation studies.

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 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

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.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

No other information

Conclusions:

Interpretation of results (migrated information):
negative

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:

A Mammalian cell gene mutation test with Lithium chloride is not available. Consequently, read across was applied using study results obtained from Lithium hydroxide.

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro assays in bacteria

A bacteria reverse mutation test according to OECD Test Guideline (TG) and GLP principles with lithium chloride is not available. Read across was applied using study results obtained from lithium hydroxide: Lithium hydroxide was tested in the Salmonella typhimurium reverse mutation assay according to OECD TG 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 tryptophane-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 at the limit concentration of 5000 ug/plate, indicating some bacteriotoxicity. 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.

A non-GLP, bacterial reverse mutation test was carried out using lithium chloride (purity not provided), using the four strains of Salmonella typhimurium TA 1535, TA1537, TA 98, TA 100 both in the presence and absence of S9-metabolic activation up to a concentration of 10000 µg lithium chloride/mL (Haworth S. et al., 1983). Concurrent positive controls were used. The test method was similar to OECD TG 471 and the test was rated as Klimisch (KL) score 2. The results were negative for mutagenicity in all the tested strains with and without metabolic activation.

Ref: Haworth S, Lawlor T, Mortelmans K, Speck W, Zeiger E (1983) Salmonella mutagenicity test results for 250 chemicals. Environ Mutagen 5, Suppl 1: 3–142.

 

In vitro assays in mammalian cells according to OECD guideline and GLP principles

In vitro chromosome aberration test

A chromosome aberration test according to OECD TG and GLP principles with lithium chloride is not available. Read across was applied using study results obtained from lithium hydroxide: The effect of lithium hydroxide on the induction of chromosome aberrations in culture peripheral human lymphocytes in the presence and absence of a metabolic activation system (Aroclor-1254 induced rat liver S9-mix) was investigated according to OECD TG 473 and EU method B.10 (NOTOX, 2000). In the absence of S9-mix lithium hydroxide was tested up to 560 µg/mL for a 3 h treatment time with a 24 h fixation time in experiment 1A and up to 375 µg/mL in experiment 1C. In the presence of 1.8 % (v/v) S9-fraction lithium hydroxide was tested up to 560 µg/mL for a 3 h treatment time with a 24 h fixation time in experiment 1A and up to 400 µg/mL in experiment 1C. In both experiments 1A and 1C, lithium hydroxide did not induce a statistically or biologically significant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix.

In experiment 1B lithium hydroxide was tested at 300, 350, 400, 450, 500 and 550 µg/mL culture medium with and without S9-mix (3 h treatment, 24 h fixation time). Because of the high cytotoxicity in cultures treated with 350 µg/mL lithium hydroxide and upwards in the presence and absence of S9-mix, the test was not used for evaluation but a further experiment (1C) was performed.

In the second experiment lithium hydroxide was tested up to 350 µg/mL for a 24 hours continuous treatment time and up to 400 µg/mL for a 48 hours continuous treatment time (both without S9-mix). In the second experiment lithium hydroxide was tested up to 450 µg/mL for a 3 h treatment time with a 48 h fixation time with S9-mix. In the absence of S9-mix, at the 24 hours continuous treatment time, lithium hydroxide induced statistically significant increases in the number of cells with chromosome aberrations at the lowest tested concentration of 275 ug/mL (only when gaps were included) and at the highest cytotoxic concentration of 350 µg/mL both when gaps were included and excluded. At the intermediate concentration of 300 µg/mL lithium hydroxide did not induce a statistically significant increase in the number of cells with chromosome aberrations. Since the increase of chromosome aberrations at 275 µg/mL was observed only when gaps were included and furthermore the increase was within the historical control data range and revealed no dose-response-relationship, the increase was not considered biologically relevant. At the continuous treatment time of 48 hours exposure of cells to 350, 375 or 400 µg/mL lithium hydroxide did not induce a significant increase in the number of cells with chromosome aberrations. In the presence of S9-mix, lithium hydroxide did not induce a statistically or biologically significant increase in the number of cells with chromosome aberrations.

Finally, it is concluded that this test is considered valid and that lithium hydroxide is not clastogenic under the experimental conditions of this test. Positive control chemicals mitomycin C and cyclophosphamide indicated that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

In vitro gene mutation assay

A mammalian cell gene mutation test according to OECD TG and GLP principles with lithium chloride is not available. Read across was applied using study results obtained from lithium hydroxide monohydrate: 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 TG 476 and the EU method B.17 (LPT, 2010). 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 10^6 clonable cells in the experiments without metabolic activation, and from 68.23 to 82.61 per 10^6 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 10^6 clonable cells (3 hours exposure) and 50.42 to 92.34 per 10^6 clonable cells (24 hours exposure) in the experiments without metabolic activation and 75.88 to 105.59 per 10^6 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.

