Sodium selenate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Ames Test with Sodium selenite:

In a reverse gene mutation assay in bacteria according to OECD guideline 471, strainsTA98, TA100, TA1535 and TA1537of S. typhimurium and Escherichia coli WP2 uvrA negative results were obtained with and without mammalian metabolic activation.

Mammalian cell gene mutation assay with Sodium selenite:

An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK^+/-3.7.2 C cells at the tk locus to test the potential of Sodium seleniteto cause gene mutation and/or chromosome damage. Treatment was performed for 3 hours with and without metabolic activation (±S9 mix) and for 24 hours without metabolic activation (-S9 mix).

	Concentration µg/mL	large colonies	small colonies	Total no. of colonies	Limit of biological Relevance  (control + 126)	Relative total growth	Mutation frequency
Assay 1 without S9   3-hour exposure	5	81	105	186	209	7	206.3*
	2.50	90	89	179	209	47	204.5*
	1.25	49	25	74	209	102	83.2
	0.625	51	49	100	209	112	110.6
	Vehicle control (water)	42	41	83		100	78.1
Assay 2 with S9   3-hour exposure	1.25	135	165	300	234	4	368.5*
	1	107	85	192	234	37	241.4*
	0.75	74	67	141	234	86	150.9
	0.5	71	54	125	234	81	131.2
	0.25	62	28	90	234	86	103.1
	0.125	64	35	99	234	83	126.1
	Vehicle control (water)	70	38	108		100	102.0
Assay 3 with S9   3-hour exposure	1	183	175	358	225	19	460.3*
	0.75	92	115	207	225	52	249.0*
	0.5	82	39	121	225	92	114.6
	0.25	61	46	107	225	80	100.3
	0.125	82	38	120	225	76	104.1
	Vehicle control (water)	64	35	99		100	94.2
Assay 3 without S9   24-hour exposure	3	249	138	387	230	13	445.3*
	2	207	134	341	230	39	415.4*
	1	96	54	150	230	147	143.1
	0.5	96	45	141	230	95	173.9
	Vehicle control (water)	77	27	104		100	118.7

As outlined in the table above, in assay 1 with 3-hour exposure without metabolic activation, statistical significant increase of mutant frequency was observed, however no biological relevant increase was observed taking into account the sum of total cell count and the Global evaluation factor of 126.

In assay 2, 3- hour exposure with metabolic activation, as well an statistical relevant increase of mutant frequency was observed, the total number of colonies was exceeding the biological limit of relevance (total cell count plus Global evaluation factor of 126), however at a concentration with very high cytotoxicity (Relative Total Growth, RTG of 4 %).

In assay 3, 3-hour exposure with metabolic activation statistical significant and biological relevant increase was observed at the highest concentration of 1 µg/mL, RTG 19 %. The effects were not reproduced in assay 1.

In assay 3, 24 - hour exposure without metabolic activation, at the two highest concentrations (2 and 3 µg/mL) a statistical significant and biological relevant increase was observed at RTG values of 39 and 13 %, respectively.

 

Chromosomal Aberration Test with Sodium selenite:

In the following a matrix of the test results is given which includes all relevant data thought to be helpful for the adequate evaluation of the in vitro assay (for details see robust study summary and study report): In particular it is emphasized that the onset and degree of cytotoxicity is additionally outlined and also data for breaks and exchanges of chromatids and chromosomes are included in this matrix, because it has been commented that breaks (besides fragments which have not been reported in this case) and also exchanges may play an indicative role for proper interpretation of positive study results (see for review [1]).

Assay 1 without metabolic activation (-S9)

Time of Treatment / Sampling: 3h / 20h

Concentration (μg/mL)	Relative Survival^#/ Cytotoxicity (%)	Pooled Breaks (chromatid and chromosome)	Pooled exchanges (chromatid and chromosome)	Mean % aberrant cells^##
Negative (solvent) control	100 / 0	3	1	2.0
100μg/mL	38 / 62	13	24	29.1***
75μg/mL	69 / 31	8	18	10.0**
50μg/mL	87 / 13	3	0	1.5
Positive control	86 / 14	14	2	7.5*

Assay 1 with metabolic activation (+S9)

