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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity. Weight of evidence. All in vitro and in vivo studies for the substance yielded negative results. Based on the available data, the test item is deemed to be non-mutagenic.

Genetic toxicity in vitro (weight of evidence):

- Method similar to OECD 471 (no GLP). The test item did not induce an increase in the number of revertants in any strain (S. typhimurium TA1535, TA97, TA98, TA100) at any dose level, up to 10000 μg/plate, either with or without metabolic activation [Zeiger, 1987].

- Method similar to OECD 471 (no GLP). The test item up to 8197 μg/plate did not induce an increase in the number of revertants in S.typhimurium TA98 at any dose level [MacGregor, 1978].

- Method similar to OECD 471 (no GLP). The test item up to 200 μg/plate did not induce an increase in the number of revertants in any strain (S. typhimurium TA1535, TA97, TA98, TA100) at any dose level [Brown, 1979].

- Method of Ames, similar to OECD 471 (no GLP). The test item up to 200 mg/plate did not induce an increase in the number of revertants in any strain (S. typhimurium TA98, TA100)at any dose level [Batzinger, 1977].

Genetic toxicity in vivo: Key study. Method similar to OECD 474 (no GLP). The test item did not induce significant increases in the micronucleus frequency in the bone marrow of Swiss-Webster mice treated with up to 5000 mg/kg test item (6 m/f mice per dose, 4 dose levels, oral administration in two doses 24h apart) [MacGregor, 1983].

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1987.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only 4 strains tested.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
The laboratories were supplied with the chemicals, which were coded by the NTP chemical repository (Radian Corp., Austin, TX), along with information on the physical characteristics of the chemicals, their solubility in different solvents, and safety and decontamination information. Supplier: Res.Organic/inorganic.
Target gene:
histidine requiring gene
Species / strain:
S. typhimurium TA 1535
Details on mammalian cell lines (if applicable):
All strains were obtained from Dr. Bruce Ames (University of California, Berkeley) and were stored as recommended [Maron and Ames, 19831. Prior to their use for mutagenicity assays, all cultures were grown overnight with shaking at 37°C in Oxoid broth, and their phenotypes were analyzed.
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix.
Species / strain:
S. typhimurium TA 98
Details on mammalian cell lines (if applicable):
All strains were obtained from Dr. Bruce Ames (University of California, Berkeley) and were stored as recommended [Maron and Ames, 19831. Prior to their use for mutagenicity assays, all cultures were grown overnight with shaking at 37°C in Oxoid broth, and their phenotypes were analyzed.
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix.
Species / strain:
S. typhimurium TA 100
Details on mammalian cell lines (if applicable):
All strains were obtained from Dr. Bruce Ames (University of California, Berkeley) and were stored as recommended [Maron and Ames, 19831. Prior to their use for mutagenicity assays, all cultures were grown overnight with shaking at 37°C in Oxoid broth, and their phenotypes were analyzed.
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix.
Species / strain:
S. typhimurium TA 97
Details on mammalian cell lines (if applicable):
All strains were obtained from Dr. Bruce Ames (University of California, Berkeley) and were stored as recommended [Maron and Ames, 19831. Prior to their use for mutagenicity assays, all cultures were grown overnight with shaking at 37°C in Oxoid broth, and their phenotypes were analyzed.
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix.
Test concentrations with justification for top dose:
100, 333, 1000, 3333 and 10000ug/plate.
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO.
- Justification for choice of solvent/vehicle: The solvent of choice was distilled water, followed by dimethyl sulfoxide, 95% ethanol, and acetone. The laboratory made an independent assessment of the solvent to be used.
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 2-aminoanthracene, 4-nitro-o-phenylenediamine
Details on test system and conditions:
METHOD OF APPLICATION: preincubation. The preincubation assay was performed as described previously [Haworth et al, 1983]: The test chemical, Salmonella culture, and S-9 mix or buffer were incubated at 37ºC, without shaking, for 20 min. The top agar was added, and the contents of the tubes were mixed and poured onto the surface of petri dishes that contained Vogel-Bonner medium [Vogel and Bonner, 1956 (preparation: in 670ml distilled water are dissolved, successively: 10g MgSO4·7H2O, 100g citric acid·H2O, 500g anhydrous K2HPO4, 175g NaNH4HPO4·4H2O, the final volume being 1L; this solution is diluted 50x)]. The histidine-revertant (his+) colonies arising on these plates were counted following 2 days incubation at 37ºC. The plates were hand-counted when a precipitate was present; otherwise automatic colony counters were used.
- Cell density at seeding (if applicable):

