Selenium

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

Study was started on 16 February 2010 and was completed on 6 April 2010.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read-across from a GLP guideline study with RL1

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Zinc selenite

EC Number:

237-048-9

EC Name:

Zinc selenite

Cas Number:

13597-46-1

Molecular formula:

H2O3Se.Zn

IUPAC Name:

zinc selenite

Details on test material:

- Name of test material (as cited in study report): Zinc selenite
- Molecular formula (if other than submission substance): ZnSeO3
- Molecular weight (if other than submission substance): 192.34 g/mol
- Physical state: solid, white powder
- Storage condition of test material: stored at 2-8C in the dark. The test article was kept in a tightly-closed container away from water and metals which may be attacked by an alkaline medium.

Method

Target gene:

hprt locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The master stock of L5178Y tk +/- mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co.
- Type and identity of media: RPMI 1640 media supplemented with heat inactivated horse serum (0%, 10% and 20%, respectively), 100 units/mL penicillin, 100 µg/mL streptomycin, 2.5 µg/mL Amphotericin B and 0.5 mg/mL pluronic (except for RPMI 20%) were used.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes; Each batch of cells was checked for that it was mycoplasma free.
- Periodically "cleansed" against high spontaneous background: yes; Each batch of cells was purged of TK- mutants, checked for spontaneous mutant frequency.
The cells diluted in RPMI 10 and incubated in a humidified atmosphere of 5% v/v CO2 in air.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Concentrations selected for the Mutation Experiments were based on the results of this cytotoxicity Range Finder Experiment.

Range-Finder:
- with and without metabolic activation: 3.758, 7.516, 15.03, 30.06, 60.13, 120.3, 240.5, 481.0, 962.0 and 1924 µg/mL

Experiment I:
- with metabolic activation: 4, 8*, 12, 16*, 20*, 25*, 30*, 35*, 40* and 50 µg/mL
- without metabolic activation: 10, 20*, 40*, 50, 60*, 75*, 90*, 105*, 120* and 150 µg/mL

Experiment II:
- with metabolic activation: 2.5, 5, 10*, 15*, 20*, 25*, 30*, 35*, 40* and 50* µg/mL
- without metabolic activation: 10*, 20*, 40*, 60*, 75*, 90*, 100*, 110*, 120, 150, µg/mL

* = Concentrations selected for mutation assessment.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: methyl cellulose
- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that Zinc selenite was not soluble in dimethyl sulphoxide (DMSO) or RPMI 1640 culture medium containing 5% v/v horse serum. The test article was formulated as a suspension (at 19.24 mg/mL) in 0.5% w/v methyl cellulose (0.5% MC) which was considered acceptable.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Untreated controls (UTC), comprising of treatments with culture medium in place of the vehicle, were included in the cytotoxicity Range-Finder and Experiment II.

Negative solvent / vehicle controls:

yes

Remarks:

Vehicle controls comprised treatments with 0.5% MC diluted 10 fold in the treatment medium.

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without metabolic activation

Migrated to IUCLID6: 0.1 and 0.15 µg/mL; dissolved in DMSO

Controls 2

Untreated negative controls:

yes

Remarks:

Untreated controls (UTC), comprising of treatments with culture medium in place of the vehicle, were included in the cytotoxicity Range-Finder and Experiment II.

Negative solvent / vehicle controls:

yes

Remarks:

Vehicle controls comprised treatments with 0.5% MC diluted 10 fold in the treatment medium.

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with metabolic activation

Migrated to IUCLID6: 2.0 and 3.0 µg/mL; dissolved in DMSO

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours at 37 +/- 1°C
After incubation, the cells washed and resuspended in RPMI 10 medium. Cells were transferred to flasks for growth through the expression period or were diluted to be plated for survival (scoring after 7 days).
- Expression time (cells in growth medium): Cultures were maintained for a period of 7 days during which the hprt- mutation would be expressed.
Thereafter cultures were selected to be plated for viability and 6TG resistance.
- Selection time (if incubation with a selection agent): 12 days; At the end of the expression period, the cell suspension was placed into each well of 4 x 96 well microtitre plates (384 wells at 2 x 10^4 cells/well). Plates were incubated at 37 +/-1ºC in a humidified incubator gassed with 5% v/v CO2 in air until scoreable and wells containing clones were identified and counted.

SELECTION AGENT (mutation assays): 6-thioguanine (6TG)

NUMBER OF REPLICATIONS: Each treatment, in the absence or presence of S9 mix, was performed in duplicate cultures (single cultures only used for positive control treatments).

EVALUATION: Wells containing viable clones were identified by eye using background illumination and counted.

DETERMINATION OF CYTOTOXICITY
- Method: relative survival:
Single cultures only were used and positive controls were not included. The final treatment volume was 20 mL. Following treatment, cells were washed with tissue culture medium and resuspended in RPMI 10. Cells were plated into each well of a 96 well microtitre plate for determination of relative survival. The plates were incubated at 37 +/-1ºC in a humidified incubator gassed with 5% v/v CO2 in air for 7 days. Wells containing viable clones were identified by eye using background illumination and counted.

OTHER: Plating efficiency (PE), percentage relative survival (% RS) and mutant frequency (MF) were determined.

Evaluation criteria:

For valid data, the test article was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:
1. The mutant frequency at one or more concentrations was significantly greater than that of the negative control (p<0.05).
2. There was a significant concentration relationship as indicated by the linear trend analysis (p<0.05).
3. The effects described above were reproducible.
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis.

