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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-12-06 to 2018-03-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
2016-07-29
Deviations:
no
GLP compliance:
yes
Type of assay:
other: in vitro mammalian cell gene mutation test

Test material

Constituent 1
Chemical structure
Reference substance name:
Tripotassium hexacyanocobaltate
EC Number:
237-742-1
EC Name:
Tripotassium hexacyanocobaltate
Cas Number:
13963-58-1
Molecular formula:
C6CoN6.3K
IUPAC Name:
tripotassium hexacyanocobalttriuide
Test material form:
solid: particulate/powder
Details on test material:
- State of aggregation: light yellow powder
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored at 15 - 25 °C, protected from light

Method

Target gene:
HPRT
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Remarks:
clone 3.7.2C
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Dr Donald Clive, Burroughs Wellcome Co.
- Doubling time: average doubling time of L5178Y tk+/- (3.7.2C) cells is 10-12 hours.

For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated at 37±1ºC. When the cells were growing well, subcultures were established in an appropriate number of flasks.

MEDIA USED
- Type and identity of media: RPMI 1640 media supplied containing L-glutamine and HEPES were prepared as follows:
RPMI A: 0 % v/v horse serum (heat activated), 100 units/mL penicillin, 100 µg/mL streptomycin, 2.5 µg/mL amphotericin B, 0.2 mg/mL pyruvic acid and 0.5 mg/mL pluronic
RPMI 10: 10 % v/v horse serum (heat activated), 100 units/mL penicillin, 100 µg/mL streptomycin, 2.5 µg/mL amphotericin B, 0.2 mg/mL pyruvic acid and 0.5 mg/mL pluronic
RPMI 20: 20 % v/v horse serum (heat activated), 100 units/mL penicillin, 100 µg/mL streptomycin, 2.5 µg/mL amphotericin B and 0.2 mg/mL pyruvic acid
RPMI 5 consisted of RPMI 10 diluted with RPMI A [prepared as RPMI 10 but with no serum added] to give a final concentration of 5% serum

- Periodically checked for Mycoplasma contamination: yes
- Periodically 'cleansed' against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix (contained 10 % S-9 homogenate): 100 µmoles sodium phosphate buffer (pH 7.4); 5 µmoles glucose-6-phosphate (disodium); 4 µmoles ß-nicotinamide adenine dinucleotide phosphate (disodium); 8 µmoles MgCl2; 33 µmoles KCl; water (to volume); 100 µL S-9
Test concentrations with justification for top dose:
Range-finder: 62.5, 125, 250, 500, 1000 and 2000 µg/mL (with and without metabolic activation; 3 hour treatment)
Mutation experiment: 200, 400, 600, 800, 1200, 1600 and 2000 µg/mL (with and without metabolic activation; 3 hour treatment)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: purified water
Preliminary solubility data indicated that tripotassium hexacyanocobaltate(III) was soluble in purified water at concentrations up to at least 22.02 mg/mL. The solubility limit in culture medium was in excess of 2202 µg/mL, as indicated by a lack of any visible precipitation at this concentration after approx. 24 hours’ incubation at approx. 37 ºC.

Test article stock solutions were prepared by formulating tripotassium hexacyanocobaltate(III) under subdued lighting in purified water, with the aid of vortex mixing, to give the maximum required concentration. The stock solutions were membrane filter-sterilised (Pall Acrodisc 32 mm filter, 0.2 µm pore size) and subsequent dilutions were made using purified water. The test article solutions were protected from light and used within approximately 1 hour of initial formulation.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
purified water
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 4-nitroquinoline 1-oxide
Details on test system and experimental conditions:
RANGE-FINDING/SCREENING STUDIES:
- treatment of cell cultures for the cytotoxicity range-finder experiment was as described below for the Mutation Experiment. However, single cultures only were used and positive controls were not included. The final treatment volume was 20 mL.
- following 3 hour treatment, cells were centrifuged, washed with tissue culture medium and resuspended in RPMI 10.
- cell concentrations were adjusted to 8 cells/mL and, for each concentration, 0.2 mL was plated into each well of a 96-well microtitre plate for determination of relative survival.
- plates were incubated at 37±1ºC in a humidified incubator gassed with 5±1% v/v CO2 in air for 9 days.
- wells containing viable clones were identified by eye using background illumination and counted.
- osmolality and pH measurements on post-treatment media were taken in the cytotoxicity range-finder experiment