Time of Treatment / Sampling: 3h / 20h

Concentration (μg/mL)	Relative Survival# / Cytotoxicity (%)	Pooled Breaks (chromatid and chromosome)	Pooled exchanges (chromatid and chromosome)	Mean % aberrant cells##
Negative (solvent) control	100 / 0	0	1	0.5
2.0 μg/mL	47 / 53	2	30	24.0***
1.75 μg/mL	77 / 23	2	10	7.5***
1.5 μg/mL	81 /19	7	18	12.0***
Positive control	58 / 42	7	41	62.5***

 

Assay 2 without metabolic activation (-S9)

Time of Treatment / Sampling: 20h / 28h

Concentration (μg/mL)	Relative Survival^#/ Cytotoxicity (%)	Pooled Breaks (chromatid and chromosome)	Pooled exchanges (chromatid and chromosome)	Mean % aberrant cells^##
Negative (solvent) control	100 / 0	1	0	0.5
10μg/mL	43 / 57	2	4	3.0
5μg/mL	47 / 53	2	9	5.5**
2.5μg/mL	86 / 14	4	12	6.5**
Positive control	59 / 41	14	24	19.5***

 

Assay 2 with metabolic activation (+S9)

Time of Treatment / Sampling: 20h / 28h

Concentration (μg/mL)	Relative Survival# / Cytotoxicity (%)	Pooled Breaks (chromatid and chromosome)	Pooled exchanges (chromatid and chromosome)	Mean % aberrant cells##
Negative (solvent) control	100 / 0	1	0	0.5
1.75 μg/mL	27 / 73	6	7	8.0***
1.5 μg/mL	61 / 39	4	12	7.5***
1.25 μg/mL	96 / 4	2	8	4.0*
Positive control	48 / 52	10	9	81.1***

#: compared to the negative (solvent) control

##: excluding gaps

*: p<0.05 comparing numbers of aberrant cells excluding gaps with corresponding negative (solvent) control

**: p<0.01 comparing numbers of aberrant cells excluding gaps with corresponding negative (solvent) control***: p<0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative (solvent) control

The following is noted from the test results:

(1) Longer periods of treatment (3 hours in assay 1 versus 20 hours in assay 2) significantly increase cytotoxicity to the marker cells (i.e. reduction of test concentrations by a factor of 10 to 20) and additional treatment with a system for metabolic activation (i.e. S9) further increases cytotoxicity (i.e. decrease of survival of cells) even though not as pronounced as without the addition of S9.

(2) For assay 1 without the addition of S9 in general a correlation can be observed for a significant increase of aberrant cells with a strong increase of cytotoxicity which is approximately 4 times that of the positive control. It is noted that at the lowest concentration of 50 µg/mL where only little cytotoxicity was observed (similar to the positive control) the number of aberrant cells (contrary to expectation) are below negative (solvent) control.Counts for pooled breaks correlate well with the observed cytotoxicity; as is the case for pooled exchanges which are at the highest test concentration approximately 10 times above positive control.

(3) For assay 1 with the addition of S9 pronounced cytotoxicity is observed already with the lowest test concentration of 1.5 µg/mL and a significant increase of aberrant cells follows (even though only up to 30 % of the positive control) with a high count for breaks.It is noted that no typical concentration-effect curve can be observed because initial increase at the lowest test concentration of 1.75 µg/mL is followed by a sharp decrease of aberrant cell counts (together with a decrease of breaks) and thereafter (at 2.0 µg/mL) again an increase can be observed but the count for breaks stays at low level.

Counts for pooled exchanges also correlate well with the non-proportional counts for aberrant cells.

(4) For assay 2 without the addition of S9 an inverse concentration-effect curve has been obtained which does not correlate with the again observed increase in cytotoxicity already with the lowest test concentration of 2.5 µg/mL. The counts for breaks are low and there is obviously no correlation with cytotoxicity and count for aberrant cells. Further, there is also an inverse relationship of counts for pooled exchanges with increase of the test concentration.

(5) For assay 2 with the addition of S9 a positive concentration-response curve has been obtained with increasing test concentrations which correlates with the count for breaks but not with counts for exchanges; absolutely the positive response is only weak because at near absence of cytotoxicity it is only 5 % of the positive control whereas it is only 10 % at the highest concentration of 1.75 µg/mL which is extremely cytotoxic (only 27 % survival).

 

Summarizing the obtained results as explained in detail indicates that Selenium seems to be extremely cytotoxic to mammalian cells under in vitro conditions once a certain threshold has been exceeded, in particular when “activated” by a metabolic system and at longer periods of exposure.

