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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:
2004
Report date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
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): N-[[4-([(cyclopropylamino}carbonyl]phenyl]sulfonyl]-2-methoxybenzamide
- Analytical purity: 97.4%
- Lot/batch No.: 08466/0013

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen
- Age at study initiation: 6-12 weeks
- Weight at study initiation: 36-46 g
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: individually in type II cages; bedding of soft wood granules, type BK8/15 (J. Rettenmaier & Soehne, Fuellstoff-Fabriken, 73494 Ellwangen-Holzmuehle) was used
- Diet (ad libitum): fixed-formula feed 3883 (10 mm cubes), produced according to specification by Provimi Kliba SA, CH-4303 Kaiseraugst
- Water (ad libitum): tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-22
- Humidity (%): 43-48
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: 0.5% aqueous Cremophor emulsion; Cyclophosphamide was dissolved in physiological saline solution
- Justification for choice of solvent/vehicle: suitability for intraperitoneal application
- Amount of vehicle (intraperitoneal): 20 mL/kg
- Lot/batch no. (if required): Fluka, batch 398261/134099
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was suspended in 0.5% aqueous Cremophor emulsion, using a rotation mixer for 3 minutes, and formed milky-white suspensions. The suspensions were stirred with a magnetic mixer during administration
Duration of treatment / exposure:
48 hours
Frequency of treatment:
two intraperitoneal injections separated by 24 hours
Post exposure period:
24 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
500, 1000, 2000 mg/kg bw per injection
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide in the form of Endoxan 100 mg injection vials of dry substance (Baxter Oncology GmbH), batch 3B102
- Justification for choice of positive control(s): proven cytostatic agent and known clastogen with bifunctional alkylation action.
- Route of administration: intraperitoneal (1 injection); 24 hour treatment
- Doses / concentrations: 20 mg/kg bw

Examinations

Tissues and cell types examined:
Bone marrow cells (erythrocytes)
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Based on the range-finder study with doses of 1000, 2000 and 4000 mg/kg, each applied to groups of 3 males and 3 females, a dose of 2000 mg/kg was chosen as MTD for males.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
Test animals received 2 intraperitoneal injections 24 hours apart, 24 hours after the last application the animals were sacrificed and the bone marrow cells were isolated. The positive control substance was injected only once for 24 hours.

DETAILS OF SLIDE PREPARATION:
Bone marrow smears were prepared according to the method of Schmid (Mutat Res 31: 9-15; 1975. DFG; Kommission fuer Mutagenitaetsfragen; Mitteilung III, 53-61). At least one intact femur was prepared from each sacrificed animal. The bone marrow cells were flushed out from the femur with fetal calf serum. After centrifugation the supernatant was discarded and a homogeneous cell suspension prepared from the pellet. One drop of this suspension was then placed on a well-cleaned slide and spread out with a suitable object. The slides were dried overnight and stained automatically on the following day with an Ames Hema-Tek Slide Stainer from the Miles Company. Unspecific background staining was subsequently removed with methanol, then the slides were rinsed with deionised water and left to dry. Thereafter the slides were fixed with xylene, embedded in covering agent and covered with a covering glass. Slides were not evaluated until the covering agent had dried.

METHOD OF ANALYSIS:
Coded slides were evaluated using a light microscope at a magnification of about 1000. Micronuclei appear as stained chromatin particles in the anucleated erythrocytes. They can be distinguished from artifacts by varying the focus. Normally, 2000 polychromatic erythrocytes were counted per animal. The incidence of cells with micronuclei was established by scanning the slides in a meandering pattern.
Evaluation criteria:
A test was considered positive if there was a relevant and significant increase in the number of polychromatic erythrocytes showing micronuclei in comparison to the negative control.
A test was considered negative if there was no relevant or significant increase in the rate of micronucleated polychromatic erythrocytes. A test was also considered negative if there was a significant increase in that rate which, according to the laboratory's experience was within the range of historical negative controls.
In addition, a test was considered equivocal if there was an increase of micronucleated polychromatic erythrocytes above the range of attached historical negative controls, provided the increase was not significant and the result of the negative control was not closely related to the data of the respective treatment group. A test was also considered equivocal, if its result was implausible. In both cases, normally a second test will be performed.

An assay was considered acceptable if the figures of negative and positive controls were within the expected range, in accordance with the laboratory's experience and/or the available literature data.
Statistics:
The test substance group(s) with the highest mean (provided this superceded the negative control mean) and the positive control were checked by Wilcoxon's nonparametric rank sum test with respect to the number of polychromatic erythrocytes having micronuclei and the number of normochromatic erythrocytes. A variation was considered statistically significant if its error probability was below 5% and the treatment group figure was higher than that of the negative control.
The rate of normochromatic erythrocytes containing micronuclei was examined if the micronuclear rate for polychromatic erythrocytes was already relevantly increased. In this case, the group with the highest mean was compared with the negative control using the one-sided chi²-test. A variation was considered statistically significant if the error probability was below 5% and the treatment group figure was higher than that of the negative control.
In addition, standard deviations (1s ranges) were calculated for all the means.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
compound-related symptoms until sacrifice: apathy, roughened fur, loss of weight, spasm, twitching and slitted eyes; one of the treated males died during the test period due to the acute toxicity of 2000 mg/kg test substance
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
The pilot test was performed in the laboratory which conducted the main study using animals of the same source, strain and age. Groups consisting each of three males and three females received two intraperitoneal injections separated by 24 hours.

