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Genetic toxicity in vitro

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Lithium hydroxide was found to be non-mutagenic in three in vitro tests (AMES test, chromosome aberration test, in vitro mammalian cell gene mutation test).Two additional publications support the findings of the studies. As lithium hydroxide is formed when lithium comes in contact with water / aqueous solutions, it can be assumed that the results obtained in these studies with lithium hydroxide are applicable for lithium (read-across approach). Thus, lithium can be considered as not genotoxic / mutagenic.

Additional information

Reverse mutation assay – read-across from lithium hydroxide

 Salmonella typhimurium reverse mutation assay (Ames test) is not available for the test substance lithium. Consequently, a read-across approach was applied as lithium has similar properties to lithium hydroxide which is formed when lithium gets in contact with water.

In the presented study 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, twofold, 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. It was concluded that lithium hydroxide is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Based on these results and read-across approach lithium is expected to be not mutagenic in an Ames test.

 

In vitro Mammalian Chromosome Aberration – read-across from lithium hydroxide 

An in vitro chromosome aberration (CA) study is not available for the test substance lithium. Consequently, a read-across approach was applied using data obtained in this study, as lithium has similar properties to lithium hydroxide which is formed when lithium gets in contact with water (as is the case in this study).

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 µg/mL 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 the second experiment lithium hydroxide was tested up to 450 µg/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 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 350 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. 

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 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 confounding 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. 

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

Based on these results and in a read-across approach lithium is expected to be not mutagenic/ clastogenic in an in vitro chromosome aberration test.

 

In vitro Mammalian Cell Gene Mutation – read-across from lithium hydroxide

 

An in vitro mammalian cell gene mutation study is not available for the test substance lithium. Consequently, a read-across approach was applied as lithium has similar properties to lithium hydroxide which is formed when lithium gets in contact with water.

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

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

Based on these results and in read-across approach lithium is expected to be not mutagenic in an in vitro mammalian cell gene mutation test.

 

Publication: Genetic toxicity in vitro - read-across from lithium carbonate

 

The author evaluated the results of studies on genotoxicity performed in bacteria, mice, rats, V79 cells and human EUE fibroblasts to be inconclusive on the contrary the results obtained from lymphocytes of patients treated with lithium carbonate. The tests in bacteria and mice were negative while high concentrations of Li2CO3 (3 mg/mL) slightly inhibited DNA synthesis in V79 Chinese hamster cells and human EUE fibroblasts, an effect that was lessened by the addition of S9 fraction. Lithium carbonate (500 ppm) to the drinking water of 6-month old rats for 3, 6 or 12 months slightly reduced unscheduled DNA synthesis induced in blood lymphocytes by the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine, indicating a potential "protective" mechanisms.

Lithium carbonate added at the start of a 72 h culture period at concentrations equivalent to 0.1, 1.0 or 10 g distributed in the body of a 70 kg person did not increase structural chromosome aberrations. Also at toxic doses of lithium, cytogenetic changes in peripheral blood lymphocytes of patients treated with lithium salts were not observed. No aberrations were found in 19 lithium-treated manic-depressive patients compared with 23 controls. Moreover, negative results were found in peripheral lymphocytes of 70 patients treated with various lithium salts (17 with lithium carbonate). This result was confirmed in 16 manic-depressive patients who had been given lithium carbonate from 2 weeks to more than 2 years (seven of them for more than a year) which did not show aberrations in their lymphocytes.

The reported data (secondary sources), in particular those in humans, support the negative findings of the three in-vitro tests with lithium hydroxide, presented above as a read-across to lithium.

 

Publication: Genetic toxicity in vivo

The authors summarized the results of in vitro and in vivo mutagenicity , DNA damage , CA and SCE tests with various lithium salts. Several studies reported genotoxic effects of various lithium compounds at high doses (equivalent to therapeutic doses or higher), whereas many other studies have failed to demonstrate an effect. The Nordic Expert Group stated that considering the chemical properties of the lithium compounds it is unlikely that they act as direct mutagens. A possible explanation to the genotoxicity observed might be a secondary effect of increased cell survival caused by lithium’s inhibition of GSK3.

Based on the conclusion of the authors, lithium hydroxide (formed following contact of lithium with water) and lithium could be considered not genotoxic or clastogenic substances.


Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genetic toxicity, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.

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