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Genetic toxicity in vitro

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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
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
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
Target gene:
his
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
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.
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
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.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
test performed accoding to the original protocol of B.N. Ames
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: dehydrated alfalfa was purchased from commercial source.
- Expiration date of the lot/batch: not available
- Purity test date: not available

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: stable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not available

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: 60 g of plant material was shaken in 300 ml distilled acetone for 48 hours. The liquid portion was filter-sterilized using a Seitz cellulose filter and then stored until refrigeration until used.
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: test solutions were prepared each day from the stock solution. Initial volumes of stock solution were rotary-evaporated to dryness and then reconstituted to a new volume with filter-sterilized distilled acetone to provide samples of original concentration or increased concentration of plant material. The reconstitution scheme was 21 ml stock solution evaporated to dryness and reconstituted to new volumes of 21, 12, 6 or 3 ml, which was 100, 57.1, 28.6 and 14.3% of the original volume or 1, 2, 3, and 7 times the original concentration of plant material. These were expressed as relative concentrations of 1, 2, 3, and 7, respectively.
- Final preparation of a solid: not applicable


OTHER SPECIFICS: none
Target gene:
His
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
other: deficient in excision repair of DNA damage (uvrB)
Remarks:
susceptible to frameshift mutagens
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
other: deficient in excision repair of DNA damage (uvrB)
Remarks:
susceptible to base-pair substitution mutagens
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Relative concentratios of 1, 2, 3 and 7
Untreated negative controls:
yes
Remarks:
acetone
Positive controls:
yes
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: none
- Exposure duration: 24 hours

NUMBER OF REPLICATIONS: Triplicate plates were used for each determination and all positive (revertant number of at least twice the revertant control number) or toxic (revertant number of 0.5 or less than that of the revertant control number) responses were repeated at least twice.
Evaluation criteria:
Test results were considered positive when the number of revertants induced by exposure to the test compound was as great or greater than twice the natural spontaneous mutation rate
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
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:
not specified
Untreated negative controls validity:
valid
Positive controls validity:
valid

The natural mutation rates of the cultures unexposed to alfalfa extract were 14 and 74 per plate for TA 98 and TA 100 without the S9 mix and 20 and 73 with the S9 mix.

Conclusions:
In an in vitro gene mutation test in bacteria performed according to the original Ames protocol an acetone extract of alfalfa was concluded to have negative mutagenic potential in the presence and absence of metabolic activation.
Executive summary:

The acetone extract of alfalfa was tested in the Ames assay in two Salmonella strains, TA98 and TA100 with and without metabolic activation (S9). It was tested at four different concentrations, 1, 2, 3 and 7 times the original concentration of stock solution prepared from the plant material. Alfalfa extract did not increase the number of revertants in either Salmonella strain.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
test performed according to original protocol of B.N. Ames.
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: phytopreparation of alfalfa extract
- Expiration date of the lot/batch: not available
- Purity test date: not available

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: not available
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: stable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not available

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: 5 – 50 000 µg/plate
- Final preparation of a solid: not applicable

OTHER SPECIFICS: none
Target gene:
His
Species / strain / cell type:
S. typhimurium TA 1537
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9
Untreated negative controls:
yes
Remarks:
water
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
N-ethyl-N-nitro-N-nitrosoguanidine
other: 2,7-diamino-4,9-dioxi-5,10-dioxo-4,5,9,10-tetrahydro-4,9-diazoprene (DDDTDP), 1,6-dinitropyrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: not specified
- Exposure duration: not specified

NUMBER OF REPLICATIONS: 2
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:
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

See attached additional information on results.

Conclusions:
In an in vitro gene mutation test in bacteria performed according to the original Ames protocol a phyto-preparation derived from alfalfa extract was concluded to have negative mutagenic potential in the presence and absence of metabolic activation.
Executive summary:

A phyto-preparation derived from alfalfa extract was tested in the Ames assay in TA 98, TA 100 and TA 1537 strains with and without metabolic activation (S9) at concentrations up to 50'000 µg/plate. The phyto-preparation did not increase the number of revertants in the presence and absence of metabolic activation. In addition, there were no signs of cytotoxicity even at the highest concentration. 

