Tripotassium hexacyanocobaltate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay/Ames test: negative (OECD 471; GLP)

In vitro mammalian cell micronucleus test: negative (OECD 487; GLP)

In vitro mammalian cell gene mutation test: negative (OECD 476; GLP)

Link to relevant study records

Reference

Reference 1

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

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

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

Target gene:

HPRT

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.

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.

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-12-18 to 2018-02-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2016-07-29

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

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

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: human (peripheral)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: two healthy human donors (non-smoking; not heavy drinkers of alcohol; no known virus infection or exposure to high levels of radiation or hazardous chemicals; no medication)

- Cell cycle length: 13 ± 2 hours

- Sex, age and number of blood donors:
Range-finder: two males of the age of 35 years
Micronucleus experiment: two males of the age of 32 and 35 years, respectively

- Whether whole blood or separated lymphocytes were used: for each experiment, an appropriate volume of whole blood was drawn from the peripheral circulation into heparinised tubes one day of culture initiation. Blood was stored refrigerated and pooled using equal volumes from each donor prior to use.

Cytokinesis block (if used):

Cyto-B (formulated in DMSO)

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:
- 7.256, 12.09, 20.16, 33.59, 55.99, 93.31, 155.5, 259.2, 432.0, 720.0, 1200 and 2000 µg/mL (with and without metabolic activation; 3 hour treatment + 21 hour recovery)
- 7.256, 12.09, 20.16, 33.59, 55.99, 93.31, 155.5, 259.2, 432.0, 720.0, 1200 and 2000 µg/mL (without metabolic activation; 24 hour treatment + 24 recovery)

Micronucleus experiment:
- 50.00, 100.0, 250.0, 500.0, 1000, 1500 and 2000 µg/mL (with and without metabolic activation; 3 hour treatment + 21 hour recovery)
- 50.00, 100.0, 250.0, 500.0, 1000, 1500 and 2000 µg/mL (without metabolic activation; 24 hour treatment + 24 recovery)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: purified water
Preliminary solubility data indicated that tripotassium hexacyanocobaltate(III) was soluble in water for irrigation (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 the lack of precipitation which persisted following 24 hours of incubation at 37±1ºC.

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 treatment 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 2.5 hours of initial formulation.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

sterile purified water

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Vinblastine

Details on test system and experimental conditions:

BLOOD CULTURES:
- whole blood cultures were established in sterile disposable centrifuge tubes by placing 0.4 mL of pooled heparinised blood into 7.6 mL pre-warmed (in an incubator set to 37±1°C) HEPES-buffered RPMI medium containing 10% (v/v) heat inactivated foetal calf serum and 0.52% penicillin / streptomycin, so that the final volume following addition of S-9 mix/KCl and the test article in its chosen vehicle was 10 mL.
- mitogen phytohaemagglutinin (PHA, reagent grade) was included in the culture medium at a concentration of approximately 2% of culture to stimulate the lymphocytes to divide.
- blood cultures were incubated at 37±1°C for approx. 48 hours and rocked continuously.

RANGE-FINDING/SCREENING STUDIES:
- following the preparation of the blood cultures, as described above, S-9 mix or sodium phosphate buffer (1 mL/culture) was added appropriately.
- cultures were treated with the test article or vehicle (1 mL/culture; vehicle control: 2 cultures/treatment; test article: 1 culture/treatment).
- positive control treatments were not included.
- final culture volume was 10 mL.
- cultures were incubated at 37±1°C for the designated exposure time (3 hours treatment + 21 hours recovery (with and without S-9 mix) or 24 hours treatment + 24 hours recovery (without S-9 mix)).
- osmolality and pH measurements on post-treatment incubation medium were taken in the cytotoxicity range-finder experiment.
- slides from the cytotoxicity range-finder experiment were examined for proportions of mono-, bi- and multinucleate cells, to a minimum of 200 cells per concentration. From these data the replication index (RI) was determined.
- RI, which indicates the relative number of nuclei compared to vehicle controls was determined using the formulae as follows: RI = [number binucleate cells + 2(number multilucleate cells)]/(total number of cells in treated cells)
- Relative RI (expressed in terms of percentage) for each treated culture was calculated as follows: relative RI (%) = RI of treated cultures/RI of vehicle controls) x 100
Cytotoxicity (%) is expressed as (100 – Relative RI).
- selection of random fields was observed from enough treatments to determine whether chemically induced cell cycle delay or cytotoxicity had occurred.
- suitable range of concentrations was selected for the Micronucleus Experiment based on these toxicity data.