MUTATION ASSAY
1) Treatment of cell cultures:
- at least 10E7 cells in a volume of 16 mL of RPMI 5 were placed in a series of sterile disposable 50 mL centrifuge tubes.
- for all treatments 2 mL vehicle or test article, or 0.2 mL of positive control solution (+1.8 mL purified water) was added.
- S-9 mix or 150 mM KCl was added.
- each treatment, in the absence or presence of S-9, was in duplicate (single cultures only used for positive control treatments) and the final treatment volume was 20 mL.
- after 3 hours’ incubation at 37 ± 1 °C with agitation, cultures were centrifuged, washed with the appropriate tissue culture medium, centrifuged again and resuspended in 20 mL RPMI 10 medium.
- cell densities were determined using a Coulter counter and, the concentrations adjusted to 2 x 10E5 cells/mL.
- cells were transferred to flasks for growth throughout the expression period or were diluted to be plated for survival as described below.

2) Plating for survival:
- following adjustment of the cultures to 2 x 10E5 cells/mL after treatment, samples from these were diluted to 8 cells/mL.
- then, 0.2 mL of the final concentration of each culture was placed into each well of 2 x 96-well microtitre plates (192 wells, averaging 1.6 cells/well).
- plates were incubated at 37 ± 1 ºC in a humidified incubator gassed with 5 ± 1 % v/v CO2 in air until scoreable (7 days).
- wells containing viable clones were identified by eye using background illumination and counted.

3) Expression period:
- cultures were maintained in flasks for a period of 7 days during which the hprt- mutation would be expressed.
- sub-culturing was performed as required with the aim of retaining an appropriate concentration of cells/flask.
- from observations on recovery and growth of the cultures during the expression period, the following cultures were selected to be plated for viability and 6-thioguanine (6TG) resistance: 0, 200, 400, 600, 800, 1200, 1600, 2000 µg/mL (with and without metabolic activation) as well as positive controls.

4) Plating for viability:
- at the end of the expression period, cell concentrations in the selected cultures were determined using a Coulter counter and adjusted to give 1 x 10E5 cells/mL in readiness for plating for 6-thioguanine (6TG) resistance. Samples from these were diluted to 8 cells/mL.
- then, 0.2 mL of the final concentration of each culture was placed into each well of 2 x 96-well microtitre plates (192 wells averaging 1.6 cells/well).
- plates were incubated at 37 ± 1 ºC in a humidified incubator gassed with 5±1% v/v CO2 in air until scoreable (9 days).
- wells containing viable clones were identified by eye using background illumination and counted.

5) Plating for 6-thioguanine (6TG) resistance:
- at the end of the expression period, the cell densities in the selected cultures were adjusted to 1 x 10E5 cells/mL.
- 6TG (1.5 mg/mL) was diluted 100-fold into these suspensions to give a final concentration of 15 µg/mL.
- then, 0.2 mL of each suspension was placed into each well of 4 x 96-well microtitre plates (384 wells at 2 x 10E4 cells/well).
- plates were incubated at 37±1ºC in a humidified incubator gassed with 5±1% v/v CO2 in air until scoreable (14 days)
- wells containing clones were identified as above and counted.

