In vitro and in vivo genotoxicity assessment of ferric ferrocyanide and potassium-cobalt ferrocyanide

Highlights

• Fe-ferrocyanide and K-Co-ferrocyanide are cesium decontaminant agents in human.

• in vitro genotoxic profiles of both agents are similar with positive responses.

• in vivo, Fe-ferrocyanide induced genotoxicity.

• It is expected that K-Co-ferrocyanide is an in vivo genotoxicant.

• The use of both decontaminants must follow a benefit/risk approach.

Abstract

Ferric hexacyanoferrate(II) (Fe₄[Fe(CN)₆]₃), i.e. Prussian blue (PB) has been used for many years to remove from the body the two toxic isotopes of cesium and thallium following irradiation.

Recently, potassium cobalt hexacyanoferrate(II) (K₂COFe(CN)₆), which has shown a better efficacy for decontamination, is also being considered for use to enhance the elimination of cesium isotopes. In view to its preclinical and clinical development, in vitro and in vivo GLP-compliant genotoxicity studies were carried out on this product as well as on PB for comparison. Several tests dissecting the main events leading to genotoxicity, i.e. mutagenicity and chromosomal aberrations, both structural and quantitative were implemented.

In vitro, no mutagenic effect was observed in the Ames test but both compounds were positive in the mouse lymphoma assay on TK locus and induced clastogenic effects in the in vitro chromosomal aberrations test on human lymphocytes, either in absence or in presence of metabolic activation.

K-Co-ferrocyanide was also assayed in vivo in the mouse bone marrow micronucleus assay and PB was assessed for DNA fragmentation in the rodent Comet assay in both glandular stomach and colon. In the in vivo micronucleus mouse bone marrow, K-Co-ferrocyanide did not display any genotoxic activity up to 2000 mg/kg/d (x2) by oral route. In opposite, PB induced a significant increase in DNA fragmentation both in the glandular stomach and in the colon of rat treated 3 times with intake ranging from 2000 to 500 mg/kg. PB should be considered as an in vivo mutagen as well as Potassium cobalt hexacyanoferrate(II) since the in vitro genotoxicity profiles of both ferrocyanides are quite similar.

Their use as cesium/ thallium decontamination agents in human should be assessed following a benefit/risk approach to enable a robust decision-making.

Introduction

Ferric hexacyanoferrate(II), known as Prussian blue (PB), has the empirical formula Fe₄[Fe(CN)₆]₃. It was probably synthesized for the first time by the paint maker Diesbach in Berlin in 1704, and it was one of the first synthetic pigments. Prussian blue is available as Radiogardase® in Germany and France. The use of Prussian blue against thallium poisoning and as a decorporation agent for ¹³⁴cesium and ¹³⁷cesium has been investigated since the 1960s. It was recommended in the treatment of thallium intoxication in the 1970s and it is now normally used. The disaster at the Chernobyl nuclear reactor in 1986 led to further studies on the elimination of radioactive cesium. In 1987, following Goiania accident in Brazil, Prussian blue was used in the management of a large scale radiation disaster [1]. It is used as an orally ingested drug to enhance the excretion of isotopes of cesium and thallium from the body by means of ion exchange: thallium ions are excreted into the intestine and reabsorbed mainly in the colon into blood to be excreted again into the intestinal tract while cesium is excreted into the intestinal tract in the bile to be reabsorbed into portal blood and transported to the liver to again be excreted via bile (enterohepatic circulation). Therefore, orally administered Prussian blue is able to take over these two toxic metal ions in the intestine, stopping the reabsorption from the gastrointestinal tract and favoring their fecal excretion. Prussian blue is not intestinally absorbed in significant amounts, and can be considered safe and effective for the treatment of internal contamination with radioactive or nonradioactive thallium, and with radioactive cesium.

Potassium cobalt hexacyanoferrate(II), formula K₂COFe(CN)₆, has also been investigated as a cesium decontamination agent. In vivo, on rats, this compound proved to be at least as efficient as PB and even more efficient according some studies [2] to enhance the excretion of isotopes of cesium from the body. This result, associated with a total insolubility, meaning no absorption in gastrointestinal tract, made Potassium cobalt hexacyanoferrate(II) suitable for a potential clinical use.

To investigate the opportunity of further preclinical and clinical development of this compound, it was then necessary to assess its genotoxicity profile and to compare it with the reference clinical standard PB. For this, the bacterial reverse mutation assay (Ames test), the mutation assay at the TK locus in L5178Y mouse lymphoma cells and the in vitro chromosomal aberrations test on peripheral blood lymphocytes were carried out. Moreover, the in vitro Unscheduled DNA Synthesis (UDS) assay was performed with potassium-cobalt ferrocyanide salts. Furthermore, the in vivo mouse bone marrow micronucleus test and the Comet Assay in rat were implemented for Ferric ferrocyanide exclusively.