MICRONUCLEUS EXPERIMENT
- following the preparation of the blood cultures, as described above, immediately prior to treatment, all positive control cultures had 0.9 mL culture medium added to give a final pre-treatment volume of 8.9 mL.
- S-9 mix or KCl (1 mL/ culture) was added appropriately.
- cultures were treated with the test article, vehicle, or positive controls (1 mL/culture or 0.1 mL/culture for positive control cultures; vehicle control: 4 cultures/treatment; test article: 2 culture/treatment; positive control: 2 cultures/treatment).
- final culture volume was 10 mL.
- cultures were incubated at 37±1°C for the designated exposure time (3 hours treatment + 21 hours recovery (with and without S-9 mix) or 24 hours treatment + 24 hours recovery (without S-9 mix)).

For removal of the test article, cells were pelleted (approx. 300 g, 10 minutes), washed twice with sterile saline (pre-warmed in an incubator set to 37 ± 1°C), and resuspended in fresh pre-warmed medium containing foetal calf serum and penicillin / streptomycin. Approx. 1 hour after test article removal, Cyto-B (formulated in DMSO) was added to post wash-off culture medium to give a final concentration of 6 µg/mL per culture.

HARVESTING
- after the designated recovery times after treatment (21 or 24 hours), harvesting of the cells was conducted immediately
- cultures were centrifuged at approx. 300 g for 10 minutes, the supernatant removed and discarded.
- cells were resuspended in 4 mL (hypotonic) 0.075 M KCl at 37±1°C for 4 minutes to allow cell swelling to occur.
- cells were fixed by dropping the KCl suspension into fresh, cold methanol/glacial acetic acid (7:1, v/v).
- fixative was changed by centrifugation (approx. 300 g, 10 minutes) and resuspension. This procedure was repeated as necessary (centrifuging at approximately 1250 g, 2-3 minutes) until the cell pellets were clean.

METHOD OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
- lymphocytes were kept in fixative at 2 - 8 °C prior to slide preparation for a minimum of 3 hours to ensure that cells were adequately fixed.
- cells were centrifuged (approx. 1250 g, 2 - 3 minutes) and resuspended in a minimal amount of fresh fixative (if required) to give a milky suspension.
- several drops of cell suspension were spread onto multiple clean, dry microscope slides.
- slides were air-dried then stored protected from light at room temperature prior to staining.
- slides were stained by immersion in 12.5 µg/mL Acridine Orange in phosphate buffered saline (PBS), pH 6.8 for approx. 10 minutes and washed with PBS (with agitation) for a few seconds.
- quality of the staining was checked.
- slides were air-dried and stored protected from light at room temperature.
- immediately prior to analysis 1 2 drops of PBS were added to the slides before mounting with glass coverslips.
- slides were examined for replication index (RI) to a minimum of 500 cells per culture to determine whether chemically induced cell cycle delay or toxicity had occurred.
- highest concentration selected for micronucleus analysis following all treatment conditions was the highest concentration tested (2000 µg/mL).
- slides from the highest selected concentration and two lower concentrations were taken for microscopic analysis.
- positive control concentrations analysed did not exceed the cytotoxicity limits for the test article concentration selection.

SLIDE ANALYSIS:
Scoring was carried out using fluorescence microscopy.
Binucleate cells were only included in the analysis if all of the following criteria were met:
1. the cytoplasm remained essentially intact, and
2. the daughter nuclei were of approximately equal size.
A micronucleus was only recorded if it met the following criteria:
1. the micronucleus had the same staining characteristics and a similar morphology to the main nuclei, and
2. any micronucleus present was separate in the cytoplasm or only just touching a main nucleus, and
3. micronuclei were smooth edged and smaller than approximately one third the diameter of the main nuclei.
For each treatment regime, two vehicle control cultures were analysed for micronuclei.
Slides from the positive control treatments were checked to ensure that the system was operating satisfactorily. One concentration from each positive control, which gave satisfactory responses in terms of quality and quantity of binucleated cells and numbers of micronuclei, was analysed.
One thousand binucleate cells from each culture (2000/concentration) were analysed for micronuclei. The number of cells containing micronuclei and the number of micronuclei per cell on each slide was recorded. The microscope stage co-ordinates of the first six micronucleated cells were recorded.
Nucleoplasmic bridges between nuclei in binucleate cells were recorded during micronucleus analysis to provide an indication of chromosome rearrangement.

ACCEPTANCE CRITERIA:
The assay was to be considered valid if the following criteria were met:
1. the binomial dispersion test demonstrated acceptable heterogeneity (in terms of binucleate cells with micronuclei frequency) between replicate cultures, particularly where no positive responses were seen
2. the frequency ofbinucleate cells with micronuclei in vehicle controls fell within the 95th percentile of the current observed historical vehicle control (normal) ranges
3. the positive control chemicals induced statistically significant increases in the proportion of cells with micronuclei. Both replicate cultures at the positive control concentration analysed under each treatment condition demonstrated binucleate cells with micronuclei frequencies that clearly exceeded the normal range
4. a minimum of 50% of cells had gone through at least one cell division (as measured by binucleate + multinucleate cell counts) in vehicle control cultures at the time of harvest
5. the maximum concentration analysed under each treatment condition met the criteria specified as in "METHOD OF SLIDE PREPARATION AND STAINING TECHNIQUE USED" above.

