Iron tris(2-ethylhexanoate)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From April 20 to 07 May, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 1997

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Iron tris(2-ethylhexanoate)

IUPAC Name:

Iron tris(2-ethylhexanoate)

Method

Target gene:

Histidine locus for Salmonella
Tryptophan locus for E.Coli

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 tissue fraction

Test concentrations with justification for top dose:

5000, 2500, 1250, 625 and 313 µg/plate for TA1535, WP2 uvrA, TA98 with and without S9
5000, 2500, 1250, 625 and 313 µg/plate for TA100 without S9
5000, 2500, 1250, 625, 313 and 156 µg/plate for TA100 with S9
5000, 2500, 1250, 625, 313 and 156 µg/plate for TA1537 with and without S9

The maximum concentration of the test item to be used in the main experiments was selected as the concentration which elicits a moderate toxicity.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: ethanol is compatible with the survival of the bacteria and the S9 metabolic activity. The test item was found to be soluble at 500 mg/mL. This result permitted a maximum concentration of 5000 µg/plate to be used in the toxicity test.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Solvent

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

Remarks:

Absence of S9

Controls 2

Untreated negative controls:

yes

Remarks:

Solvent

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

Presence of S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in the first main assay a plate-incorporation method was used. In the second a pre-incubation method was used.

DURATION
The prepared plates were inverted and incubated for approximately 72 hours at 37°C. After this period of incubation, plates were held at 4°C for 24 hours or immediately scored by counting the number of revertant colonies on each plate.

NUMBER OF REPLICATIONS: three replicate plates were used at each test point.

Results and discussion

Test results

Species / strain:

other: TA1535, TA1537, TA98, TA100 and WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Solubility

Solubility of the test item was evaluated in a preliminary trial using DMSO and ethanol. These solvents were selected since they are compatible with the survival of the bacteria and the S9 metabolic activity. The test item was found to be soluble in ethanol at 500mg/mL after vortexing. This result permitted a maximum concentration of 5000µg/plate to be used in the toxicity test.

Toxicity test

The test item was assayed in the toxicity test at a maximum dose level 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.

Toxicity, as indicated by thinning of the background lawn and/or reduction in revertant numbers,was observed with TA1535, TA1537 and TA100 tester strains at the highest or two highest dose levels both in the absence and presence of S9 metabolism. A more pronounced toxic effect was observed with TA100 tester strain in the presence of S9 metabolism, where a dose related reduction in revertant colonies was observed from 500µg/plate onwards. No relevant increase in revertant numbers was observed at any concentration tested.

Main Assays

Two Main Assays were performed. The maximum concentration of the test item to be used in the main experiments should be determined taking into consideration cytotoxicity and solubility in the final treatment mixture.

The number of lower dose levels included in each treatment series was selected in order to have a sufficient number of analysable concentrations.

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 the following dose levels:

5000, 2500, 1250, 625 and 313 µg/plate for TA1535, WP2 uvrA, TA98 with and without S9

5000, 2500, 1250, 625 and 313 µg/plate for TA100 without S9

5000, 2500, 1250, 625, 313, 156 and 78.1 µg/plate for TA100 with S9

5000, 2500, 1250, 625, 313 and 156 µg/plate for TA1537 with and without S9.

Toxicity, as indicated by thinning of the background lawn from slight to moderate, was observed with TA1535 and TA1537 tester strains at the highest or two highest dose levels both in the absence and presence of S9 metabolism and with TA100 tester strain only in its absence. As no relevant increase in revertant numbers was observed at any concentration tested, a Main Assay II was performed using the pre-incubation method for all treatments. The dose-range was slightly modified to take into account the toxicity results of Main Assay I.

The test item was assayed at the following dose levels:

5000, 2500, 1250, 625 and 313 µg/plate for TA1535, WP2 uvrA, TA98 with and without S9

5000, 2500, 1250, 625 and 313 µg/plate for TA100 without S9

5000, 2500, 1250, 625, 313 and 156 µg/plate for TA100 with S9

5000, 2500, 1250, 625, 313 and 156 µg/plate for TA1537 with and without S9

Toxicity, as indicated by thinning of the background lawn from slight to moderate, was observed with TA1535, TA1537 and TA100 tester strains at the highest or two highest dose levels both in the absence and presence of S9 metabolism.

No precipitation of the test item was observed at the end of the incubation period at any concentration tested, in any experiment, in the absence or presence of S9 metabolism. No relevant increase in the number of revertant colonies was observed in the plate in corporation pre-incubation assay,at any dose level, with any tester strain, in the absence or presence of S9 metabolism. The sterility of the S9 mix and of the test item solutions was confirmed by the absence of colonies on additional agar plates spread separately with these solutions. Marked increases in revertant numbers were obtained in these tests following treatment with the positive control items,indicating that the assay system was functioning correctly.

Analysis of results

Acceptance criteria

The assay was considered valid if the following criteria were met:

1. Mean plate counts for untreated and positive control plates should fall within 2 standard deviations of the current historical mean values.

2. The estimated numbers of viable bacteria/plate should fall in the range of 100 – 500 millions for each strain.

3. No more than 5% of the plates should be lost through contamination or other unforeseen event.

Criteria for outcome of the assays

For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.

Evaluation

Results show that mean plate counts for untreated and positive control plates fell within the normal range based on historical control data.

The estimated numbers of viable bacteria/plate (titre) fell in the range of 100 - 500 million for each strain. No plates were lost through contamination or cracking. The study was accepted as valid.

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

Applicant's summary and conclusion

Conclusions:

Not genotoxic

Executive summary:

Method

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.

Observations

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.

Conclusion

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.