2-ethylhexanoic acid, manganese salt

Endpoint summary

Additional information

Read-across approach

Selected endpoints for the human health hazard assessment are addressed by read-across, using a combination of data on the metal cation and the organic acid anion. This way forward is acceptable, since metal carboxylates are shown to dissociate to the organic anion and the metal cation upon dissolution in aqueous media. No indications of complexation or masking of the metal ion through the organic acid were apparent during the water solubility and dissociation tests (please refer to the water solubility and dissociation in sections 4.8 and 4.21 of IUCLID). Once the individual transformation products of the metal carboxylate become bioavailable (i.e. in the acidic environment in the gastric passage or after phagocytosis by pulmonary macrophages), the “overall” toxicity of the dissociated metal carboxylate can be described by a combination of the toxicity of these transformation products, i.e. the metal cation and carboxylate anion according to an additivity approach.

 

2-ethylhexanoic, manganese salt is the manganese metal salt of 2-ethylhexanoic acid, which readily dissociates to the corresponding divalent manganese cation and 2-ethylhexanoic acid anions. The manganese cation and the 2-ethylhexanoic acid anion are considered to represent the overall toxicity of 2-ethylhexanoic, manganese salt in a manner proportionate to the free acid and the metal (represented by one of its readily soluble salts). 

 

A detailed justification for the read-across approach is added as a separate document in section 13 of IUCLID.

 

Genetic toxicity

No genetic toxicity study with 2-ethylhexanoic acid, manganese salt is available, thus the genetic toxicity will be addressed with existing data on the dissociation products as detailed in the table below.

 

Table: Summary of genetic toxicity data of 2-ethylhexanoic acid, manganese salt and the individual constituents.

	Manganese sulfate (CAS# 7785-87-7)	2-ethylhexanoic acid (CAS# 149-57-5)	2-ethylhexanoic, manganese salt (CAS#15956-58-8)
In vitro gene mutation in bacteria	Negative	Negative	Negative (read-across)
In vitro cytogenicity in mammalian cells or in vitro micronucleus test	Negative	Negative	Negative (read-across)
In vitro gene mutation study in mammalian cells	Negative	Negative	Negative (read-across)

 

Manganese sulfate

The registration dossier for manganese sulfate contains a set of genetic toxicity studies which were conducted with the read-across substance manganese dichloride. No genotoxic effects were observed in a (i) bacterial reverse mutation assay (ii) in vitro mammalian chromosome aberration test and (iii) in vitro mammalian cell gene mutation test.

 

Manganese (information taken from IEH, 2004)

The Salmonella typhimurium reversion test measures mutagenicity by analysing the reversion of histidine-dependent mutants of S. typhimurium bacteria to the histidine-dependent wild type, which appear as colonies. As many compounds require metabolic activation before they show mutagenic activity, the assays are performed in the presence and absence of a suitable metabolising system. The fraction of rat liver, termed S9, is commonly used for this purpose.

There are a number of strains of S. typhimurium, and TA97, TA98, TA100, TA102, TA1535 and TA1537 have been used in in vitro mutagenicity studies with manganese chloride and manganese sulphate. Both compounds were mutagenic, without activation in S. typhimurium TA102; manganese (II) ion was the major mutagenic species (De Meo et al., 1991). As S. typhimurium TA102 identifies mutagenicity derived from compounds that produce ROS (Levin et al., 1982), the result may provide some information about mechanism of action. Other studies in S. typhimurium have produced conflicting results with manganese sulphate. It was not mutagenic in TA97, TA98, TA100, TA1535 and TA1537 in the presence or absence of metabolic activation (Mortelmans et al., 1986; NTP, 1993); however, Pagano and Zeiger (1992) reported it was mutagenic in TA97. In S. typhimurium TA98, TA102, TA1535 and TA1537, with no metabolic activation, manganese chloride was only mutagenic in strain TA1537 at concentrations of 8.8–52.8 mg Mn/ℓ (reported in the paper as 20–120 ppm; Wong, 1988). However, no data were provided on positive controls. Studies providing negative results in the absence of S9 mix should be treated with caution. Some compounds are only mutagenic after metabolic activation by the Phase 1 and Phase 2 metabolising systems present in the S9 liver homogenate (Levin et al., 1982).