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 for the Micronucleus Experiment were selected based on the results of this cytotoxicity Range-Finder Experiment.

Evaluation criteria:

The test article was considered to induce clastogenic and/or aneugenic events if:
1. a statistically significant increase in the frequency of binucleate cells with micronuclei at one or more concentrations was observed
2. an incidence of binucleate cells with micronuclei at such a concentration that exceeded the normal range in both replicates was observed
3. a concentration-related increase in the proportion of binucleate cells with micronuclei cells was observed (positive trend test).
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Evidence of a concentration-related effect was considered useful but not essential in the evaluation of a positive result (Scott et al., 1990)*.

*Reference:
- Scott D, Dean B J, Danford N D and Kirkland D J (1990). Metaphase chromosome aberration assays in vitro. Basic Mutagenicity Tests; UKEMS recommended procedures. Kirkland D J (Ed), pp 62 - 86.

Statistics:

After completion of scoring, the numbers of binucleate cells with micronuclei (MNBN cells) in each culture were obtained.
The proportions of MNBN cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test (Richardson et al., 1989)*.
The proportion of MNBN cells for each treatment condition were compared with the proportion in vehicle controls by using Fisher's exact test (Richardson et al., 1989). A Cochran-Armitage trend test was applied to each treatment condition. Probability values of p≤0.05 were accepted as significant.

*Reference:
- Richardson C, Williams D A, Allen J A, Amphlett G, Chanter D O and Phillips B (1989). Analysis of data from in vitro cytogenetic assays. In "Statistical Evaluation of Mutagenicity Test Data", (UKEMS Guidelines Sub-committee Report, Part III), Ed D J Kirkland, Cambridge University Press, pp 141-154

Key result

Species / strain:

lymphocytes: human (peripheral)

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmilality (range-finder study): no marked changes in osmolality or pH were observed at the highest concentration tested (2000 µg/mL) as compared to the concurrent vehicle controls.

RANGE-FINDING/SCREENING STUDIES:
- cytotoxicity of the test item (based on replication index) was observed as follows:
7.256 to 2000 µg/mL (without metabolic activation; 3 hour treatment + 21 recovery period): cytotoxicity was between 0 % to 11 %
7.256 to 2000 µg/mL (with metabolic activation; 3 hour treatment + 21 recovery period): cytotoxicity was between 0 % to 10 %
7.256 to 2000 µg/mL (wihout metabolic activation; 24 hour treatment + 24 recovery period): cytotoxicity was between 0 % to 36 %
Please also refer to the field "Attached background material" below

MICRONUCLEUS EXPERIMENT
- cytotoxicity of the test item (based on replication index) was observed as follows:
50.00 to 2000 µg/mL (without metabolic activation; 3 hour treatment + 21 recovery period): cytotoxicity was between 0 % to 6 %
50.00 to 2000 µg/mL (with metabolic activation; 3 hour treatment + 21 recovery period): cytotoxicity was between 0 % to 6 %
50.00 to 2000 µg/mL (wihout metabolic activation; 24 hour treatment + 24 recovery period): cytotoxicity was between 0 % to 3 %

NOTE: Only the three highest concentrations were further evaluated for micronucleus analysis

Treatment of cells with tripotassium hexacyanocobaltate(III) in the absence and presence of S-9 resulted in frequencies of micronucleated binucleate cells, which were similar to and not significantly (p ≤ 0.05) higher than those observed in concurrent vehicle controls for all concentrations analysed. The micronucleated binucleate cells frequency of all tripotassium hexacyanocobaltate(III) treated cultures fell within 95th percentile of the current observed normal ranges.
No test article related increases in cells with Nucleoplasmic bridges were observed.

