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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

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

Data source

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

Materials and methods

Test guideline
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

Test material

1
Chemical structure
Reference substance name:
Silybum marianum, ext.
EC Number:
283-298-7
EC Name:
Silybum marianum, ext.
Cas Number:
84604-20-6
Molecular formula:
Not available - UVCB substance
IUPAC Name:
Extract of Chardon marie without support
Test material form:
liquid
Details on test material:
Name: Extract of Chardon marie without support
Batch/Lot number: VDO06205A
Appearance: Light yellow brown cloudy oil
Purity: Considered as 100%
Retest date: 13 July 2019
Storage conditions: Refrigerated
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.


Name: Extract of Chardon marie without support
Batch/Lot number: ABT01059A
Appearance: Brown/orange turbid oil
Purity: Considered as 100%
Retest date: 19 July 2018
Storage conditions: Refrigerated
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.

Reference: MPB-693061 / MPF-00024688
Batch number: VDO06126E
Provider: Pierre Fabre
Reference: GD 1508
Certificate of analysis: Yes
MSDS: Yes
Type: Raw material
Form: Liquid
Nature: Complex mixture
Colour: Yellowish
Storage conditions: Stored at room temperature
Requested test conditions: To be tested at 100 % (as is)
Sent quantity: 10 g
Expiry / Retest date: 27 November 2016
Specific details on test material used for the study:
No further details specified in the study report.

Test animals

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.

Administration / exposure

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

Results and discussion

Effect levels
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.

Applicant's summary and conclusion

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.