[No public or meaningful name is available]

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Remarks:

in vitro cytotoxicity assay

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From January 10 to February 01, 2019

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guidance document No. 129, "Guidance Document on using cytotoxicity test to estimate starting doses for accurate oral systemic toxicity tests".

GLP compliance:

yes (incl. QA statement)

Test type:

other: in vitro basal cytotoxicity assay

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

[(2-hydroxyacetyl)oxy](oxo) vanadium 2-hydroxyacetate]

IUPAC Name:

[(2-hydroxyacetyl)oxy](oxo) vanadium 2-hydroxyacetate]

Test material form:

solid

Results and discussion

Effect levels

Effect levels 1

Dose descriptor:

other: IC50

Effect level:

1.647 other: µg/mL

Based on:

test mat.

Remarks on result:

other: mean value of two experimets

Effect levels 2

Dose descriptor:

LD50

Remarks:

for rats

Effect level:

127 mg/kg bw

Based on:

test mat.

Remarks on result:

other: value extrapolated from mean IC50

Any other information on results incl. tables

PRELIMINARY TEST

The preliminary test was performed to determine the relevant concentration range at which cytotoxicity is obtained. The test item was dissolved in DMEM₀at 200 mg/mL after 45 minutes of magnetic stirring. From that formulation, seven serial dilutions were prepared in DMEM₀ with a geometric dilution factor of 10. Using a treatment volume of 50% (50 μL in 50μL of culture medium), the concentrations tested in this preliminary test were: 0.01, 0.1, 1, 10, 100, 1000, 10000 and 100000 µg/mL in DMEM₀.

 

The following results were obtained after 48 hours incubation:

.            the concentrations ≥ 10000 µg/mL were unreadable due to a strong coloration of the bottom of the wells (linked to the intrinsic coloration of the test item),

.            a strong cytotoxicity associated with rounded cells and a decrease in cell viability (decrease in NRU) was noted at concentrations ≥ 10 µg/mL,

.            test item was also suspected to be volatile since vehicle control neighboring the highest tested concentration were at 0% viability instead of 100%, as usual. It was therefore decided to add a plate sealer on the top of the plates after treatment of the test item and positive control in the main experiments.

 

These results were taken into account to select a more appropriate test item concentration range for the main tests.

 

MAIN TEST

First experiment

The test item was first dissolved in DMEM₀at 2 mg/mL after 25 minutes of magnetic stirring and was then 10-fold diluted inDMEM₀to reach the concentration of 0.2 mg/mL. From that formulation, seven serial dilutions were prepared in DMEM₀ with a geometric dilution factor of 3.16. Using a treatment volume of 50% (50 μL in 50 μL of culture medium), the final concentrations tested in this main test were: 0.03, 0.1, 0.32, 1, 3.17, 10.01, 31.65 and 100 µg/mL in DMEM₀.

 

All acceptance criteria were fulfilled. This test was therefore considered as valid.

 

The following results were obtained after 48 hours incubation:

.            strong cytotoxicity associated with rounded cells and decrease in cell viability (decrease in NRU) were noted at concentrations ≥ 3.17 µg/mL,

. the calculated IC₅₀was 1.502 µg/mL,

.            the LD₅₀was therefore estimatedto be 123 mg/kg.

 

Second experiment

The same test item concentrations as those used in the first experiment were selected for the treatment of the second experiment.

All acceptance criteria were fulfilled. This test was therefore considered as valid.Results of the microscopic observation of the plates treated with the test item are outlined in Table 3. A summary of the NRU results obtained for the test item, the vehicle and positive controls are outlined in Table 4. Individual results of these plates are presented in Appendix 2. The graphs obtained after the analysis with the GraphPad Prism from the test item and positive control-treated plates are presented in Figures 3 and 4, respectively.

 

The following results were obtained after 48 hours incubation:

.            strong cytotoxicity associated with rounded cells and decrease in cell viability (decrease in NRU) were noted at concentrations ≥ 3.17µg/mL,

.            the calculated IC₅₀was 1.791 µg/mL,

.            the LD₅₀was therefore estimated to be 131 mg/kg.

 

Final interpretation

According to the results obtained in both experiments, the calculated mean IC₅₀was 1.647 µg/mL. Therefore, based on the mean IC₅₀, the mean estimated LD₅₀in rats was 127 mg/kg.

Applicant's summary and conclusion

Interpretation of results:

other: Category 3 based on CLP criteria

Conclusions:

IC50 = 1.647 µg/mL
LD50 (estimated) = 127 mg/kg in rats

Executive summary:

Method

The objective of this study was to evaluate the basal cytotoxicity of the test item, using the 3T3 NRU test (Neutral Red Uptake). The design of the study is based on the OECD Guidance document No. 129 for "Guidance Document on using cytotoxicity test to estimate starting doses for accurate oral systemic toxicity tests". The NRU assay is performed in a dose-response format allowing the calculation of the concentration of the test item that reduces Neutral Red Uptake (NRU) by 50% (IC50). The IC50 value is used in a linear regression equation to estimate the oral LD50 value in rats. The assay evaluates the cytotoxicity of the test item applied to mouse fibroblasts (Balb/c 3T3, clone A 31).

Results

In both experiments, the tested concentrations were: 0.03, 0.1, 0.32, 1, 3.17, 10.01, 31.65 and 100 µg/mL in DMEM₀. The following results were obtained after 48 hours incubation:

• strong cytotoxicity associated with rounded cells and decrease in cell viability (decrease in NRU) were noted at concentrations ≥ 3.17µg/mL,
• the calculated IC50 were 1.502 µg/mL and 1.791 µg/mL in the first and second experiments, respectively,
• these results correspond to estimated LD50 of 123 mg/kg and 131 mg/kg, in the first and second experiments, respectively.

Conclusion 

Under the experimental conditions of this study and after treatment of cells for 48 hours, the test item, is considered as cytotoxic in this in vitro test system. The mean IC50 was estimated to be 1.647 µg/mL and the corresponding LD50 for rats was estimated to be 127 mg/kg according to OECD Guidance No. 129.