Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 January to 15 January 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Remarks:
Brown-yellow viscous liquid
Details on test material:
Test item: Fenugreek Absolute
Batch No.: 2709379
Identity: 100 % UVCB Substance
Appearance: Brown-yellow viscous liquid
Storage conditions: Ambient temperature (10 °C to 30 °C), in original aluminium bottle, dark
Expiry date: October 24, 2018

Method

Target gene:
Histidine and tryptophan for S. typhimurium and E. coli, respectively.
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
No applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Obtained from Molecular Toxicology Incorporated, Boone, NC, USA (MolTox™) and prepared according to the method of Ames et al from male Sprague Dawley rats induced with a single dose of Aroclor 1254.
Test concentrations with justification for top dose:
In order to determine the toxicity of Fenugreek Absolute to the test bacteria, an initial plate incorporation experiment was carried out using TA98 and WP2 uvrA in the presence and absence of S 9 mix. The dose levels used were 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. Based on the range finder observations, dose levels for the main experiment were chosen to include the guideline regulatory maximum dose level of 5000 µg/plate.
Vehicle / solvent:
Dimethylsulphoxide (DMSO), a commonly used vehicle for which extensive historical control data is available; at concentrations up to 50 mg/mL.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
Remarks:
Without S9
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (2AA)
Details on test system and experimental conditions:
Salmonella typhimurium and Escherichia coli strains were obtained from Molecular Toxicology Incorporated, Boone, NC, USA (MolTox™) and used in the Bacterial Reverse Mutation test.
The Salmonella typhimurium and Escherichia coli strains are defective in DNA repair (Δuvr B , A- respectively); this confers extra sensitivity to DNA damage. The Salmonella strains have a defective lipopolysaccharide barrier on the cell wall (rfa-) which affords greater permeability to large molecules. The strains TA98 and TA100 also contain a plasmid (pKM101) which enhances error prone repair and confers ampicillin resistance.
The strains are tested routinely for:
1. Auxotrophic control of growth, (histidine or tryptophan dependence).
2. Deep rough mutation (rfa) of the bacterial cell wall (cell membrane permeability, crystal violet sensitivity).
3. Plasmid presence (Ampicillin resistance).
4. Absence of uvrA or uvrB repair enzyme systems (sensitivity to UV irradiation).
TA1535, TA100 and WP2 uvrA are reverted to prototrophy by base substitution mutagens. TA1537 and TA98 are reverted by frame shift mutagens; TA100 can also be affected by these on occasion.
Before the experiment, 25 mL quantities of nutrient broth were inoculated with 100 µL from a freshly thawed vial of the appropriate strain, and incubated overnight at 37 °C to give a pure culture at approximately 10^9 cells per mL.
Rationale for test conditions:
Range finder: As all the Salmonella strains and the Escherichia strain show a similar toxic response to most chemicals, only one strain of each species, typically TA98 and WP2 uvrA, was tested by plate incorporation in this experiment.
A range of concentrations up to a maximum of 5000 μg/plate was used, with duplicate plates at each concentration, both with and without S-9.
The plates examined for integrity of the background lawns after approximately 48 hours ± 1 hour incubation.
Experiment I: Mutation experiment, plate incorporation : The mutation experiment was carried out using fresh bacterial cultures for each experiment.
The test item was tested at sufficient concentration levels to provide at least 5 analysable concentrations, with triplicate Petri dishes prepared for each control and experimental point. The following was added to sterile disposable tubes containing 2 mL of L-histidine: D-biotin (Salmonella strains) or L-tryptophan (Escherichia strain) supplemented top agar. The contents of each tube was mixed and added to a Petri dish containing minimal agar. When the top agar has set, the Petri dishes will be inverted and incubated at 37 °C ± 3 °C for approximately 66 hours ± 1 hour.

Experiment II: Mutation experiment, preincubation: The mutation experiment was carried out using fresh bacterial cultures for each experiment.
The test item was tested at sufficient concentration levels to provide 5 analysable concentrations, with triplicate Petri dishes prepared for each control and experimental point. Each tube was fitted with a sterile cap and incubated at approximately 37 °C with shaking, for 20 minutes.
At the end of this period, 2 mL of top agar (L-histidine: D-biotin for Salmonella strains or L-tryptophan for Escherichia strain) was added to each tube, the contents mixed and added to Petri dishes containing minimal agar. When the top agar has set, the Petri dishes are inverted and incubated at 37 °C ± 3 °C for approximately 66 hours ± 1 hour.
Evaluation criteria:
A test was considered to be positive if the test item induced dose related statistically significant increases in numbers of revertants over the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system, compared with negative Controls scored in 2 separate experiments. Biological relevance of the result should be considered. Statistical significance should not be the only determining factor for a positive response.

