Silybum marianum, ext.

Endpoint summary

Description of key information

Under the experimental conditions of the study, due to the test item limit of solubility (i.e. 100 μg/mL in DMEM0 containing 0.5% ethanol) and the fact that no cytotoxicity was achieved after the preliminary test, this study was stopped at this stage. It was not possible to derive any IC50 and therefore any LD50.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: Not specified

Principles of method if other than guideline:

The objective of this study was to evaluate the basal cytotoxicity of the test item, applied to mouse fibroblasts (Balb/c 3T3, clone A 31 and using the. Cytotoxicity is measured as the inhibition of the capacity to take up the vital dye, Neutral Red (NR). NR readily penetrates cell membranes by non-diffusion and accumulates in the cell lysosomes. Damage to the lysosomal membrane leads to irreversible lysosome fragility. Damage to lysosomes by a test item results in a decrease in the uptake and accumulation of NR, allowing the quantification by spectrophotometry of viable, damaged or dead cells. The NRU assay is performed in a dose-response format allowing the calculation of the concentration of the test item that reduces the neutral red uptake (NRU) by 50% (IC50), when applicable.

GLP compliance:

no

Remarks:

The study was stopped, no treatment of main test was performed and the GLP status was removed.

Test type:

other: 3T3 NRU Test

Limit test:

no

Specific details on test material used for the study:

No further details specified in the study report.

Species:

mouse

Strain:

Balb/c

Sex:

not specified

Details on test animals or test system and environmental conditions:

Mouse fibroblasts (Balb/c 3T3, clone A 31) - no further details specified in the study report.

Route of administration:

other: Not applicable

Vehicle:

ethanol

Details on oral exposure:

Not applicable

Doses:

During the assay, eight concentrations of test item were tested using six replicates per concentration. These concentrations were spaced using a decimal geometric dilution factor of 10. Eight concentrations of the positive control were also applied to the cells in parallel.

No. of animals per sex per dose:

Not specified

Control animals:

yes

Remarks:

Positive control in parallel

Details on study design:

The objective of the study was to evaluate the basal cytotoxicity of the test item, applied to mouse fibroblasts (Balb/c 3T3, clone A 31 and using the 3T3 NRU test. Cytotoxicity is measured as the inhibition of the capacity to take up the vital dye, Neutral Red (NR). NR readily penetrates cell membranes by non-diffusion and accumulates in the cell lysosomes. Damage to the lysosomal membrane leads to irreversible lysosome fragility. Damage to lysosomes by a test item results in a decrease in the uptake and accumulation of NR, allowing the quantification by spectrophotometry of viable, damaged or dead cells.
A solubility assay was performed in order to evaluate the solubility of the test item among the vehicles DMEM0, DMSO or ethanol.
A solubility evaluation in DMEM0 containing 1% of vehicle was also performed once solution was achieved in a vehicle.
A preliminary test was performed prior to the main tests to determine the relevant concentration range at which cytotoxicity is obtained. During this assay, eight concentrations of test item were tested using six replicates per concentration. These concentrations were spaced using a decimal geometric dilution factor of 10. Eight concentrations of the positive control were also applied to the cells in parallel.

Statistics:

Not specified

Key result

Dose descriptor:

LD50

Remarks on result:

not determinable because of methodological limitations

Mortality:

Not specified

Clinical signs:

other: Not specified

Gross pathology:

Not specified

Other findings:

Solubility
The test item was found not soluble in culture medium at 2, 20 and 200 mg/mL, in DMSO at 20 and 200 mg/mL and in ethanol at 200 mg/mL.
A solution was reached once test item was prepared in ethanol at 20 mg/mL after at least 5 minutes of vortex. A solution was still obtained after a 100-fold dilution in culture medium. As a result, ethanol was the retained vehicle and the highest final tested concentration to be used in the preliminary test was 100 μg/mL.

Preliminary test
Due to test item limit of solubility and therefore the low tested concentration (i.e. 100 μg/mL in culture medium containing 0.5% ethanol), no cytotoxicity was reached at the end of this preliminary test. Since the aim of this study cannot be achieved (i.e. no IC50 and therefore no LD50 can be determined), it was agreed with the Sponsor to stop the study as it is. Indeed, solubility is a limitation to this type of assay.
Consequently this study was stopped, no treatment of main test was performed and the GLP status was removed.

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the experimental conditions of the study, due to the test item limit of solubility (i.e. 100 μg/mL in DMEM0 containing 0.5% ethanol) and the fact that no cytotoxicity was achieved after the preliminary test, this study was stopped at this stage. It was not possible to derive any IC50 and therefore any LD50.

Executive summary:

The objective of this study was to evaluate the basal cytotoxicity of the test item, applied to mouse fibroblasts (Balb/c 3T3, clone A 31 and using the 3T3 NRU test. Cytotoxicity is measured as the inhibition of the capacity to take up the vital dye, Neutral Red (NR). NR readily penetrates cell membranes by non-diffusion and accumulates in the cell lysosomes. Damage to the lysosomal membrane leads to irreversible lysosome fragility. Damage to lysosomes by a test item results in a decrease in the uptake and accumulation of NR, allowing the quantification by spectrophotometry of viable, damaged or dead cells. The NRU assay is performed in a dose-response format allowing the calculation of the concentration of the test item that reduces the neutral red uptake (NRU) by 50% (IC50), when applicable. The IC50 value could then be used in a linear regression equation to estimate the oral LD50 value in rats. However, and due to test item limit of solubility, the objective of this study cannot be achieved since no cytotoxicity was reached after the preliminary test.

 

Under the experimental conditions of this study, due to the test item limit of solubility (i.e. 100 μg/mL in DMEM0 containing 0.5% ethanol) and the fact that no cytotoxicity was achieved after the preliminary test, this study was stopped at this stage. It was not possible to derive any IC50 and therefore any LD50.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

K2

Additional information

The objective of this study was to evaluate the basal cytotoxicity of the test item, applied to mouse fibroblasts (Balb/c 3T3, clone A 31 and using the 3T3 NRU test. Cytotoxicity is measured as the inhibition of the capacity to take up the vital dye, Neutral Red (NR). NR readily penetrates cell membranes by non-diffusion and accumulates in the cell lysosomes. Damage to the lysosomal membrane leads to irreversible lysosome fragility. Damage to lysosomes by a test item results in a decrease in the uptake and accumulation of NR, allowing the quantification by spectrophotometry of viable, damaged or dead cells. The NRU assay is performed in a dose-response format allowing the calculation of the concentration of the test item that reduces the neutral red uptake (NRU) by 50% (IC50), when applicable. The IC50 value could then be used in a linear regression equation to estimate the oral LD50 value in rats. However, and due to test item limit of solubility, the objective of this study cannot be achieved since no cytotoxicity was reached after the preliminary test.

 

Under the experimental conditions of this study, due to the test item limit of solubility (i.e. 100 μg/mL in DMEM0 containing 0.5% ethanol) and the fact that no cytotoxicity was achieved after the preliminary test, this study was stopped at this stage. It was not possible to derive any IC50 and therefore any LD50.