DURATION
- Exposure duration: 48h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth; other: A toxicity assay was performed to determine the appropriate dose range, by using TA100 or the system developed by Waleh (1982), see 'attached background material'.
- Any supplementary information relevant to cytotoxicity: Toxic concentrations were those at which a decrease in the number of his-f colonies was seen or at which there was a clearing in the density of the background lawn. Experiments were repeated at least 1 week following the initial trial.

- OTHER: The S-9 fractions of Aroclor 1254-induced, male Sprague-Dawley rat and male Syrian hamster livers were prepared as described previously [Haworth et al, 1983].
Evaluation criteria:
Data was evaluated as described by Haworth et al., 1983. An individual trial was judged mutagenic (+) if a dose-related increase over the corresponding solvent control was seen, and it was judged weakly mutagenic (+W) if a low-level dose response was seen. A trial was considered questionable (?) if a dose related increase was judged insufficiently high to justify a call of " + W," if only a single dose was elevated over the control, or if a non-dose-related increase was seen. A chemical was judged to be mutagenic (+), or weakly mutagenic (+W), if it produced a reproducible, dose-related increase in his+ revertants over the corresponding solvent controls in replicate trials. A chemical was considered to be questionable (?) if a reproducible increase of hist revertants did not meet the criteria for either a " + " or " + W," or if only single doses produced an increase in his+ revertants in repeat trials.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes

Table 1. Neohesperidin dihydrochalcone (lab: MIC, solvent: DMSO).

Dose

TA 100

TA 1535

NA

(-)

10% HLI

(-)

30% HLI

(-)

10% RLI

(-)

30% RLI

NA

(-)

10% HLI

(-)

30% HLI

(-)

10% RLI

(-)

30% RLI

μg/plate

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

0.000

139

9.8

71

0.9

112

6.7

82

3.8

100

9.0

45

3.9

10

1.5

14

2.4

10

2.3

19

1.0

100.000

136

10.8

72

3.3

104

2.3

75

1.9

93

3.8

48

7.5

10

1.8

16

2.5

8

1.8

13

1.2

333.000

127

2.5

75

3.5

95

4.8

89

0.7

115

11.9

33

4.4

11

1.9

11

3.0

12

1.7

12

1.7

1000.000

128

5.2

84

4.6

92

8.1

84

4.5

104

6.1

40

4.9

11

1.2

15

2.2

8

3.2

15

2.7

3333.000

119

2.5

74

3.7

99

1.5

76

6.8

99

2.0

43

4.8

12

1.0

10

0.9

13

2.2

13

2.1

10000.000

119

2.8

71

2.3

106

5.9

78

3.5

88

1.2

41

5.9

10

1.2

9

1.2

11

1.0

17

4.1

POS

1238

19.9

1232

46.7

460

25.5

1077

23.8

421

8.0

884

33.4

149

14.5

168

8.7

128

10.5

106

0.6

 

Dose

TA 97

TA 98

NA

(-)

10% HLI

(-)

30% HLI

(-)

10% RLI

(-)

30% RLI

NA

(-)

10% HLI

(-)

30% HLI

(-)

10% RLI

(-)

