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Toxicological information

Acute Toxicity: oral

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Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
6 March to 15 March, 2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: ICCVAM - Recommended Test Method Protocol BALB/c 3T3 NRU Cytotoxicity Test Method
Version / remarks:
2006
Qualifier:
according to
Guideline:
other: ENV/JM/MONO(2010)20 – series of Testing and Assessment No. 129
Version / remarks:
2010
Principles of method if other than guideline:
Test method under evaluation
GLP compliance:
yes
Test type:
other:
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous

Administration / exposure

Route of administration:
other: in vitro method

Results and discussion

Effect levels
Dose descriptor:
other: IC50
Effect level:
504 other: µg/mL
Based on:
other: cytotoxic effects to Balb/c 3T3 cells

Any other information on results incl. tables

Solubility

Solubility of the test item was evaluated in a preliminary trial using Chemical Dilution Medium. A clear solution was obtained at 200 mg/mL (250 mg/mL, test item as received).

Since solutions in this solvent should be added to DMEM culture medium in ratio 1:1, this result permitted a maximum concentration of 100 mg/mL to be used in the preliminary dose-range finder experiment.

Preliminary dose-range finder experiment

A preliminary range-finder experiment was undertaken in order to select appropriate dose levels for the Main Assay. In this experiment, the test item was assayed at a maximum dose level of 100000 μg/mL and at a wide range of lower dose levels: 0.010, 0.100, 1.00, 10.0, 100, 1000 and 10,000 μg/mL. No precipitation of the test item was observed by the end of treatment at any concentration tested. Dose related reduction of the cell layer was noted

starting from 1000 μg/mL; changes in cell morphology were observed at the three highest dose levels, where mild to severe reduction in neutral red uptake (NRU) was noted. The calculated IC50 value was 862 μg/mL.

Optical density values of NR extract at 530 nm are presented in Table 1 (in "Illustrations" in "Overal remarks, attachments"). 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.

The mean value of optical density, the standard deviation and the percentage over the solvent control value for each test point are presented in Table 1 (in "Illustrations" in "Overal remarks, attachments").

Main Assay

Based on the results obtained, the Main Assay was performed using the following concentrations: 3.16, 10.0, 31.6, 100, 316, 1000, 3160 and 10,000 μg/mL. No precipitation of the test item was noted at any dose level. Dose-related reduction of cell layer and change in cell morphology were noted at the four highest dose levels. Marked toxicity was observed at 10,000 and 3160 μg/mL, reducing NRU to 1 and 10% of the negative control, respectively.

Mild toxicity (NRU = 35 % of the negative control value) was observed at the next lower concentration of 1000 μg/mL, slight reduction in neutral red uptake was noted at 316 and 100 μg/mL (63 % and 68 %, respectively); while no relevant toxicity was observed at the remaining concentrations tested. The calculated IC50 value was 504 μg/mL.

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. The mean value of optical density, the standard deviation and the percentage over the solvent control value for each test point, together with the IC50 value and its variance are presented in Table 2.

Negative control cultures gave optical density values higher than the maximum acceptable value based on the range established by international community, but within RTC historical control range (OD530 NRU lower than 2.065).Dose related toxicity was observed after treatment with the positive control, with a calculated IC50 value of 45.2 μg/mL, coherent with the range established by the international scientific community, indicating the correct functioning of the assay system.

Analysis of results

In the preliminary dose-range finder experiment, the calculated IC50 value was 862 μg/mL. In the Main Assay, a narrow dose spacing was used in order to cover the relevant concentration range around the IC50 value, with several points of a graded effect. The calculated IC50 value was 504 μg/mL. On the basis of these results, it is concluded that the test item is cytotoxic under the reported experimental conditions.

Conclusion

The potential in vitro cytotoxicity of the test item was evaluated on Balb/c 3T3 cells. Negative and positive controls gave the expected results, indicating the correct functioning of the assay system. Dose-related toxicity was noted after treatment with the test item, with a calculated IC50 value of 504 μg/mL. It can therefore be concluded that the test item produced cytotoxic effects to Balb/c 3T3 cells, under the reported experimental conditions.

Applicant's summary and conclusion

Interpretation of results:
other: Category 4 for Acute Toxicity (300 < ATE ≤ 2000 mg/kg), according to the CLP Regulation (EC) No. 1272/2008
Conclusions:
The LD50 of the test item was calculated to be 457.3 mg/kg
Executive summary:

The potential in vitro cytotoxicity of the test item was evaluated in an experimental study on Balb/c 3T3 cells, according to the ICCVAM Recommended Test Method Protocol BALC/C 3T3 NRU Cytotoxicity Test Method (2006) and the ENV/JM/MONO (2019)20 Series of Testing and Assessment No. 129 (2010). A preliminary range-finding test was performed on concentrations ranging from 0.01 µg/mL to 100 mg/L (based on results obtained from a preliminary solubility test) which found reduction in neutral red uptake (NRU), dose-related reduction of the cell layer and changes in cell morphology at concentrations of 1 mg/L and above. Based on the preliminary test results, the main assay was performed using concentrations of 3.16, 10, 31.6, 100, 316, 1000, 3160 and 10,000 µg/mL.

Negative and positive controls gave the expected results, indicating the correct functioning of the assay system. Marked toxicity was observed at the highest two doses: NRU was reduced to 1 % of the negative control at 10,000 µg/mL, and 10 % of the negative control at 3160 µg/mL. Mild toxicity was observed at the next lower concentration, slight reduction in NRU was noted at 316 µg/mL and 100 μg/mL (63% and 68%, respectively); no relevant toxicity was observed at the remaining concentrations tested. Therefore, based on these values, dose-related toxicity was noted after treatment with the test item, and the IC50 value was calculated to be 504 μg/mL. The acute toxicity estimate (ATE) was extrapolated using the following formula, provided in the ICCVAM Recommended Test Method Protocol BALC/C 3T3 NRU Cytotoxicity Test Method (2006), which predicts a corresponding median lethal dose (LD50) value:

log LD50 (mmol/kg) = 0.439 log IC50 (mM) + 0.621

where:

- IC50 = 504 µg/mL

- molecular weight of the test item = 207 g/mol

The corresponding log LD50 value was found to be 0.7926 mmol/kg, equivalent to LD50 value of 457.3 mg/kg.