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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a reliable GLP OECD Guideline 471 study, Formononetin was tested in a bacterial reverse mutation assay in Salmonella typhimurium (strains TA 98, TA 100, TA 1535 and TA 1537) and Escherichia coli WP2 uvrA with and without metabolic activation (S9). The results show that Formononetin did not cause a positive increase in the mean number of revertants per plate with any of the tester strains either in the presence or absence of S9.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 January 2008 - 02 July 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Batch No. of test material: 07-170-FIL-2/3
- Expiration date of the batch: 31 May 2010
- Purity: 98.4%
- Storage: Room temperature with desiccant and protected from light
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Liver microsomal enzymes (S9 homogenate)
Test concentrations with justification for top dose:
Test concentrations in the initial mutagenicity assay: 33.3, 100, 333, 1000, 2000 and 5000 µg/ plate (with and without S9 mix)

Test concentrations in the confirmatory mutagenicity assay: 10.0, 33.3, 100, 333, 1000, 2000 and 5000 µg/ plate (with and without S9 mix)

Since no cytotoxicity was observed in the dose range-finding study (10 doses of test article from 6.67 to 5000 µg/plate using tester strains TA100 and WP2uvrA in both the presence and absence of S9 mix with one plate per dose), the highest dose level of test article used in the mutagenicity assay was 5000 µg per plate.
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of vehicle: Based on information supplied by the Sponsor
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: ICR-191 [1]; 2-aminoanthracine [2]
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Incubation duration: 52 ± 4 hours at 37 ± 2°C

NUMBER OF REPLICATIONS: All doses of the test article, the vehicle controls and the positive controls were plated in triplicate.

STAINING TECHNIQUE USED: To demonstrate the presence of the rfa wall mutation, Salmonella typhimurium tester strain cultures exhibited sensitivity to crystal violet.

NUMBER OF CELLS EVALUATED: The density of tester strain cultures was greater than or equal to 0.5 x 10^9 bacteria per mL and/or had reached a target density demonstrated to produce cultures with at least 0.5 x 10^9 bacteria per mL

DETERMINATION OF CYTOTOXICITY
Cytotoxicity is detectable as a decrease in the number of revertant colonies per plate and/or by a thinning or disappearance of the bacterial background lawn.

EXAMINATIONS:
The condition of the bacterial background lawn was evaluated both macroscopically and microscopically (using a dissecting microscope) for indications of cytotoxicity and test article precipitate. Evidence of cytotoxicity was scored relative to the vehicle control plate and was recorded along with the revertant counts for all plates at that dose level. Lawns were scored as normal (N), reduced (R), obscured by precipitate (O), macroscopic precipitate present (P), absent (A), or enhanced (E); contaminated plates (C) were also noted.
Revertant colonies were counted by automated colony counter and/or by hand.
Evaluation criteria:
Tester Strains TA98, TA100, and WP2uvrA: For a test article to be considered positive, it had to produce at least a 2-fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate had to be accompanied by a dose response to increasing concentrations of the test article.

Tester Strains TA1535 and TA1537: For a test article to be considered positive, it had to produce at least a 3-fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate had to be accompanied by a dose response to increasing concentrations of the test article.
Statistics:
For all replicate platings, the mean revertants per plate and the standard deviation were calculated.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated 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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated 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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated 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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: At 100 mg/mL, the most concentrated stock dilution prepared for the mutagenicity assay, the test article was observed to form a light orange, transparent, nonviscous solution.
- Precipitation: The test article remained in solution at all succeeding dilutions prepared for the mutagenicity assay.

RANGE-FINDING/SCREENING STUDIES: Ten doses of test article, from 6.67 to 5000 µg per plate, were tested. No indications of cytotoxicity were observed with either of the tester strains in the presence or absence of S9 mix as evidenced by no dose-related decreases in the number of revertants per plate. Normal bacterial background lawns were observed with both strains in the presence of S9 mix at all doses. In the absence of S9 mix, normal bacterial background lawns were observed at doses up to and including 1000 µg per plate. At doses ≥3330 µg per plate, the lawns were obscured by test article precipitate.

MUTAGENICITY ASSAY RESULTS:
In the initial mutagenicity assay (Table 1), all data were acceptable, and no positive increases in the mean number of revertants per plate were observed with any of the tester strains in either the presence or absence of S9 mix.
The results of the initial mutagenicity assay were used to select the doses tested in the confirmatory mutagenicity assay. The doses tested with all tester strains were 10.0, 33.3, 100, 333, 1000, 2000, and 5000 µg per plate in the presence and absence of S9 mix. A dose of 10.0 µg per plate was added to include another non-precipitating dose.
In the confirmatory assay, the mean vehicle control values for tester strain TA1537 in the presence and absence of S9 mix were originally not within the acceptable range specified for this strain in the protocol. For this reason, the data generated with TA1537 in this trial were not used to evaluate the mutagenicity of the test article. The test article was re-tested with tester strain TA1537 in the presence and absence of S9 mix and all data were acceptable and no positive increases in the mean number of revertants per plate were observed (as shown in Table 2). All other data generated were acceptable, and no positive increases in the mean number of revertants per plate were observed with any of the other tester strains in either the presence or absence of S9 mix (Table 2).

HISTORICAL CONTROL DATA
All historical control data was considered acceptable.

Table 1: Mutagenicity Assay Results – Summary

 

 

Mean revertants per plate with standard deviation

Back-ground lawna

 

Dose/plate

(µg)

TA98

TA100

TA1535

TA1537

WP2uvrA

 

 

Mean

S.D.