30% RLI

μg/plate

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

0.000

96

3.7

99

5.9

142

6.7

101

5.8

133

6.1

16

3.0

34

5.8

30

3.0

32

2.8

44

2.7

100.000

85

5.7

89

0.6

124

8.8

116

5.2

152

6.4

18

2.2

31

0.7

35

0.3

32

2.3

39

4.7

333.000

88

7.5

85

4.1

130

4.2

91

8.4

138

11.9

17

2.3

37

2.9

30

0.6

27

1.2

41

0.7

1000.000

91

2.4

94

5.2

125

3.1

102

5.0

134

9.2

20

2.3

27

4.9

33

5.5

26

2.7

37

5.0

3333.000

82

2.0

88

2.7

129

3.7

110

9.4

134

10.3

23

1.7

30

4.1

39

0.9

36

1.9

41

4.1

10000.000

97

4.0

88

6.0

146

9.3

96

4.8

147

9.2

19

0.0

28

2.3

34

3.3

31

0.6

42

2.0

POS

797

8.4

634

17.4

378

13.0

558

18.0

290

12.6

1931

52.0

1212

25.4

418

16.1

814

37.6

366

20.2

* NA: without metabolic activation, HLI: Aroclor 1254- induced hamster liver homogenate; RLI: Aroclor 1254 -induced rat liver homogenate; (-): negative.

Conclusions:
The test item was found to be non-mutagenic.
Executive summary:

The ability of the test item to induce mutation was assessed by the bacterial reverse mutation test (Ames test), performed according to the method described by Haworth, similar to OECD 417. Four histidine dependent strains of Salmonella typhimurium (TA1535, TA97, TA98, TA100) were exposed to 0, 100, 333, 1000, 3333, and 10000 μg/plate of test item in the presence and absence of S9 metabolic activation (10 and 30% Aroclor 1254- induced hamster liver homogenate and rat liver homogenates), by the preincubation method. Under the experimental conditions used, the test item did not induce an increase in the number of revertants in any strain at any dose level, either with or without metabolic activation. Therefore, the test item was not mutagenic.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1978.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only 1 strain tested (TA98), only 3 doses tested.
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: Compounds were prepared by selective methylation of quercetin or synthesized by established methods [10,19]. After repeated crystallization, the purity and identity of each compound was confirmed by elementary analysis and by UV or NMR spectra. The absence of quercetin was confirmed by thin-layer chromatography (TLC).
Target gene:
histidine requiring gene.
Species / strain:
S. typhimurium TA 98
Details on mammalian cell lines (if applicable):
Presence of the R-factor plasmid was confirmed by cross-streaking strains TA98 and TA1538 on ampicillin and the presence of the rfa character by
crystal violet growth inhibition, as described by Ames et al.
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix.
Test concentrations with justification for top dose:
820, 1640 and 8197 nmol/plate.
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO.
Negative controls:
no
Solvent controls:
yes
Remarks:
DMSO.
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: quercetin, aflatoxin B1.
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation): The compound was dissolved in DMSO and added in a volume of 0.1mL after filter sterilization. An equivalent volume of solvent was added to the control plates. Each determination was carried out in duplicate.

NUMBER OF REPLICATIONS: 2
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes

Table 1. Mutagenicity of substituted flavones and related compounds in salmonella typhimurium strain TA98

Test Compound

nmol/plate

Mutants/plate

Control (b)

- S9

+ S9

35

Hesperetin dihydrochalcone

50

(7)

6

-

167

3

(4)

500

1

(6)

1667

(3)

(8)

36

Neohesperidin dihydrochalcone

820

(3)

11

At 1660 nanomoles/plate,

+ 10 μg 2-aminofluorene,

> 1000 with S-9.

1640

1

9

8197

(3)

18

(a) Tabular values are the mean of replicate determination minus the spontaneous control value.

(b) Concurrently determined responses to quercetin (corrected for spontaneous revertants).

