Iron(III) manganese(II) hexacyanoferrate(II) sodium salts

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:

Experimental phase 18 March 2020 til 24 April 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

August 1998

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Guidelines of 31 March 2011

Deviations:

no

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: ICH S2(R1) guideline adopted June 2012 (ICH S2(R1)

Version / remarks:

2012; 77:33748-33749

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Iron(III) manganese(II) hexacyanoferrate(II) sodium salts

EC Number:

847-973-5

Cas Number:

2363126-51-4

Molecular formula:

NaxMny1Fey2[Fe(CN)6]z X = 1-2; Y1 = 0.5-0.9; Y2 = 0.1-0.5; Z = 0.67-1.0

IUPAC Name:

Iron(III) manganese(II) hexacyanoferrate(II) sodium salts

impurity 1

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Water

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

See section test material

Method

Target gene:

Salmonella typhimurium
Strains - Genotype - Type of mutations indicated
TA1537 - his C 3076; rfa-; uvrB-: - frame shift mutations
TA98 - his D 3052; rfa-; uvrB-;R-factor - frame shift mutations
TA1535 - his G 46; rfa-; uvrB-: - base-pair substitutions
TA100 - his G 46; rfa-; uvrB-;R-factor - base-pair substitutions

Escherichia coli
Strains - Genotype - Type of mutations indicated
WP2uvrA - trp-; uvrA-: - base-pair substitution

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:

- source of S9: Moltox (Lot no, 4146), expiry date 19 Sept 2021

- method of preparation of S9 mix
protein level was adjusted to 20 mg/mL
The S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test.
S9 5.0 mL 1.65 M KCl/0.4 M MgCl2 1.0 mL 0.1 M Glucose-6-phosphate 2.5 mL 0.1 M NADP 2.0 mL 0.2 M Sodium phosphate buffer (pH 7.4) 25.0 mL Sterile distilled water 14.5 mL

- concentration or volume of S9 mix and S9 in the final culture medium
0.5 mL per 100 mL medium

- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability)
Sterility tested in triplicate for the whole test system agar, S9, test item

Test concentrations with justification for top dose:

The dose range for Experiment 1 (plate incorporation) was based on OECD TG 471 and was 1.5 to 5000 μg/plate.
The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and ranged between 0.05 and 5000 μg/plate, depending on bacterial strain type and presence or absence of S9-mix.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:
Identity: dimethyl formamide
Supplier: Acros Organics
Batch number, (purity), expiry: 1871962, (99.97%), Apr 2022

- Justification for choice of solvent/vehicle: three solvents tested, DMF found to be best

- Justification for percentage of solvent in the final culture medium:

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene (2AA)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 3 x
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
- Test substance added in medium; in agar

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection):
3 d

Evaluation criteria:

1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
5. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).

A test item is considered non-mutagenic (negative) in the test system if the above criteria are not met.1.Pw4IU6

Statistics:

Statistical significance was confirmed by using Dunnett’s Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Results and discussion

Test results

Test results 1

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

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

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

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

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

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES (if applicable):
provided, valid

STUDY RESULTS
- Concurrent vehicle negative and positive control data
Provided, valid
For all test methods and criteria for data analysis and interpretation:

Ames test:
- Signs of toxicity
: negative
- Individual plate counts: yes
- Mean number of revertant colonies per plate and standard deviation
: see results table


HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data:
provided, valid
- Negative (solvent/vehicle) historical control data: provided, valid

Applicant's summary and conclusion

Conclusions:

In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) the test item did not induce an increase in the frequency of revertant colonies that met the criteria for a positive result, either with or without metabolic activation (S9-mix). Under the conditions of this testthe test item was considered to be non-mutagenic.

Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with suspensions of the test item using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 (plate incorporation) was based on OECD TG 471 and was 1.5 to 5000 μg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 5 to 5000 μg/plate. Seven test item concentrations were selected in Experiment 2 in order to ensure the study achieved at least four non-toxic dose levels as required by the test guideline, and were selected based on the lack of cytotoxicity noted in Experiment 1 and the potential for a change in the cytotoxicity of the test item following the change in test methodology from plate incorporation to pre-incubation.

There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Two statistically significant values were noted (TA100 at 5 and 15 μg/plate in the absence and presence of metabolic activation (S9-mix), respectively), however as the maximum fold increase was only 1.3 times the concurrent vehicle controls and the mean colony count was within the in-house historical vehicle/untreated control range for the strain the responses were considered of no biological relevance. Therefore, these values have not been highlighted in Table 2 and Table 3 as they did not meet the required criteria for a positive response.

Similarly, no biologically relevant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method). A statistically significant value was noted (TA100 at 150 μg/plate in the presence of metabolic activation (S9-mix)), however as the maximum fold increase was only 1.2 times the concurrent vehicle control and the mean colony count was within the in-house historical vehicle/untreated control range for the strain the response was considered of no biological relevance. Therefore, this value has not been highlighted in Table 5 as it did not meet the required criteria for a positive response.

The vehicle (dimethyl formamide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method).

Based on the results of Experiment 1, the same maximum dose level (5000 μg/plate) was employed in the second mutation test (pre-incubation method). Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix).

A fine test item precipitate (blue and particulate in appearance) was noted at and above 1500 μg/plate in both the presence and absence of metabolic activation (S9-mix) in Experiments 1 and 2. This observation did not prevent the scoring of revertant colonies.