A test was considered to be negative if the test item produced no greater increases in revertants, than may be expected from normal variation in the negative Control number of revertants, for any strain, in either experiment.
Statistics:
Mean, standard deviation (s.d.) and Dunnett's t-statistic (t) were calculated for each group and bacterial strain

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Toxicity Range finder : No toxicity presents. The only effect is a precipitation at the top dose (5000 µg/plate).
Experiment 1: All negative Controls gave mean counts of spontaneous revertant colonies within expected historical negative Control ranges obtained in this laboratory.
All positive Controls induced marked increases in the number of revertant colonies and gave mean counts of induced revertant colonies which were within expected historical positive Control ranges. The response to the positive Controls demonstrates that the bacteria were sensitive to the mutagens and that the S 9 mix was able to metabolise the pro-mutagen, 2AA, to a mutagen.
Fenugreek Absolute was tested up to the regulatory maximum dose level of 5000 µg/plate in all strains in the presence and absence of S 9 mix, under plate incorporation conditions.
Precipitation was observed at 5000 µg/plate in all strains in the presence and absence of S 9 mix.
Calculated t values were compared with reference critical values and, in the event that the calculated values exceeded the critical values, they were to be highlighted in the appropriate tables, to indicate statistically significant increases in colony counts. None of the calculated t values in this experiment exceeded the critical values.
There were no dose-related or statistically significant increases in revertant numbers observed in any strain at any dose of Fenugreek Absolute, in the presence or absence of S 9 mix, under plate incorporation conditions.


Experiment 2: All negative Controls gave mean counts of spontaneous revertants within expected ranges obtained in this laboratory.
All positive Controls gave mean counts of induced revertants within expected ranges obtained in this laboratory. The response to the positive Controls indicated that the bacteria were sensitive to the mutagens and that the S 9 mix was able to metabolise the pro-mutagen, 2AA, to a mutagen.
Fenugreek Absolute was tested up to the regulatory maximum dose level of 5000 µg/plate in all strains in the presence and absence of S 9 mix, under pre-incubation conditions.
Precipitation was observed at 5000 µg/plate in all strains in the presence and absence of S 9 mix.
Calculated t values were compared with reference critical values and, where the calculated values exceeded the critical values, were highlighted in the appropriate tables, indicating statistically significant increases in colony counts. None of the calculated t values in this experiment exceeded the critical values.
There were no dose-related statistically significant increases in revertant numbers observed in any strain at any dose of Fenugreek Absolute, in the presence or absence of S 9 mix, under pre incubation conditions.

Historical control data: The negative and positive Controls from the mutation experiment were compared with previously established ranges of these parameters for this laboratory. Control data were accepted for this study as they were within the minimum and maximum values recorded within the previous 2 year period.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not reported
- Effects of osmolality: Not reported
- Evaporation from medium: Not reported
- Water solubility: Not reported
- Precipitation: The test item precipitated in the overlay agar at the highest investigated concentration.
- Other confounding effects: Not reported

MUTAGENICITY RESULTS
See 'Any other information on results incl. tables'

Any other information on results incl. tables

Table 1 : Mean Number of Revertants Per Plate – Experiment 1 – Plate Incorporation

Strain

% S-9
mix v/v

Dose level of test item (µg/plate)

PC

0

50

150

500

1500

5000

TA1535

0

19.7

12.7

15.3

13.7

17.0

13.0
ppt 

659.7

TA1537

0

9.3

5.3

7.7

6.7

2.3

5.3
ppt 

681.0

TA98

0

20.0

19.0

25.0

21.3

19.7

23.7
ppt 

94.7

TA100

0

82.7

89.3

95.3

97.0

96.0

91.3
ppt 

588.7

WP2uvrA

0

18.0

19.0

21.7

22.3

22.7

22.7
ppt 

552.3

Strain

% S-9
mix v/v

Dose level of test item (µg/plate)

