Registration Dossier

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:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
May 26, 1983
GLP compliance:
yes
Type of assay:
other: Mammalian Erythrocyte Micronucleus Test

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
mouse
Strain:
Swiss
Remarks:
CD-1 (SPF-quality)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
On arrival and prior to final assignment to study, all animals have undergone a detailed clinical examination to ensure selected animals were in a good state of health.
-Source: Charles River Wiga GmbH, Sulzfeld, FRG
-Age at start of treatment: approx. 8 weeks
-Body weight at start of treatment: females 20-25 g, males 26-32 g
-Identification: unique cage number and a mark on the tail (for a unique animal number)
-Acclimatisation: at least 6 days under laboratory conditions
-Allocation: allocated to treatment groups as they came to hand from delivery boxes
-Housing: in groups of 5 per sex in polycarbonate cages
-Bedding: Purified sawdust (Woody Clean, from the Broekmann Institute, Someren, The Netherlands).
-Diet: standard laboratory animal diet (RMH-B, pellet diameter 10 mm, Hope Farms, Woerden, The Netherlands), feed (analysed for contaminants by the manufacturer) withheld overnight prior to dosing until approximately 3-4 h after administration of the test substance
-Water: Tap water, ad libitum

ENVIRONMENTAL CONDITIONS
-Temperature: 21±3 °C
-Humidity: 40-70 %
-Air changes: 7.5 air changes per hr
-Photoperiod: 12 hrs dark /12 hrs artificial fluorescent light

Administration / exposure

Route of administration:
other: oral intubation
Vehicle:
The test substance was suspended in Milli-RO water (Millipore Corp., Bedford, Mass., USA), concentrations were prepared directly prior to use
Details on exposure:
OBSERVATIONS
-Systemic toxic signs: daily
-Body weights: day 1 of test (just prior to dosing).

DOSAGE SELECTION/PILOT STUDY
Selection of an adequate dose for the test was based on a pilot study. Depending on the available toxicity data 1 to 5 dose groups, each comprising 3 males and 3 females, received a single dose of the test substance. Generally, the study duration is 3 d, however this might be changed if considered necessary. During this period mortality and physical condition has been recorded daily.
Frequency of treatment:
Single dosing
Doses / concentrations
Dose / conc.:
3 000 mg/kg bw/day (nominal)
Remarks:
Dosing volume: 10 ml/kg bw
No. of animals per sex per dose:
Preliminary study: 3 males and 3 females per group
Main study: 5 males and 5 females per treatment group
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide (CP; CAS no. 50-18-0; Endoxan, Asta-Werke, F.R.G,) at 50 mg/kg bw dissolved in 0.9 % NaCl (Merck) in Milli-RO water.
The route and frequency of administration and the volume administered were consistent with those of the test substance. Solutions were prepared on day of administration. The stability of CP at room temperature is good. At 20°C only 1% of CP is hydrolysed per day in aqueous solution.

Examinations

Tissues and cell types examined:
Erythrocytes in the bone marrow
Details of tissue and slide preparation:
SLIDES PREPARATION
The animals were sacrificed by cervical dislocation at 24, 48 and 72 h after dosing of the test substance and the vehicle and at 48 h after dosing of the positive control. Both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible. The bone was flushed with approximately 2 ml of foetal calf serum. The cell suspension was collected and centrifuged at 1000 rpm (approximately 100 g) for 5 min. The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed by aspiration with the serum. A drop of the cell suspension was placed on the end of a slide which was previously cleaned (24 h immersed in a 1:1 mixture of 96 % ethanol/ether and cleaned with a tissue) and marked. The drop was spread by moving a clean slide with round-whetted sides at an angle of 45 ° over the slide with the drop of bone marrow suspension. The preparations were then air-dried and thereafter fixed for 5 min in 100 % methanol and air-dried overnight. Two slides were prepared per animal.

