Diosmin

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro. Weight of evidence: Four Ames tests for the analogue substance neohesperidin dihydrochalcone, all of them similar to OECD 471 (no GLP), were all negative. Based on the available information for the read-across approach, the target substance is deemed to be non-mutagenic.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

equivalent or similar to guideline

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

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.

- Name of test material (as cited in study report): neohesperidin dihydrochalcone
- Molecular formula (if other than submission substance): C28H36O15
- Molecular weight (if other than submission substance): 612.5764
- Smiles notation (if other than submission subs.): CC1C(O)C(O)C(O)C(OC2C(O)C(O)C(CO)OC2Oc2cc(O)c(C(=O)CCc3ccc(OC)c(O)c3)c(O)c2)O1
- InChl (if other than submission subs.): InChI=1/C28H36O15/c1-11-21(34)23(36)25(38)27(40-11)43-26-24(37)22(35)19(10-29)42-28(26)41-13-8-16(32)20(17(33)9-13)14(30)5-3-12-4-6-18(39-2)15(31)7-12/h4,6-9,11,19,21-29,31-38H,3,5,10H2,1-2H3
- Structural formula attached as image file (if other than submission substance): see Fig. in attached report.

Target gene:

histidine requiring gene

Species / strain / cell type:

S. typhimurium TA 1535

Details on mammalian cell type (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 / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (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 / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (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 / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 97

Details on mammalian cell type (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 / cell type 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 / solvent:

- 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.

Untreated negative controls:

no

Negative solvent / vehicle 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 experimental 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 / choice of top concentrations:

no cytotoxicity

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

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
See "attached justification".

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

Based on the read-across approach, the target substance was found to be non-mutagenic.

Executive summary:

The ability of the analogue substance neohesperidin dihydrochalcone 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. Based on the read-across approach, the target substance was found to be non-mutagenic.

Reference 3

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

Qualifier:

equivalent or similar to guideline

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

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).

- Name of test material (as cited in study report): neohesperidin dihydrochalcone (No 36)
- Molecular formula (if other than submission substance): C28H36O15
- Molecular weight (if other than submission substance): 612.5764
- Smiles notation (if other than submission subs.): CC1C(O)C(O)C(O)C(OC2C(O)C(O)C(CO)OC2Oc2cc(O)c(C(=O)CCc3ccc(OC)c(O)c3)c(O)c2)O1
- InChl (if other than submission subs.): InChI=1/C28H36O15/c1-11-21(34)23(36)25(38)27(40-11)43-26-24(37)22(35)19(10-29)42-28(26)41-13-8-16(32)20(17(33)9-13)14(30)5-3-12-4-6-18(39-2)15(31)7-12/h4,6-9,11,19,21-29,31-38H,3,5,10H2,1-2H3
- Structural formula attached as image file (if other than submission substance): see Fig. in attached report.

Target gene:

histidine requiring gene.

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (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 / cell type 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 / solvent:

- Vehicle(s)/solvent(s) used: DMSO.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO.

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: quercetin, aflatoxin B1.

Details on test system and experimental 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 / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

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.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
See "attached justification".

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

Based on the read-across approach, the target substance is not mutagenic.

Executive summary:

The ability of the analogue substance neohesperidin dihydrochalcone 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 analogue substance was found to be not mutagenic. Based on the read-across approach, the target substance is not mutagenic.

Reference 5

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

Qualifier:

equivalent or similar to guideline

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

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)
OTHER SPECIFICS:
- Name of test material (as cited in study report): neohesperidin dihydrochalcone
- Molecular formula (if other than submission substance): C28H36O15
- Molecular weight (if other than submission substance): 612.5764
- Smiles notation (if other than submission subs.): CC1C(O)C(O)C(O)C(OC2C(O)C(O)C(CO)OC
2Oc2cc(O)c(C(=O)CCc3ccc(OC)c(O)c3)c(O)c2)O1
- InChl (if other than submission subs.): InChI=1/C28H36O15/c1-11-21(34)23(36)25(38)27(40-11)43-26-
24(37)22(35)19(10-29)42-28(26)41-13-8-16(32)20(17(33)9-13)14(30)5-3-12-4-6-18(39-2)15(31)7-12/
h4,6-9,11,19,21-29,31-38H,3,5,10H2,1-2H3
- Structural formula attached as image file (if other than submission substance): see Fig. in attached
report.