PC

0

50

150

500

1500

5000

TA1535

10

14.7

12.0

12.7

15.0

14.3

14.3
ppt 

165.0

TA1537

10

10.7

9.7

10.0

6.0

8.0

8.3
ppt 

183.7

TA98

10

25.3

24.7

32.7

27.7

32.0

32.7
ppt 

654.7

TA100

10

100.3

101.7

95.0

97.3

59.7

83.7
ppt 

1697.7

WP2uvrA

10

31.3

37.3

25.3

28.7

24.3

30.7
ppt 

150.3

PC:

positive control

ppt:

compound precipitation


 Table 2 :Mean Number of Revertants Per Plate – Experiment 2 – Pre-incubation

Strain

% S-9
mix v/v

Dose level of test item (µg/plate)

PC

0

50

150

500

1500

5000

TA1535

0

13.3

14.3

12.0

14.0

14.0

13.0
ppt 

527.0

TA1537

0

7.3

7.3

5.7

3.3

5.5

6.0
ppt 

527.7

TA98

0

16.3

18.0

17.0

19.0

17.3

21.0

ppt

123.0

TA100

0

70.7

90.3

82.0

90.7

86.0

82.0
ppt 

522.3

WP2uvrA

0

28.3

25.3

25.0

30.7

28.3

32.7
ppt 

1444.3

Strain

% S-9
mix v/v

Dose level of test item (µg/plate)

PC

0

50

150

500

1500

5000

TA1535

10

10.0

14.0

10.3

9.3

13.7

14.7
ppt 

126.0

TA1537

10

11.7

13.3

10.7

8.7

5.0

5.0
ppt 

176.7

TA98

10

23.7

29.7

31.7

25.7

26.0

29.3
ppt 

1063.0

TA100

10

81.3

94.7

103.3

75.7

66.3

68.0
ppt 

1536.0

WP2uvrA

10

31.0

35.7

29.7

30.7

36.0

39.0
ppt 

180.7

PC:

positive control

ppt:

compound precipitation

 

 

Applicant's summary and conclusion

Conclusions:
There were no biologically relevant or statistically significant increases in revertant numbers observed in any strain at any dose of Fenugreek Absolute, in the presence or absence of S 9 mix, under plate incorporation or pre-incubation conditions.
Fenugreek Absolute showed no mutagenic potential in the Bacterial Reverse Mutation Assay under the conditions of this test.
Executive summary:

Study Objective: The purpose of the bacterial reverse mutation test was to assess the mutagenic potential of the test item by its ability to induce reverse mutation in the specified bacterial strains from auxotrophic growth to prototrophy. Fenugreek Absolute was tested in vitro, for its ability to induce mutations in 4 histidine dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100 and 1 tryptophan dependent auxotrophic mutant of Escherichia coli, WP2 uvrA.

Study Design: A range-finder experiment was performed to determine the cytotoxic range of the test item under plate incorporation conditions in TA98 and WP2 uvrA, in the presence and absence of S 9 mix (a liver post mitochondrial fraction derived from the livers of Aroclor 1254 treated rats). Mutation tests were then performed, using the plate incorporation method and pre-incubation method, in both the presence and absence of S 9 mix. The bacteria were exposed to the test item dissolved in dimethylsulphoxide (DMSO); the latter was also the negative Control. The positive Control chemicals were sodium azide (TA1535 and TA100), 9 aminoacridine (TA1537), 2 nitrofluorene (TA98) and 4 nitroquinoline-N-oxide (WP2 uvrA) in the absence of S 9 mix and 2-aminoanthracene (2AA) in the presence of S 9 mix (all strains).

The doses of Fenugreek Absolute used in Experiment 1 under plate incorporation conditions and Experiment 2 under pre-incubation conditions were 50, 150, 500, 1500 and 5000 µg/plate in all strains in the presence and absence of S 9 mix.

Results: All negative Controls gave mean counts of spontaneous revertant colonies within expected historical negative Control ranges obtained in this laboratory. All positive Controls induced marked increases in the number of revertant colonies and gave mean counts of induced revertant colonies which were within historical positive Control ranges. The responses to the positive Controls demonstrates that the bacteria were sensitive to the mutagens and that the S 9 mix was able to metabolise the pro-mutagen, 2AA, to a mutagen.

In Experiments 1 and 2, Fenugreek Absolute was tested up to the regulatory maximum dose level (5000 µg/plate) in all strains in the presence and absence of S 9 mix.

Precipitation was noted at 5000 µg/plate in all strains in the presence and absence of S-9 mix in both Experiments.

There were no biologically relevant or statistically significant increases in revertant numbers observed in any strain at any dose of Fenugreek Absolute, in the presence or absence of S 9 mix, under plate incorporation or pre-incubation conditions.

Conclusion: Fenugreek Absolute showed no mutagenic potential in the Bacterial Reverse Mutation Assay under the conditions of this test.