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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Potasssium ferricyanide was found to be not mutagenic in an Ames test performed according to OECD test guideline. According to REACH Annex VIII and XI (section 1), performance of an in vitro chromosome aberration study (required in section 8.4.2) and an in vitro gene mutation study (required in section 8.4.3) is not considered necessary, since adequate data from an in vivo carcinogenicity study performed with sodium ferrocyanide are available showing no neoplastic findings in rats after 2 years of exposure (see summary of the study in the respective paragraphs). This result is read across to potassium ferricyanide (read across hypothesis is attached in IUCLID Section 13). Based on this data, the substance can not be genotoxic in vivo, and thus no further in vitro study is required.

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:
26 March 2018 - 11 April 2018
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:
21 July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
31 May 2008
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
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:
rat liver S9-mix induced by Aroclor 1254 (500 mg/kg bw)
Test concentrations with justification for top dose:
Direct plate assay
Dose-range finding test (without and with S9; tester strains TA100 and WP2uvrA): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate (reported as part of experiment 1)
First experiment (without and with S9; tester strains TA1535, TA1537 and TA98): 52, 164, 512, 1600 and 5000 μg/plate

Pre-incubation assay
Second experiment (without and with S9, tester strains TA1535, TA1537, TA 98, WP2uvrA and TA100): , 52, 164, 512, 1600, 5000 μg/plate
Vehicle / solvent:
- Vehicle used: Milli-Q water
- Justification for choice of vehicle: a previously performed solubility test showed that the test item was dissolved in water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene
Remarks:
For details on positive control substances, see Table 1 and Table 2
Details on test system and experimental conditions:
Two individual experiments were performed. The dose range-finding study with tester strains TA100 and WP2uvrA was reported as part of the first experiment. The first experiment was a direct plate assay. The second experiment was a pre-incubation assay and was performed to obtain more information about the possible mutagenicity of the test item.

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period (second experiment): 30 ± 2 minutes at 70 rpm at 37 ± 1°C
- Exposure duration: 48 ± 4 h (in the dark at 37.0 ± 1.0 °C)

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain.

Methods: The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (10^9 cells/mL) of one of the tester strains, 0.1 ml of a dilution of the test item in Milli-Q water and either 0.5 ml S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0°C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.

DETERMINATION OF CYTOTOXICITY
- Method: the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.
- Other: precipitation of the test item was recorded

COLONY COUNTING
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

ACCEPTABILITY CRITERIA
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at Charles River Den Bosch.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.
Evaluation criteria:
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.
Key result
Species / strain:
S. typhimurium TA 1535
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 applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
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 applicable
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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 applicable
Positive controls validity:
valid
Additional information on results:
- There were no increases in the number of revertant colonies indicating a positive repsonse for inducing mutagenicity.
- The negative and strain-specific positive control values were within the laboratory historical control data ranges
- In both the first and second assay, criteria for a negative response were met for all tester strains with and without metabolic activation.
Conclusions:
Based on the results of an Ames test, performed according to OECD guideline 471 and GLP principles, Potassium ferricyanide is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with and without metabolic activation.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Potassium ferricyanide was tested in an Ames test performed according to OECD test guideline 471. Salmonella typhimurium TA98, TA100, TA1535, and TA1537, and Escherichia coli strain WP2uvrA in the presence or absence of metabolic activation (S9) were exposed to the substance up to and including 5000 µg/plate. The results show that potassium ferricyanide is not mutagenic in the S. typhimurium and E. coli reverse mutation assay. Further data are available for its analogue sodium ferrocyanide. A long-term exposure study via feed did not show carcinogenicity. Since no carcinogenicity was observed up to the highest concentration (450 mg/kg bw/day) after exposure for two years, it is concluded that sodium ferrocyanide is not genotoxic. This result is read across to potasium ferricyanide. Further testing for genotoxic properties is not necessary.


Justification for classification or non-classification

Based on the available data, potassium ferricyanide was found not to be genotoxic and does not need to be classified for mutagenicity according to CLP Regulation (No) EC 1272/2008.