 

Non-GLP in vitro and in vivo assays were reported in the CLH report Proposal for Harmonised Classification and Labelling Based on Regulation (EC) No 1272/2008, Annex VI, Part 2 for lithium carbonate, lithium chloride and lithium hydroxide.

In vitro studies

In vitro gene mutation

A non-GLP gene mutation assay (HGPRT) was cited in Slameňová, et al., 1986. The test method was similar to OECD TG 476 and the study was given the KL score rating of 2. In this test, lithium carbonate (purity not provided) was tested using V79 cells at test concentrations 0, 1500, 2000, 2500, 3000 µg/mL (282-564 µg Li/mL) both in the presence and absence of S9 metabolic activation. Expression time was 6 and 8 days. An untreated control was included in the study and the results were compared to mutagenic activity of B(a)P. No positive control was used. Cytotoxicity was observed at the highest concentration (564 µg Li/mL). In the absence of S9, average number of 6-TG mutants per 100000 cells were partially increased at the respective dose groups as follows: 0.2 (control group), 0.3, 1.1 (weak effect), 0.4 (very weak effect), 0.4. In the presence of S9, average number of 6-TG mutants per 100000 cells were partially increased at the respective dose groups as follows: 0.4 (control group), 0.2, 0.1, 0.9 (very weak effect), 0.8. Increased mutation rates reported for lithium carbonate in the HGPRT-assay by Slameňová et al. (1986) revealed no dose response relationship.

Ref: Slameňová, D.; Budayová, E.; Gábelová, A.; Morávková, A.; Pániková, L. (1986) Results of genotoxicity testing of mazindol (degonan), lithium carbonicum (contemnol) and dropropizine (ditustat) in Chinese hamster V79 and human EUE cells Mutation Research - Genetic Toxicology, 169, 171-177

In vitro comet assay (DNA strand breaks)

In a non-GLP study, lithium chloride and lithium carbonate (purity not provided) were tested in a Comet assay using AA8 CHO cells at the concentrations of 1-20 mM (42.4-848 µg/mL; 7-140 µg Li/mL) and 1-10 mM (73.9 - 739 µg/mL; 13.9-139 µg Li/mL) respectively, using either 3 h or 24 h treatment in the absence of S9 metabolic activation. Untreated and positive controls were included. Results were expressed as tail moment. Cytotoxicity was observed at concentrations ≥ 70 µg Li/mL. The test guideline was not mentioned but the study was performed according to the original protocol of Singh et al. (1988), and was rated as KL score 2. Under the conditions of the study, the test substance was tested negative.

Ref: Pastor, N.; Kaplan, C.; Domínguez, I.; Mateos, S.; Cortés, F. (2009) Cytotoxicity and mitotic alterations induced by non-genotoxic lithium salts in CHO cells in vitro Toxicology In vitro, 23, 432-438

In another non-GLP DNA strand breaks (alkaline elution) assay cited in Slameňová, et al., 1986, lithium carbonate (purity not provided) was tested using Human EUE cells at concentrations of 150, 250, 500 µg/mL (28-94 µg Li/mL), 1 h treatment in the absence of S9 metabolic activation. The test guideline followed was not mentioned and the study was rated as KL score 2. No information on controls used are given. The test substance was positive (- S9 mix) at the highest concentration tested (94 µg Li/mL). Concentrations higher than 900 µg/mL lithium carbonate (169 µg Li/mL) caused an extensive loss of EUE cells.

Ref: Slameňová, D.; Budayová, E.; Gábelová, A.; Morávková, A.; Pániková, L. (1986) Results of genotoxicity testing of mazindol (degonan), lithium carbonicum (contemnol) and dropropizine (ditustat) in Chinese hamster V79 and human EUE cells Mutation Research - Genetic Toxicology, 169, 171-177.

In vitro micronucleus assay

A non-GLP in vitro micronucleus (MN) study was cited in Pastor et al., 2009. In this test, lithium chloride and lithium carbonate (purity not provided) at concentrations of 5-20 mM lithium chloride (212-848 μg LiCl/mL, 35-139 µg Li/mL) and 2.2-10 mM lithum carbonate (163-739  µg Li2CO3/mL, 31-139 µg Li/mL) were tested using AA8 CHO cells. Cells were treated for 3 h in the absence of S9 metabolic activation. Untreated and vehicle controls were included. No positive control was used. The test method was similar to OECD TG 487 and was rated as KL score 2. Two thousand binucleated cells from both control and lithium-treated preparations were scored blind, and classified as normal or micronucleated cells. All the experiments were carried out in triplicate. Cytotoxicity was observed at concentrations ≥ 70 µg Li/mL. Under the test conditions, a concentration-dependent induction of micronuclei by lithium chloride and lithium carbonate was observed,  most of them kinetochore positive.

