Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1998-06-04 - 1998-12-31
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-compliant study comparable to OECD TG 474. The reliable study is acceptable for assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report date:
1998

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
(12/96 final draft [adopted on 21st July 1997])
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
429-460-4
EC Name:
-
Cas Number:
7078-98-0
Molecular formula:
C21 H26 O
IUPAC Name:
2,6-di-tert-butyl-4-(phenylmethylidene)cyclohexa-2,5-dien-1-one

Test animals

Species:
mouse
Strain:
other: Crl:CD-l(ICR) BR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, NC or Portage, MI, U.S.A.
- Age at study initiation: 9 weeks old
- Weight at study initiation:
26.2 - 34.8 g (males), 24.3 - 28.6 g (females) - first dose range-finding study;
30.9 - 37.2 g (males), 20.9 - 27.8 g (females) - second dose range-finding study;
31.3 - 34.3 g (males) - third dose range-finding study
29.1 - 37.1 g (males) - first MN study;
32.2 - 36.4 g (males) - second MN study).
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: separated by gender, up to seven animals per cage
- Diet: Commercial diet, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 64° F - 79° F (18 - 26 °C)
- Humidity: 30 - 70 %
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 1998-07-22 To: 1998-12-03

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle/solvent used: corn oil
- Justification for choice of solvent/vehicle: Because of difficulty passing the test item mixed with 0.5% methylcellulose through a 23-gauge needle, corn oil was selected.
- Concentration of test material in vehicle: 10 – 75 mg/mL (Dose Range-Finding Study 2)
- Amount of vehicle: Dosing volume 20 mL/kg
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Prior to dosing, the top stock of the test item was prepared by adding the appropriate volume of the vehicle, corn oil, to a preweighed quantity of the test item. The stock was mixed by stirring with a spatula for about 2 minutes, sonicating for about 10 minutes, and stirring on a stirplate for about 3 minutes. The remaining stocks were prepared by dilution from the top stock.
- Dosing Stocks for Micronucleus Study 1: 11.25, 22.5, 45.0 mg/mL [225, 450, 900 mg/kg bw]
- Dosing Stocks for Micronucleus Study 2: 25.0, 37.5 mg/mL [500, 750 mg/kg bw]
Duration of treatment / exposure:
single treatment
Frequency of treatment:
once
Post exposure period:
Micronucleus Study 1: 24 and 48 hours
Micronucleus Study 2: 48 hours
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
225, 450, 900 mg/kg bw (MN study 1)
Basis:
other: intraperitoneal injection
Remarks:
Doses / Concentrations:
500, 750 mg/kg bw (MN study 2)
Basis:
other: intraperitoneal injection
No. of animals per sex per dose:
6 males/6 females per dose and sampling time,
except:
- 10 satellite animals at 900 mg/kg bw
- 16 satellite animals at 750 mg/kg bw
Control animals:
yes, concurrent vehicle
Positive control(s):
- Name: Cyclophosphamide (CP)
- Justification for choice of positive control: known as substance to produce micronuclei in vivo at exposure levels expected to give a detectable increase over background (according to OECD TG 474)
- Route of administration: orally
- Frequency of administration: once
- Doses / concentrations: 80 mg/kg bw
- Solubilized in: sterile deionized water

Examinations

Tissues and cell types examined:
Polychromatic erythrocytes (PCE) in the bone marrow of the mouse.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Results of three range-finding studies with the test item

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
Approximately 18 hours before treatment with the test article the animals received no food but water ad libitum. At the beginning of the treatment the animals (including the controls) were weighed and the individual volume to be administered was adjusted to the animal's body weight. The animals received the test article, the vehicle or the positive control substance once. Sampling of the bone marrow was done 24 or 48 hours after treatment.

