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Diss Factsheets

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: other: damage to the chromosomes or the mitotic apparatus
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1994-04-05 to 1994-04-26
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: well performed GLP study following a former OECD guideline version

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report date:
1994

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
Adoted 1983
Deviations:
yes
Remarks:
Only one dose level tested, scoring of 1000 instaed of 2000 erythrocytes
Principles of method if other than guideline:
Only one dose level tested, untreated controls missing, scoring of 1000 instaed of 2000 erythrocytes
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
1,3-dihydro-5-nitro-2H-benzimidazol-2-one
EC Number:
202-282-2
EC Name:
1,3-dihydro-5-nitro-2H-benzimidazol-2-one
Cas Number:
93-84-5
Molecular formula:
C7H5N3O3
IUPAC Name:
5-nitro-1,3-dihydro-2H-benzimidazol-2-one
Details on test material:
- Name of test material (as cited in study report): 5-Nitrobenzimidazolon TF
- Other names: Nitrolon
- Molecular Formula: C7H5N3O3
- Lot/batch No.: 617/2, dated May 24th, 1990
- Composition of test material, percentage of components: Nitrolon and Water
- Content: 88% (m/m)
- Certificate of Analysis: N0 04814, Dated December 6th, 1990, Analytisches Laboratorium, Dr. R. Fischbach
- Stability and homogenity in vehicle: stable for five hours, Pharma Development Corporate Toxicologie, Dr. H-J. Pletsch, dated April 5th, 1994
- Storage stability: stable until October 1995, guaranteed by the sponsor
- Storage conditions: dark at approximately 20°C
- Appearance: olive-green powder
- pH-value in water: 6.8

On the day of the experiment the test substance was suspended in starch mucilage at an appropriate concentration.The preparation for application was kept homogenous by a magnetic stirrer until the completion of dosing.

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga GmbH, Sandhofer Weg 7, 97633 Sulzfeld
- Number of animals: 70 (35 males / 35 females)
- Age at study initiation: 7 weeks
- Weight at study initiation: males, median weight: 35.1g (/27-41g), females, median weight: 27.1g (24-30g)
- Assigned to test groups randomly: [no/yes, under following basis: ] yes, randomisation schedule 94.0129 and 94.0130
- Animal identification: fur marking with KMnO4 and cage numbering

- Housing: In air-conditioned rooms in Macrolon cages Type 2 (one male per cage) and Type 3 (five females per cage) on softwood granulate. Because of increased aggressivity the male animals were placed in single cages
- Diet (e.g. ad libitum): rat/mice diet Altromin 1324 (Altromin-GmbH, Lage/Lippe), ad libitum
- Water (e.g. ad libitum): tap water in plastic bottles, ad libitum
- Acclimation period: 7d


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 50 +/- 20%
- Photoperiod (hrs dark / hrs light): 12hrs dark/light

:

Administration / exposure

Route of administration:
oral: unspecified
Vehicle:
- Vehicle(s)/solvent(s) used: starch mucilage at appropriate concentrations
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment the test substance was suspended in starch mucilage at an appropriate concentration.The preparation for application was kept homogenous by a magnetic stirrer until the completion of dosing.
Frequency of treatment:
one time application
Post exposure period:
12, 24 and 48hrs
Doses / concentrations
Remarks:
Doses / Concentrations:
5-Nitrobenzimidazolon TF was administered orally
Basis:
other: nominal dose: 1500 mg/kg bw
No. of animals per sex per dose:
5 male and 5 female per dose and killing-time
Control animals:
yes, concurrent vehicle
other: no other negative control
Positive control(s):
Cyclophosphamide (Endoxan(R))
- Route of administration: once orally
- Doses / concentrations: 50mg/kg body weight