ACCEPTANCE CRITERIA:
The assay was considered valid if all of the following criteria were met:
1. the mutant frequency (MF) in the vehicle control cultures was considered acceptable for addition to the laboratory historical negative control database
2. the MF in the concurrent positive controls induced responses that were comparable with those generated in the historical positive control database and gave a clear, unequivocal increase in MF over the concurrent negative control
3. the test was performed with and without metabolic activation
4.adequate numbers of cells and concentrations were analysable.
Rationale for test conditions:
A maximum concentration of 2000 µg/mL was selected for the cytotoxicity range-finder experiment in order that treatments were performed up to the maximum recommended concentration according to current regulatory test guidelines. Concentrations selected for the mutation experiment were based on the results of this cytotoxicity range-finder experiment.
Evaluation criteria:
For valid data, the test article was considered to be mutagenic in this assay if:
1. the mutant frequency (MF) 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. if both of the above criteria were fulfilled, the results should exceed the upper limit of the last 20 studies in the historical negative control database (mean MF +/- 2 standard deviations).
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis.
Statistics:
From the zero term of the Poisson distribution the probable number of clones/well (P) on microtitre plates in which there are EW empty wells (without clones) out of a total of TW wells is given by: P = -ln (EW/TW).
Cloning Efficiency (CE) in any given culture is therefore: CE = P/No of cells plated per well
and as an average of 1.6 cells/well were plated on all survival and viability plates,
CE = P/1.6.
Percentage Relative Survival (%RS) in each test culture was determined by comparing plating efficiencies in test and control cultures thus:
%RS = [CE (test)/CE (control)] x 100.
To take into account any loss of cells during the 3 hour treatment period, percentage relative survival values for each concentration of test article were adjusted as follows:
Adjusted %RS = %RS x (Post-treatment cell concentration for test article treatment / Post-treatment cell concentration for vehicle control)
All %RS values were adjusted as described above.
Mutant Frequency (MF) is usually expressed as "mutants per 10E6 viable cells". In order to calculate this, the cloning efficiencies of both mutant and viable cells in the same culture were calculated: MF = [CE (mutant)/CE (viable)] x 10E6.
From the formulae given and with the knowledge that 2 x 10E4 cells were plated/well for mutation to 6-thioguanine (6TG) resistance,
CE (mutant) = P (mutant)/2 x 10E4
CE (viable) = P (viable)/1.6
where, in each case, P = -ln (EW/TW).
Therefore,
MF = [P (mutant)/2 x 10E4] x [1.6/P (viable)] x 10E6
= {-ln [EW/TW (mutant)]/-ln [EW/TW (viable)]} x 80.
Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines (Robinson et al., 1990). 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
Key result
Species / strain:
mouse lymphoma L5178Y cells
Remarks:
clone 3.7.2C
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
please refer to the field "Additional information on results" below.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
- highest concentration to give >10% relative survival (RS) was 2000 µg/mL, which gave 66% and 87% RS in the absence and presence of S-9, respectively.
- the remaining concentration had the following relative survival:
62.5 µg/mL: 98 % RS (without metabolic activation) and 102 %RS (with metabolic activation)
125 µg/mL: 93 % RS (without metabolic activation) and 100 %RS (with metabolic activation)
250 µg/mL: 110 % RS (without metabolic activation) and 132 %RS (with metabolic activation)
500 µg/mL: 96 % RS (without metabolic activation) and 108 %RS (with metabolic activation)
1000 µg/mL: 83 % RS (without metabolic activation) and 100 %RS (with metabolic activation)
- no marked changes in osmolality or pH were observed in the range-finder at the highest concentration tested (2000 µg/mL) as compared to the concurrent vehicle controls.

MUTATION EXPERIMENT:
1) Toxicity:
- seven days after treatment all concentrations in the absence and presence of S-9 were selected to determine viability and 6TG resistance.
- highest concentration analysed was 2000 µg/mL, which gave 89% and 106% RS in the absence and presence of S-9, respectively.
- the remaining concentration had the following relative survival:
200 µg/mL: 91 % RS (without metabolic activation) and 106 %RS (with metabolic activation)
400 µg/mL: 93 % RS (without metabolic activation) and 91 %RS (with metabolic activation)
600 µg/mL: 106 % RS (without metabolic activation) and 109 %RS (with metabolic activation)
800 µg/mL: 100 % RS (without metabolic activation) and 98 %RS (with metabolic activation)
1200 µg/mL: 99 % RS (without metabolic activation) and 95 %RS (with metabolic activation)
1600 µg/mL: 89 % RS (without metabolic activation) and 102 %RS (with metabolic activation)
2000 µg/mL: 89 % RS (without metabolic activation) and 106 %RS (with metabolic activation)

2) Mutation:
- following 3 hour treatment in the absence and presence of S-9 no statistically significant increases in mutant frequency (MF), compared to the vehicle control, were observed following treatment with tripotassium hexacyanocobaltate(III) at any concentration analysed. All concentrations were within the vehicle control ranges generated by the last twenty experiments performed in this laboratory (1.15 to 6.61 mutants per 10E6 viable cells in the absence of S-9 and 2.40 to 6.62 mutants per 10E6 viable cells in the presence of S-9).
- both vehicle and positive control were within the historical control ranges.
- a statistically significant (p≤0.05) linear trend was observed in the presence of S-9 however, in the absence of any statistically significant increases in MF for any treated culture this observation was considered not biologically relevant.

Please also refer to the field "Attached background material" below

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
Please also refer to the field "Attached background material" below

Applicant's summary and conclusion

Conclusions:
It is concluded that tripotassium hexacyanocobaltate(III) did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to 2000 µg/mL, the maximum concentration for this type of study according to OECD Guideline 476, for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9).
According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance should not be considered to have a mutagenic potential.