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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Extract from the seeds of Trigonella foenum-graecum (Fabaceae) obtained by extraction with polar solvents
EC Number:
950-727-0
Molecular formula:
Fenugreek extract
IUPAC Name:
Extract from the seeds of Trigonella foenum-graecum (Fabaceae) obtained by extraction with polar solvents
Specific details on test material used for the study:
Identification/Synonym FENUGREEK EXTRACT
Substance type UVCB substance
EC No. 950-727-0
Recommended storage Ambient (18-20°C) and protected from temperatures below 4°C; dry (< 70% rh);
conditions to be stored under dark conditions

Method

Target gene:
Tryptophan and histidine
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- Rat (Sprague Dawley)
- Mixing of S9 tissue fraction and cofactors
- quality controls of S9
- enzymatic activity
- sterility
- metabolic capability
Test concentrations with justification for top dose:
TOXICITY TEST WITH AND WITHOUT METABOLIC ACTIVATION:
5000; 1580; 500; 158 and 50.0 μg/plate

Main Assay I and II
5000; 2500; 1250; 625 and 313 μg/plate
Vehicle / solvent:
Sterile water and Dimethylsulfoxide (DMSO)
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Preliminary toxicity test one plate for each concentration with and without S9
- Main assay I and II
- negative and positive controls with and without S9
- Three replicate plates per test point

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: bacteria (1-5 x 10^8 cells / mL).

TREATMENT AND HARVEST SCHEDULE:
- Main Assay II: Preincubation period, 37°C for 30 min
- Exposure duration: 72 hours at 37°C

METHODS FOR MEASUREMENTS OF GENOTOXICIY

Plate count (number of colonies)
Rationale for test conditions:
Guideline study
Evaluation criteria:
For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.
Statistics:
Regression lines
Correlation co-efficient (r)
Students "t" Test

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: 50.0 mg/mL (maximum concentration of 5000 µg/plate)

RANGE-FINDING/SCREENING STUDIES:
Toxicity test: 5000; 1580; 500; 158 and 50.0 μg/plate
Main assay I and II: 5000; 2500; 1250; 625 and 313 μg/plate

Applicant's summary and conclusion

Conclusions:
It is concluded that the test item does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.

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