Statistics:

Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines. The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

No statistically significant increases in mutant frequency were observed following treatment with zinc selenite at any concentration analysed

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

When tested up to precipitating concentrations in Experiment I without S9 mix, statistically significant increases in mutant frequency (MF) were observed at 75, 90 and 105 µg/mL. A statistically significant linear trend was observed and there was evidence of a concentration-related increase in MF at concentrations up to and including 75 µg/mL (but not at the higher concentrations). However, with the exception of the cultures treated at 75 µg/mL, all MF values in treated cultures were within three times the current historical mean vehicle control MF, therefore the biological relevance of these observations was inconclusive. When tested up to toxic concentrations in Experiment II, no significant increases in MF were observed at any concentration analysed and there was no significant linear trend. The data in the absence of S9 mix in Experiment I were not concentration-related and, importantly, were not reproduced between experiments over a similar range of concentrations. Overall, it may be considered that the evaluation criteria for a positive result were not fulfilled, particularly due to the lack of reproducibility.

In the presence of S9 mix, no significant increases in MF were observed in Experiments I and II, respectively, at any concentration analysed. A weak but significant linear trend was observed in Experiment II but in the absence of any marked increases in MF at any concentration analysed, this observation was not considered biologically relevant.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: No marked changes in osmolality or pH were observed in the absence of S9 mix in the cytotoxicity Range-Finder Experiment at the highest concentration tested (1924 µg/mL), compared to the concurrent vehicle control. No marked changes in osmolality or pH were observed in the presence of S9 mix in Experiment I at the highest concentration analysed (40 µg/mL), compared to the concurrent vehicle controls.
- Water solubility and precipitation: The nominal solubility limit in culture medium was considered to be approximately 1924 µg/mL, although persistent precipitate (haze) was observed at 1924 and 962 µg/mL, either 3 hours after test article addition or at the time of test article addition.

RANGE-FINDING/SCREENING STUDIES: In the cytotoxicity Range-Finder Experiment, ten concentrations were tested in the absence and presence of S9mix ranging from 3.758 to 1924 µg/mL (equivalent to 10 mM at the highest concentration tested). Following the 3 hour treatment incubation period, precipitate was observed at the highest 5 concentrations tested in the absence of S9 mix (120.3 to 1924 µg/mL) and at the highest three concentrations tested in the presence of S9 mix (481 to 1924 µg/mL). As a precaution (due to the nature of the test article formulation), all cultures were plated for survival. The highest concentrations to provide >10% RS were 60.13 µg/mL in the absence of S9 mix and 15.03 µg/mL in the presence of S9 mix, which gave 30% and 25% RS, respectively.

COMPARISON WITH HISTORICAL CONTROL DATA: yes; positive controls were compared with historical means.

ADDITIONAL INFORMATION ON CYTOTOXICITY: no further data

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

It is concluded that Zinc selenite did not induce biologically relevant increases in mutation at the hprt locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included treatments up to precipitating and toxic concentrations in Experiments I and II, respectively, in the absence and presence of a rat liver metabolic activation system (S9 mix).

Executive summary:

Zinc selenite was assayed for the ability to induce mutation at the hypoxanthine‑guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells.

A 3 hour treatment incubation period was used for all experiments. In the cytotoxicity Range-Finder Experiment, 10 concentrations were tested in the absence and presence of S9 mix, ranging from 3.758 to 1924 µg/mL (equivalent to 10 mM at the highest concentration tested).

According to the results from the Range-Finder, in Experiment I 10 concentrations, ranging from 10 to 150 µg/mL in the absence of S9 mix and from 4 to 50 mg/mL in the presence of S9 mix, were tested.

In Experiment II 10 concentrations, ranging from 10 to 150 µg/mL in the absence of S9 mix, and from 2.5 to 50 mg/mL in the presence of S9 mix were tested. Although the maximum concentrations analysed were similar in the absence and presence of S9 mix in the two experiments, the solubility and toxicity profiles were different.

Vehicle and positive control treatments were included in each Mutation Experiment.

In the absence of S9 mix, the maximum concentrations analysed in Experiments I and II were 120 and 110 µg/mL, limited by precipitate and by toxicity, respectively. When tested up to precipitating concentrations in Experiment I, statistically significant increases in mutant frequency (MF) were observed at 75, 90 and 105 µg/mL. A statistically significant linear trend was observed and there was evidence of a concentration-related increase in MF at concentrations up to and including 75 µg/mL (but not at the higher concentrations). However, with the exception of the cultures treated at 75 µg/mL, all MF values in treated cultures were within three times the current historical mean vehicle control MF, therefore the biological relevance of these observations was inconclusive. When tested up to toxic concentrations in Experiment II, no significant increases in MF were observed at any concentration analysed and there was no significant linear trend.

The data in the absence of S9 mix in Experiment I were not concentration-related and, importantly, were not reproduced between experiments over a similar range of concentrations. Overall, it may be considered that the evaluation criteria for a positive result were not fulfilled, particularly due to the lack of reproducibility.

In the presence of S9 mix, the maximum concentrations analysed in Experiments I and II were 40 and 50 µg/mL. When tested up to precipitating and toxic concentrations in Experiments I and II, respectively, no significant increases in MF were observed at any concentration analysed. A weak but significant linear trend was observed in Experiment II but in the absence of any marked increases in MF at any concentration analysed, this observation was not considered biologically relevant.