Registration Dossier

Toxicological information

Genetic toxicity: in vivo

Currently viewing:

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:
2000-10-09 - 2001-02-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Remarks:
Experimental Toxicology and Ecology, BASF AG
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): Isovaleraldehyde
- Purity: 99.3 % (GC)
- Batch No.: Continuous production, Kol 4321 Seit.
- Test substance No.: 00/0558-1
- Date of manufacture: 27 Jul 2000
- Physical state: Colorless liquid
- Storage : Room temperature (N2 conditions)

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Deutschland
- Age at study initiation: 5 - 8 weeks
- Weight at study initiation: mean ca. 28 g
- Assigned to test groups randomly: yes
- Housing: housed individually in Makrolon cages, type MI
- Diet (e.g. ad libitum): ad libitum, Kliba Haltungsdiät, Provimi Kliba SA, Kaiseraugst, Switzerland
- Water (e.g. ad libitum): ad libitum, drinking water from bottles
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, DMSO was selected as the vehicle, which had been demonstrated to be suitable in the in vivo micronucleus test and for which historical data are available.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
All test substance formulations were prepared immediately before administration
Duration of treatment / exposure:
single injection
Frequency of treatment:
one single application
Post exposure period:
24, 48 h
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 25, 50, 100 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (CPP), 20 mg/kg body weight for clastogenic effects
Vincristirie Sulphate (VCR), 0,15 mg/kg body weight for aneugenic effects
- Justification for choice of positive control(s): The stability of CPP and VCR is well-defined under the selected conditions, since both positive control articles are well-defined clastogens and aneugens respectively
- Route of administration: intraperitoneally

Examinations

Tissues and cell types examined:
polychromatic and normochromatic erythrocytes of bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
In a pretest for the determination of the acute intraperitoneal toxicity, deaths were observed down to a dose of 150 mg/kg body weight. 100 mg/kg body weight were survived by all animals but led to evident signs of toxicity such as piloerection, squatting posture, and the general state of the animals was poor, however, showing no distinct symptomatic differences between the male and female animals.
Thus, only male animals were used for the cytogenetic investigations.
Therefore, a dose of 100 mg/kg body weight was selected as the highest dose in the present cytogenetic study. 50 mg/kg and 25 mg/kg body weight were administered as further doses.


TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The animals were treated once and samples of bone marrow were taken 24 hours and 48 hours after the treatment
- The low dose group was given 25 mg test substance/kg body weight or 4 ml/kg body weight of a solution with a concentration of 0.625 g/100 ml.
- The intermediate dose group was given 50 mg test substance/kg body weight or 4 ml/kg body weight of a solution with a concentration of 1.25 g/100 ml.
- The top dos~: groups were given 100 mg test substance/kg body weight or 4 ml/kg body weight of a solution with a concentration of 2.5 g/100 ml

DETAILS OF SLIDE PREPARATION:
The bone marrow was prepared according to the method described by SCHMID (1976):
- The two femora were prepared by dissection and removing all soft tissues.
After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube using a cannula filled with fetal calf serum which was at 37°C (about 2 ml/femur). The suspension was mixed thoroughly with a pipette, centrifuged at 300 x g for 5 minutes, the supernatant was removed and the precipitate was resuspended in about 50 pl fresh FCS. 1 drop of this suspension was dropped onto clean microscopic slides, using a Pasteur pipette. Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained.

METHOD OF ANALYSIS:
In general, 2000 polychromatic erythrocytes (PCE) from each of the male animals of every test group are evaluated and investigated for micronuclei (MN). The normochromatic erythrocytes (NCE) which occur are also scored.
Evaluation criteria:
The test chemic ;al is to be considered positive in this assay if the following criteria are met:
- A dose-relatod and significant increase in the number of micronucleated polychromatic erythrocytes at any of the intervals.
- The proportion of cells containing micronuclei exceeded both the values of the concurrent negative control range and the negative historical control range.
A test substance is generally considered negative in this test system if:
- There was no significant increase in the number of micronucleated polychromatic erythrocytes at any dose above concurrent control frequencies and at any time.
- The frequen,-ies of cells containing micronuclei were within the historical control range.
Statistics:
The statistical evaluation of the data was carried out using the program system MUKERN (BASF Aktiengesellschaft) .
The number of micronuclei in polychromatic erythrocytes was analyzed. A comparison of the dose group with the vehicle control was carried out using the Wilcoxon test for the hypothesis of equal medians. Here, the relative frequencies of cells with micronuclei of each animal were used. If the results of this test were significant, labels (* for p <= 0.05, ** for p >= 0.01) were printed with the group means in the tables.
This test was performed one-sided.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
squatting, poor general state, piloerection
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 150, 100 mg/kg bw
- Clinical signs of toxicity in test animals: piloerection, squatting posture, and the general state of the animals was poor

Any other information on results incl. tables

The administration of the test substance led to evident signs of toxicity:
- 25 mg/kg bw:
 1, 2 and 4 h: squatting posture; 1 d: no symptoms
- 50 mg/kg bw:
 1 h: squatting posture; 2 and 4 h: squatting posture and  poor general state; 1 d: no symptoms
- 100 mg/kg bw:
 1 h: squatting posture and poor general state, 2 and 4 h:
 squatting posture, poor general state and piloerection;
 1 d: poor general state and piloerection

Results:

Test group Interval: 24 h Interval: 48 h
Polychromatic erythrocytes Normocytes / total amount polychromatic erythrocytes Cells with micronuclei (%) Polychromatic erythrocytes Normocytes / total amount polychromatic erythrocytes Cells with micronuclei (%)
Dose (mg/kg bw) polychromatic normochromatic polychromatic normochromatic
vehicle control 10000 3396 1.3 0.3 100000 2974 1.7 1.0
25 10000 3997 1.3 1.5
50 10000 3764 2.2 2.9
100 10000 3769 1.9 1.6 10000 4193 0.9 1.0
20, cyclophosphamide  10000 3092 21.5** 3.6
0.15, vincristine 10000 4859 49.7** 2.3

* p<=0.05; ** p<= 0.01

There was no increase in the number of polychromatic erythrocytes containing either small or large micronuclei.
The rate of microneuclei was nearly the range of the concurrent negative
 control in all dose groups and within the range of the historical control data.
No inhibition of erythropoiesis, determined from the ratio of
 polychromatic to normochromatic erythrocytes, was detected.

The result for the negative control was within the historical control range.

Both of the positive control chemicals, i.e. cyclophosphamide for clastogenic effects and vincristine for induction of spindle poison effects, induced the expected increases in the rate of polychromatic erythrocytes containing small or large micronuclei.


Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The test substance had no chromosome-damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.