Iron tris(2-ethylhexanoate)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Not mutagenic

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test

The test item was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy, according to the OECD guideline 471. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbital and 5,6-benzoflavone. The test item was used as a solution in ethanol.

The test item was assayed in the toxicity test at a maximum concentration of 5000 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 µg/plate. No precipitation of the test item was observed at the end of the incubation period, at any concentration tested, in the absence or presence of S9 metabolism. Neither toxicity, nor relevant increases in revertant numbers were observed with any tester strain, at any dose level, in the absence or presence of S9 metabolism.

On the basis of the results obtained in the preliminary toxicity test, in Main Assay I, using the plate incorporation method, the test item was assayed at 5000, 2500, 1250, 625 and 313 µg/plate with all tester strains.

No toxicity was observed at any dose level with any tester strain, in the absence or presence of S9 metabolic activation.

As no relevant increase in revertant numbers was observed at any concentration tested, a Main Assay II was performed using the same concentrations and including a pre-incubation step for all treatments. Neither toxicity, nor relevant increase in the number of revertant

colonies was observed at any dose level, with any tester strain, in the absence or presence of S9 metabolism.

No precipitation of the test item was observed at the end of the incubation period, at any concentration, in any experiment.

The test item did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of S9 metabolism.

It is concluded that the test item does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.

In vitro micronucleus test in human lymphocytes

The test item was assayed for the ability to induce micronuclei in human lymphocytes, following in vitro treatment in the absence and presence of S9 metabolic activation according to the OECD guideline 487.

Three treatment series were included in the study. A short treatment, where the cells were treated for 3 hours, was performed in the absence and presence of S9 metabolism. The harvest time of approximately 32 hours, corresponding to approximately two cell cycle

lenghts, was used. A long term (continuous) treatment was also performed only in the absence of S9 metabolism, until harvest at 31 hours.

The test item was used as a solution in ethanol. Selection of dose levels for treatment was based on the solubility of the test item in the final treatment medium.

For the 3-hour treatment series, both in the absence and presence of S9 metabolism, the test item was assayed at the maximum concentration of 1000 μg/mL and at the following dose levels: 667, 444, 296, 198, 132, 87.8, 58.5, and 39.0 μg/mL. For the continuous treatment in the absence of S9 metabolism, the additional dose level of 26.0 μg/mL was employed.

Each treatment series included appropriate negative controls (untreated and solvent controls).

In addition, positive controls were included for the short term and the long term treatment series (Cyclophosphamide and Colchicine, respectively).

Two cell cultures were prepared at each test point.

The actin polymerisation inhibitor cytochalasin B was added prior to the targeted mitosis to allow the selective analysis of micronucleus frequency in binucleated cells. The cytokinesisblock proliferation index CBPI was calculated in order to evaluate cytotoxicity.

Dose levels for the scoring of micronuclei were selected with the aim to evaluate the test item concentrations at adequate levels of cytotoxicity, covering a range from the maximum (55 ± 5%) to slight or no toxicity. In the absence of cytotoxicity, the highest treatment level was selected as the highest dose level for scoring.

In the short term treatment series, no relevant toxicity was observed at any dose level, in the absence or presence of S9 metabolic activation, while using the continuous treatment in the absence of S9 metabolic activation, a dose related cytotoxic effect was noticed. Based on the results obtained, the following concentrations were selected for the scoring of micronuclei:

S9	Treatment time (hours)	Harvest time (hours)	Dose level (μL/mL)	Cytotoxicity (%)
+	3	32	1000, 444 and 198	1, 1 and 1
-				6, 2 and -10
-	31	31	667, 296 and 132	55, 24 and 11

One thousand binucleated cells per culture were scored to assess the frequency of micronucleated cells.

Following treatment with the test item, no statistically significant increase in the incidence of micronucleated cells over the control value was observed at any dose level, in any treatment series. All the results obtained were inside the distribution of historical control data and no dose effect relationship was indicated by a linear trend analysis.

Statistically significant increases in the incidence of micronucleated cells were observed following treatments with the positive controls Cyclophosphamide and Colchicine, indicating the correct functioning of the test system.

It is concluded that the test item does not induce micronuclei in human lymphocytes after in vitro treatment, under the reported experimental conditions.

In vitro gene mutation chinese hamster V79 cells

The test item was examined for mutagenic activity by assaying for the induction of 6-thioguanine resistant mutants in Chinese hamster V79 cells after in vitro treatment according to the OECD guideline 476. A Main Assay was performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and

betanaphthoflavone. Based on solubility data, the maximum practicable concentration of the test item in the final treatment medium was 500 µg/mL using ethanol (EtOH) as solvent.

On the basis of this result, a preliminary cytotoxicity assay was performed both in the absence and presence of S9 metabolic activation, using the maximum dose level of 500 µg/mL and a wide range of lower dose levels: 250, 125, 62.5, 31.3,15.6, 7.81, 3.91 and 1.95 µg/mL.

In the absence of S9 metabolism, slight reduction of relative survival (RS) was noted at the three highest dose levels, while no relevant toxicity was observed at the remaining concentrations tested. In the presence of S9 metabolism, no relevant toxicity was seen at any concentration tested. Precipitation was noted from 31.3 µg/mL onwards, in the absence of S9 metabolism and at the two highest dose levels in its presence.

A Main Assay for mutation to 6-thioguanine resistance was performed. Cells were treated for 3 hours, both in the absence and presence of S9 metabolism and maintained in growth medium for 8 days to allow phenotypic expression of induced mutation. The maximum concentration to be assayed was selected on the basis of solubility results. The following dose levels were used:

62.5, 31.3, 15.6, 7.81 and 3.91 µg/mL without metabolic activation;

500, 250, 125, 62.5 and 31.3 µg/mL with meabolic activation.

No toxicity was observed at any concentration tested, in the absence or presence of S9 metabolism. Upon addition of the test item to the culture and by the end of treatment, precipitation was noted at the two highest dose levels, in both treatment series. No relevant increases in mutant numbers or mutant frequency were observed following treatment with the test item at any dose level, in the absence or presence of S9 metabolism.

Negative and positive control treatments were included in each mutation experiment in the absence and presence of S9 metabolism. Marked increases were obtained with the positive control treatments, indicating the correct functioning of the assay system.

It is concluded that the test item does not induce gene mutation in Chinese hamster V79 cells after in vitro treatment in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), for the purpose of the classification for germ cell mutagenicity, substances are allocated in one of two categories in consideration of the fact that they are:

- substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans or substances known to induce heritable mutations in the germ cells of humans or

- substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

The test substance did not show any reasons of concern in the tests performed.

In conclusion, the substance does not meet the criteria to be classified for genetic toxicity according to the CLP Regulation (EC 1272/2008).