Iron tris(2-ethylhexanoate)

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Remarks:

in vitro cytotoxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From May 22 to 31, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted according to accepted guideline but used in a weight of evidence approach.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: ENV/JM/MONO(2010)20 – series of Testing and Assessment No. 129 – Guidance document on using cytotoxicity tests to estimate starting doses for acute oral systemic test.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Iron tris(2-ethylhexanoate)

IUPAC Name:

Iron tris(2-ethylhexanoate)

Test animals

Species:

other: in vitro study

Administration / exposure

Route of administration:

other: in vitro study

Vehicle:

ethanol

Results and discussion

Preliminary study:

A preliminary dose-range finder experiment was undertaken in order to select appropriate dose levels for theMain Assay. In this experiment, the test item was assayed at a maximum dose level of 250 µg/mL and at a wide range of lower dose levels: 25.0, 2.50, 0.250, 0.0250, 0.00250, 0.000250 and 0.0000250 µg/mL. No precipitation of the test item was noted upon addition of the test item to the cultures at any concentration tested. Microscopic evaluation of cultures showed an increase in cell growth after treatment with the test item at 0.250, 2.50 and 25.0 µg/mL; at these concentrations a marked increase in neutral red uptake was
also observed. Reduction of the cell layer and change in cell morphology were noted at 250 µg/mL, where mild reduction in neutral red uptake was also seen. However, at this concentration, precipitation of the test item was observed by the end of treatment. After making the appropriate blank correction, mean values of absorbance, standard deviation and percentages over the solvent control values were calculated for each dilution of test item and positive control.

Effect levels

Dose descriptor:

other: IC50

Remarks on result:

other: not determinable due to precipitation of the compound at the highest dose levels

Any other information on results incl. tables

Solubility

Solubility of the test item was evaluated in a preliminary trial using ethanol (EtOH), which is the most appropriate solvent as indicated by the Sponsor. This solvent is known to be cytotoxic in the selected assay system at a concentration greater than 0.5% (volume fraction).

Based on this statement, ethanol has to be present at a constant volume equal to or lower than 0.5% (v/v) in the solvent/vehicle control and test item treatment media. A clear preparation was obtained in EtOH at the concentration of 200 mg/mL, after mixing with vortex. Solutions at 200 and 100 mg/mL, when added to Chemical DilutionMedium (CDM) in the ratio of 1:100, gave plenty precipitate. An opaque mixture with slight precipitate was obtained after addition of solution at 50.0 mg/mL to CDM. This suspension was considered suitable for treatment. Since solutions/suspensions in CDM should be added to DMEM culture medium in ratio 1:1, this result permitted a maximum final concentration of 250 µg/mL to be used in the preliminary dose-range finder experiment.

Main Assay

Based on the results obtained, in order to assay the test item at soluble concentrations, a Main Assay was performed using the following dose levels: 125, 50.0, 20.0, 8.00, 3.20, 1.28, 0.512 and 0.205 µg/mL; a smaller dilution factor was used in order to focus on the maximum practicable concentration. Moreover, in the preliminary experiment the mean OD value obtained for the solvent/vehicle control fell in the lower part of the historical distribution range for negative controls; therefore, in order to avoid toxicity due to the solvent, a reduced final percentage of solvent/vehicle was used for treatment. No precipitation of the test item was noted upon addition of the test item to the cultures at any concentration tested.

Treatment at concentrations between 0.512 and 20.0 µg/mLyielded an increased cell growth, as indicated by microscopic evaluation and neutral red uptake. Slight reduction of the cell layer compared to the negative control, was noted at 50.0 µg/mL, while reduction of the cell layer and change in cell morphology were noted at the highest dose level, where mild reduction in neutral red uptake was also seen. However, at this concentration precipitation of the test item was observed by the end of treatment. Optical density values of NR extract at 530 nm are presented in Table 2. After making the appropriate blank correction, mean values of absorbance, standard deviation and percentages over the solvent control values were calculated for each dilution of test item and positive control.

Negative control cultures gave acceptable optical density values (0.183 ≤OD530 NRU ≤ 0.769). Dose related toxicity was observed after treatment with the positive control, with a calculated IC50 value of 45.6 µg/mL, indicating the correct functioning of the assay system.

Applicant's summary and conclusion

Interpretation of results:

other: Not cytotoxic under the reported experimental conditions

Conclusions:

The test item was not considered to be cytotoxic, under the reported experimental conditions. However, solubility is a limiting factor in achieving sufficient cytotoxicity for the calculation of the IC50 value.

Executive summary:

Method

The potential in vitro cytotoxicity of the test item has been evaluated on Balb/c 3T3 cells. Cell cultures were treated with different concentrations of the test item. At the end of treatment time, measurement of neutral red uptake was performed to assess cytotoxicity and cell layers were examined in order to evaluate changes in cell morphology. Test item solutions were prepared using Ethanol.

A preliminary range-finder experiment was undertaken in order to select appropriate dose levels for the Main Assay. Based on the results obtained in a preliminary solubility trial, the test item was assayed at a maximum dose level of 250 µg/mL and at a wide range of lower dose levels: 25.0, 2.50, 0.250, 0.0250, 0.00250, 0.000250 and 0.0000250 µg/mL.

Observations

Reduction of the cell layer and change in cell morphology were noted at 250 µg/mL, where slight reduction in neutral red uptake was also observed. However, at this concentration precipitation of the test item was seen.

Since cytotoxicity should be evaluated at dose levels without visible precipitate, the Main Assay was performed using the following concentrations spaced by a factor of 2.5: 125, 50.0, 20.0, 8.00, 3.20, 1.28, 0.512 and 0.205 µg/mL. Precipitation of the test item was observed by the end of treatment at the highest dose level. Slight reduction of cell layer was noted at 50 µg/mL, while reduction of the cell layer and change in cell morphology were noted at 125 µg/mL. At these concentrations mild reduction in neutral red uptake was also observed.

Negative and positive control treatments were included in the Main Assay. Negative control cultures gave acceptable optical density values (0.183 ≤ OD≤ 0.769). Dose related toxicity was observed after treatment with the positive control, with a calculated IC50 value of 45.6 µg/mL, indicating the correct functioning of the assay system.

Conclusion

The test item was not considered to be cytotoxic, under the reported experimental conditions. However, solubility is a limiting factor in achieving sufficient cytotoxicity for the calculation of the IC50 value.