Tricobalt bis(orthophosphate)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 10, 2022 - May 31, 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21 July 1997, corrected 26 June 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Tricobalt bis(orthophosphate)

EC Number:

236-655-6

EC Name:

Tricobalt bis(orthophosphate)

Cas Number:

13455-36-2

Molecular formula:

Co3(PO4)2

IUPAC Name:

tricobalt bis(orthophosphate)

impurity 1

Reference substance name:

Dicobalt diphosphate

EC Number:

238-689-7

EC Name:

Dicobalt diphosphate

Cas Number:

14640-56-3

Molecular formula:

Co2P2O7

IUPAC Name:

dicobalt diphosphate

impurity 2

Reference substance name:

cobalt phosphate octahydrate

Cas Number:

10294-50-5

Molecular formula:

Co3(PO4)2.8H2O

IUPAC Name:

cobalt phosphate octahydrate

Test material form:

solid: particulate/powder

Method

Target gene:

Not applicable

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

metabolic activation by a microsomal fraction of rat liver (S9-mix 10%v/v)

Test concentrations with justification for top dose:

- Concentrations for assay n°1: 50, 150, 500, 1500 and 5000 µg/plate
- Concentrations for assay n°2: 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

Solutions were prepared in ethanol.

Controls

Controls 1

Untreated negative controls:

yes

True negative controls:

yes

Remarks:

Absolute negative control containing no test item corresponding to the spontaneous reversion rate

Controls 2

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

Remarks:

Vehicle used to solubilize test item. Test item was completely soluble in ethanol at all concentrations tested.

Controls 3

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Remarks:

Solvent used to solubilize positive controls

Controls 4

Negative solvent / vehicle controls:

yes

Remarks:

NaCl 0.15 M

Remarks:

Solvent used to solubilize positive controls

Controls 5

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Stock solution was formulated in DMSO.

Controls 6

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Stock solution was formulated in NaCl 0.15 M.

Controls 7

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Stock solution was formulated in DMSO.

Controls 8

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Stock solution was formulated in NaCl 0.15M.

Controls 9

Positive controls:

yes

Positive control substance:

other: 2-anthramine: for TA1535, TA1537, TA98, TA100 and E. Coli with S9

Remarks:

Stock solution was formulated in DMSO.

Details on test system and experimental conditions:

PREPARATION OF STOCK SOLUTION
-Test item is completely soluble in ethanol at all concentrations tested.
- Stock solution for each assay was prepared extemporaneously at 100 mg/mL in ethanol.
- Test item is tested at 5000, 1500, 500, 150 and 50 µg/plate.
- Samples were taken under constant stirring.

BACTERIA
- Strains of Salmonella typhimurium and Eschericia coli are purchased from Moltox. They are maintained in laboratory.
- The genotype of bacterial strains was checked.

EXPERIMENTS
- Triplicate plates test material.
- Negative, positive and vehicle controls were included in triplicate with and without metabolic activation.
- S9 fraction, microsome fraction, prepared from Sprague Dawley rat liver homogenate, was provided by Moltox USA (S9 Moltox-11-101.5-4477 validated on 11.10.2021 - expiry date: 13.07.2023).
- For experiments without metabolic activiation: 0.1 mL bacterial culture, 50 µL each dilution of the originial solution or control and 0.5 mL of phosphate buffer were added to 2 mL overlay agar maintained super cooled in 45°C containing 10%v/v of a L-Histidine-D-Biotine solution (0.5 mM) for Salmonella Typhimurium strains or 5%v/v of nutrient broth n°2 to which are added 5 µL of a L-Tryptophane solution at 2 mg/L for Escherichia coli strain. Plates are incubated at 37°C over 48-72 hour period.The pre-incubation phase is performed for the second assay if the first assay is negative.
- For experiments with metabolic activation: protocol similar to that described above, except that 500 µL of S9-mix fraction is quickly added before pouring the mixture onto the plates. Since the first assay was negative, the pre-incubation test was performed for the second assay.

COLONY COUNTING
- After a 48-72 hour incubation period at 37°C, revertant colonies are counted in each plate.

Evaluation criteria:

The following validity criteria were checked to validate each experiment:
- the bacteriostatic activity of the highest concentration tested shall be equal to or less than 75%,
- the spontaneous reversion rate of the absolute negative control shall comply with the historical values of the laboratory,
- in presence of the solvent the spontaneous reversion rate shall comply with the historical values of the absolute negative control of the laboratory,
- the mean number of revertant colonies obtained for each strain and the corresponding positive control with and/or without metabolic activation shall comply with the historical values of the laboratory,
- negative and positive values should not show significant difference with the historical values of the laboratory (+/- 2 standard deviations).

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not valid

Additional information on results:

There is no difference between the number of spontaneous reversions, the number of reversions obtained for positive controls (with and without metabolic activation), and the mean of corresponding experimental historical values obtained in the laboratory.
There is no evidence of any increase in the number of revertant colonies in the presence of the test item without and with metabolic activaiton in Salmonella typhimurium TA1535, TA1537, TA98, TA100 strains and Escherichia coli WP2(uvrA)(pKM101) strain.
Results are confirmed in an independant experiment.

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative
In the assay conditions, solutions prepared from test item do not induce any mutagenic change in Salmonella typhimurium TA1535, TA1537, TA98, TA100 and Eschirichia coli WP2(urA-)(pKM101) without, or with metabolic activation, according to the OECD guidelines n°471.