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

Conclusions:

It is concluded that tripotassium hexacyanocobaltate(III) did not induce micronuclei in cultured human peripheral blood lymphocytes when tested to 2000 µg/mL, an acceptable maximum concentration for in vitro micronucleus studies according to current regulatory test guidelines, in both the absence and presence of S-9.
According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance should not be considered to have a mutagenic potential.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-03-16 to 2017-04-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997-07-21

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2015-09-14

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature

Target gene:

TA98: his D3052
TA100 and TA1535: his G 46
TA1537: his C 3076
E. coli WP2 uvrA: trp-

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (containing approx. 10 % S9): sodium-ortho-phosphate buffer (100 mM, pH 7.4); NADP (4 mM); glucose-6-phosphate (5 mM); MgCl2 (8 mM); KCl (33 mM)

Test concentrations with justification for top dose:

Pre-experiment/Experiment 1: 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate (with and without metabolic activation)
Experiment 2: 33, 100, 333, 1000, 2500, and 5000 µg/plate (with and without metabolic activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: the solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (without metabolic activation) & 2-aminoanthracene (with metabolic activation))

Details on test system and experimental conditions:

METHOD OF APPLICATION: Pre-experiment/Experiment 1: in agar (plate incorporation); Experiment 2: preincubation

EXPERIMENTAL PERFROMANCE
Experiment 1:
- the following materials were mixed in a test tube and poured onto the selective agar plates: 100 μL test solution at each dose level (solvent or positive control), 500 μL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation), 100 μL bacteria suspension, and 2000 μL overlay agar
- after solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

Experiment 2:
- 100 μL test solution (solvent or positive control), 500 μL S9 mix / S9 mix substitution buffer and 100 μL bacterial suspension were mixed in a test tube and incubated at 37 °C for 60 minutes.
- after pre-incubation 2.0 mL overlay agar (45 °C) was added to each tube.
- mixture was poured on minimal agar plates.
- after solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

After incubation, revertant colonies were counted.

In parallel to each test a sterile control of the test item was performed. Therefore, 100 μL of the stock solution, 500 μL S9 mix / S9 mix substitution buffer were mixed with 2.0 mL overlay agar and poured on minimal agar plates.

NUMBER OF REPLICATIONS: triplicates

DETERMINATION OF CYTOTOXICITY
To evaluate the toxicity of the test item a pre-experiment was performed with all strains used. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. This pre-experiment was conducted as plate incorporation test.
Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
The pre-experiment is reported as main experiment I, since the following criteria are met:
Evaluable plates (>0 colonies) at five concentrations or more in all strains used.

Rationale for test conditions:

In the pre-experiment the concentration range of the test item was 3 – 5000 μg/plate. The pre-experiment is reported as experiment 1. Since no toxic effects were observed 5000 μg/plate were chosen as maximal concentration.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

A statistical analysis of the data is not mandatory.

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, and TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no precipitation of the test item occurred up to the highest investigated dose.

CYTOTOXICITY:
The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used.
No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

EXPERIMENT 1 & EXPERIMENT 2:
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with potassium hexacyanocobaltate (III) at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Please also refer for results to the field "Attached background material" below.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
Please refer to the field "Any other information on results incl. tables" below.

Table 1: Historical data

Strain		without S9 mix				with S9 mix
		Mean	SD	Min	Max	Mean	SD	Min	Max
	Solvent control	12	2.5	6	25	12	2.5	7	26
TA 1535	Untreated control	12	3.1	6	28	12	2.9	7	26
	Positive control	1130	143.1	334	1816	388	58.2	176	668
	Solvent control	10	2.2	6	19	13	3.5	7	30
TA1537	Untreated control	11	2.7	5	21	14	4.0	7	31
	Positive control	82	12.7	43	157	191	60.8	83	434
	Solvent control	25	4.4	13	43	34	6.2	15	58
TA 98	Untreated control	27	4.9	12	43	37	6.5	11	57
	Positive control	378	73.7	211	627	3949	771.8	360	6586
	Solvent control	156	26.0	78	209	148	32.3	73	208
TA 100	Untreated control	176	23.6	79	217	172	25.4	85	218
	Positive control	1966	293.2	498	2767	3798	830.4	536	6076
	Solvent control	41	5.6	27	63	50	6.8	28	72
WP2uvrA	Untreated control	42	5.8	30	63	52	6.8	36	88
	Positive control	798	362.7	319	4732	378	112.6	167	1265

Mean = mean value of revertants/plate
SD = standard deviation

Min = minimal value/Max = maximal value

Conclusions:

The substance tested non-mutagenic under the conditions of the study.
According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance should not be considered to have a mutagenic potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Genetic toxicity in vitro

The substance was not observed to be mutagenic in a reliable bacterial reverse mutation assay (OECD 471), a reliable in vitro mammalian cell micronucleus test (OECD 487), and a reliable in vitro mammalian cell gene mutation test (OECD 476).

Justification for classification or non-classification

Genetic toxicity in vitro

The substance should not be considered to have a mutagenic potential based on a bacterial reverse mutation assay (OECD 471), an in vitro mammalian cell micronucleus test (OECD 487) and an in vitro mammalian cell gene mutation test (OECD 476). The substance does not require classification according to Regulation (EC) No 1272/2008 and subsequent adaptations.