4-allylveratrole

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

11 July 1988 - 13 October 1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Unnamed

Year:

2000

Reference 2

Reference Type:

publication

Title:

Micronucleated Erythrocyte Frequency in Peripheral Blood of B6C3F1 Mice from Short-Term, Prechronic, and Chronic Studies of the NTP Carcinogenesis Bioassay Program

Author:

Witt K L

Year:

2000

Bibliographic source:

Environmental and Molecular Mutagenesis 36:163-194

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Remarks:

(Food and Drug Administration (FDA) Good Laboratory Practice)

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

4-allylveratrole

EC Number:

202-223-0

EC Name:

4-allylveratrole

Cas Number:

93-15-2

Molecular formula:

C11H14O2

IUPAC Name:

1,2-dimethoxy-4-(prop-2-en-1-yl)benzene

Test material form:

liquid

Test animals

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Simonsen Laboratories, Inc. (Gilroy, CA)
- Age at study initiation: 6 weeks (female), 7 weeks (male)
- Weight at study initiation: 21.1-23.2 g (males) and 18.4-19.5 g (females)
- Housing: 1 per cage; Cages: Polycarbonate (Lab Products, Inc., Maywood, NJ), changed weekly; Bedding: Sani-Chips® (P.J. Murphy Forest Products Corp., Montville, NJ), changed weekly; Racks: Stainless steel (Lab Products Inc., Maywood, NJ), changed and rotated every 2 weeks.
- Diet (e.g. ad libitum): NIH-07 open formula pelleted diet (Zeigler Brothers, Inc., Gardners, PA), available ad libitum, changed weekly.
- Water (e.g. ad libitum): Tap water (Birmingham municipal supply) via automatic watering system (Edstrom Industries, Inc., Waterford, WI), available ad libitum.
- Acclimation period: 11 days (female) or 10 days (male)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72 ± 3ºF
- Humidity (%): 50% ± 15%
- Air changes (per hr): minimum of 10/hour
- Photoperiod (hrs dark / hrs light): 12 hours/day room fluorescent light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 0.5% aqueous methylcellulose
- Justification for choice of solvent/vehicle: not reported.
- Concentration of test material in vehicle: 1, 3, 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw /day
- Lot/batch no. (if required): 874544
- Purity: USP/FCC grade

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The vehicle was prepared by mixing methylcellulose and heated, deionized water with a magnetic stirrer to form a 0.5% solution, which was then cooled. Methyleugenol was slowly added to the 0.5% methylcellulose and mixed for 2 minutes using a homogenizer fitted with an anaerobic generator. The anaerobic generator was removed and the mixture was stirred with a magnetic stirrer for 1 hour.

Duration of treatment / exposure:

14 weeks

Frequency of treatment:

5 days per week

Post exposure period:

None

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

(vehicle control)

Doses / concentrations 2

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

30 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 5

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Doses / concentrations 6

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Positive control(s):

No

Examinations

Tissues and cell types examined:

peripheral blood

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Doses were selected for the 14-week repeated dose toxicity study (NTP TR491, 2000) at the end of which peripheral blood samples were taken for this micronucleus study.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Peripheral blood smears were obtained from B6C3F1 male and female mice after 14 weeks of treatment. Smears were immediately prepared and fixed in absolute methanol.

DETAILS OF SLIDE PREPARATION: The methanol-fixed slides were stained with a chromatin-specific fluorescent dye mixture of Hoechst 33258/pyronin Y (MacGregor et al., 1983) and coded.

METHOD OF ANALYSIS: Slides were scored at 630x or 1000x magnification by epifluorescence microscopy. Criteria for identification of MN were those of Schmid [1976] with the additional requirement that MN exhibit the fluorescence emission characteristic of the fluorescent stain used (orange with green [540nm] excitation with Hoechst/pyronin stain). Polychromatic erythrocytes (PCE) were scored by direct manual counting. Normochromatic erythrocytes (NCE) were scored using a semiautomated method described by Jauhar et al. [1988], in which cell counts were determined by counting a subfield of approximately 1/16th of the full microscope field. Micronucleus frequency scores were based on 10,000 NCE per sample, and the percentage of PCE among the total erythrocyte population was based on the number of PCE among approximately 5,000 erythrocytes.
A set of fixed slides from mice maintained on 0.2% urethane in drinking water was available for random selection of three slides per micronucleus experiment. These slides from urethane-treated mice were stained, coded, and scored along with the experimental slides, thereby providing a positive control for staining and scoring.