With almost all test concentrations (possibly with the only exception of one, i.e. 1.25 µg/mL in assay 2 with S9) a positive response was only observed at cytotoxic concentrations but without a clear concentration-response curve in 2 out of 4 assays.

Counts for breaks do not correlate well in 2 out of the 4 assays, and for counts for exchanges a correlation is only seen in 1 out of 4 assays.

 

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 Selenium (i.e. Sodium selenite) but rather to its ability for generation of cytotoxic species.

 

Further, in 2 out of 4 assays (assay 1 without S9 and assay 2 with S9) cytotoxicity correlated well with chromatid (and chromosome) breaks.

Breaks have been associated with the observed cytotoxicity of a test substance to marker cells such, that detected chromosomal aberration is thought to be of a secondary nature (see for review [1]).

For the exchanges which also have been associated with cytotoxicity only for the assay 1 without S9 a clear positive correlation with increasing test concentrations was observed.

 

The results of this test for chromosomal aberration/clastogenicity seem to be more adequately explained by an artefactual response of the marker cell system due to a pronounced cytotoxicity (at high enough concentrations) which is a unique property of the Selenium moiety due to its abiliy for redox cycling and to its presumed generation of reactive oxygen species.

   

It is therefore expected that at appropriate concentrations, i.e. absence of cytotoxicity, Sodium selenite would not be specifically clastogenic under in vitro conditions.

Therefore 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 from in vivo data 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 (see below).

Conclusion for the Need on in vivoTesting of Sodium selenite:

 

The above rationalized assumption fits well with the observation that under in vivo conditions with whole animals “it is likely that the increases in chromosome damage observed at almost exclusively at very high and near lethal doses resulted from the general toxicity of the selenium compounds tested and are not due to a specific mutagenic effect” (citation from [1]).

 

This position is also expressed by the recently published documentation on the German OEL (Occupational Exposure Limit: see German MAK Commission: Toxikologisch-arbeitsmedizinische Begründungen von MAK-Werten “Selen und seine anorganischen Verbindungen”, 2011, available at:http://onlinelibrary.wiley.com/book/10.1002/3527600418).

 

Therefore further in vivo testing for clastogenic activity is not 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 (see ATSDR2003, available at: http://www.atsdr.cdc.gov/toxprofiles/tp.asp?id=153&tid=28).

 

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) [2]). 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) [3]).

 

The following statement was drawn from the conclusion of the German MAK Commission:

“In vitro Selenium and its inorganic compounds are genotoxic. In experimental animals positive results were observed in micronucleus and chromosome aberration assays at doses near the LD₅₀. Concerning germ cell mutagenicity, negative data were obtained from a chromosome aberration assay in spermatocytes. The clastogenic potential observed in in vitro studies is relevant in vivo only at elevated doses. Up to 1.5 mg Selenium/kg no genotoxic effects were observed in reliable in vivo studies.”

For further synoptic reading see above mentioneddocumentation on the German OEL from the German MAK Commission.

 

Therefore the available data from in vivo testing with experimental animals does not give any indication for clastogenic activity provided (cyto)toxicity is cautiously avoided. This observation is in agreement with the fact that data from the human-being has also not indicated for any positive response with regard to clastogenicity.

 

 

References:

 

[1] Fahrig, R.: Expert Report: Genotoxicity and carcinogenicity of elemental selenium and selenium compounds, prepared for Selenium-Tellurium Developmental Association (STDA), April 17, 2000

[2] Norppa et al. (1980): Chromosomal effects of sodium selenite in vivo, in: Aberrations and sister chromatid exchanges in human lymphocytes, Hereditas 93, 93-96

[3] Itoh and Shimada (1996): Micronucleus induction by chromium and selenium, and suppression by metallothionein, Mutation Research, 367, 233-236

 

Justification for read-across:

The physico-chemical behavior of elemental Selenium (once it has formed an ion from its metal state), Sodium selenite, Sodium selenate and Selenium dioxide 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 [Se2-], 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 sodium selenite. It is concluded that additional testing of elemental Selenium, Sodium selenate and Selenium dioxide is not necessary and scientifically not meaningful.

Justification for selection of genetic toxicity endpoint
Reliable data are available from the full in-vitro test set required under REACH regulation. Additionally data are available from an in-vivo chromosome aberration study.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on all available information, inorganic Se substances are devoid of any relevant genotoxic potency.