Conclusions:
The test item is not mutagenic.
Executive summary:

The ability of the test item to induce mutation was assessed by the bacterial reverse mutation test (Ames test), performed according to the method described by Ames, similar to OECD 417. An histidine dependent strain of Salmonella typhimurium (TA98) was exposed to 820, 1640, and 8197 μg/plate of test item in the presence and absence of S9 metabolic activation. Under the experimental conditions used, the test item did not induce an increase in the number of revertants at any dose level, either with or without metabolic activation. Therefore, the test item was not mutagenic.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1979
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reference:
Composition 0
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only 4 strains tested.
Principles of method if other than guideline:
Study followed the Salmonella/mammalian microsome assay of Ames (see attached background material), with certain modifications of the routine-assay procedures to allow for nonmicrosomal enzymic activation of the flavonoid compounds (greater S9 fraction).
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: Nutrilite Products, Buena Park, CA.
- Purity was assessed by TLC using polyamide plates (polyamide-6 UV2s4, 0.1 ram, Brinkmann) developed with chloroform : methanol : 2-butanone : acetone, 20:10:5:1 (v/v)
Target gene:
Histidine dependent gene.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell lines (if applicable):
CELLS USED
- Source of cells: Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538, TA98 were obtained from Dr. Bruce N. Ames (University of California, Berkeley).
- Methods for maintenance in cell culture if applicable: Manipulation of these tester strains was carried out as recommended by Ames et al. [1]. Frozen permanents of each tester strain and the S-9 fractions (see below) were stored in sterile plastic vials under liquid nitrogen (Linde LR-30). Broth cultures of the tester strains were prepared in nutrient broth containing 0.5% NaCl. Five 25-ml Delong culture flasks, each containing 10 ml broth, were inoculated from frozen permanents of the respective tester strains and incubated 14-16h, 37ºC, 300 rpm in a New Brunswick G24 gyrotory incubator shaker.
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 (Aroclor 1254 induced rat-liver microsome preparations)
Test concentrations with justification for top dose:
Various concentrations up to up to 200 μg/plate.
Vehicle:
- Vehicle(s)/solvent(s) used: Test agents were dissolved in dimethyl sulfoxide (DMSO) or sterile water.
Negative controls:
no
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
ethylmethanesulphonate
methylmethanesulfonate
other: anthragallol, 2-anthramine
Details on test system and conditions:
METHOD OF APPLICATION:
- Preincubation: Up to 0.1 ml of a solution of the test agent in DMSO or water was combined with 0.1 ml of a culture of the tester strain and 0.5 ml of S9-mix in a sterile screwcap tube (16 X150 mm, Bellco) in an ice bath. The tube was then transferred to a water bath at 37ºC and incubated with occasional hand agitation for 30 min. Then 2.5 ml molten top agar (45ºC) were quickly added, mixed, and plated and incubated as usual. (method from Yahagi et al).
- Plate incorporation: Plates containing up to 200 μg test agent contained in a sterile 0.25-inch concentration disk applied to the top agar, were incubated 3 days at 35ºC.
- Test System: Qualitative plate tests (and in some cases, spot tests) were performed on the test agent. 1-2 days prior to the assay the bottom agar medium (Vogel - Bonner E plus 1.5% agar) was prepared, sterilized by autoclaving, tempered for 1 h at 50ºC, and 20 ml dispensed into Optilux (Falcon No. 1001) disposable plastic petri dishes with a Technomat automatic petri dish filler type 121 (Manostat, New York, NY). The top agar was prepared at the same time, autoclaved and tempered before 10 ml of sterile 0.5 mM L-histidine-0.5 mM d-biotin solutions are added per 100 ml agar. The complete top agar medium was dispensed (2.5 ml/tube) into new sterile disposable screw cap glass culture tubes (16 × 150 mm, Bellco Glass, Vineland, NJ). Immediately prior to pouring the top agar, 0.1 ml of the broth culture, 0.2 ml or less of the test solution, and, in the case of liver activation, 0.25 ml S9-mix were added. When present, 0.2 ml of cell-free extract was added.

