Sodium selenite

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Background

 

A read-across category-approach is used for the assessment of the toxicological properties of selenium and selenium compounds. The following Se-substance are included in the category:

• Se-metal (massive, powder)
• Disodium selenate
• Disodium selenite
• Selenium dioxide / selenious acid
• Zinc selenite
• Barium selenite

A detailed rationale for the read-across hypothesis has been outlined in the read-across report that was generated according to the principles laid out in the Read-Across Assessment Framework (RAAF). In summary, the physico-chemical behavior of elemental selenium (once it has formed an ion-from its metal state), disodium selenite, disodium selenate and selenium dioxide/selenious acid is the same with regard to their metabolic fate. All selenium compounds (organic and inorganic, including elemental selenium), do share the very same metabolic fate in that after their resorption, reduction to the selenide moiety [Se^2-], which is the single common precursor for its further metabolic conversion, takes place.

Therefore, there seems to be good evidence that different selenium moieties will behave very similar also for their ability to form reactive species which may play a decisive role in the generation of cytotoxicity followed likewise by unspecific and secondary clastogenicity and read-across can be made from the available data for disodium selenite. It is concluded that additional testing for each individual member of the proposed Se-category is not necessary and scientifically not meaningful.

In the case of inorganic salts like barium selenite and zinc selenite, uptake is always associated with a dissolution of the substance, i.e. dissociation into the metal cation (Zn²⁺, Ba²⁺) and the selenite anion (SeO₃^2-). It can safely be assumed that the selenium/selenite moiety of barium/zinc selenite is generally of higher toxicological relevance than the zinc/barium cations. Therefore, the subsequent assessment of the toxicity of barium/zinc selenite focuses on the selenium moiety. As no in vivo toxicokinetic data or in vitro bioaccessibility data are available for a comparative assessment of relative bioavailability of various selenite substances, water solubility is adopted as a surrogate for bioavailability. Disodium selenite is readily soluble, with a water solubility of 800-900 g/L at 20°C. Barium selenite and zinc selenite, on the other hand, are poorly soluble salts (water solubility at 20°C of 66.7 mg/L and 16 mg/L, respectively, i.e. a difference of four/five orders of magnitude). Based on that, an intrinsically very conservative read-across from highly soluble forms to the poorly soluble barium/zinc selenite is proposed as the latter are assumed to have a lower solubility. It should also be noted that selenite anions in the tests with disodium selenite are formed under most physiological relevant conditions (i.e. neutral pH), thus facilitating unrestricted read-across between the various substances. In slightly acid conditions (pKa:8.32) the hydrogen selenite ion (HSeO₃^-) is formed whereas in more acidic conditions (pKa:2.62) the formation of selenious acid is observed (H₂SeO₃). Based on such existing equilibrium conditions, read-across between selenites, hydrogen selenites and selenious acid (solubility of 1670 g/L at 20°C) is justified.

 

Read-across from sodium selenite and selenious acid to barium/zinc selenite

Based on a comparison between toxicity reference values of zinc compounds and selenium compounds, it can safely be assumed that the selenium/selenite moiety of zinc selenite is generally of higher toxicological relevance than the zinc cations. Comparing the DNELs for the zinc/barium ion itself with the zinc/barium levels that are associated with the DNELs for barium/zinc selenite (based on selenite-data) indicated significantly higher values (in the range of factor 10 to 20) for the DNELs derived for the barium/zinc ion itself. Therefore, the subsequent assessment of the toxicity of barium/zinc selenite focuses on the selenium moiety.

The Table below gives an overview of reliable in vitro studies that assessed the mutagenic properties of various Se-compounds.

Genotoxic properties of different Se-compounds: available in vitro data 

Test substance	Test Method	Result	Reference
Disodium selenite	OECD 473	Positive - clastogenic	Hargitai, 2013a
Disodium selenite	OECD 471	Negative	Hargitai, 2013b
Disodium selenite	OECD 476	Positive	Hargitai, 2013c
Disodium selenite	No guideline study	Negative – clastogenic effects where cytotoxicity was noted	Slapsyte, 2003
Se-metal	OECD 471	Negative	Hargitai, 2012
Zinc selenite	OECD 476	Negative	Lloyd, 2010

 

Genetic toxicity in vivo

Description of key information

The Table below gives an overview of reliable in vivo studies that assessed the mutagenic properties of various Se-compounds. The table reports the test substance, test method, test result and reference

 

Genotoxic properties of different Se-compounds: available in vivo data 

Test substance	Test Method	Result	Reference
Disodium selenite	In vivo	Negative	Norppa, 1980
Selenious acid	In vivo – micronucleus assay	Negative	Itoh, 1996

 

Additional information

Overview of relevant and reliable data

 

A comprehensive literature search has been conducted by the registrant in factual data bases and secondary literature sources. Studies performed with organic selenium compounds were excluded from evaluation as the bioavailability is much higher than for inorganic selenium compounds.