Examinations

Tissues and cell types examined:
Both femora were removed and the bones freed of muscle tissue. Bone marrow cells were collected therefrom.
Details of tissue and slide preparation:
In conformity with the test procedure the animals were killed by carbon dioxide asphyxiation 12, 24 or 48 hours after application. For each animal, about 3 ml foetal bovine serum was poured into a centrifuge tube. Both femora were removed and the bones freed of muscle tissue. The proximal ends of the femora were opened and the bone marrow flushed into the centrifuge tube. A suspension was formed. The mixture was then centrifuged for 5 minutes at approx. 1200 rpm and almost all the supernatant discarded. One drop of the thoroughly mixed sediment was smeared on a cleaned slide, identified by project code and animal number and air-dried for about 12 hours.
Evaluation criteria:
1000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded, not the number of individual micronuclei. As a control measure 1000 mature erythrocytes were also counted and examined for micronuclei. In addition, the ratio of polychromatic to normochromatic erythrocytes was determined. All bone marrow smears for evaluation were coded to ensure that the group which they belong to remained unknown to the investigator. The number of polychromatic erythrocytes with micronuclei occurring in the 1000 polychromatic erythrocytes counted, and the number of normocytes with micronuclei occurring in the 1000 normocytes counted, were evaluated statistically.
Both biological and statistical significances were considered together in evaluation. A substance is considered as positive if there is a significant increase in the number of micronucleated polychromatic erythrocytes for at least one of the time points. A test substance producing no significant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.
Statistics:
A Wilcoxon-Test (one-sided) was evaluated to check the validity of the study. This is done by comparing the number of polychromatic erythrocytes with micronuclei in the positive control with the negative control.

A Wilcoxon-Test (one-sided) is calculated for each measurement group (12h, 24h, 48h) and for polychromatic and normochromatic erythrocytes. These tests are performed sequentially with a multiple level of significance of 5%.

The presupposition to make any of the following comparisons is a difference between the positive control and the negative control (24h). This is tested with a Wilcoxon-Test (two-sided) with 5 %-level of significance. Wilcoxon-Tests (two-sided) are performed sequentially for the ratio of polychromatic erythrocytes for each measurement group (12h, 24h, 48h) at a multiple level of significance of 5 %. Actual data were also compared with historical controls

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Remarks:
See "Remarks on results including tables and figures"
Toxicity:
yes
Remarks:
See "Addtional information on results"
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
All animals survived after application of 1500 mg per kg bodyweight. The following signs of toxicity were observed: spontaneous activity decreased, palpebreal fissure narrow, palpebreal fissure very narrow, urine yellowish, unsteady gait, coat bristling, squatting posture, lacrimation blood colored, general condition poor, forward crawling. During the whole study duration clinical signs of toxicity had been observed. The dissection of the animals revealed the following macroscopic findings: content of intestinum partial yellowish coloured.

Any other information on results incl. tables

Mice were treated with 1500 mg Nitrobenzimidazolon TF per kg bodyweight to study the induction of micronuclei in bone marrow cells.

The incidence of micronucleated polychromatic and normochromatic erythrocytes in the dose groups of 5 -Nitrobenzimidazolon TF was within the normal range of the negative control groups. No statistically significant increase of micronucleated polychromatic erythrocytes has been observed. The number of normochromatic erythrocytes with micronuclei did not differ from the values of the simultaneous control animals for each of the three killing times investigated. The ratio of polychromatic erythrocytes to normocytes remained essentially unaffected by the test compound.

Cyclophosphamide (Endoxan®) induced a marked and statistically significant increase in the number of polychromatic erythrocytes with micronuclei in both males and females indicating the sensitivity of the test system. Summarising it can be stated that 5 -Nitroimidazolon TF did not lead to a substantial increase of micronucleated polychromatic erythrocytes and is not mutagenic in the micronucleus test. Summary of findings and individual data are attached.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
5-Nitrobenzimidazolon TF is not mutagenic in the micronucleus test.
Executive summary:

5 -Nitrobenzimidazolon TF was tested in the micronucleus test. The test compound was suspended in starch mucilage and dosed once orally at 1500 mg per kg bodyweight to male and female mice, upon the results of the previously conducted dose range finding assay (see preliminary study). According to the test procedure the animals were killed 12, 24 or 48 hours after administration.

A pretest revealed the substance is toxic is toxic to mice at dose 2000 mg / kg bw so the test was run with 1500 mg / kg bw, at this concentration no clinical signs were observed.

Endoxan® was used as positive control substance and was administered once orally at a dose of 50 mg per kg bodyweight.

The number of polychromatic and normochromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained unaffected by the treatment with 5 -Nitrobenzimidazolon TF and was statistically not different from the control values.

Endoxan® induced in both males and females a marked statistically significant increase in the number ofpolychromatic cells with micronuclei, indicating the sensitivity of the test system. The ratio of polychromaticerythrocytes to normocytes was not changed to a significant extent.

Under the conditions of the present study the results indicate that 5-Nitrobenzimidazolon TF is not mutagenic in the micronucleus test.