Lithium hexafluorophosphate(1-)

Endpoint summary

Additional information

LiPF6

Ames test (Salmonella + E. Coli) negative results have been obtained.

  

HF

Using either in vitro or in vivo assays for genotoxicity, exposure to HF will result in target cell exposure only to fluoride (ionic F- with some organically bound fluoride). For this reason, test data from assays using sodium fluoride are relevant for assessment of HF genotoxicity (HF: EU Risk Assessment Report, 2001); however this EU RAR also notes that HF itself has given a negative result in a bacterial (Salmonella) mutation assay.

 

US NTP Technical Report 393 (1990) describes genotoxicity tests of sodium fluoride using bacteria (Salmonella typhimurium TA 1535, 1537, 98 and 100) and cultured mammalian cells (mouse lymphoma L5178Y; Chinese hamster ovary, SCE and chromosome aberration assay). Tests were conducted with and without rat liver derived S9 metabolic activating system. Among assays nominally detecting gene mutation, wholly negative results were obtained in bacteria but using L5178Y cells, two separate laboratories found positive results (both with and without S9 in the one laboratory testing both). However the observed L5178Y mutant colonies were principally small in size, suggesting chromosomal damage as the underlying mechanism. This was supported by the observation of induced chromosome aberrations in one test laboratory (at 400 µg/ml and higher concentration, with S9 only), but a second laboratory which tested at concentrations below 400 µg/ml found no such effect. One laboratory (but not the other) reported a positive SCE test result.

Clastogenic activity of NaF has also been investigated by other workers, using in vitro and in vivo assays. In cultured human lymphocytes, NaF caused chromosome damage in cultures without S9 and (to a lesser degree) with S9, but only at 20 and 40 µg/ml (Albanese, 1987): this worker reviewed other reports of NaF clastogenicity and noted a clear threshold effect, with no genotoxicity below 10µg/ml. He also dosed rats orally with NaF at 500 and 1000 mg/kg and scored bone marrow cells for micronucleated polychromatic erythrocytes: no significant increase in micronucleated cell frequency was seen (both 24 and 48h post-dose, but at 48h only 1/5 mice given 1000 mg/kg survived). This absence of activity in vivo was confirmed by Zeiger et al (1994): mice exposed to NaF in drinking water at up to 400 ppm F-(a concentration proving lethal to 3/16 mice) were examined for micronucleated peripheral erythrocytes (in blood samples) after 1 and 6 weeks and for chromosome aberrations (in bone marrow cells) after 6 weeks. No evidence of clastogenic activity was found, although fluoride uptake was demonstrated by measurement of F- in bones: at 400 ppm F- in water (average intake 75µg/kg/day) humerus F- concentration was raised to 7302 ppm, compared to 947 ppm in untreated controls.

 

Fluoride (F-)

This is addressed in the HF text above. 

 

Lithium

The genotoxicity of lithium has been reviewed by various workers. One expert group report summarised most studies of chromosome damage in patients receiving lithium therapy as showing no significant increase in chromosome aberrations or SCEs, and noted a reported weight of evidence conclusion in bacteria (Nordic Expert Group for Criteria Documentation of Health Risks from Chemicals 131, 2002). Another review (Aral and Vecchio-Sadus, 2008) lists negative results in bacterial mutation tests (lithium chloride in Bacillus stains, trilithium citrate in Salmonella strains and E.coli) and the host-mediated assay (trilithium citrate: E. coli and intraperitoneally treated mice), plus a negative Drosophila sex-linked lethal test (trilithium citrate). One report of increased single-strand breaks in DNA and slightly increased gene mutation in cultured V79 cells (only in the absence of S9 activating system) was cited in this review, but only questionable mutagenic activity in vitro was concluded; in another cited study, no increase in chromosome aberrations was seen in cultured human lymphocytes exposed to lithium carbonate at concentrations stated equivalent to distribution of up to 10g in a 70kg patient body. Bille et al (1975) investigated haematological parameters and bone marrow cell chromosome morphology in patients treated with lithium (900 – 1500 mg/day) for two months to 10 years: no differences in % cells with chromosome aberrations or % hyperdiploid cells was seen between this test group and controls. They also examined bone marrow cell aspirates from rats dosed intraperitoneally with lithium (3 x 86 mg/day), finding a similar absence of effect. Lithium bromide has been shown to be inactive in bacterial mutation assays (Salmonella typhimurium TA 1535, 1537, 98 and 100 plus E. coli WP2 uvrA, up to 5 mg/plate: Japan MHW, 2002).