Target gene:

Histidine dependent gene.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (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 / cell type 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 / solvent:

- Vehicle(s)/solvent(s) used: Test agents were dissolved in dimethyl sulfoxide (DMSO) or sterile water.

Untreated negative controls:

no

Negative solvent / vehicle 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 experimental 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 / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

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.

Reference 6

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
See "attached justification".

Reason / purpose for cross-reference:

read-across source

Type of assay:

bacterial reverse mutation assay

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: no mutagenic potential (based on read-across from analogue substance).

Conclusions:

Based on the read-across approach, the test item is not mutagenic.

Executive summary:

The ability of the analogue substance neohesperidin dihydrochalcone 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 analogue substance was not mutagenic. Based on the read-across approach, the test item is not mutagenic.

Reference 7

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

Qualifier:

equivalent or similar to guideline

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

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.

- Name of test material (as cited in study report): neohesperidin dihydrochalcone
- Molecular formula (if other than submission substance): C28H36O15
- Molecular weight (if other than submission substance): 612.5764
- Smiles notation (if other than submission subs.): CC1C(O)C(O)C(O)C(OC2C(O)C(O)C(CO)OC2Oc2cc(O)c(C(=O)CCc3ccc(OC)c(O)c3)c(O)c2)O1
- InChl (if other than submission substance): InChI=1/C28H36O15/c1-11-21(34)23(36)25(38)27(40-11)43-26-24(37)22(35)19(10-29)42-28(26)41-13-8-16(32)20(17(33)9-13)14(30)5-3-12-4-6-18(39-2)15(31)7-12/h4,6-9,11,19,21-29,31-38H,3,5,10H2,1-2H3
- Structural formula attached as image file (if other than submission substance): see Fig. attached.

Target gene:

Histidine requiring gene of S. typhimurium strains TA98 and TA 100.

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type 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 / solvent:

No data.

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Details on test system and experimental 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 / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

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.

Reference 8

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
See "attached justification".

Reason / purpose for cross-reference:

read-across source

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine requiring gene of S. typhimurium strains TA98 and TA 100.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

other: Read-across from analogue substance.

Conclusions:

Based on the read-across approach, the target substance is not mutagenic.

Executive summary:

A study on the mutagenic activity of the analogue substance neohesperidin dihydrochalcone was performed, following the method of Ames, similar to OECD TG 471. Mutagenic activities of the test substance 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. Therefore, the analogue substance is considered to be non mutagenic under test conditions. Based on the read-across approach, the target substance is not mutagenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro. Weight of evidence:

- Read-across from supporting substance (structural analogue or surrogate). Source: Key study. Method similar to OECD 471 (no GLP). The analogue substance neohesperidin dihydrochalcone 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]. Based on the read-across approach, the target substance is not mutagenic.

- Read-across from supporting substance (structural analogue or surrogate). Source: Key study. Method similar to OECD 471 (no GLP). The analogue substance neohesperidin dihydrochalcone 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]. Based on the read-across approach, the target substance is not mutagenic.

- Read-across from supporting substance (structural analogue or surrogate). Source: Key study. Method similar to OECD 471 (no GLP). The analogue substance neohesperidin dihydrochalcone 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]. Based on the read-across approach, the target substance is not mutagenic.

- Read-across from supporting substance (structural analogue or surrogate). Source: Key study. Method of Ames, similar to OECD 471 (no GLP). The analogue substance neohesperidin dihydrochalcone 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]. Based on the read across approach, the target substance is not mutagenic.

Justification for classification or non-classification

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