Mean

S.D.

Mean

S.D.

Mean

S.D.

Mean

S.D.

 

Microsomes: Rat liver

 

 

 

 

 

 

 

 

 

 

 

Vehicle control

17

3

96

14

14

4

5

3

17

3

N

Test article

33.3

16

4

109

10

8

2

5

3

16

3

N

100

26

1

101

10

9

2

5

2

17

8

N

333

19

8

107

9

8

2

5

1

17

1

N

1000

15

1

81

11

13

6

4

2

15

2

NP

2000

17

6

96

13

8

4

3

2

10

3

OP

5000

17

4

87

7

6

2

4

2

9

3

OP

Positive controlb

497

20

1247

212

140

23

101

5

310

43

N

Microsomes: None

 

 

 

 

 

 

 

 

 

 

 

Vehicle control

11

5

85

4

13

4

6

1

10

2

N

Test article

33.3

16

4

85

10

12

3

6

2

15

5

N

100

15

3

74

2

10

3

4

4

14

5

N

333

13

3

86

4

14

5

5

2

15

5

NP

1000

13

1

71

6

14

5

4

2

17

5

OP

2000

8

3

74

7

7

2

3

1

12

3

OP

5000

8

3

72

14

7

2

3

1

8

4

OP

Positive controlc

333

22

1245

60

808

33

316

49

272

75

N

aBackground Lawn Evaluation Codes:

N = normal; R = reduced; O = obscured; A = absent; P = precipitate

 

bTA98: benzo[a]pyrene: 2.5 μg/plate

TA100: 2-aminoanthracene: 2.5 μg/plate

TA1535: 2-aminoanthracene: 2.5 μg/plate

TA1537: 2-aminoanthracene: 2.5 μg/plate

WP2uvrA: 2-aminoanthracene 25.0 μg/plate

 

cTA98: 2-nitrofluorene: 1.0 μg/plate

TA100: sodium azide: 2.0 μg/plate

TA1535: sodium azide: 2.0 μg/plate

TA1537: ICR-191: 2.0 μg/plate

WP2uvrA: 4-nitroquinoline-N-oxide: 1.0 μg/plate

Table 2: Mutagenicity Assay Confirmatory Results – Summary

 

 

Mean revertants per plate with standard deviation

Back-ground lawna

 

Dose/plate

(µg)

TA98

TA100

TA1535

TA1537 *

WP2uvrA

 

 

Mean

S.D.

Mean

S.D.

Mean

S.D.

Mean

S.D.

Mean

S.D.

 

Microsomes: Rat liver

 

 

 

 

 

 

 

 

 

 

 

Vehicle control

20

10

123

8

13

2

4

3

19

3

N

Test article

10.0

22

3

135

28

13

4

3

2

16

3

N

33.3

23

5

135

10

12

3

5

2

21

5

N

100

33

6

115

7

12

4

5

2

15

8

N

333

26

10

140

6

13

7

5

2

10

5

NP

1000

24

6

138

8

11

1

5

2

15

1

NP

2000

21

5

114

14

15

4

4

2

14

7

OP

5000

16

1

98

15

8

3

2

1

8

3

OP

Positive controlb

430

41

1347

276

179

17

84

8

447

37

N

Microsomes: None

 

 

 

 

 

 

 

 

 

 

 

Vehicle control

14

5

107

7

10

2

2

2

15

5

N

Test article

10.0

17

3

85

16

10

2

4

3

18

2

N

33.3

18

6

97

6

10

1

3

3

21

4

N

100

13

5

89

10

9

3

5

1

10

3

N

333

15

4

86

9

15

2

4

2

13

2

NP

1000

15

4

89

7

15

3

7

2

14

5

NP

2000

11

5

78

8

10

3

4

0

6

2

OP

5000

8

2

75

17

8

71

3

2

10

2

OP

Positive controlc

1988

81

1140

71

920

 

212

8

315

99

N

aBackground Lawn Evaluation Codes:

N = normal; R = reduced; O = obscured; A = absent; P = precipitate

 

bTA98: benzo[a]pyrene: 2.5 μg/plate

TA100: 2-aminoanthracene: 2.5 μg/plate

TA1535: 2-aminoanthracene: 2.5 μg/plate

TA1537: 2-aminoanthracene: 2.5 μg/plate

WP2uvrA: 2-aminoanthracene 25.0 μg/plate

 

cTA98: 2-nitrofluorene: 1.0 μg/plate

TA100: sodium azide: 2.0 μg/plate

TA1535: sodium azide: 2.0 μg/plate

TA1537: ICR-191: 2.0 μg/plate

WP2uvrA: 4-nitroquinoline-N-oxide: 1.0 μg/plate

 

* The mean vehicle control values for tester strain TA1537 were not within the acceptable range for this strain. For this reason, the data generated in the trial with tester strain TA1537 were not used to evaluate the mutagenicity of the test article and were retested. The re-test results are provided in the table.

 

Conclusions:
The results of the Salmonella-Escherichia coli/ Mammalian-Microsome Reverse Mutation Assay with a Confirmatory Assay indicate that under the conditions of this study, the test article, Formononetin, did not cause a positive increase in the mean number of revertants per plate with any of the tester strains either in the presence or absence of microsomal enzymes prepared from Aroclor™-induced rat liver (S9).
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

The result of an in vitro gene mutation study in bacteria was negative. Therefore, it is concluded that Formononetin is not genotoxic and does not warrant classification for mutagenicity in accordance with Regulation (EC) No. 1272/2008.