Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report date:
2000

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
3-(4-hydroxy-4-methylpentyl)cyclohex-3-ene-1-carbaldehyde; 4-(4-hydroxy-4-methylpentyl)cyclohex-3-ene-1-carbaldehyde
EC Number:
915-617-9
Molecular formula:
C13H22O2
IUPAC Name:
3-(4-hydroxy-4-methylpentyl)cyclohex-3-ene-1-carbaldehyde; 4-(4-hydroxy-4-methylpentyl)cyclohex-3-ene-1-carbaldehyde
Test material form:
liquid

Test animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague Dawley, Inc., Frederick, MD, USA.
- Age at study initiation: 6 - 8 weeks.
- Weight at study initiation: pilot toxicity study: males 27.9 - 32.6 g, females 25.6- 27.8 g; toxicity study: males 26.3 - 29.1 g, females 24.7 - 27.7 g; micronucleus assay: males 28.1 - 33.3 g, females 26.0 - 30.4 g.
- Assigned to test groups randomly: no, according to a computer-generated program based on distribution according to body weight.
- Housing: up to 5/cage in polycarbonate cages with heat-treated hardwood chips for bedding.
- Diet: certified laboaratory rodent chow (Harlan TEKLAD certified Rodent 7012C), ad libitum.
- Water: tap water (Washington Buburban Sanitary Commission, Potomac Plant), ad libitum.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperatur (°F): 72 ± 3
- Humidity (%): 50 ± 20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle used: corn oil
- Justification for choice of solvent/vehicle: based on information provided by the sponsor and compatibility of the vehicle with the test system animals
- Concentration of test material in vehicle: 100 mg/mL
- Amount of vehicle: 20 mL/kg bw
Duration of treatment / exposure:
Single administration
Frequency of treatment:
Once
Post exposure period:
For Group 2 and 3 and positive control: 24 hours
For Group 1 and 4: 24 and 48 hours
Doses / concentrationsopen allclose all
Dose / conc.:
225 mg/kg bw/day (actual dose received)
Remarks:
Group 2: Low dose
Dose / conc.:
450 mg/kg bw/day (actual dose received)
Remarks:
Group 3: Mid-dose
Dose / conc.:
900 mg/kg bw/day (actual dose received)
Remarks:
Group 4: High-dose
No. of animals per sex per dose:
Group 1: 10
Group 2 and 3 and positive control: 5
Group 4: 15
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: intraperitoneal
- Doses / concentrations: 50 mg/kg bw

Examinations

Tissues and cell types examined:
Bone marrow erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Based on the results of the pilot study (dose levels 1, 10, 100 and 1000 mg/kg bw (males) and 2000 mg/kg bw (males and females)) and the preliminary toxicity study (dose levels 200, 400, 600 or 800 mg/kg bw, males and females).

DETAILS OF SLIDE PREPARATION:
At the scheduled sacrifice times, mice 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 ca. 1 mL fetal bovine cerum. The bone marrow cells were pelleted by centrifugation at ca. 100 x g for 5 min 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 slides were prepared from each mouse. The slides were fixed in methanol, stained with May-Gruenwald-Giemsa and permanently mounted.

SCORING FOR MICRONUCLEI:
Using oil immersion, 2000 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 2000 polychromatic erythrocytes was enumerated. The proportion of polychromatic erythrocytes to total erythrocytes was also recorded per 1000 erythrocytes.
Evaluation criteria:
The test substance was considered to give a positive response if a dose-responsive increase in micronucleated polychromatic erythrocytes was observed and one or more doses were statistically elevated relative to the vehicle control (p ≤ 0.05) 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 substance was considered negative if no statistically significant increase in micronucleated polychromatic erythrocytes above the concurrent vehicle control was observed at any sampling time.
Statistics:
Kastenbaum-Bowman tables, based on the binomial distribution

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
DOSE RANGE
- In the pilot study: 1, 10, 100 and 1000 mg/kg bw (males) and 2000 mg/kg bw (males and females)
- In the preliminary toxicity study: 200, 400, 600 or 800 mg/kg bw, males and females

CLINICAL SIGNS OF TOXICITY IN TEST ANIMALS
- In the pilot study, mortality was observed in 1/2 male mice at 1000 mg/kg and in 5/5 male and 5/5 female mice at 2000 mg/kg bw. Clinical signs included lethargy in males at 1000 mg/kg bw, piloerection and irregular breathing in males at 1000 mg/kg bw and in males and females at 2000 mg/kg bw. An animal in hunched position was observed at 1000 mg/kg bw and crusty eyes, irregular breathing and prostration were observed in males and females at 2000 mg/kg bw.
- In the toxicity study, no mortality was observed in any animal at any dose level. Clinical signs included lethargy and piloerection in males and females at 400, 600 and 800 mg/kg bw.
In the micronucleus assay, mortality was observed in 1/15 females at 900 mg/kg bw. Clinical signs included lethargy and piloerection in males and females at 225, 450 and 900 mg/kg bw and irregular breathing in males and females at 900 mg/kg bw.

GENOTOXICITY RESULTS
- Evidence of cytotoxicity in tissue analyzed: slight to moderate reductions (up to 31 %) in the ratio of polychromated erythrocytes to total erythrocytes were observed in the test article-treated groups relative to the respective controls. These reductions suggest bioavailability of the substance to the bone marrow target.
- Induction of micronuclei (for Micronucleus assay): no significant increase in micronucleated polychromatic erythrocytes in test article-treated group relative to the respective vehicle control group was observed in male or female mice at 24 or 48 hr after dosing (p > 0.05).

Applicant's summary and conclusion

Conclusions:
The test substance did not induce an increase in the number of polychromated micronucleated erythrocytes in bone marrow in the in vivo micronucleus assay with mice following intraperitoneal administration.
Executive summary:

The ability of the test substance to induce micronuclei in vivo was investigated in a micronucleus test with mice, performed according to a protocol similar to OECD Guideline 474 in compliance with GLP. Groups of male and female ICR mice (vehicle control: 10/sex/dose, low and mid dose groups and positive control group: 5/sex/dose, high dose group: 15/sex/dose) were administered the test substance at dose levels of 0 (corn oil), 225, 450 and 900 mg/kg bw by a single intraperitoneal administration. Animals of low and mid-dose groups and positive control animals were sacrificed 24 hours post-administration, high dose and vehicle control groups were sacrificed 24 and 48 hours post-administration, and bone marrow slides were prepared. Mortality was observed in 1/15 females at 900 mg/kg bw. Clinical signs included lethargy and piloerection in males and females at 225, 450 and 900 mg/kg bw and irregular breathing in males and females at 900 mg/kg bw. Slight to moderate reductions (up to 31 %) in the ratio of polychromated erythrocytes to total erythrocytes were observed in the test substance-treated groups relative to the respective controls. These reductions suggest bioavailability of the substance to the bone marrow target. No significant increase in micronucleated polychromatic erythrocytes in the test substance-treated group relative to the respective vehicle control group was observed in male or female mice at 24 or 48 hr after dosing (p > 0.05). The substance was thus concluded to give negative results in this micronucleus test in vivo