Another non-mammalian genotoxicity test uses a mutation gene conversion assay in yeast, Saccharomyces cerevisiae. S. cerevisiae strain D7 requires the medium supplement, tryptophan, to grow. The basis of the mutagenicity assay is the conversion of differentially inactive alleles to wild-type alleles, by mutagenic agents on medium lacking tryptophan; the only colonies that grow are wild-type. Manganese sulphate was positive in this assay without an exogenous metabolic activation system (Singh, 1984).

The other mutagenicity assays that have been used to test the genotoxicity of manganese compounds have been performed in vitro using mammalian cells. The mouse lymphoma assay using L5178Y mouse lymphoma cells has been used to detect point mutations and clastogenic (chromosomal breakage) effects in a target gene, thymidine kinase. Manganese chloride was mutagenic in mouse lymphoma cells, in the absence of an S9 metabolising system (Oberly et al., 1982); using a range of concentrations, an increase in mutation frequency up to approximately sevenfold over solvent controls was observed. In contrast, in studies by Umeda and Nishimura (1979), manganese chloride was not clastogenic in cultured FM3A cells in the absence of S9. These studies suggest observed responses are very specific to cell type.

Manganese (II) sulphate (MnSO4) caused sister chromatid exchanges and chromosomal aberrations in Chinese hamster ovary cells in the absence of S9, and only sister chromatid exchanges in the presence of S9 (NTP, 1993).

A limited number of studies have looked at the genotoxicity of manganese compounds in vivo using fruit flies and rodents. In Drosophila melanogasta, the fruit fly, manganese chloride did not induce somatic cell mutations (Rasmuson, 1985), and manganese sulphate fed at 4500 mg Mn/kg bw (cited as 12 500 mg MnSO4/kg) or injected at 360 mg Mn/kg bw (1000 mg MnSO4/kg) did not produce sexlinked recessive lethal mutations (Rasmuson, 1985). In one study, oral doses of manganese chloride did not produce chromosomal aberrations in the bone marrow cells or spermatogonia of rats (Dikshith & Chandra, 1978). However, in another study, oral administration of manganese sulphate (36.9, 72.9 and 219.6 mg Mn/kg bw; presented in the paper as 10.25, 20.25 and 61 mg MnSO4/100g bw) or potassium permanganate (22.8, 45.5 and 133 mg Mn/ kg bw; i.e. 6.5, 13 and 38 mg KMnO4/100g bw) to rats for three weeks induced micronuclei and chromosomal aberrations in bone marrow cells and sperm-head abnormalities (Joardar & Sharma, 1990). The authors suggested that the effects were mediated by manganese (II) ions, in view of their affinity for chromosomal components (Joardar & Sharma, 1990). However, the effects were only produced at high concentrations of manganese (II) ions and may have been secondary to cytotoxicity. Manganese sulphate did not induce heritable translocations in mice, following dietary administration for 7 weeks, or dominant lethal mutations in rats, following administration by gavage once a day for 1 to 15 days (NTP, 1993).

 

Manganese (information taken from SCOEL, 2011)

Although evidence is limited, the carcinogenicity, mutagenicity, genotoxicity, and reproductive toxicity profiles for manganese and its compounds do not suggest that these aspects are key to an evaluation of occupational exposure standards.

 

2-ethylhexanoic acid

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 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, manganese salt

2-ethylhexanoic acid, manganese salt is not expected to be genotoxic, since the two constituents manganese and 2-ethylhexanoicacid have not shown gene mutation potential in a range of in vitro test systems. Thus, 2-ethylhexanoic acid, manganese salt is not classified according to regulation (EC) 1272/2008 as genetic toxicant. Further testing is not required. For further information on the toxicity of the individual constituents, please refer to the relevant sections in the IUCLID and CSR.

Justification for selection of genetic toxicity endpoint
Read-across information

Short description of key information:
2-ethylhexanoic acid, manganese salt is not expected to be genotoxic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

2-ethylhexanoic acid, manganese salt is not expected to be genotoxic, since the two constituents manganese and 2-ethylhexanoicacid have not shown gene mutation potential in a range of in vitro test systems. Thus, 2-ethylhexanoic acid, manganese salt is not classified according to regulation (EC) 1272/2008 as genetic toxicant.