STAINING OF THE BONE MARROW SMEARS
The slides were stained for 3 min in undiluted May-Grünwald solution followed by 2 min in May-Grünwald solution diluted 1:1 with Sorensen buffer pH 6.8. Thereafter slides were rinsed in this buffer and stained for 25 min in 5 % (v/v) Giemsa solution in Sorensen buffer pH 6.8. The preparations were rinsed for 1 min in running tap-water and blotted dry between filter paper. The dry slides were cleared by dipping them in xylol before they were embedded in DePeX and mounted with a coverslip.

ANALYSIS OF THE BONE MARROW SMEARS FOR MICRONUCLEI
All slides were randomly coded before examination. An adhesive label with study identification number and code was stuck over the marked slide. At first the slides were screened at a magnification of 100 X for regions of suitable technical quality, i.e. where the cells were well spread, undamaged and well stained. Slides were scored at a magnification of 1000 X. The number of micronuclei was counted in 1000 polychromatic erythrocytes. The ratio polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated.

ACCEPTABILITY OF ASSAY
A micronucleus test is considered acceptable if it meets the criteria:
a) The positive control substance induced a statistically significant (Wilcoxon Rank Sum Test, two-sided test at P < 0.05) increase in the frequency of micronuclei.
b) The incidence of micronuclei in the control animals should reasonably fall within the laboratory historical control data range.
Evaluation criteria:
A test substance is considered in the micronucleus test:
-positive if it induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test; two-sided test at p < 0.05) increase in the frequency of micronuclei (at any dose or at any sampling time) in the combined data for both sexes or in the data for male or female groups separately.
-negative if none of the tested concentrations or sampling times showed a statistically significant (p < 0.05) increase in the incidence of micronuclei neither in the combined data for both sexes nor in the data for male or female groups alone.
The preceding criteria are not absolute and other extenuating factors may enter into the final evaluation decision.
Statistics:
Wicoxon rank-sum test.
Number of micronuclei per 1000 polychromatic erythrocytes; treatment/control comparison

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
PILOT STUDY/DOSE SELECTION
In a preliminary study 12 animals (3 males and 3 females per group) were dosed orally with 3000 and 1000 mg/kg bw (groups A and B, respectively). Higher concentrations could not be dosed because of aggregation of the test substance in suspension. Animals of group A and B did not show any signs of reaction to treatment. Based on the results of this pilot study 3000 mg/kg body weight was selected as an appropriate dose for the Micronucleus Test.

MICRONUCLEUS TEST
The mean number of micronuclei scored in the test substance-treated groups was compared with the corresponding control groups. No increase in the frequency of micronuclei was observed.
The incidence of micronuclei in the control animals was found to be in the range of historical data (0.75 ± 0.97; mean ± standard deviation, N = 700). The groups that were treated with Cyclophosphamide showed a decrease in the ratio of polychromatic to normochromatic erythrocytes, which reflects a toxic effect of this compound on the erythropoiesis. The positive control substance induced in both sexes a statistically significant increase in the number of micronuclei.

Applicant's summary and conclusion

Conclusions:
The substance can be considered as not mutagenic in the Micronucleus Test under the experimental conditions.
Executive summary:

The test substance was tested for genetic toxicity in vivo in the Micronucleus Test in mice, according to the OECD Guideline 474 (1983). Three groups (each 5 males and 5 females) received a single oral dose of 3000 mg/kg bw. Bone marrow was sampled at 24, 48 and 72 h after dosing. Corresponding vehicle treated groups served as negative controls. Bone marrow from a positive control group, treated with a single oral dose of cyclophosphamide (CP) at 50 mg/kg bw, was harvested at 48 h after dosing only. The test substance was found to respond negatively in the Micronucleus Test, whereas the positive control substance (CP) produced a statistically significant increase in the incidence of micronuclei in polychromatic erythrocytes.

It is concluded that the substance can be considered as not mutagenic in the Mouse Micronucleus Test under the experimental conditions.