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Toxicological information

Genetic toxicity: in vivo

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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
Study period:
From 25 FEB 2003 to 01 APR 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD 474), GLP compliant

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report date:
2003

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
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
4,4'-[(3,3'-dichloro[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[2,4-dihydro-5-methyl-2-(p-tolyl)-3H-pyrazol-3-one]
EC Number:
239-898-6
EC Name:
4,4'-[(3,3'-dichloro[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[2,4-dihydro-5-methyl-2-(p-tolyl)-3H-pyrazol-3-one]
Cas Number:
15793-73-4
Molecular formula:
C34H28Cl2N8O2
IUPAC Name:
4,4'-[(3,3'-dichlorobiphenyl-4,4'-diyl)didiazene-2,1-diyl]bis[5-methyl-2-(4-methylphenyl)-2,4-dihydro-3H-pyrazol-3-one]
Test material form:
not specified

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Strain specifics: NMRI BR mice (SPF)
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6-8 weeks
- Weight at study initiation: males: 30.2-32.8g, females: 26.2-27.6 g
- Housing: in groups of five per sex
- Diet (e.g. ad libitum): standard diet, ad libitum (Altromin, code VRF1)
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3 °C
- Humidity (%): 30-70 %
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: 1% (w/v) carboxymethylcellulose
- Amount of vehicle (if gavage or dermal): 10 ml/kg body weight
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item concentrations were treated with ultra-sonic waves to obtain a homogeneous suspension,. Dosing was performed withing 4 h after preparation.
Duration of treatment / exposure:
Single intraperitoneal administration
Frequency of treatment:
See above
Post exposure period:
24 hours (all dose groups, negative control), 48 hours (highest dose group, positive control)
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 500, 1000, 2000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
5 males and 5 females per sampling time in each treatment group
Control animals:
yes, concurrent vehicle
Positive control(s):
50 mg/kg body weight cyclophosphamide dissolved in physiological saline, single intraperitoneal injection

Examinations

Tissues and cell types examined:
2000 polychromatic erythrocytes from the femoral bone marrow of each animal
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
based on results of dose range finding studies; 3 males and 3 females received 2000 mg/kg bw and were observed for 4 days

DETAILS OF SLIDE PREPARATION:
bone marrow suspension preparations were air dried, fixed for 5 min in 100% methanol and air-dried overnight; slides were automatically stained using the "Wright-stain-procedure" in an an "Ames" HEMA-tek slide stainer; no further information on staining procedure

METHOD OF ANALYSIS:
- number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes
- ratio polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes
Evaluation criteria:
A micronucleus test is considered acceptable if it meets the following 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 micronucleated polychromatic erythrocytes.
b) The incidence of micronucleated polychromatic erythrocytes in the control animais should reasonably be within the laboratory historical control data range (mean ± three times the standard deviation): Males: 1.2%0 ± 3.5% indicated are means for n=199. Females: 1.3%0 ± 4.1%0 indicated are means for n=125).

A test substance is considered positive in the micronucleus test if:
- lt 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 micronucleated polychromatic erythrocytes (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.

A test substance is considered negative in the micronucleus test if:
- None of the tested concentrations or sampling times showed a statistically significant (P < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes neither in the combined data for both sexes nor in the data for male or female groups separately.
Statistics:
- Wilcoxon Rank Sum Test; two-sided test
- Averages and standard deviations were calculated

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
for details see below
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- no mortality occurred
- ataxia and lethargy were observed in all animals 20 min after dosing, >/= 2 hours after dosing all animals showed lethargy and hunched posture, rough coat was only observed in one animal on day 2 and 3 after dosing; no abnormalities were observed in any animal on day 4

RESULTS OF DEFINITIVE STUDY
- No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of treated animals compared to the vehicle treated animals.
- The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the historical solvent control data range.
- Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes
- The animals of the groups which were treated with test item showed no decrease in the ratio of polychromatic to normochromatic erythrocytes, which reflects a lack of toxic effects of this compound an the erythropoiesis. The animals of the groups treated with cyclophosphamide showed a decrease in the ratio of polychromatic to normochromatic erythrocytes.

Toxicity:

The animals of the negative and positive control groups showed no abnormalities.

2000 mg/kg body weight group:
During the first 1.5 hour after dosing all animals were lethargic, 19 animals showed ataxia, three male and two female animals also had a rough coat.
Within 18 hours after dosing all animals had a rough coat, eight male and five female animals also had a hunched posture.
Within 42 hours after dosing all males and one female animal still had a rough coat, one male animal also had a hunched posture. All other female animals had recovered from the treatment.

1000 mg/kg body weight group:
During the first 1.5 hour after dosing all animals were lethargic, two males and one female animal also had a rough coat.
Within 18 hours after dosing all animals had a rough coat, four male and two female animals also had a hunched posture.

500 mg/kg body weight group:
During the first 1.5 hour after dosing all animals were lethargic. Within 18 hours after dosing all male and three female animals had a rough coat. All other female animals had recovered from the treatment.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The test item was not mutagenic in an in vivo mouse micronucleus test under the conditions of the test (single intraperitoneal application of up to 2000 mg/kg bw).
Executive summary:

The test item was tested in the Micronucleus Test in mice, to evaluate its genotoxic effect on erythrocytes in bone marrow.

Six groups each comprising 5 males and 5 females, received a single intraperitoneal injection.

Two groups were dosed with 2000 mg/kg body weight, one group was dosed with 1000 mg/kg bodyweight and one group was dosed with 500 mg/kg body weight. After dosing the animals of the dose levels of 2000, 1000 and 500 mg/kg body weight showed the following toxic signs: ataxia, lethargy, rough coatand hunched posture.

A vehicle treated group served as negative control, a group treated with a single intraperitoneal injectionof cyclophosphamide (CP) at 50 mg/kg body weight served as positive control.

Bone marrow of the groups treated with test item was sampled 24 or 48 hours after dosing. Bonemarrow from the negative control group was harvested at 24 hours after dosing only and bone marrow from the positive control group was harvested at 48 hours after dosing only.

Cyclophosphamide, the positive control substance, induced a statistically significant increase in thenumber of micronucleated polychromatic erythrocytes.

No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with test item.

The groups that were treated with test item showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, which reflects a lack of toxic effects of this compound on the erythropoiesis. The group that was treated with cyclophosphamide showed a decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls.

The test item was not mutagenic in the micronucleus test under the experimental conditions described in this report.

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