Ref: Pastor, N.; Kaplan, C.; Domínguez, I.; Mateos, S.; Cortés, F. (2009) Cytotoxicity and mitotic alterations induced by non-genotoxic lithium salts in CHO cells in vitro Toxicology In vitro, 23, 432-438.

In vitro Chromosome aberration assays

A non-GLP in vitro chromosome aberration assay was cited in Pastor et al., 2009. In this test, lithium chloride and lithium carbonate (purity not provided) at concentrations of 1-30 mM (42.4-1272 µg LiCl/mL; 7-209 µg Li/mL and 73.9-2217 µg Li2CO3/mL; 13.9-417 µg Li/mL, respectively) were tested using AA8 CHO cells. The cells were treated for 3 h following a 12 h growth phase in the absence of S9 metabolic activation. Negative and positive controls were included. The test method was similar to OECD TG 473 and was rated as KL score 2. 200 metaphases were examined and the experiments were carried out in triplicate. Cytotoxicity was observed at concentrations ≥ 70 µg Li/mL. Under the test conditions, test substance was negative (- S9 mix) for chromosome aberrations.

Ref: Pastor, N.; Kaplan, C.; Domínguez, I.; Mateos, S.; Cortés, F. (2009) Cytotoxicity and mitotic alterations induced by non-genotoxic lithium salts in CHO cells in vitro Toxicology In vitro, 23, 432-438.

In a non-GLP chromosome aberration study by De La Torre, 1976, lithium chloride (purity not provided) was tested in Phytohemagglutinin-stimulated lymphocyte cultures of a healthy human donor at concentrations of 0, 50, 100, 150 µg lithium chloride/mL (8.2-25 µg Li/mL). An untreated control was included. The test method was similar to OECD TG 473 and was rated as KL score 2. Metabolic activation was not mentioned, and a positive control was not used. Under the test conditions, the test substance was positive for chromosome aberrations: increase in breaks (7.9%, 4.5%, 10.9% compared to 1.2% in the control) and gaps (14.4%, 14%, 20.5% compared to 0.8% in the control) in all groups, increased deletion from 100 µg (2.2%, 4.2%) and translocation (0.6%, 1.7%) was observed.

Ref: De La Torre, R.; Krompotic, E.; Kowlessar, L. (1976) The in vivo and in vitro effects of lithium on human chromosomes and cell replication Teratology, 13, 131-138.

No increase in structural chromosome aberrations in peripheral human blood lymphocytes were seen in a non-GLP study after treatment with lithium carbonate for 72 h with concentrations equivalent to 0.1, 1.0 and 10 g lithium carbonate distributed in the body of a 70 kg person (no information on number of cells screened, no positive controls). The study was rated as KL 4.

Ref: Timson, J.; Price, D.J. (1971) Lithium and mitosis The Lancet, 2, 93.

In vitro anaphase anomaly study

In a non-GLP in vitro anaphase anomaly study, lithium chloride (purity not provided) and lithium carbonate (puritiy not provided) up to 10 mM (739 µg/mL; 139 µg Li/mL) were tested using AA8 CHO cells. Cells were treated for 3 h in the absence of S9 metabolic activation. Details of concentrations and number of cells evaluated were not provided. Negative controls were included. After treatment, cells in anaphase were analysed for any alterations of normal chromosome segregation. No test guideline was mentioned, and the test was given a KL score of 2. Cytotoxicity was observed at concentrations ≥ 70 µg Li/mL. Under the test conditions, the test substances were tested positive with anomalous anaphases: multipolar anaphases and lagging chromosome.

Ref: Pastor, N.; Kaplan, C.; Domínguez, I.; Mateos, S.; Cortés, F. (2009) Cytotoxicity and mitotic alterations induced by non-genotoxic lithium salts in CHO cells in vitro Toxicology In vitro, 23, 432-438.

