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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07-25 February 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
The tester strain WP2uvrA was accidentally additionally tested in the dose range finding test.
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
The tester strain WP2uvrA was accidentally additionally tested in the dose range finding test.
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Remarks:
Alfalfa, ext was observed to be a very thick dark coloured paste at room temperature.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: AL01
- Expiration date of the lot/batch: 10.09.2019
- Purity test date: not available

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: refrigerated
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: water solubility 5.35 mg/L at 25°C
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: diluted in Milli-Q water, the stock solution was treated with ultrasonic waves to obtain a homogeneous suspension
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable

OTHER SPECIFICS: none

Method

Target gene:
his
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Species / strain / cell type:
E. coli WP2 uvr A
Remarks:
Tested only in direct plate assay
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate (TA 100, WP2uvrA; direct plate assay, dose-range finding)
52, 164, 512, 1600 and 5000 µg/plate (TA 98, TA 102, TA1535, TA 1537; direct plate assay)
52, 164, 512, 1600 and 5000 µg/plate (TA 98, TA 100, TA 102, TA1535, TA 1537; pre-incubation assay)
Vehicle / solvent:
Milli-Q water
Controls
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191 (-S9 mix), tert-butyl hyperoxide (-S9 mix), 2-aminoanthracene (+S9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: agar plates, 1st experiment: direct plate assay, 2nd experiment: pre-incubation assay

DURATION
- Preincubation period: 30 ± 2 minutes at 70 rpm at 37 ± 1°C (2nd experiment)
- Exposure duration: 48 ± 4 h at 37 ± 1°C (1st and 2nd experiment)

NUMBER OF REPLICATIONS: triplicates

DETERMINATION OF CYTOTOXICITY
- Method: visual inspection
Rationale for test conditions:
In accodrance with the OECD Testing Guideline 471.
Evaluation criteria:
A test item is considered negative (not mutagenic) in the test if:
1) The total number of revertants in the tester strains TA100, TA102 and WP2uvrA is not greater than two times the concurrent control, and the total number of revertants in tester strain TA1535, TA1537 and TA98 is not greater than three times the concurrent control.
2) The negative response should be reproducible in at least one independently repeated experiment.

A test item is considered positive (mutagenic) in the test if:
1) The total number of revertants in the tester strains TA100, TA102 and WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strain TA1535, TA1537 and TA98, is greater than three times the concurrent control.
2) In case a second experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
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
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
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
Untreated negative controls validity:
valid
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
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Remarks:
Only direct plate assay
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
In a Bacterial Reverse Mutation assay, conducted according to OECD test guideline 471 and to GLP, Alfalfa, ext. was concluded to have negative mutagenic potential.
Executive summary:

Bacterial Reverse Mutation assay conducted according to OECD test guideline 471 and to GLP was performed to determine mutagenic potential of Alfalfa, ext.

In the dose-range finding study, the test item was initially tested up to concentrations of 5000 µg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Results of this dose-range finding test were reported as part of the first mutation assay.

In the first mutation experiment, the test item was tested at a concentration range of 52 to 5000 µg/plate in the strains TA1535, TA1537, TA98 and TA102. The test item did not precipitate on the plates at these dose levels. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. 

In the second mutation experiment, the test item was tested at a concentration range of 52 to 5000 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and TA102 in the pre-incubation assay. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. 

In this study, acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

The test item did not induce any increase in the number of revertant (His+) colonies in any of the five Salmonella tester strains and in the number of revertant (Trp+) colonies in E. coli tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

In conclusion, based on the results of this study it is concluded that Alfalfa ext. is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.