During this search the most comprehensive and recent review document " Toxicological Profile for Selenium”, ATSDR - U.S. Department of Health and Human Services, Public Health Service (2003)" has been identified and is used as the key source of relevant data on selenium and selenium compounds, because it contains an evaluation of the toxicity information performed by a renowned scientific body. More recent reviews, e.g. the work conducted for the Canadian Soil Quality Guidelines / Sudbury Soil Study, have also been screened for additional data. The underlying assumption is that the key literature considered by renowned international organisations such as ATSDR has usually already been subjected to a reliability assessment. Nevertheless all key references identified by ATSDR have been screened for use in the REACH dossiers according to Klimisch and with respect to the requirements for risk assessment.

In addition, after the evaluation of the ATSDR review and further other secondary publications, it was suggested that a follow-up endpoint specific literature search should be conducted in bibliographic databases, restricting the search to references published since the beginning of 2000. The results of this search have also been screened for use in the REACH dossiers.

Literature cited in the ATSDR

The ATSDR document identified a set of 27 references on genetic toxicity which can be subdivided in

·      17 references on in vitro test systems, of which 5 references are on bacterial tests systems and 12 on mammalian cell test systems, and

·      10 references on in vivo tests all on clastogenic effects.

All references cited in the ATSDR were acquired in full text and evaluated with regard to relevance and reliability. A small subset of references was added which were considered relevant during the literature search, mainly based on the test substance used. All studies identified by ATSDR as key references concerning tests with selenites or selenious acid were included in the IUCLID as study records. Following the quality and reliability screening, those references which were assessed as not adequate, relevant or reliable by expert judgement during screening procedure were assigned to "disregarded study", and rated as "not reliable" (RL=3 or 4), with the rationale being included in the endpoint study record.

 

Evaluation of relevant references

A concise summary of the main test results is provided here; more details can be found in the individual study records.

 

In vitro data:

 

A.    One well described study (Slapsyte, 2003; not cited in ATSDR) on the induction of chromosome aberrations on isolated human lymphocytes following treatment with disodium selenite (Na₂SeO₃), reports no biological relevant increase in chromosome aberrations following 24h exposure. The maximum dose was limited by cytotoxicity. Clastogenic effects were reported at top concentrations for which a significant cytotoxicity was observed.

 

B.    Negative results were obtained in a reverse gene mutation assay in bacteria (S. typhimurium,E.coli), using disodium selenite as test substance. The test was conducted according to OECD guideline 471, with and without mammalian metabolic activation. strainsTA98, TA100, TA1535 and TA1537 of WP2 uvrA.

 

C.   A state of the art, guideline and GLP compliant in vitro gene mutation test was conducted with zinc selenite (Lloyd, 2010). It is concluded that zinc selenite did not induce increases in mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to precipitating and toxic concentrations in two independent experiments, with and without rat liver metabolic activation system (S9 mix).

 

D.   The potential of disodium selenite to cause gene mutation and/or chromosome damage was investigated in anin vitromammalian cell assay in mouse lymphoma (3/24h exposure, with and/or without metabolic activation). Statistical relevant increases of mutant frequency was observed in most of the exposures, though in some tests no biological relevant increase was observed taking into account the sum of total cell count and the Global evaluation factor of 126. In addition, effects were sometimes noted at a concentration with very high cytotoxicity. The results indicate that disodium selenite seems to be extremely cytotoxic to mammalian cells underin vitroconditions once a certain threshold has been exceeded, in particular when “activated” by a metabolic system and at longer periods of exposure.

 

E.    The lack of an in vitro gene mutation test in bacteria constitutes a formal data gap. However, tests on the mutagenic potential of selenite in bacteria is considered dispensable for principal considerations, since inorganic metal compounds are frequently negative in this assay due to limited capacity for uptake of metal ions (Guidance on information requirements and chemical safety assessment, Chapter R.7a, p. 387; HERAG facts sheet mutagenicity, Chapter 2.1).