 

In vivo studies

Chromosome aberration studies

In a non-GLP in vivo chromosome aberration (CA) assay in Sobti et al., 1989, lithium chloride (purity not provided) was tested using mice (Lacca strain) at doses of 0, 0.212, 2.125, 21.25 mg/kg bw (0, 0.035, 0.35, 3.5 mg Li/kg bw) by single gavage (vehicle olive oil) 72 h before bone marrow preparation. Bone marrow cells were assessed for aberrations and positive results were seen at the respective doses as follows: mean CAs 2.66, 9.0, 10.0, 14.66 in the respective dose groups. In the same study lithium carbonate (purity not provided) was tested at doses of 0, 1.2, 12, 120 mg/kg bw (0, 0.23, 2.3, 23 mg Li/kg bw) by single gavage (vehicle olive oil) 72 h before bone marrow preparation. Bone marrow cells were assessed for aberrations and positive responses were seen without clear dose response with mean CAs of 5.66, 14.0, 13.0, 20.33 in the respective dose groups.

The test guideline followed was not mentioned. Number of animals per dose and cells per animal studied were not provided. No positive control was included and negative control values were higher than in other published reports. The study was rated as KL score 3.

Ref: Sobti, R.C.; Sharma, M.; Gill, R.K. (1989) Frequency of sister chromatid exchanges (sces) and chromosome aberrations (cas) caused by three salts of lithium (in vivo) Cytologia, 54, 245-248.

In a non-GLP in vivo sister chromatid exchange assay, lithium chloride (purity not provided) was administered at doses of 0, 0.212, 2.125, 21.25 mg/kg bw (0, 0.035, 0.35, 3.5 mg Li/kg bw) to mice (Lacca strain) by single gavage (vehicle olive oil) 72 h before bone marrow preparation. Bone marrow cells were assessed, and the test substance was negative. In the same study, lithium carbonate (purity not provided) was administered at doses of 0, 1.2, 12, 120 mg/kg bw by single gavage (vehicle olive oil) 72 h before bone marrow preparation. Bone marrow cells were assessed, and the test substance was negative. Only slight increases compared to control were observed, but these were not statistically significant. The test guideline followed was not mentioned, and the study was rated as KL score 3: Number of animals per dose and cells per animal studied not provided, no positive control included and negative control values higher than in other published reports.

Ref: Sobti, R.C.; Sharma, M.; Gill, R.K. (1989) Frequency of sister chromatid exchanges (sces) and chromosome aberrations (cas) caused by three salts of lithium (in vivo) Cytologia, 54, 245-248.

In a non-GLP study in vivo chromosome aberration study, lithium (no further details) was administered intraperitoneally to female Lister rats at a test dose of 86 mg/day for three days. Rats were sacrificed at 12 and 24 hours after the last injection and the bone marrow cells were evaluated for chromosome aberrations. The values from the treated animals did not differ from the control values. The guideline followed was not mentioned and the study was rated as KL score 3: Very few details given on method used, no control was used.

Ref: Bille, P.E.; Jensen, M.K.; Kaalund Jensen, J.P.; Poulsen, J.C. (1975) Studies on the haematologic and cytogenetic effect of lithium Acta Medica Scandinavica, 198, 281-286.

Human data

There are a few human data available reporting on the potential of lithium to induce chromosomal damage in psychiatric patients. Mainly negative results were described: Chromosome aberrations were analyzed from peripheral blood lymphocytes obtained from patients receiving chronic (2 weeks to 10 years) lithium carbonate treatment for psychiatric disorders and compared to negative controls; the observations for CAs were negative (Matushima et al., 1986, De La Torre et al., 1976, Jarvik et al 1971, Tureki et al, 1994). Friedrich and Nielsen (1969) reported an increase in mean chromosome breaks (not statistically significant) and hypodiploid cells (statistically significant) in 3 psychiatric patients treated with lithium. However, hypodiploid cells were only increased in one patient, i.e. the patient with the highest total dose. An insufficient number of patients and cells were analysed and details on the method used is missing.

Overall conclusion

The in vitro tests performed according to the respective OECD TG and GLP (bacteria reverse mutation test, chromosome aberration test, mouse lymphoma cell mutation test) with well-defined purity did not indicate a genotoxic potential for lithium chloride. However, positive results were reported in some of the published non-GLP studies, like a dose-related increase in micronuclei in CHO cells, most likely due to aneugenicity and possibly influenced by cytotoxicity (Pastor et al., 2009).

As a concern for genotoxicity cannot be fully ruled out and there are no acceptable in vivo tests a combined in vivo micronucleus test (OECD TG 474) combined with the in vivo comet assay (OECD TG 489) is proposed.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
Based on available data on genetic toxicity, the test item is not classified and labelled for genetic toxicity according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighteenth time in Regulation (EU) 2022/692. However, a concern for chromosome damage in vitro was identified and an in vivo genetic toxicity study is proposed.