Registration Dossier

Diss Factsheets

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: Well performed GLP and OECD guideline study

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)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
4-(3-methyl-5-oxo-2-pyrazolin-1-yl)benzenesulphonic acid
EC Number:
201-901-3
EC Name:
4-(3-methyl-5-oxo-2-pyrazolin-1-yl)benzenesulphonic acid
Cas Number:
89-36-1
Molecular formula:
C10H10N2O4S
IUPAC Name:
4-(3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzene-1-sulfonic acid

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Strain specifics: Hoe: NMRI
- Source: Charles River Wiga GmbH, Sandhofer Weg 7, D-97633 Sulzfeld
- Age at study initiation: 7 weeks
- Weight at study initiation: males: 31-38 g, mean 34.9 g, females: 23-31 g, mean 26.2 g
- Housing: in groups of five (females) in Macrolon type 3 cages; one male per cage (Macrolon type 2); all in fully air-conditioned room, softwood granulate;
- Diet (e.g. ad libitum): rat/mouse diet Altromin 1324, ad libitum
- Water (e.g. ad libitum): tap water in plastic bottles, ad libitum
- Acclimation period: at least 5 days

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: starch mucilage
- Concentration of test material in vehicle: 20 % (w/v)
- Amount of vehicle (if gavage or dermal): 10 mL/kg body weight
Duration of treatment / exposure:
One single oral administration
Frequency of treatment:
One single oral administration
Post exposure period:
12 hours (dosed group, negative control and positive control), 24 hours (dosed group and negative control) and 48 hours (dosed group and negative control)
Doses / concentrations
Remarks:
Doses / Concentrations:
2000
Basis:
nominal conc.
mg/kg body weight
No. of animals per sex per dose:
5 males and 5 females (10 animals) per dose group
Control animals:
yes, concurrent vehicle
Positive control(s):
50 mg/kg body weight cyclophosphamide (Endoxan(R), 5 males and 5 females)

Examinations

Tissues and cell types examined:
1000 polychromatic erythrocytes from the femoral bone marrow of each animal
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Animals were killed 12, 24 or 48 h after application

DETAILS OF SLIDE PREPARATION:
Removal of femoral bones and bone marrow from the proximal ends flushed into centrifuge tube containing about 3 ml foetal bovine serum, centrifugation (5 min at 1200 rpm), one drop of thoroughly mixed sediment smeared on a cleaned slide, air-dried for approx. 24 h, staining (methanol, May-Grünwalds solution, Giemsa) and coating with Entellan

METHOD OF ANALYSIS:
Number of cells with micronuclei and ratio of polychromatic to normochromatic erythrocytes
Evaluation criteria:
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:
Wilcoxon (paired, one-sided, increase) for number of cells and Wilcoxon (paired, two-sided) for the ratio

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
Only orange discoloured urinewas observed.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: limit dose of 2000 mg/kg body weight
- Clinical signs of toxicity in test animals: orange coloured urine, increase of spontaneous activity
- Other: lethality: 0 out of 3 males and 0 out of 3 females

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no statistically significant increase of micronucleated polychromatic erythrocytes in dosed animals
- Ratio of PCE/NCE (for Micronucleus assay): the ratio remained essentially unaffected by the test compound
- Statistical evaluation: see above

Any other information on results incl. tables

Number of polychromatic erythrocytes containing micronuclei (%) and mutagenic index

SEX

DOSE

SAMPLE TIME

ERYTHROCYTES WITH MICRONUCLEI

POLY (MEAN)

%

MUTAGENIC INDEX

MALE

CONTROL

12 H

0.2

1.0

2000

12 H

0.1

0.6

FEMALE

CONTROL

12 H

0.1

1.0

2000

12 H

0.1

0.8

 

MALE

CONTROL

24 H

0.1

1.0

2000

24 H

0.2

1.1

POSITIVE CONTROL

24 H

4.1

29.0

FEMALE

CONTROL

24 H

0.2

1.0

2000

24 H

0.2

1.4

POSITIVE CONTROL

24 H

3.6

22.8

 

MALE

CONTROL

48 H

0.2

1.0

2000

48 H

0.1

0.8

FEMALE

CONTROL

48 H

0.1

1.0

2000

48 H

0.1

1.2

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Oral administration of the test item did not lead to a substantial increase of micronucleated polychromatic erythocytes and was not mutagenic in the in vivo mouse micronucleus test
Executive summary:

The test item was tested in the micronucleus test. The test compound was suspended in starch mucilage and dosed once oral at 2000 mg per kg bodyweight to male and female mice, upon the results of the previously conducted dose range finding assay. According to the test procedure the animals were killed 12, 24 or 48 hours after administration.

EndoxanR was used as positive control substance and was administered 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 unaffectedby the treatment with the test item and was statistically not different from the control values.

EndoxanR induced in both males and females a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity af the system.

The results indicate that, under the conditions of the present study, the test item is not mutagenic in the micronucleus test.