 

These results leave the impression that they are not consistent with a plausible and causal relationship of a specific mode of action of sodium selenite with regard to chromosomal aberration/clastogenicity, rather this behavior may be better explained by the observation that selenite can undergo redox cycles because it may be either oxidized or reduced, such that, beyond a certain threshold (i.e. high enough concentration of selenium in the in vitro cell system) reactive oxygen species may be generated which are known for their cytotoxicity (for example H₂O₂ is known to induce programmed cell death (apoptosis). Defense mechanism of the cells may be overwhelmed then. If so, the integrity of the marker system may have been damaged to the extent that the observed effects are secondary; they therefore would not be attributable to any unique cytogenic action of delenium (i.e. diodium selenite) but rather to its ability for generation of cytotoxic species. Hence, at appropriate concentrations that do not cause cytotoxicity, sodium selenite would not be specifically clastogenic under in vitro conditions.

 

It seems to be more appropriate to assume an indirect, secondary mode of action of sodium selenite at concentrations above a certain threshold which leads to “cytotoxicity” to the marker cells and hence unspecific induction of chromosomal aberrations.This is more in line with the observation fromin vivodata (see below) that only at toxic doses (close to the LD₅₀) clastogenicity can be observed and it is also more easily plausible from the fact that clastogenicity has not been measured in humans.

 

 

In vivo data:

Itoh, (1996) described the induction of micronuclei in the bone marrow of mice following i.p. exposure towards selenious acid (H₂SeO₃). A significant increase in micro-nucleated cells was observed, with a clear positive finding in the maximum dose of 5 mg/kg. All lower dose levels were in the range of the control (0.0-0.3% MNPCE). However, there was no clear justification for the dose selection, and a description of the toxic effects is lacking. As stated below one might speculate that the maximum dose already showed signs of near-lethal toxicity, making this positive finding of questionable biological significance. Due to reporting deficiencies this reference is only used as supportive information.

 

From studies with humans it could be shown that patients exposed from 0.004 to 0.05 mg selenite for 1 to 13 months did not produce chromosomal aberrations in lymphocytes (Norppa et al., 1980).

Also, no genotoxicity was observed in valid studies after human exposure for up to 1.5 mg Selenium/kg body weight, Itoh and Shimada (1996).

 

 

Conclusion

The findings in the in vivo test for selenites (Itoh, 1996) indicate that positive findings are only obtained at very high, near lethal doses, and are caused by the general toxicity of the administered selenite and are not due to a specific mutagenic effect. This position is also expresssed by the recently published documentation on the German Occupational Exposure Limit (OEL) (MAK Commission, 2011). In addition, zinc selenite did not show a significant or dose-dependent increase in mutations in cultured mouse lymphoma cells (L5178Y) up to the maximum dose of 120 µg/mL. Furthermore, an overwhelming database of positive findings in in vitro tests was identified during the literature search and in the ATSDR. However, especially for metal compounds false positive findings have repeatedly been published which, can be attributed to osmolality or pH instead of genotoxic effects exerted by the metal ion itself and are therefore of limited biological relevance.

Also, the risk assessment document published by the European Food Safety Authority (EFSA, 2006) summarise the genotoxic effects of selenium compounds as follows: “In vivo, only toxic amounts were shown to be active, keeping in mind the central role of hydrogen selenide in the metabolism of most selenium compound it is likely that overproduction of this and other auto-oxidisable selenium metabolites could promote the formation of DNA reactive oxygen radicals. [...] Genotoxicity has been seen in a number of in vitro systems and also in vivo at toxic doses. It is likely, however, that these effects may be related to the generation of reactive oxygen radicals, being dose dependent and showing a threshold in vivo and not occurring at nutritionally adequate intakes.”

Based on the above given information it can be concluded thatin vivoclastogenic effects may occur at high, nearly lethal doses which are not attributable to exposure conditions for workers, consumers and humans exposed via the environment. Thus, this effect is considered of no biologcal relevance for humans.

Further in vivo testing for clastogenic activity is not considered necessary from a scientific point of view as it would not give a deeper insight into seleniums`s potency for adverse effects onto chromosomes.It has been also observed that below a certain cytotoxic threshold selenium and selenium compounds exert even antimutagenic activity.

 

 

 

 

Secondary reference

MAK Commission, 2011.Toxikologisch-arbeitsmedizinische Begründungen von MAK-Werten “Selen und seine anorganischen Verbindungen”, at:http://onlinelibrary.wiley.com/book/10.1002/3527600418).

Selenium, In: European Food Safety Authority (EFSA) (2006) Tolerable upper intake levels for vitamins and minerals, page 65f.

Justification for classification or non-classification

In a weight of evidence it has to be concluded that in vivo clastogenicity is only observed at extremely high, nearly lethal doses. Thus, this effect is considered of no biological relevance.

The classification criteria according to regulation (EC) 1272/2008 as germ cell mutagen are not met, no classification is required.

Further testing of in vivo genetic toxicity tests is not considered necessary.