Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: other: Incidence of micronucleated polychromatic erythrocytes
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP study and range finding for OECD method 474 for mouse micro nucleus study. The protocol has been written to comply with EEC Directive 79/831 Annex V, B.12 Mutagenicity (Micronucleus Test), June, 1989; OECD Guideline 474 (Genetic Toxicology: Micronucleus Test); EPA Health Effects Testing Guidelines, Subpart 798.5395 (In Vivo Mammalian Bone Marrow Cytogenetics Tests: Micronucleus assay). Fed. Register. vol. 50. September, 1985 with revisions Fed. Register. vol. 52, May, 1987, and Notification No. t 18 of the Pharmaceutical Affairs Bureau, Ministry of Health and Welfare, Japan, February 15, 1984.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
other: micronucleus/cytogenetic assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
AHTN

Test animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
ICR mice were obtained from Harlan Sprague Dawley, Inc., Frederick, MD. At the initiation of the study, the mice were 6 to 8 weeks old. Males, 28.1-37.2 grams at randomization Females, 24.5-31.0 grams at randomization.
The mice were housed in an AAALAC-accredited facility with a controlled environment of 20-27°C average 23°C, 50±20% relative humidity, and a 12 hour light/dark cycle. Mice of the same sex were housed up to five per cage in polycarbonate cages which were maintained on stainless steel racks equipped with automatic watering manifolds and were covered with filter material. Heat-treated hardwood chips were used for bedding. Mice had free access to certified laboratory rodent chow which had been analyzed for environmental contaminants (Harlan TEKLAD certified Rodent 7012C) and to tap water (Washington Suburban Sanitary Commission, Potomac Plant). There are no contaminants in the feed which are expected to influence the study. The water meets USEPA drinking water standards and is monitored at least annually for levels of organophosphorus pesticides, metals, and coliform and other contaminants.

Administration / exposure

Route of administration:
intraperitoneal
Duration of treatment / exposure:
24, 48 and 72 hours (see table 1)
Frequency of treatment:
single treatment
Post exposure period:
72 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
400, 800 and 1600 mg/kg body weight
Basis:
other: Intraperitoneal
No. of animals per sex per dose:
goups of 5 male and 5 female
Control animals:
yes, concurrent vehicle
Positive control(s):
Gyclophosphamide (CP, CAS 6055-19-2), was obtained from Sigma Ghemical Company and was dissolved in sterile distilled water at a concentration of 3 mg/ml.

Examinations

Tissues and cell types examined:
Immediately following sacrifice, the femurs were exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing fetal bovine serum. The bone marrow cells were transferred to a capped centrifuge tube containing approximately 1 ml fetal bovine serum. The bone marrow cells were pelleted by centrifugation and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were resuspended and a small drop of bone marrow suspension was spread onto a clean glass slide. Two to four slides were prepared from each mouse. The slides were fixed in methanol, stained and permanently mounted.
Details of tissue and slide preparation:
Cells were treated and well spread and stained. 1000 polychromatic erythrocytes were scored for the presence of micronuclei which are defined as round, darkly staining nuclear fragments, having a sharp contour with diameters usually from 1/20 to 1/5 of the erythrocyte. The number of micronucleated normochromatic erythrocytes in the field of 1000 polychromatic erythrocytes was enumerated. The proportion of polychromatic erythrocytes to total erythrocytes was also recorded per 1000 erythrocytes.
Evaluation criteria:
The incidence of micronucleated polychromatic erythrocytes per 1000 polychromatic erythrocytes was determined. Statistical significance was determined using the Kastenbaum-Bowman tables which are based on the binomial distribution (Kastenbaum and Bowman, 1970; Mackey). The test article was considered to induce a positive response if a treatment-related increase in micronucleated polychromatic erythrocytes was observed and one or more doses were statistically elevated relative to the vehicle control (p≤0.05, Kastenbaum-Bowman Tables) at any sampling time. If a single treatment group was significantly elevated at one sacrifice time with no evidence of a dose-response, the assay was considered a suspect or unconfirmed positive and a repeat assay recommended. The test article was considered negative if no statistically significant increase in micronucleated polychromatic erythrocytes above the concurrent vehicle control was observed at any sampling time.
Criteria for a Valid Test
The mean incidence of micronucleated polychromatic erythrocytes must not exceed 5/1000 polychromatic erythrocytes (0.5%) in the vehicle control. The incidence of micronucleated polychromatic erythrocytes in the positive control group must be significantly increased relative to the vehicle control group (p≤0.05, Kastenbaum-Bowman Tables).
Statistics:
Kastenbaum, M.A. and K.O. Bowman. 1970. Tables for determining the statistical significance of mutation frequencies. Mutation Res. 9:527-549.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative

Any other information on results incl. tables

Reductions of up to 23% in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test article-treated groups relative to their respective vehicle controls. The reduction in the frequency of polychromatic erythrocytes in the bone marrow suggest that there was toxicity and bioavailability of the test article to the bone marrow target tissue.

The number of micronucleated polychromatic erythrocytes per 1000 polychromatic erythrocytes in test article treated groups was not statistically increased relative to their respective vehicle control in either male or female mice, regardless of dose level or bone marrow collection time (table 2).

Gyclophosphamide induced a significant increase in micronucleated polychromatic erythrocytes in both male and female mice.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
All criteria for a valid test were met. Under the conditions of the assay described in this report, AHTN did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow and was concluded to be negative in the micronucleus test using male and female ICR mice.
Executive summary:

ICR mice were dosed with 400, 800, or 1600 mg/kg bw AHTN in corn oil by intraperitoneal injection. The high dose was selected to be 80% of the estimated intraperitoneal LD50 . The positive control was cyclophosphamide. Bone marrow was collected at 24, 48 and 72 hr after dosing and examined for micronucleated polychromatic erythrocytes (PCE). No mortality occurred. Clinical signs consisted of lethargy at all dose levels and diarrhoea at 1600 mg/kg bw. Moderate reductions (up to 23%) in the ratio of PCE to total erythrocytes were observed in some treated groups suggesting toxicity and bioavailability to the bone marrow target. The positive control induced a significant increase in micronucleated PCE in both male and female mice at 24 hr (the only harvest time for this group). No significant increase in micronucleated PCE in AHTN-treated groups relative to the respective vehicle control group was observed in male or female mice at 24, 48 or 72 hr after dose administration.