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:
other information
Study period:
FROM 4 OCTOBER 1996 to NOVEMBER 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Reliability 1 is assigned because the study is conducted according to OECD TG 474, without deviations that influence the quality of the results, in compliance with GLP.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997
Reference Type:
publication
Title:
Genotoxicity Tests with|6-Acetyl-1,1,2,4,4,7- hexamethyltetraline and|1,3,4,6,7,8-Hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-gamma|--benzopyran.
Author:
Api, A.M., and San, R.H.C.
Year:
1999
Bibliographic source:
Mutation Research, 446: 67-81.

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): HHCB
- Physical state: Clear, colorless liquid
- Lot/batch No.: ES 7953-1
- Storage condition of test material: RT, protected from exposure to light

Test animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague Dawley, Inc., USA
- Age at study initiation: 6-8 wks
- Weight at study initiation: (tox study; males: 30.1-34.9 gr; females: 24.4-28.6 gr, Micronucleus assay; males: 28.1-37.2 gr, Micronucleus assay; 24.5-31 gr).
- Assigned to test groups randomly: yes
- Housing: Mice of the same sex were housed up to five per cage in polycarbonate cages which were maintained on stainless racks equipped with automatic watering manifolds and were covered with filter material.
- Diet (ad libitum): Certified laboratory chow which was analysed for environmental contaminants (Harlan TEKLAD certified rodent 7012C).
- Water (ad libitum): tap water (Washington Suburban Sanitary Commistion, Potomac plant).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23±2°C
- Humidity (%): 50±20%
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: Corn oil
- Justification for choice of solvent/vehicle: Chosen by the sponsor
- Concentration of test material in vehicle: max. 250 mg/mL
- Amount of vehicle (if gavage or dermal): 20 mL/kg
Details on exposure:
OTHER: mice were observed after dose administration for clinical signs of a chemical effect.
Duration of treatment / exposure:
single exposure
Frequency of treatment:
Single
Post exposure period:
24-48-72 hrs
Doses / concentrations
Remarks:
Doses / Concentrations:
376 mg/kg, 750 mg/kg and 1500 mg/kg bw
Basis:
nominal conc.
IP
No. of animals per sex per dose:
5 females, 5 males / dose
Control animals:
yes, concurrent vehicle
yes, historical
Positive control(s):
cyclophosphamide
- Route of administration: IP
- Doses / concentrations: 60 mg/kg

Examinations

Tissues and cell types examined:
Micronucleated Polychromatic Erythrocytes
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
In the micronucleus assay, male and female mice were dosed with 376, 750 or 1500 mg test article/kg body weight. Bone marrow cells, collected 24, 48 and 72 hours after treatment, were examined microscopically for micronucleated polychromatic erythrocytes.

DETAILS OF SLIDE PREPARATION:
At the scheduled sacrifice times, up to five mice per sex per treatment were sacrificed by CO2 asphyxiation. 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 I ml fetal bovine serum. The bone marrow cells were pelleted by centrifugation at approximately 100 x g for five minutes and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were resuspended by aspiration with a capillary pipet 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 with May-Gruenwald-Giemsa and permanently mounted.

METHOD OF ANALYSIS:
Slides were coded using a random number table by an individual not involved with the scoring process. Using medium magnification, an area of acceptable quality was selected such that the cells were well spread and stained. Using oil immersion, 1000 polycbromatic 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 mean incidence of micronucleated polychromatic erythrocytes (PCEs) must not exceed 5/1000 PCEs (0.5%) in the vehicle control. The incidence of PCEs in the positive control group must be significantly increased relative to the vehicle control group (p<0.05).
Statistics:
The test article was considered to induce a positive response if a treatment-related increase in PCEs was observed and one or more doses were statistically elevated relative to the vehicle control (<0.05) at any sampling time.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Mortality at 4/5 male mice and 5/5 female mice at 3 gr/kg
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): NO
- Appropriateness of dose levels and route: The dose level and route seems to be appropriate
- Statistical evaluation: The test article was considered to induce a positive response if a treatment-related increase in PCEs was observed and one or more doses were statistically elevated relative to the vehicle control (<0.05) at any sampling time.

Any other information on results incl. tables

Table 1: Summary of bone marrow micronucleus study using HHCB.

Treatment  Sex Time (hrs) Micronucleated polychromatic erythrocytes (number/1000 PCEs) ± Stdev
Corn oil
20 mL/kg bw M 24 1 ± 0.71
F 24 0.4 ± 0.55
HHCB
376 mg/kg bw M 24 0.6 ± 0.89
F 24 0.2 ± 0.45
750 mg/kg bw M 24 0.2 ± 0.45
F 1.2 ± 1.3
1500 mg/kg bw M 24 0.4 ± 0.55
F 24 0.6 ± 0.55
CP
60 mg/kg bw M 24 43.6 ± 9.29
F 24 37.4 ± 15.73
Corn oil
20 mL/kg bw M 48 0.6 ± 0.89
F 48 1 ± 1.22
376 mg/kg bw M 48 1 ± 1
F 48 0.4 ± 0.55
750 mg/kg M 48 1.6 ± 1.52
F 48 0.2 ± 0.45
1500 mg/kg M 48 1.2 ± 1.1
F 48 0.8 ± 0.84
Corn oil
20 mL/kg bw M 72 0.6 ± 0.89
F 72 0.8 ± 0.84
376 mg/kg bw M 72 1.2 ± 1.3
F 72 1 ± 1
750 mg/kg bw M 72 1.4 ± 0.55
F 72 1.2 ± 1.64
1500 mg/kg bw M 72 0.6 ± 0.55
F 72 1 ± 0.71

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, HHCB did not induce a significant increase in the incidence of PCEs in bone marrow and was concluded to be negative in the micronucleus test using male and female ICR mice.
Executive summary:

In the present micronucleus test according to OECD guideline 474, groups of 5 male (28.1-37.2 g) and 5 female ICR mice (24.5-31.0 g) were dosed with 0, 376, 750, or 1500 mg/kg bw HHCB (in corn oil - purity >99%) by intraperitoneal injection at a constant volume of 20 ml/kg bw. The high dose was selected to be approximately 70% of the estimated intraperitoneal LD50. The positive control was cyclophosphamide. Bone marrow was harvested at 24, 48 and 72 hr after dosing and examined for micronucleated polychromatic erythrocytes (PCE). No mortality was seen. Lethargy was observed in all animals on 1500 mg/kg bw, in 4/15 males and 4/15 females on 750 mg/kg, and 1/15 males and 0/15 females on 376 mg/kg bw. Moderate reductions (up to 25%) in the ratio of PCE to total erythrocytes were observed in groups on 1500 mg/kg bw after 48 and 72 hrs indicating 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 HHCB-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.