DURATION
- Preincubation period: 30 min (preincubation method).
- Exposure duration: 72h

OTHER:
- Rat-liver homogenate fractions: Female Sprague Dawley rats (Simonsen Laboratories, Gilroy, CA) were maintained on Simonsen White laboratory diet. Removal of livers and subsequent preparation of the mammalian liver homogenate S9 fraction (9000 X g supernatant) and the S9-mix (0.2 ml S9 fraction per ml) was prepared as described by Ames. Aroclor induced S9 was prepared from animals injected i.p. 5 days prior to sacrifice with about 0.5 ml of a corn oil solution of Aroclor 1254 (400 mg/ml) to give a dose of 500 mg/kg bw.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Additional information on results:
his+ revertants per nmole: <0.01 revertants per nmol test agent.
his+ revertants per plate/ug: 0 (200) revertants per plate less background for a given quantity (ug) test agent.
Conclusions:
No biologically significant increase in the number of revertants was noted in any strain at any dose level, either with or without metabolic activation. Therefore, the test item was not mutagenic.
Executive summary:

The ability of the test item to induce mutation was assessed by the bacterial reverse mutation test (Ames test), performed according to the method described by Ames, similar to OECD 417. Four histidine dependent strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100) were exposed to various concentrations up to 200 μg/plate of test item in the presence and absence of S9 (Aroclor 1254 induced rat liver homogenate fraction) metabolic activation, by the preincubation method or the plate incorporation method. Under the experimental conditions used, the test item did not induce an increase in the number of revertants in any strain at any dose level, either with or without metabolic activation. Therefore, the test item was not mutagenic.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1977.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only 2 strains tested.
Principles of method if other than guideline:
Method by Ames (see 'attached background material').
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: donated by Dr. R. Horowitz.
Target gene:
Histidine requiring gene of S. typhimurium strains TA98 and TA 100.
Species / strain:
S. typhimurium TA 98
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9.
Species / strain:
S. typhimurium TA 100
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9.
Test concentrations with justification for top dose:
40, 120, and 200 mg/plate.
Vehicle:
No data.
Negative controls:
not specified
Solvent controls:
not specified
True negative controls:
not specified
Positive controls:
not specified
Details on test system and conditions:
METHOD OF APPLICATION: Direct assays of the compounds were conducted at several concentrations, both in the absence and presence of a rat liver microsomal fraction (S9). All determinations were conducted in duplicate.

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified

Table 1. In vitro mutagenic activities.

Substance

Amount (mg/plate)

Number of revertants in excess of controls

TA 100

TA 98

- S9

+ S9

- S9

+ S9

NHDC

40

0

0

0

0

120

0

0

0

0

 

Table 2. Revertants induced by urines excreted per mouse over 24h after treatment.

Compound and preparation

Number of revertants in excess of controls

TA 100

TA 98

-b-glucuronidase

+b-glucuronidase

-b-glucuronidase

+b-glucuronidase

NHDC

0

0

0

0

 

Conclusions:
The test item was found to be non mutagenic.
Executive summary:

A study on the mutagenic activity of the test item was performed, following the method of Ames, similar to OECD TG 471. Mutagenic activities of the test substance at up to 200mg/plate were determined directly on Salmonella typhimurium strains TA98 and TA100 (with and without metabolic activation) directly, as well as in a host-mediated assay, and in the urines of rats after oral administration of the test item. In all cases, the test substance was found to be non mutagenic. Therefore, the test item is considered to be non mutagenic under test conditions.

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

Genetic toxicity in vivo

Link to relevant study records
Reference
Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1983.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
bone marrow sampling time 6h after last dose instead of 18h.
GLP compliance:
no
Type of assay:
micronucleus assay
Test material information:
Composition 1
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Synthesized by Nutrilite Products, Buena Park, CA.
- Purity: The NMR spectrum was consistent with a purity of ca. 99%.
Species:
mouse
Strain:
Swiss Webster
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Simonsen Laboratories, Gilroy, CA.
- Age at study initiation: approximately 6 weeks. Animals were age-matched within each experiment.
- Weight at study initiation: 20-32 g
- Assigned to test groups randomly: yes
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: 2% acacia (gum arabic) in water.
- Amount of vehicle (if gavage): 10 ml/kg body weight
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Oral dosing was by gavage in 2% acacia (gum arabic) in water.
Duration of treatment / exposure:
48h
Frequency of treatment:
Two doses 24h apart.
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
5 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
6 animals per dose, 12 in negative and positive controls.
Control animals:
yes, concurrent vehicle
Positive control(s):
- triethylenemelamine, TEM, lot 2075-J0710, Lederle Laboratories, Pearl River, NY.
- Route of administration: oral
- Doses / concentrations: positve controls were dosed in a volume of saline corresponding to that employed for the test compounds in each experiment.
Tissues and cell types examined:
Bone marrow.
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Swiss-Webster mice were given 2 doses 30 and 6 h prior to sacrifice. Bone marrow was sampled 6 h after the second of 2 doses given 24 h apart. Bone marrow from both femurs was suspended in fetal bovine serum and smears were made according to Schmid (1976).

