Registration Dossier

Toxicological information

Genetic toxicity: in vivo

Currently viewing:

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian bone marrow chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Ltd, Margate, UK
- Age at study initiation: 36-43 days
- Weight at study initiation: 25-33g male, 20-28g female
- Assigned to test groups randomly: yes
- Fasting period before study: not starved before dosing
- Housing: groups of no more than three animals of the same sex
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-20
- Humidity (%): 52-59
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12

IN-LIFE DATES: From: 1996-10-23 To: 1996-12-31

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: CMC (carboxymethyl cellulose) 1%
- Concentration of test material in vehicle: 5, 10 and 20 mg/ml
- dose volume: 20 ml/kg
- The test article preparations were protected from light and used within approximately 2 hours of initial formulation
Duration of treatment / exposure:
24h and 48h respectively
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
actually injected
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Remarks:
actually injected
Dose / conc.:
400 mg/kg bw/day (actual dose received)
Remarks:
actually injected
No. of animals per sex per dose:
5 per sex and dose
Control animals:
yes, concurrent vehicle
Positive control(s):
- cyclophosphamid. dissolved in saline, 40 mg/kg bw single dose, intraperitonally, on the second day of dosing

Examinations

Tissues and cell types examined:
both femours removed, bone marrow smear examined
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: range finder

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Test article and vehicle treated mice were killed in groups, 24 or 48 hours after the second administration; CPA-treated mice were killed 24 hours after the single dose. Mice were killed by asphyxiation with carbon dioxide in the same order as they were dosed.

DETAILS OF SLIDE PREPARATION: The tubes containing bone marrow-serum-mix were centrifuged and the serum was aspirated to leave one or two drops and the cell pellet. The pellet was mixed into this small volume of serum in each tube using a Pasteur pipette and from each tube one drop of
suspension was placed on the end of each of two slides labelled with the appropriate study number, sampling time, sex, date of preparation and tag number. The latter served as a code so analysis could be conducted "blind". A smear was made from the drop by drawing the end of a clean slide along the labelled slide. Slides were allowed to air-dry and were fixed for 5 minutes in absolute methanol before being stained. One slide from each set of two was then taken, the other was kept in reserve. After rinsing several times in water, slides were stained for 10 minutes in filtered Giemsa stain diluted 1:6 (v/v) in distilled water. Stained slides were rinsed, and allowed to dry thoroughly before clearing in xylene for 3 minutes. When dry, the slides were mounted with coverslips.

METHOD OF ANALYSIS: ratio of PCE/NCE for each animal and the mean for each group was calculated, compared with historical negative control ranges, compared with the numbers in vehicle control groups, further statistical test (for linear trend) was used to evaluate possible dose-response relationships
Evaluation criteria:
The test article was to be considered as positive in this assay if:
1) a statistically significant increase in the frequency of micronucleated PCE occurred at least at one dose, at one sampling time, and
2) the frequency of micronucleated PCE at such a point exceeded the historical vehicle control range.
Statistics:
- inter-individual variation in the numbers of micronucleated PCE was estimated by means of a heterogeneity x2 test
- numbers of micronucleated PCE in each treated group (males and females, separately and combined) were compared with the numbers in vehicle
control groups by using a 2 x 2 contingency table to determine x2 (Improbability values of p < 0.05 were to be accepted as significant
- statistical test (for linear trend) was used to evaluate possible dose-response relationships

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
decrease in PCE/NCE ratio
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 250 - 2000 mg/kg bw
- Clinical signs of toxicity in test animals: lethargy, hunched posture, tremors, prostration, twisting, coldness, yellow feces, eye closure
- mortalities: no mortalities at 250 mg/kg bw, 1-2 animals per sex 500 - 1600 mg/kg bw, no survivors at 1800 and 2000 mg/kg bw

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): frequencies of micronucleated PCE were similar to the values for vehicle control groups at both sampling times
- Ratio of PCE/NCE (for Micronucleus assay): Mice treated with the test substance generally exhibited group mean ratios of PCE to NCE which were lower than those in concurrent controls indicating exposure of the target tissue to the test article
- Statistical evaluation: no instances of statistically significant increases in micronucleus frequency for any of the groups receiving the test article at either sampling time

Any other information on results incl. tables

Eye closure was apparent at 100 and 200 mg/kg/day. Prostration, hunched appearance, piloerection, coldness, eye closure and twisting were seen among animals dosed at 400 mg/kg/day. Three males and two females receiving this dose were either found dead or were killed in extremis prior to sampling. Loss of body weight was seen at all dose levels.

Applicant's summary and conclusion

Conclusions:
It is concluded that the test item did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of mice treated up to 400 mg/kg/day, a dose at which limited mortality and cytotoxicity were apparent.
Executive summary:

Test item was assayed in vivo in a mouse bone marrow micronucleus test at three dose levels. The choice of dose levels was based on an initial toxicity range-finding study in which the test article, made up in 1% (w/v) methyl cellulose, was administered to mice intraperitoneally. The test article was administered once daily on two consecutive days to groups of three male and three female mice at doses covering the range 250 to 2000 mg/kg/day. Observations were made over a 4 day period following the second administration and signs of toxicity recorded. For the micronucleus test, test substance was made up as described and administered at 100, 200 and 400 mg/kg/day to groups of five male and five female mice killed 24 or 48 hours after the second administration. Several animals receiving the highest dose died prior to the scheduled sampling times indicating that it would not have been possible to administer the test article at an appreciably higher dose.

The negative (vehicle) control in the study was 1 % (w/v) methyl cellulose also administered intraperitoneally once daily on two consecutive days. Groups of five male and five female mice treated with this were killed and sampled 24 or 48 hours after the second administration. Cyclophosphamide (CPA), the positive control, was dissolved in physiological saline and administered intraperitoneally as a single dose at 40 mg/kg to groups of five male and five female mice which were killed after 24 hours. All positive control animals exhibited increased numbers of micronucleated polychromatic erythrocytes (PCE) such that the micronucleus frequency in the positive control group was significantly greater than in concurrent controls (2x2 contingency x2 test).

Slides from all dose groups were analysed. Negative (vehicle) control mice exhibited normal group mean ratios of PCE to NCE (normochromatic erythrocytes) and normal frequencies of micronucleated PCE within historical negative control ranges. Mice treated with the test substance exhibited group mean ratios of PCE to NCE which were generally lower dian those in concurrent controls indicating exposure of the target tissue to the test article. Frequencies of micronucleated PCE, however, were similar to the values for vehicle control groups at both sampling times. There were no instances of statistically significant increases in micronucleus frequency for any of the groups receiving the test article at either sampling time.

It is concluded that the test article did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of mice treated up to 400 mg/kg/day, a dose at which limited mortality and cytotoxicity were apparent.