- Dose range: 1000, 2000, 4000 mg/kg bw

- Solubility: The test substance was suspended in 0.5% aqueous Cremophor (Fluka, batch 398261/134099) emulsion, using a rotation mixer for 3 minutes, and formed milky-white suspensions. The suspensions were stirred with a magnetic mixer during administration and injected intraperitoneally.

- Clinical signs of toxicity in test animals: In males the following symptoms were recorded for up to at least 24 hours after the second application, starting at 1000 mg/kg: apathy, roughened fur, loss of weight, spasm, difficulty in breathing, slitted eyes and closed eyes. 3 of 3 males died in the 4000 mg/kg group. In females the following symptoms were recorded for up to at least 24 hours after the second application, starting at 1000 mg/kg: apathy, roughened fur, loss of weight, spasm, periodically stretching of body, difficulty in breathing, slitted eyes and reduced body-temperature. 2 of 3 females died in the 4000 mg/kg group.

- Rationale for exposure: Based on these findings, 2000 mg/kg test substance were chosen as MTD for males. Due to the results of the dose range finder it was concluded, that there were no substantial differences between sexes in toxicity. Therefore, no females were used. Intraperitoneal application is one of the common routes recommended by the guideline.
- Harvest times: 24 hours after last treatment
- High dose with and without activation: activation not required, in vivo study

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): No biologically important or statistically significant variations existed for males between the negative control and the groups treated intraperitoneally with the test substance, with respect to the incidence of micronucleated polychromatic erythrocytes. The incidence of these micronucleated cells was 2.4/2000 (1s=2.1) in the negative control, and 2.4/2000 (1s=1.5), 2.2/2000 (1s=1.6) and 3.8/2000 (1s=1.8) in the treatment groups (500, 1000 and 2000 mg/kg bw, respectively).

- Ratio of PCE/NCE (for Micronucleus assay): The ratio of polychromatic to normochromatic erythrocytes in males was altered by the treatment with the test substance, being 2000: 1734 (1s=748) in the negative control, 2000: 2141 (1s=701) in the 500 mg/kg group, 2000: 3065 (1s=781) in the 1000 mg/kg group and 2000: 3018 (1s=1389) in the 2000 mg/kg group. Relevant variations were thus noted for males. This finding demonstrates relevant systemic exposure of the males to the test substance.

- Appropriateness of dose levels and route: After two intraperitoneal administrations of 500, 1000 and 2000 mg/kg test substance, treated males showed the following compound-related symptoms until sacrifice, starting at 500 mg/kg bw: apathy, roughened fur, loss of weight, spasm, twitching and slitted eyes. These symptoms demonstrate relevant systemic exposure of males to the test substance. One of treated males died during the test period, due to the acute toxicity of 2000 mg/kg test substance. No symptoms were recorded for the control groups. No animals died in these groups.

- Statistical evaluation: There was no biologically significant variation between the negative control and the test substance groups in the number of micronucleated normochromatic erythrocytes, since normochromatic erythrocytes originated from polychromatic ones. As expected, relevant variations were not observed. The results gave no indications of clastogenic effects for male mice after two intraperitoneal treatments with doses up to and including 2000 mg/kg bw.

Any other information on results incl. tables

Summary of results of the micronucleus test in mice with the test substance:

 

Experimental groups

Number of evaluated PCE

Number of NCE / 2000 PCE

MNNCE / 2000 NCE

MNPCE / 2000 PCE

Negative control

10000

1734 ± 748

1.3 ± 1.9

2.4 ± 2.1

2 x 500 mg/kg

10000

2141 ± 701

1.7 ± 1.5

2.4 ± 1.5

2 x 1000 mg/kg

10000

3065 # ± 781

2.3 ± 1.3

2.2 ± 1.6

2 x 2000 mg/kg

10000

3018 # ± 1389

2.4 ± 1.6

3.8 ± 1.8

Cyclophosphamide 20 mg/kg

10000

1788 ± 514

3.1 ± 1

26 ± 6.7*

 

*P<0.01 in non-parametric Wilcoxon ranking test

#: considered biologically relevant

PCE: polychromatic erythrocytes

NCE: normochromatic erythrocytes

MNNCE: micronucleated normochromatic erythrocytes

MNPCE: micronucleated polychromatic erythrocytes

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative

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