2-ethylhexanoic acid, iron salt

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No genetic toxicity study with 2-ethylhexanoic acid, iron salt is available, thus the genetic toxicity will be addressed with existing data on the individual assessment entities iron and 2-ethylhexanoic acid. 2-ethylhexanoic acid, iron salt is not expected to be genotoxic, since the two moieties iron and 2-ethylhexanoic acid have not shown gene mutation potential in bacteria and mammalian cells as well as in in vivo micronucleus tests conducted with ferric chloride and ferrous sulphate.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Iron

In vitro- Ferric

An Ames test with Salmonella typhimurium strains TA97a, TA98, TA100, TA102, TA1535, TA1537, TA1538 was conducted with FeCl3 x 6H2O dose levels up to 10000 µg Fe/plate. The cytotoxicity of the test substance was determined with and without metabolic activation (rat liver S9 mix) prior to the mutagenicity test in order to determine appropriate testing concentrations. All tests were perforrned in triplicates. As indication of a positive effect doubling of the mutant frequency was used. A substance is considered positive if it shows induction of at least doubling of mean number of revertants per plate in at least one tester strain, accompanied by a clear dose response. In strains TA 1537 and 1538, an increase in revertants of less than threefold would be confirmed in a repeat experiment. The Ames test showed neither with nor without metablic activation an increase in the number of revertants. FeCl3 x 6H2O is negative for mutagenicity in presence and in the absence of metabolic activation under the conditions of this test system.

A L5178Y TK+/- mouse lymphoma cell assay was conducted with FeCl3 x 6H2O. The cytotoxicity of the test substance was determined with and without metabolic activation (rat liver S9 mix) prior to the mutagenicity test in order to determine the appropriate testing concentrations. The mutagenicity assay was performed in duplicates. As indication of a positive effect doubling of the mutant frequency was used. The mouse lymphoma cells showed without metablic activation no increase in the number of induced mutants. In the presence of S9 at marked increase in cytotoxicity, there was a dose-related mutagenic response. FeCl3 x 6H2O is negative for mutagenicity in presence and absence of metabolic activation under the conditions of this test system.

 

In vitro- Ferrous

A L5178Y TK+/- mouse lymphoma cell assay was conducted with FeSO4 x 7H2O. The cytotoxicity of the test substance was determined with and without metabolic activation (rat liver S9 mix) prior to the mutagenicity test in order to determine the appropriate testing concentrations. The mutagenicity assay was performed in duplicates. As indication of a positive effect doubling of the mutant frequency was used. The mouse lymphoma cells showed with and without metablic activation a weak increase in the number of induced mutants at cytotoxic concentrations. FeSO4 x 7H2O is negative for mutagenicity in absence and presence of metabolic activation under the conditions of this test system.

 

In vivo- Ferric

Groups of 4 - 9 C57BL/6J female mice received a single dose of ferric chloride either orally or intrarectally (2, 6.5 and 13 mg Fe/kg by both routes) at a volume of 0.2 ml in saline. The lowest dose tested was equivalent to a therapeutic dose in iron deficiency anaemia. The iron compounds were administered to both fasting and non-fasting mice (oral administration only). Samples of fore stomach, duodenum and colon were taken following oral administration and colon samples after intrarectal administration. The samples were examined for micronulei and nuclear aberration postulating that micronuclei are a specific sign of genetic damage while nuclear aberrations are more unspecific effects related to cellular toxicity. Ferric chloride did not significantly increase the incidence of micronuclei in the stomach, duodenum or colon following oral administration and only marginally in the colon after intrarectal administration. In the stomach, duodenum and colon nuclear aberrations indicating cellular toxicity were increased in fasting mice receiving oral ferric chloride. Following intrarectal administration ferric chloride showed cellular toxicity to the colon producing increase in nuclear aberrations. Since nuclear aberrations are more unspecific effects related to cellular toxicity ferric chloride does not appear to have a significant genotoxic effect under the conditions of this test system.

 

In vivo- Ferrous

Groups of 4-9 C57BL/6J female mice received a single dose of ferrous sulphate either orally or intrarectally (2, 6.5 and 13 mg Fe/kg by both routes) at a volume of 0.2 ml in saline. The lowest dose tested was equivalent to a therapeutic dose in iron deficiency anaemia. The iron compound were administered to both fasting and non-fasting mice (oral administration only). Samples of fore stomach, duodenum and colon were taken following oral administration and colon samples after intrarectal administration. The samples were examined for micronulei and nuclear aberration postulating that micronuclei are a specific sign of genetic damage while nuclear aberrations are more unspecific effects related to cellular toxicity. Ferrous sulphate did not significantly increase the incidence of micronuclei in the stomach, duodenum or colon following oral administration and only marginally in the colon after intrarectal administration. In the colon nuclear aberrations indicating cellular toxicity were increased in fasting and non-fasting mice receiving oral ferrous sulphate and marginally in the duodenum of fastening mice. Following intrarectal administration ferrous sulfate showed cellular toxicity to the colon producing increase in nuclear aberrations. Since nuclear aberrations are more unspecific effects related to cellular toxicity ferrous sulphate does not appear to have a significant genotoxic effect under the conditions of this test system.

2-ethylhexanoic acid

in vitro

2-ethylhexanoic acid was negative in the bacterial Ames test with S. typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 and E. coli WP2 uvr A (Jung et al., 1982; Zeiger et al., 1988; Warren et al., 1982), as well as in a HPRT locus assay with mammalian CHO cells (Schulz et al., 2007). In cultured human lymphocytes, 2-ethylhexanoic acid induced a minimal increase in frequency of sister-chromatid exchanges (below 1.5 fold increase at concentrations of the test substance of 0.63 to 2.5 mM; Sipi et al., 1992), which is not considered significant.

 

in vivo

In an in vivo micronucleus assay with mice, 2-ethylhexanoic acid was administered by gavage up to the maximum tolerated oral dose of 1600 mg/kg/day. No bone marrow toxicity was observed, nor did the test substance induce any bone marrow micronuclei (Holstrom et al., 1994).

2-ethylhexanoic acid, iron salt

2-ethylhexanoic acid, iron salt is not expected to be genotoxic, since the two assessment entities iron and 2-ethylhexanoic acid have not shown gene mutation potential in bacteria and mammalian cells as well as in in vivo micronucleus tests conducted with both iron species, ferric chloride (trivalent iron) and ferrous sulphate (divalent iron). Further testing is not required. For further information on the toxicity of the individual assessment entities, please refer to the relevant sections in the IUCLID and CSR.

Justification for classification or non-classification

2-ethylhexanoic acid, iron salt is not expected to be genotoxic, since the two assessment entities iron and 2-ethylhexanoic acid have not shown gene mutation potential in bacteria and mammalian cells as well as in in vivo micronucleus test. Thus, 2-ethylhexanoic acid, iron salt is not to be classified according to regulation (EC) 1272/2008 for germ cell mutagenicity.