DETAILS OF SLIDE PREPARATION: Slides were air-dried, fixed in absolute methanol, and stained with filtered Wright-Giemsa stain as described elsewhere (Schlegel and MacGregor, 1982; see 'attached background material').

METHOD OF ANALYSIS: The incidence of micronuclei in polychromatic and normochromatic erythrocytes, and the ratio of polychromatic to normochromatic erythrocytes, were scored at 1000 x under oil immersion. The polychromatic/normochromatic erythrocyte ratio was based on a minimum of 500 erythrocytes.
Evaluation criteria:
The polychromatic/normochromatic erythrocyte ratio and the percentage of polychromatic erythrocytes were based on a minimum of 500 erythrocytes.
Statistics:
Micronucleus frequencies in polychromatic erythrocytes of treated groups were compared with concurrent control values using both the negative binomial comparison described by Mackey and MacGregor (1979) and the binomial comparison described by Kastenbaum and Bowman (1970). Micronucleus frequencies in normochromatic erythrocytes were compared with concurrent control group values by the binomial comparison of Kastenbaum and Bowman (1970). The negative binomial test was set up to determine whether or not a 3-fold or greater increase over the spontaneous value in all comparable control groups was observed at a type 2 ( fl ) error of 0.10 ( Mackey and MacGregor , 1979).
The Krulkal-Wallis 1-way analysis of variance by ranks (Siegel, 1956) was used to test for differences in the percentage of polychromatic erythrocytes among groups. When the analysis of variance for the concurrent negative control and all dosage groups of a single test agent was significant at P <0.05, subsequent pairwise comparis on were made to determine which individual dosage groups differed from the concurrent control group.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Additional information on results:
Neohesperidin dihydrochalcone did not increase the micronucleus frequency in bone marrow erythrocytes 6h after the second of 2 doses 24 h apart.

Table 1. Incidence of micronucleated erythrocytes in bone marrow of male mice exposed to plant flavonols (a).

Dose

(mg/kg)

N

Micronucleated

PCE/1000 PCE

Micronucleated

PCE/1000 PCE

PCE (%)

Mortality

(%)

5000

6

1.3

(6152)

2.5

(4363)

59

0

1000

6

0.5

(6056)

0.4

(2829)

68

0

500

6

3.1#,*

(6136) (c)

1.5*

(4694) (c)

57

0

500

6

1.4

(6399)

0.3

(3435)

65

0

200

6

0.3

(6126)

0.0

(2211)

73

0

2% acacia

12

1.1

(12413)

0.6

(7135)

64

0

TEM, 0.25

12

16**

(11736)

1.3

(6923)

62

0

(a)   Swiss-Webster mice (20-32g) were given 2 doses 30 and 6h prior to sacrifice.

(c) Only 1 animal of the group was affected; group value is not significant if this animal (14 micronucleated PCE/1000 PCE, 3 micronucleated NCE/245 NCE) is not included.

Numbers of cells upon which each micronucleus frequency is based are given in parentheses. Significance levels of groups differing from the concurrent control are as follows: # indicates no decision possible at α = 0.05, β = 0.1; * denotes P < 0.005, ** denotes P < 0.01.