Evaluation criteria:

The micronucleus results were tabulated as the mean frequency of micronucleated erythrocytes per 1000 cells per animal, plus or minus the standard error of the mean among animals within a treatment group. An individual trial was considered positive if the trend test P value was less than or equal to 0.025 or if the P value for any single dose group was less than or equal to 0.025 divided by the number of dose groups.

Statistics:

The frequency of micronucleated cells among NCE or PCE was analyzed by a statistical software package that tested for increasing trend over exposure groups using a one-tailed Cochran-Armitage trend test, followed by pairwise comparisons between each exposure group and the control group [Margolin and Risko, 1988; Margolin et al., 1990]. In the presence of excess binomial variation, as detected by a binomial dispersion test, the binomial variance of the Cochran-Armitage test was adjusted upward in proportion to the excess variation. Pairwise comparisons between each treatment group and the concurrent solvent control group were performed using an unadjusted one-tailed Pearson x2 test that incorporated the calculated variance inflation factor for the study.
In this test, an individual trial was considered positive if the trend test P value was less than or equal to 0.025 or if the P value for any single dose group was less than or equal to 0.025 divided by the number of dose groups. A final call of positive for micronucleus induction was preferably based on reproducibly positive trials (as noted above). Ultimately, the final call was determined by the scientific staff after considering the results of statistical analyses, the reproducibility of any effects observed, and the magnitudes of those effects. The percentage of PCEs among total erythrocytes was determined by an analysis of variance on ranks (classed by sex), and individual dosed groups were compared with the concurrent solvent control with a t-test on ranks.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not examined

Additional information on results:

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no increase in the frequency of micronucleated NCEs.
- Ratio of PCE/NCE (for Micronucleus assay): no alteration of the percentage of PCEs among total erythrocytes.

Any other information on results incl. tables

Table 1: Micronucleated Erythrocyte Frequencies in Peripheral Blood of B6C3F1 Mice from NTP Subchronic Toxicity Study (a,b)

Compound	Dose (mg/kg)	Number of Mice with Erythrocytes Scored	Micronucleated NCEs/ 1,000 NCEs	P value^c	PCEs (%)
Male
Methyleugenol	0	10	0.81+-0.10		1.40+-0.05
	10	10	0.70+-0.10	0.8230	1.02+-0.06
	30	10	0.78+-0.10	0.5935	1.51+-0.08
	100	10	0.84+-0.05	0.4077	1.61+-0.11
	300	10	0.63+-0.06	0.9364	1.18+-0.12
	1000	10	0.65+-0.02	0.8992	1.16+-0.11
Trend test^d			P=0.915
ANOVA^e					P < 0.001
Female
Methyleugenol	0	9	0.46+-0.09		1.27+-0.15
	10	10	0.45+-0.06	0.5555	1.15+-0.13
	30	9	0.43+-0.06	0.6160	1.38+-0.13
	100	10	0.54+-0.08	0.2151	1.41+-0.09
	300	10	0.45+-0.08	0.5908	1.37+-0.10
	1000	9	0.63+-0.08	0.0620	1.21+-0.13
Trend test^d			P=0.027
ANOVA^e					P = 0.599

a Values are mean ± SEM. Summary conclusions presented as (male/female).

b NCE, normochromatic erythrocytes; % PCE, percentage of polychromatic erythrocytes in 5,000 total erythrocytes.

c Comparison of treated group to control, x2-test, significant at P ≤ 0.025 per number of test chemical dose groups.

d One-tailed trend test, significant at P ≤ 0.025.

e Analysis of variance, significant at P ≤ 0.025.

Applicant's summary and conclusion

Conclusions:

Methyleugenol was negative in the mammalian erythrocyte micronucleus test.

Executive summary:

An in-vivo micronucleus test was performed with methyleugenol following a method comparable to OECD TG 474. Groups of 10 male and 10 female B6C3F1 mice were administered test item dissolved in 0.5% methylcellulose in concentrations of 0, 10, 30, 100, 300, or 1,000 mg/kg bw by gavage 5 days per week during a 14-week repeated dose toxicity study. At the end of this study, peripheral blood samples were obtained from male and female mice and smears were immediately prepared and fixed in absolute methanol. The methanol-fixed slides were stained with a chromatin-specific fluorescent dye mixture of Hoechst 33258/pyronin Y and coded. Micronucleus frequency scores were based on 10,000 NCE per sample, and the percentage of PCE among the total erythrocyte population was based on the number of PCE among approximately 5,000 erythrocytes. Methyleugenol did not increase the frequency of micronucleated NCEs in peripheral blood and did not alter the percentage of PCEs among total erythrocytes (an indication of bone marrow toxicity). Thus, a negative result is obtained from the mammalian erythrocyte micronucleus test.