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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

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 (incl. certificate)
Remarks:
BASF AG, Experimental Toxicology and Ecology
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): beta-lonon R
- Test substance No.: 02/0449-1
- Physical state: Clear liquid
- Purity test date: 97.8%
- Lot/batch No.: Continuous production
- Date of manufacture: October 9, 2002
- Storage condition of test material: protected from light

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland GmbH
- Age at study initiation: about 5 - 8 weeks
- Weight at study initiation: mean weight of about 29 g
- Assigned to test groups randomly: yes, according to a randomization plan prepared with an appropriate computer program
- Housing: individually, Makrolon cages MI
- Diet (e.g. ad libitum): ad libitum, Standardized pelleted feed (Maus/Ratte Haltung "GLP", 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

OTHER:
- Feed analysis:
The feed used in the study was assayed for chemical and microbial contaminants. In view of the aim and duration of the study, contaminants occurring in commercial feed ought not to influence the test result .

- Water analysis: The drinking water is regularly assayed for chemical contaminants both by the municipal authorities of Frankenthal and the Department of Water Chemistry and the Technical Services of BASF Aktiengesellschaft as well as for microorganisms by a contract laboratory . However, in view of the aim and duration of the study, there are no special requirements exceeding the specification of drinking water.

- Analysis of bedding: The bedding is regularly assayed for contaminants (chlorinated hydrocarbons and heavy metals) . In view of the aim and duration of the study, there are no special requirements exceeding the specification of a commercial product monitored by the manufacturer.

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: olive oil
- Lot/batch no. (if required): Ph.Eur/DAB
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, olive oil 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 .
- Concentration of test material in vehicle: 2.5 g/100 ml in low dose group; 5 g/100 ml in low dose group; 7.5 g/100 ml in low dose group;
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- The stability of the test substance at 4°C in the vehicle olive oil over a period of 4 days
was verified analytically .
- For the determination of the test substance concentrations in the vehicle, 3 samples of each dose were taken from the test substance preparations, kept at room temperature until the treatment of the last animal (approximately 1 hour) and then deep-frozen until they were determined analytically .
- The determination of the concentrations in the vehicle was carried out by means of HPLC.
- The analyses were carried out at the Analytical Chemistry Laboratory of the Experimental Toxicology and Ecology of BASF Aktiengesellschaft .
Duration of treatment / exposure:
single administration
Frequency of treatment:
one application
Post exposure period:
48
Doses / concentrations
Remarks:
Doses / Concentrations:
250, 500, 750 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 CPP (Endoxan®, ASTA MEDICA, Reg . Nr. E 432-1)/kg body weight for clastogenic effects
- Vincristine Sulphate (VCR ): 0.15 mg VCR (SIGMA - V 8879)/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-established reference clastogens and aneugens respectively.
- Route of administration: once orally or intraperitoneally
- dose concentrations: 10 ml/kg bw

Examinations

Tissues and cell types examined:
polychromatic and normochromatic erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
- In a pretest for the determination of the acute intraperitoneal toxicity, deaths were observed at 2 000 mg/kg body weight. Doses of 1 000 mg/kg and 750 mg/kg body weight led to evident signs of toxicity and some animals were sacrificed moribund . 500 mg/kg body weight were survived by all animals, but led also to evident signs of toxicity.
- However, there were no distinct differences in the symptoms between males and females. Thus, only male animals were used for the cytogenetic investigations.
-Therefore, a dose of 750 mg/kg body weight was selected as the highest dose in the present cytogenetic study. 500 mg/kg and 250 mg/kg body weight were administered as further doses.


DETAILS OF SLIDE PREPARATION:
The bone marrow was prepared according to the method described by Schmid, W (1977; 1976) and Salamone M (1980):
- The two femora of the animals sacrificed by cervical dislocation 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 pI 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.
- The slides were stained in eosin and methylene blue (modified May-Grünwald solution or Wrights solution) for about 5 minutes.
- After having briefly been rinsed in purified water, the preparations were soaked in purified water for about 2 - 3 minutes.
- Subsequently, the slides were stained in Giemsa solution ( 15 ml Giemsa, 185 ml purified water) for about 15 minutes.
- After having been rinsed twice in purified water and clarified in xylene, the preparations were mounted in Corbit-Balsam.
Evaluation criteria:
The test chemical was considered positive if the following criteria were  met:
- A dose-related and significant increase in the number of micronucleated  poly-chromatic erythrocytes was observed.
- 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 was considered negative if 
- There was no significant increase in the number of micronucleated  polychromatic erythrocytes at any dose above concurrent control  frequencies.- The frequencies 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 asymptotic U test according to MANN-WHITNEY (modified rank test according to WILCOXON) was carried out to clarify the question whether there were significant differences between the control group and dose groups with regard to the micronucleus rate in polychromatic erythrocytes . The relative frequencies of cells containing micronuclei of each animal was used as a criterion for the rank determination for the U test.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid

Any other information on results incl. tables

ANALYTICAL RESULTS
The stability of the test substance at 4°C in the vehicle (homogenous  solution) over a period of 4 days was verified analytically.

MORTALITY
No mortality occurred in all groups.

CLINICAL SIGNS
The single administration of the test substance at 750 and 500 mg/kg bw  led to evident signs of toxicity in all treated animals (poor general  state, irregular respiration, squatting posture) which were reversible after 2 days. At the lowest doses only minor signs of clincal toxicity  were observed after 2 and 4 hours of administration of the test
substance (squatting posture.

EFFECT ON PCE/NCE RATIO
No inhibition of erythropoiesis, determined from the PCE/NCE ratio was  detected. The vehicle and the the positive control substances, CPP and  VCR, caused no evident signs of toxicity.

Mean number of PCEs and NCEs: 

Interval                         24 hrs   48 hrs
                      PCEs       NCEs     NCEs
Vehicle              10,000     4,594    3,439
250 mg/kg bw         10,000     3,755
500 mg/kg bw         10,000     4,646
750 mg/kg bw         10,000     2,476    2,804
CPP (20 mg/kg bw)    10,000     3,920
VCR (0.15 mg/kg bw)  10,000     5,374

Mean number of PCEs containing MN per 1,000 PCE at 24 hrs:
(differentiation between small and large micronuclei)

                     Small      Large    Total               
Vehicle               1.3        0.1      1.4
250 mg/kg bw          1.6        0.0      1.6
500 mg/kg bw          1.7        0.1      1.8
750 mg/kg bw          1.2        0.0      1.2 
CPP (20 mg/kg bw)     10.9       0.2      11.1 (p <= 0.01)
VCR (0.15 mg/kg bw)   35.7       11.5     47.2 (p <= 0.01)

Mean number of PCEs containing MN per 1,000 PCE at 48 hrs:
(differentiation between small and large micronuclei)

                     Small      Large    Total               
Vehicle               0.7        0.0      0.7
750 mg/kg bw          1.0        0.0      1.0 

STATISTICAL EVALUATION
The administration of the test substance did not lead to any statistical  significant increase in the number of
polychromatic erythrocytes containing either small or large
micronuclei. The rate of micronuclei was nearly the range of the  concurrent negative control in all dose groups and
within the range of the historical control data (mean 1.6, min. 0.3, max.  3.3, SD 0.6; n=393). The positive controls led to the expected increases  in micronuclei (either small or large).

Applicant's summary and conclusion