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EC number: 207-623-9 | CAS number: 485-72-3
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- Endpoint summary
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- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
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- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
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- Toxicity to microorganisms
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Endpoint summary
Administrative data
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
- 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
- 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. - 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).
Reference
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.
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.
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