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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Additional information

There is a Bacterial Reverse Mutation Assay available for the submission substance, performed according to the method of Ames prior to the invention of GLP and the adoption of OECD guideline 471, but according to the standards at the time and with only minor deviations from the guideline protocols, which were established in 1983.

The test was performed in Salmonella typhimurim strains TA 1535, TA 1537, TA 1538, TA 100 and TA 98 (Cuthbert, 1977). Test substance concentrations of 4, 20, 100, 500 and 2500 µg/ plate in DMSO were tested using the plate incorporation method, with and without the addition of a rat liver homogenate metabolising system (S9-mix). The substance was not mutagenic in any cell strains at dose levels up to and including 2500 µg per plate. A slight cytogenicity in form of slightly reduced revertant numbers at 2500 µg/plate was seen in some strains. The test substance was present mainly as suspension at concentrations >= 100 µg/plate, and 2500 µg/plate was already considered as a very high concentration, so omitting higher test concentrations is justified.

In addition, there is a Bacterial Reverse Mutation Assay available for the read-across substance Sodium Ferrocyanide, also performed according to the method of Ames prior to the invention of GLP and the adoption OECD guideline 471, but according to the standards at the time and with only minor deviations from the guideline protocols invented later, which was used as source for read-across. The test was also perfomed in Salmonella typhimurim strains TA 1535, TA 1537, TA 1538, TA 100 and TA 98 (Cuthbert and McGregor, 1977). Test substance concentrations of 4, 20, 100, 500 and 2500 µg/ plate in phosphate buffer pH 7.0 were tested using the plate incorporation method, with and without the addition of a rat liver homogenate metabolising system (S9-mix). The substance was not mutagenic in any cell strains at dose levels up to and including 2500 µg per plate. The substance was completely dissolved at all concentrations in all plates. Comparable to the submission substance a slight cytotoxicity at 2500 µg/plate was seen in some strains.

Both studies are considered as reliable and are in accordance with the standards at their time, but due to their age and the deviations from current guidelines the endpoint gene mutation in bacteria is assessed in a weight of evidence approach taking into account the results from the submission substance itself and the structurally-related substance Sodium Ferrocyanide in order to enhance the value of the assessment. The results demonstrate no mutagenicity up to and including a concentration of 2500 µg/plate (highest concentration tested) and slight cytotoxic effects at 2500 µg/plate for both substances, which supports the results observed for the submission substance. The similarity of the toxicological profiles of both substances is further supported by the comparable results demonstrated for the other toxicological endpoints.

 

A reliable in vitro mammalian cell gene mutation assay was performed with Ammonium iron (3+) hexakis (cyano-C) ferrate (4- ) according to OECD Guideline 476 in L5178Y mouse lymphoma cells (Nakagawa, 2010). The cell growth inhibition test was conducted at 39.4, 78.9, 158, 316, 631, 1262, 2524, and 5049 μg/mL for 3 hours in the absence and presence of rat liver S9 mix in the short-term treatment assay (–S9 mix assay and +S9 mix assay) and for 24 hours in the absence of S9 mix in the long-term treatment assay (24-hour assay). The observed increase in mutation frequency was significant and dose-dependent at concentrations above 1262 µg/mL in each treatment condition. However, precipitation of the test substance was observed at all concentrations. Moreover, the increased mutation frequencies were observed at concentrations that at the same time caused marked reduction of cell viability. Thus, it can be concluded that the observed effects are rather due to cytotoxicity and therefore do not represent direct genotoxic effects.

 

A reliable in vitro mammalian chromosome aberration study was performed with Ammonium iron (3+) hexakis (cyano-C) ferrate (4- ) according to OECD Guideline 473 in CHL/IU fibroblasts derived from the lung of a female Chinese hamster (Nakagawa, 2010). A preliminary cell growth inhibition test was conducted at 39.4, 78.9, 158, 316, 631, 1262, 2524, and 5049 μg/mL for 6 hours in the absence and presence of rat liver S9 mix in the short-term treatment assay (–S9 mix assay and +S9 mix assay) and for 24 hours in the absence of S9 mix in the long-term treatment assay (24-hour assay). A growth inhibition of >50% was only observed in the 24-hours assay at 2524 and 5049 µg/mL while at all other concentrations the growth inhibition was below 50%. Chromosomal aberrations were analyzed in the main test in which cells were treated with concentrations of 631, 1262, 2524, and 5049 μg/mL in the absence and presence of rat liver S9 mix in a short-term treatment assay. In the 24-hours assay the used concentrations were 505, 757, 1010, 1262, 1515, 1767, 2019, 2272, and 2524 μg/mL.

As a result of these tests, the incidences of cells with structural and numerical aberration were less than 5% in each treatment condition. In the +S9 mix assay, the reproducibility of the negative result was confirmed in the confirmation test where the cells were incubated with 1250, 2500, and 5000 μg/mL of the test substance. Although precipitation of the test substance was observed in the medium at the beginning and the end of the treatment in each test substance treatment group under each treatment condition, the cell growth was not inhibited more than 50%. Based on these results,ammonium iron (3+) hexakis (cyano-C) ferrate (4-) was considered not-clastogenic in mammalian cells under the conditions of this study.

 

Justification for selection of genetic toxicity endpoint

No specific study selected as individual studies addressing various mechanisms of genetic toxicity (Gene mutation in bacteria, Chromosomal aberration in mammalian cells, Gene mutation in mammalian cells) are available to satisfy the data requirements of the endpoint.

Short description of key information:

Gene mutation study in bacteria (Ames), Weight of Evidence incl. Read-across: negative

Gene mutation in mammalian cells (MLA, OECD 476): negative

Cytogenicity in mammalian cells (CA, OECD 473): negative

Genetic toxicity in vivo: no data

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

According to DSD (67/548/EC) and CLP (1272/2008/EC) classification criteria for mutagenicity, no classification is warranted for Ammonium iron (3+) hexakis (cyano-C) ferrate (4-) regarding this endpoint, and the data are therefore conclusive but not sufficient for classification.