DETAILS OF SLIDE PREPARATION:
- Extraction of bone marrow/preliminary work: At the appropriate harvest timepoints, the animals were euthanized by CO2 inhalation followed by incision of the diaphragm. The hind limb bones (tibias or femurs) were removed for marrow extraction. For each animal, the marrow flushed from the bones was combined in an individual centrifuge tube containing 3-5 mL fetal bovine serum (one tube per animal).
- Preparation of slides: Following centrifugation to pellet the tissue, the supernatant was removed by aspiration and portions of the pellet were spread on slides and air dried. The slides were fixed in methanol, stained in May-Grünwald solution followed by Giemsa, and protected by mounting with coverslips. At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
Slides were scored for micronuclei and the PCE to NCE cell ratio. The micronucleus frequency (expressed as percent micronucleated cells) was determined by analyzing the number of micronucleated PCEs from at least 2000 PCEs per animal. The frequency of PCE:NCE ratio was determined by scoring the number of PCEs and NCEs observed in the optic fields while scoring at least the first 200 erythrocytes on the slide. The historical background frequency of micronuclei in the Crl:CD-l(ICR) BR strain was about 0.0-0.4%, which is within the range reported in the published data (Salamone and Mavoumin, 1994). The criteria for the identification of micronuclei were those of Schmid (1976). The unit of scoring was the micronucleated cell, not the micronucleus; thus, the occasional cell with more than one micronucleus was counted as one micronucleated PCE, not two (or more) micronuclei.
Evaluation criteria:
Assay data analysis was performed using an analysis of variance on untransformed proportions of cells with micronuclei per animal and on untransformed PCE:NCE ratios when the variances were homogeneous. Ranked proportions were used for heterogeneous variances. If the analysis of variance was statistically significant (p < 0.05), a Dunnett's t-test was used to determine which dose groups, if any, were statistically significantly different from the vehicle control. Analyses were performed separately for each sampling time. Additionally, parametric or nonparametric tests for trend may have been employed to identify any dose-related response.
The criteria for a positive response was the detection of a statistically significant increase in micronucleated PCEs for at least one dose level, and a statistically significant dose-related response. A test item that did not induce both of these responses was considered negative. Statistical significance was not the only determinant of a positive response, the biological relevance of the results was also considered.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
The high dose produced mortality in the animals.
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDIES
- 1st dose range-finding study: The test item was dissolved in corn oil and dosed by intraperitoneal injection to 12 animals/sex at 2000 mg/kg bw (30 animals); three animals/sex received the vehicle control article, corn oil, only. The animals were anesthetized using CO2/O2 and blood samples were taken periorbitally at approximately 0.5, 2 and 4 hours postdose from 3 animals/sex/sampling time. At the 0.5-hour timepoint, blood samples were also taken from 3 animals/sex that received only the vehicle. The blood samples were centrifuged and the serum was transferred to labeled tubes and frozen. The animals were observed after dosing for toxic signs and/or mortality. The remaining 3 animals/sex were not bled; however, clinical observations for toxic signs and/or mortality were performed for 2 days.
- 2nd dose range-finding study: The test item was dissolved in corn oil and dosed by intraperitoneal injection to 3 animals/sex/dose level (30 animals). The animals were dosed at 200, 500, 900, 1200, and 1500 mg/kg bw and observed for 2 days after dosing for toxic signs and/or mortality.
- 3rd dose range-finding study: The test item was dissolved in corn oil and dosed by intraperitoneal injection to 3 animals at 900 mg/kg. In addition, 2 males were dosed with the vehicle control article, corn oil. The animals were observed after dosing for toxic signs and/or mortality. The animals were anesthetized using CO2/O2 and blood samples were taken periorbitally at 4 hours postdose. The blood samples were centrifuged and the serum was transferred to labeled tubes and frozen.
Based on the results of the dose range-finding studies, the maximum tolerated dose was estimated to be 900 mg/kg bw. Only males were used in the main studies because there were no substantial differences in clinical observations between the sexes in the dose range-finding studies.

RESULTS OF DEFINITIVE STUDY
- The test item induced signs of clinical toxicity in the treated animals.
- Ratio of PCE/NCE: There was a statistically significant decrease in the PCE:NCE ratio at 225 and 450 mg/kg bw at the 24-hour harvest timepoint, demonstrating that the test item was cytotoxic to the bone marrow.
- Induction of micronuclei: The test item did not induce a statistically significant increase in the frequency of micronucleated PCEs and is considered negative in the mouse bone marrow micronucleus assay under the conditions of this assay. As a positive control 80 mg/kg bw cyclophosphamide was used (administered per os), resulted in a distinct increase of induced micronucleus frequency under the conditions of this assay.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative