Registration Dossier

Administrative data

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

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1983
Report Date:
1983

Materials and methods

Test guideline
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

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
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%.

Test animals

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

Administration / exposure

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.
Doses / concentrationsopen allclose all
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.

Examinations

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.

Results and discussion

Test results
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.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.