Ferric ammonium citrate

Endpoint summary

Additional information

No data are available for reaction mass of ammonium iron (III) citrate and ammonium sulfate. Information is available for a substance described as ferric ammonium citrate (details of composition not known) from studies on mutagenicity to bacteria and in vitro cytogenicity to Chinese hamster lung cells. No further information is available for the registered substance. However, data are available for the related substances, ferric chloride (CAS 7720-78-7) and ferric chloride hexahydrate (CAS 10025-77-1) (the chemical species available to the test organisms are not affected by which of these two substances is tested). Both the registered substance and the read across substance contain ferric ions when in aqueous solution, and the key studies have been chosen to examine the potential for genetic toxicity of the ferric ion. The chloride counter ion of the read-across substance is not expected to contribute to the potential for genetic toxicity, as it is ubiquitous in nature and forms part of a normal diet. The non-ferric chemical species that are part of the registered substance are ammonium, citrate and sulfate. Neither ammonium nor sulfate ions are expected to contribute to the potential for genetic toxicity of the registered substance. Information on the lack of genetic toxicity of ammonium sulfate is available in the public domain (OECD SIAR (2004)). Information on the lack of potential for genetic toxicity of citrate is also available in the public domain (OECD SIAR (2001)) and moreover citrate is involved in the metabolism of eukaryote cells, and is ubiquitous in living organisms.

In vitro

Iron salts have been extensively studied in vitro, with variable results. The key studies are chosen based on the biological relevance of the results of the studies.

Bacterial mutagenicity

Ferric chloride hexahydrate (CAS 10025-77-1 FeCl3.6H2O) has been tested using the bacterial reverse mutation assay in Salmonella typhimurium strains TA97a, TA98, TA100, TA102, TA1535, TA1537 and TA1538 at dose levels up to 10000 µg Fe/plate (Dunkel (1999)). No evidence for test substance induced mutagenicity to bacterial cells was reported. It is concluded that ferric chloride is negative for mutagenicity to bacteria under the conditions of this test. Additional information on the lack of mutagenicity to bacteria of ferric ammonium citrate is given in a screening study of food additives (Ishidate (1984)). It was reported that ferric ammonium citrate does not induce reverse mutations in bacteria. The key study was selected based on date of publication and the amount of detail given in the paper of test method and results.

In vitro cytogenicity

Ferric chloride has been tested for the induction of micronuclei in vitro in a reliable study conducted according to OECD draft guideline 487 and in compliance with GLP (Schulz (2009)). No increase in the frequency of micronucleated cells was observed in Chinese hamster V79 cells in the presence or the absence of metabolic activation in either the initial or the repeat experiment. Appropriate positive and solvent controls were included and gave expected results. The substance is considered to be negative for micronuclei induction under the conditions of the test. Additional information on the in vitro cytogenicity of ferric ammonium citrate is reported in a study of chromosome aberration in Chinese hamster lung cells (Ishidate (1984)). Ferric ammonium citrate was tested in a study conducted according to a protocol that is similar to OECD 473. No test-substance induced increase in the frequency of cells with aberrations or of induction of polyploidy was observed in the absence of metabolic activation. The substance was not tested in the presence of metabolic activation. Appropriate solvent or untreated controls were included. Positive controls are not reported in the publication, but positive results were obtained with some substances tested. It was concluded that the test substance was negative for the induction of chromosome aberrations under the conditions of the test. The most recent study was chosen as key.

Mutagenicity to mammalian cells

Ferric chloride hexahydrate has been tested for mutagenicity to mammalian cells in an assay using mouse lymphoma L5178Y cells (Dunkel (1999)). The publication reported a negative response in the absence of metabolic activation and a positive dose-related response in the presence of metabolic activation. The response was associated with a marked increase in cytotoxicity (concentration range tested: 0.2-1.2 µg Fe/ml (+MA), 309-1030 µg/ml (-MA)). This study was chosen as key as it is the most recent reliable study on ferric chloride. The apparent mutagenic effect was not observed in an earlier publication which reported that ferric chloride was negative with and without metabolic activation in the mouse lymphoma forward mutation assay (McGregor (1988)). The positive result obtained in the presence of metabolic activation which was reported by Dunkel (1999) was attributed to the S9 mix reducing Fe(III) to Fe(II). The latter ion is more readily taken up by cells and may then be responsible for the formation of hydroxyl radicals which are mutagenic. It is therefore considered that the positive response observed will not occur in vivo as the uptake and transport (and therefore the exposure of cells to Fe ions) of the essential element iron is strictly regulated in mammalian metabolism.

In vivo

Ferric chloride has been tested in a study to examine the incidence of micronuclei in an assay using tissues from the gastrointestinal tract (Bianchini (1988)). The test substance is not considered to have a significant genotoxic effect on the mouse gastrointestinal tract as measured by induction of micronuclei. An increased incidence of nuclear aberrations was considered evidence of general toxicity.

The overall conclusion from in vitro studies is that ferric ions and ferric ammonium citrate are not mutagenic. This conclusion is supported by the negative result in vivo, which used a non-standard method in order to examine the effects of Fe(III) in tissue that is most directly exposed to iron in the diet.

Short description of key information:

In vitro:

Gene mutation (Bacterial reverse mutation assay / Ames test): read-across from structural analogue ferric  chloride hexahydrate (CAS 10025-77-2): negative with and without activation in all strains tested (similar to OECD TG 471) (Dunkel (1999)).

Cytogenicity in mammalian cells: in vitro micronucleus: read-across from structural analogue ferric  chloride (CAS 7705-08-0): negative in Chinese hamster V79 cells (OECD draft TG 487) (Schulz (2009)).

Mutagenicity in mammalian cells: read-across from structural analogue ferric  chloride hexahydrate (CAS 10025-77-1): positive without metabolic activation in L5178Y mouse lymphoma cells (similar to OECD TG 476) (Dunkel (1999)).

In vivo:

Micronucleus assay: mouse oral gavage study: read-across from structural analogue ferric  chloride hexahydrate (CAS 10025-77-1): negative for induction of micronuclei in tissues of the gastrointestinal tract (method of Wargovich, Medline and Bruce, 1983) (Bianchini (1988)).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available in vitro and in vivo genotoxicity data, reaction mass of ammonium iron (III) citrate and ammonium sulfate is not classified for mutagenicity according to Regulation (EC)1272/2008.