Ferrocholinate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ferric Choline Citrate is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22. Oct. 2019 (Study Plan dated); 29. Oct. 2019 (Experimental Starting Date); 15. Nov. 2019 (Experimental Completion Date)

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:

Adopted 21. Jul. 1997.

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) No. 440/2008, EU-Method B.13/14 adopted 30. May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

bacteria, other: S. typhimurium TA 97a

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 102

Species / strain / cell type:

S. typhimurium TA 1535

Metabolic activation:

with and without

Vehicle / solvent:

Demineralised water was used as vehicle control, prepared by LAUS GmbH, from an ion-exchanger, batch: 20190828 for the test item solutions

In a non-GLP pre-test, the solubility of the test item was tested in a concentration of 50 g/L in deminerali-zed water, dimethyl sulfoxide (DMSO) and acetone.
The solid test item is sufficiently soluble in demin. water, only.
Based on the non-GLP pre-test, demin. water was chosen as vehicle, because the test item was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.

Positive controls:

yes

Positive control substance:

other: 4-Nitro-1,2-phenylene diamine

Positive controls:

yes

Positive control substance:

sodium azide

Positive controls:

yes

Positive control substance:

other: 2-Amino-anthracene

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Negative solvent / vehicle controls:

yes

Remarks:

Dimethylsulfoxide (DMSO)

Negative solvent / vehicle controls:

yes

Remarks:

Demineralised water

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of independent experiments: Three replicates, with/without S9, for each solvent which was used in the test, incubation for 48 hours at 37 ±1°C.

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): The titre was determined by dilution of the overnight culture using sodium chloride solution and placing 0.1 mL on maximal-soft agar. Incubation for 48 hours at 37 ±1 °C followed. It should give a density of 109 cells/mL (at the least), two replicates with and without metabolic activation.
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk: Suspensions of bacterial cells are exposed to the test substance in the presence and in the absence of an exogenous metabolic activation system. In the plate incorporation method, these suspensions are mixed with an overlay agar and plated immediately onto minimal medium. In the pre-incubation method, the treatment mixture is incubated and then mixed with an overlay agar before plating onto minimal medium. For both techniques, after 2 or 3 days of incubation, revertant colonies are counted and compared to the number of spontaneous revertant colonies on solvent control plates.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: In the pre-incubation method, the treatment mixture is incubated and then mixed with an overlay agar before plating onto minimal medium. For both techniques, after 2 or 3 days of incubation, revertant colonies are counted and compared to the number of spontaneous revertant colonies on solvent control plates.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): 109 cells/mL, correlating to at least 100 colonies/plate after dilution.

FOR GENE MUTATION:
Five different analysable and non-toxic concentrations should be used for the evaluation of the mutagenic potential of the test item.
The colonies were counted visually and the numbers were recorded. A validated spreadsheet (attached) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control.
The mean values and standard deviations of each threefold determination were calculated as well as the increase factor of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (mean revertants less mean spontaneous revertants) was given.
No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found.
Therefore, the test item is stated as not mutagenic under the conditions of this experiment.

Key result

Species / strain:

other: S. typhimurium TA 97a

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Based on the results of this study it is concluded that Ferric Choline Citrate is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available