(*) For the 500mg/kg dose, the test group results exceeded the critical value for 5% significance due to a single mouse, which had 14 micronucleated cells among 1000 polychromatic erythrocytes and 3 micronucleated cells among 245 normochromatic erythrocytes. No other animal in this group had a value above 2 micronuclei/1000cells in either polychromatic or normochromatic erythrocytes, nor was there evidence of an increased micronucleus frequency in any other dose group in this experiment. A repeat experiment at the same dose failed to show any evidence of an increased micronucleus frequency in any of 6 additional mice.

Conclusions:
The test item was found to be non-mutagenic.
Executive summary:

An in vivo micronucleus assay was conducted to determine the mutagenicity potential of test item in Swiss-Webster male and female mice. 6 animals per dose were treated by oral administration with the test substance at concentrations of 200, 500, 1000 and 5000 mg/kg test item in two doses 24 h apart, by a method similar to OECD 474. Bone marrow samples were collected 6h after administration of the last dose, and at least 500 cells were scored per dose. Under test conditions, no consistent increases in the micronucleus frequency were observed in bone marrow from mice treated with the test substance. Therefore, the test item is considered to be non-mutagenic.

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

Additional information

Genetic toxicity in vitro:

- Weight of evidence. A bacterial reverse mutation test according to Haworth (1983), similar to OECD 471 (no GLP) was performed on the test item. Four histidine dependent strains of Salmonella typhimurium (TA1535, TA97, TA98, TA100) were exposed to 0, 100, 333, 1000, 3333, and 10000 μg/plate of test item in the presence and absence of S9 metabolic activation (10 and 30% Aroclor 1254- induced hamster liver homogenate and rat liver homogenates), by the preincubation method. Under the experimental conditions used, the test item did not induce an increase in the number of revertants in any strain at any dose level, either with or without metabolic activation. Therefore, the test item was not mutagenic.  

- Weight of evidence. A bacterial reverse mutation test according to Ames (1975), similar to OECD 471 (no GLP) was performed on the test item. An histidine dependent strain of Salmonella typhimurium (TA98) was exposed to 820, 1640, and 8197 μg/plate of test item in the presence and absence of S9 metabolic activation. Under the experimental conditions used, the test item did not induce an increase in the number of revertants at any dose level, either with or without metabolic activation. Therefore, the test item was not mutagenic.

- Weight of evidence. A bacterial reverse mutation test according to Ames (1975), similar to OECD 471 (no GLP) was performed on the test item. Four histidine dependent strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100) were exposed to various concentrations up to 200 μg/plate of test item in the presence and absence of S9 (Aroclor 1254 induced rat liver homogenate fraction) metabolic activation, by the preincubation or the plate incorporation method. The test item did not induce an increase in the number of revertants in any strain at any dose level, either with or without metabolic activation. Therefore, the test item was not mutagenic.

- Weight of evidence. A bacterial reverse mutation test according to Ames (1975), similar to OECD 471 (no GLP) was performed on the test item.

Mutagenic activities of the test item at up to 200 mg/plate were determined directly on Salmonella typhimurium strains TA98 and TA100 (with and without metabolic activation) directly, as well as in a host-mediated assay, and in the urines of rats after oral administration of the test item. In all cases, the test substance was found to be non mutagenic.

Genetic toxicity in vivo:

- Key study. An in vivo micronucleus assay was conducted to determine the mutagenicity potential of test item in Swiss-Webster male and female mice. 6 animals per dose were treated by oral administration with the test item at concentrations of 200, 500, 1000 and 5000 mg/kg in two doses 24 h apart, by a method similar to OECD 474 (no GLP). Bone marrow samples were collected 6h after administration of the last dose, and at least 500 cells were scored per dose. Under test conditions, no consistent increases in the micronucleus frequency were observed in bone marrow from mice treated with the test substance. Therefore, the test item is considered to be non-mutagenic.

Justification for classification or non-classification

Based on the available data (negative results in vitro and in vivo), it is concluded that the substance is not classified for mutagenicity in accordance with CLP Regulation (EC) No. 1272/2008.