 

Thus lithium is generally inactive in mutagenicity tests conducted in vitro or in vivo, with no evidence of chromosome damage in patients receiving lithium therapy. However there is some evidence for aneugenic activity of lithium at high exposure levels. The review of Aral and Vecchio-Sadus (2008) cites a report of increased micronucleated erythrocyte frequency in mice dosed intraperitoneally with lithium citrate (2 x 1100 mg/kg), and a detailed in vitro investigation of lithium salts found aneugenic activity in the absence of genotoxicity: both lithium chloride and lithium carbonate induced no chromosome aberrations or DNA strand breaks in exposed CHO cells, but increased the frequency of kinetochore-positive micronuclei (Pastor et al, 2009) at concentrations of 5mM and higher (corresponding to approximately 5-10mM lithium). The biological significance of this effect on chromosome assortment at cell division is uncertain, in view of the relatively high dosage/concentration required to elicit the effect. Also, a study of lithium bromide in CHO cells found neither increased polyploidy nor induction of chromosome aberrations in CHL cells at concentrations up to 870 µg/ml (Japan MHW, 2002).

 

Phosphate

Inorganic phosphate is routinely included in many of the standard protocols used for in vitro genotoxicity testing: as sodium phosphate, G6P, NADP, etc. in S9 mix activating system, potassium and ammonium phosphates in Vogel Bonner medium used in Ames test agar plates, sodium phosphate in cell culture media used for gene mutation and cytogenetic studies, etc. (see e.g. Handbook of Mutagenicity test procedures, Elsevier, 1977).  No mutagenic activity under test conditions is of course a requirement for such use.

The ubiquity of endogenous phosphate within the body and the Maximum Tolerable Daily Intake level of phosphate for man (70 mg/kg/day as P: FAO/WHO/IPCS, 1982; EFSA, 2008b) clearly demonstrate the low toxicity of phosphoric acid/phosphate. Any internal dose of these resulting from LiPF6 exposure at levels not causing rapid and overt toxicity due to HF/F- release will not be of concern for genotoxicity (P forming 20% of LiPF6, with F 75%).

Justification for selection of genetic toxicity endpoint
Lithium hexafluorophosphate is reactive and unstable in water and air. Reaction in contact with water proceeds rapidly, with release of hydrogen fluoride (hydrofluoric acid), lithium fluoride and phosphoric acid. Information is available on the genotoxicity of the ultimate hydrolysis products hydrogen fluoride, lithium/Li+, fluoride/F- and phosphoric acid/phosphate so further testing of LiPF6 itself, in addition to the bacterial mutation test already performed, is scientifically unnecessary (see separate read-across justification in Section 13). In accordance with Annex XI, 1.2 of the REACH Regulation testing is not scientifically necessary based on weight-of-evidence approach. 
In vivo testing for genotoxicity is similarly not necessary, and on humane grounds should not be performed on accordance with Article 15, 2 of Directive 2010/63/EU (as likely to involve severe pain, suffering or distress).

Short description of key information:
The substance itself proved negative in a bacterial mutation study. Considering its hydrolysis products:
[1] fluoride (tested as HF or NaF) proved negative in bacterial mutation assays. Tested as NaF, it gave positive results indicative of chromosome damage in vitro, in an L5178Y cell mutation study and a human lymphocyte cytogenetic study. In vivo, NaF produced no evidence of chromosome damage in two separate mouse studies:
- oral dosing at up to 1000 mg/kg did not increase micronucleated bone marrow cells
- administration in drinking water at up to 400 ppm did not increase micronucleated peripheral lymphocytes or bone marrow chromosome aberrations, despite analytical evidence of fluoride uptake in bones
[2] in patients receiving lithium therapy, the weight of evidence supports a conclusion that Li+ does not cause DNA damage, mutation or chromosome damage.In vitro, genotoxicity tests with lithium salts gave generally negative results in bacteria and mammalian cells., although one questionable positive result was reported. In vivo, bone marrow cell from lithium-treated rats (3 x 86 mg/day ip) and human patients (900-1500 mg/day for up to 10 years) showed no increased chromosomal aberrations. An apparent aneugenic effect of lithium salts in vitro and in vivo, in the absence of evident genotoxicity, has been reported.
[3] Inorganic phosphate is routinely included in many of the standard protocols used for in vitro genotoxicity testing and does not exhibit mutagenicity. it is not considered to possess genotoxic activity.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The hydrolysis products of LiPF6, formed rapidly on reaction with water, do not show mutagenic activity warranting classification.