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Additional information

In vitro

Gene mutation in bacteria

Silicic acid, lithium salt has been tested in the bacterial reverse mutation assay (Ames-Test) according to OECD Guideline 471 and EU Method B.13/14 using the Salmonella typhimurium strains TA1535, TA97a, TA98, TA100 and TA102 with and without metabolic activation system (Paulus, 2009). No significant increase in the number of revertant colonies was observed in test concentrations of 50 to 5017 µg silicic acid, lithium salt/plate and no signs of toxicity towards the tested strains were detected.

 

Chromosomal effects

There is no cytogenetic study with silicic acid, lithium salt available. Therefore, read-across was conducted from the structural analogue silicic acid, sodium salt. Since silicic acid, lithium salt contains depending on its molar ratio 3.4 - 7% lithium (MR: 2.8 - 6.3), read across was also performed from lithium hydroxide to consider possible genotoxic effects of bioavailable lithium.

 

The ability of silicic acid, sodium salt (aqueous solution with 36% active ingredient) to induce structural chromosomal aberrations was investigated in a guideline study in accordance with OECD guideline 473 (Schulz, 2006). The effect was studied in Chinese hamster lung fibroblasts using concentrations up to 312.5 µg active ingredient/mL both in the absence (4 h, 18 h and 28 h treatment) and in the presence of metabolic activation (two experiments with each 4 h treatment). Neither increase in breaks, fragments, deletions, exchanges or chromosome disintegrations, nor in gaps were recorded for silicic acid, sodium salt. Cytotoxicity occurred at 156.3 and 312.5 µg active ingredient/mL.

 

A chromosomal aberration test according to OECD Guideline 473 was conducted with lithium hydroxide using human lymphocytes (Bertens, 2000).

In the absence of S9-mix lithium hydroxide was tested up to 560 µg/mL for a 3 h treatment time followed by a 24 h fixation time, 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 S9-fraction lithium hydroxide was tested up to 560 µg/mL for a 3 h treatment time followed by a 24 h fixation time and up to 450 µg/mL for a 3 h treatment time followed by a 48 h fixation time.

Short-term treatment (3 h) 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 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 and revealed no dose-response-relationship, the increase was not considered biologically relevant.

Scoring of an additional 200 metaphases at the concentration of 350 µg/mL lithium hydroxide verified the statistically significant increase. However, the observed increase is 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 ug/mL without S9-mix lithium hydroxide did not induce a 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.

 

 

Gene mutation in mammalian cells

A gene mutation study in mammalian cells with silicic acid, lithium salt is not available. Consequently, read-across was applied using study results obtained from silicic acid, sodium salt and lithium hydroxide monohydrate.

 

Silicic acid, sodium salt (aqueous solution with 36% active ingredient) was assessed for mutagenic potential in a mammalian cell gene mutation test following OECD Guideline 476 (Wollny, 2009). Cultured Chinese hamster lung fibroblasts (genetic marker HPRT) were co-incubated with the test substance up to 450 and 675 µg/mL medium for 4 h and 24 h, respectively in the absence of metabolic activation, and up to 1800 µg/mL for 4 h in the presence of metabolic activation. The dose range of the experiments was limited by toxicity of the test item. No substantial and reproducible dose-dependent increase of the mutation frequency was observed in the experiments.

 

An in vitro mammalian cell assay was performed according to OECD Guideline 476 in mouse lymphoma L5178Y TK +/- cells to test the potential of lithium hydroxide monohydrate to cause gene mutation and/or chromosome damage (Flügge, 2010). 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 in concentrations of 12.5, 25, 50 100 and 200 ug/mL.

Cytotoxicity (decreased survival) was noted in the presence and absence of metabolic activation at the top concentration of 200μg/mL. However, the mutation frequencies of the cultures treated with lithium hydroxide monohydrate were within the range of the negative control values and, hence, no mutagenicity was observed according to the criteria for assay evaluation. 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.

 

 

In vivo

An in vivo test with silicic acid, lithium salt is not available. Consequently, read across was applied using study results obtained from the analogue substance disodium metasilicate.

 

In a mammalian bone marrow chromosome assay similar to OECD Guideline 475 disodium metasilicate was administered orally via feed (dose levels of 740 – 1340 mg/kg bw) to BDF1 mice (Ito et al., 1986; Saiwai et al., 1980). Samples of the bone marrow were taken 24 h after administration of an acute dose. After slide preparation 100 metaphases per animals were analyzed for chromosomal aberrations (including gaps, breaks, deletions, and exchanges). No significant increase of chromosomal aberrations in bone marrow cells of mice compared to negative control even at dose levels exceeding the MTD of 940 mg/kg bw were observed.

 

It is concluded, that there is no evidence of a genotoxic potential for silicic acid, lithium salt.


Short description of key information:
In vitro:
Gene mutation (bacterial reverse mutation assay/Ames test): S. typhimurium TA1535, TA97a, TA98, TA100 and TA102: negative with and without metabolic activation
Chromosome aberration (OECD 473): negative with and without metabolic activation (RA from sodium silicate solution and lithium hydroxide)
Gene mutation in mammalian cells (OECD 476): negative with and without metabolic activation (RA from sodium silicate solution and lithium hydroxide)

In vivo:
Chromosome aberration (OECD 475): negative (740 – 1340 mg/kg bw (7 graduated levels) orally administered via feed to mice) (RA from disodium metasilicate)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on available data on genetic toxicity for the test substance and for the read-across or surrogate substances, silicic acid, lithium salt does not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC.