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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

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

Constituent 1
Chemical structure
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
Chemical structure
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
Chemical structure
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.
Controlsopen allclose all
Untreated negative controls:
yes
True negative controls:
yes
Remarks:
Absolute negative control containing no test item corresponding to the spontaneous reversion rate
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.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Remarks:
Solvent used to solubilize positive controls
Negative solvent / vehicle controls:
yes
Remarks:
NaCl 0.15 M
Remarks:
Solvent used to solubilize positive controls
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Stock solution was formulated in DMSO.
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Stock solution was formulated in NaCl 0.15 M.
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Stock solution was formulated in DMSO.
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Stock solution was formulated in NaCl 0.15M.
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 resultsopen allclose all
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
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
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
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
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.