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
according to
Guideline:
other: original protocol for SOS Chronoplast test (Quillardet & Hofnung, 1985))
Principles of method if other than guideline:
The assay consists of incubating the tester strain with increasing concentrations of the agent to be tested. After a time for protein synthesis, beta-galactosidase activity is assayed by a colorimetric assay.
GLP compliance:
no
Type of assay:
SOS/umu assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: phytopreparation of alfalfa extract
- Expiration date of the lot/batch: not available
- Purity test date: not available

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: not available
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: stable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not available

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: up to 38'500 µg/ml
- Final preparation of a solid: not applicable

OTHER SPECIFICS: none
Target gene:
sfiA::lacZ operon fusion
Species / strain / cell type:
E. coli, other: PQ37
Metabolic activation:
not applicable
Test concentrations with justification for top dose:
The test item was tested at 20 concentrations, from 0.0734 to 38'500 µg/ml.
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: none
- Exposure duration: 2 hours

NUMBER OF REPLICATIONS: not specified
Evaluation criteria:
A concentration-dependent increase in the induction factor by at least 2 times served, as an indicator of genotoxicity.
Cytotoxicity was indicated by a decrease, by more than 50%, of the alkaline phosphatase constitutive enzyme activity.
Key result
Species / strain:
E. coli, other: PQ37
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
above 10'000 ug/ml
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No increase of galactosidase activity was observed up to 38’500 µg/ml. The SOS-induced potential (SOSIP, or the magnitude of the increase in the induction factor par unit mass of the test product) was determined at 0.0000251 per 1 µg. At concentrations above 10’000 µg/ml, the test substance deceased the alkaline phosphatase constitutive enzyme activity. The SOSIP of negative control was 4.06 per 1nmol. The minimum effective concentration of 4-nitroquinoline-1-oxide was 2.53 nmol/ml, and the maximum induction of SOS functions was observed at the maximum studied concentration of 20.2 nmol/ml.

See attached additional information on results.

Conclusions:
The phyto-preparation derived from alfalfa extract did not to induce the SOS functions in Escherichia coli at concentrations up to 38’500 µg/ml indicating that it has no DNA-damaging potential.
Executive summary:

SOS chromotest in the Escherichia coli PQ37 test strain was performed to determine the DNA-damaging potential of the phyto-preparation derived from alfalfa phyto-preparation. The test sample was tested at 20 different concentrations, from 0.0734 to 38'500 µg/ml and 4-nitroquinoline-1-oxide served as a positive control. The duration of exposure was 2 hours.

A concentration-dependent increase in the induction factor by at least 2 times served as an indicator of genotoxicity. Cytotoxicity was indicated by a decrease, by more than 50%, of the alkaline phosphatase constitutive enzyme activity.

No increase of galactosidase activity was observed up to 38’500 µg/ml. The SOS-induced potential (SOSIP) was determined at 0.0000251 per 1 µg. At concentrations above 10’000 µg/ml, the test substance deceased the alkaline phosphatase constitutive enzyme activity. The SOSIP of positive control was 4.06 per 1 nmol. 

Under the test conditions, the phyto-preparation derived from alfalfa phyto-preparation did not induce the SOS functions in Escherichia coli, indicating that it has no DNA-damaging potential.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

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. 

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. 

The acetone extract of alfalfa was tested in the Ames assay in two Salmonella strains, TA98 and TA100 with and without metabolic activation (S9). It did not increase the number of revertants in either Salmonella strain.

A phyto-preparation derived from alfalfa extract was tested in the Ames assay in TA98, TA100 and TA1537 strains with and without metabolic activation (S9) at concentrations up to 50’000 µg/plate. It did not increase the number of revertants in the presence and absence of metabolic activation.

SOS chromotest in the Escherichia coli PQ37 test strain was performed to determine the DNA-damaging potential of the phyto-preparation derived from alfalfa extract applied at 20 different concentrations (0.0734 to 38 500 µg/ml). Under the test conditions, the phyto-preparation did not to induce the SOS functions in Escherichia coli, indicating that it has no DNA-damaging potential.

Justification for classification or non-classification

Appropriate tests were performed on the target substance and suitable analogues of Alfalfa, ext. to assess its mutagenic potential. The target and analogue substances did not show any mutagenic potential under the conditions of the studies. Therefore, it is concluded that Alfalfa, ext. is not mutagenic, and it does not need to be classified for mutagenicity according to the criteria outlined in Annex I of CLP Regulation (EC) 1272/2008.