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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:
17 Jul - 18 Aug 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
(adopted 1983)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Version / remarks:
(adopted Sep 1985)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Tierzucht Schoenwalde GmbH i.G., Schoenwalde, Germany
- Age at study initiation: 8 weeks
- Weight at study initiation: mean: 38.1 g (males), 32.6 g (females)
- Assigned to test groups randomly: yes
- Housing: Makrolon cages Type 3 (five animals per cage) on softwood granulate
- Diet: rat/mice diet ssniff R/M-H (V 1534, ssniff GmbH, Soest, Germany), ad libitum
- Water: tap water in plastic bottles, ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): housing in fully air-conditioned rooms
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: sesame oil
- Concentration of test material in vehicle: 0.2, 1 and 2% (w/v)
- Amount of vehicle (if gavage or dermal): 10 mL/ kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment the test substance was suspended in sesame oil at appropriate concentrations. A magnetic stirrer was used to keep the preparations homogeneous until the completion of dosing.

Duration of treatment / exposure:
not applicable
Frequency of treatment:
single treatment
Post exposure period:
12, 24 or 48 h p.a.
Doses / concentrations
Remarks:
Doses / Concentrations:
20, 100 and 200 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5 per post exposure time point
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: oral gavage
- Doses / concentrations: 50 mg/kg bw

Examinations

Tissues and cell types examined:
Tissue: bone marrow
Cell type: erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: In a preliminary dose range finding study, oral administration of 400 mg Hoe 122006 per kg body weight resulted in mortality in male and female mice. Therefore the highest sublethal dose of 200 mg per kg body weight was selected for the main study.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): In conformity with the test procedure the animals were killed by carbon dioxide asphyxiation 12, 24 or 48 hours after treatment.

DETAILS OF SLIDE PREPARATION: Femoral bone marrow was flushed into the centrifuge tubes containing 3 mL of fetal bovine serum. A suspension was formed. The mixture was then centrifuged for 5 minutes at approx. 1200 rpm, after which almost all the supernatant was discarded. One drop of the thoroughly mixed sediment was smeared onto a cleaned slide, identified by project code and animal number and air-dried for about 12 hours. Slides were fixed with methanol and stained according to May-Grünwald-Giemsa staining.

METHOD OF ANALYSIS:
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. The number of polychromatic erythrocytes with micronuclei occurring in the 1000 polychromatic erythrocytes, and the number of normocytes with micronuclei occurring in the 1000 normocytes, were evaluated statistically.
Evaluation criteria:
Both biological and statistical significances were considered together for evaluation purposes. A substance is considered as positive if there is a significant dose related increase in the number of micronucleated polychromatic erythrocytes for at least one of the time points compared with the concurrent negative control group. Individual and/or group mean values should exceed the laboratory historical control range. A test substance producing no significant dose related increase in the number of micronucleated polychromatic erythrocytes, and the values within the historical control range, is considered not mutagenic in this system. For the assay to be valid, individual and/or group mean values for the positive control should exceed the laboratory's historical control range.
Statistics:
One-sided Wilcoxon-Test was performed for each treatment interval and for polychromatic and normochromatic erythrocytes. These tests are
performed sequentially with a multiple level of significance of 5%. Tests on lower dose groups are only performed if the higher dose group is significantly different from the control. If there is a difference between the positive and negative control (24 h) (two-sided Wilcoxon-Test with a 5%-level of significance) two-sided Wilcoxon-Tests are also performed sequentially for the ratio of polychromatic erythrocytes for each treatment
interval (12h, 24h, 48h) at a multiple level of significance of 5 %. Actual data were also compared with historical controls. Lower dose groups are only tested if the higher dose group is significantly different from the control.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
200 mg/kg bw: 24 h sampling time 1 male with spotted hepatic tissue
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 200 - 1200 mg/kg bw in 3 animals per sex and dose
- Clinical signs of toxicity in test animals:
1200 mg/kg bw: spontaneous activity decreased, squatting posture, ataxic gait, prone position, forward crawling, staggering gait, palpebral fissure narrow, irregular respiration, panting, flanks pinched in, clonic convulsions, no macroscopic findings were observed; mortality: 2/3 males, 3/3 females
1000 mg/kg bw: spontaneous activity decreased, squatting posture, unsteady gait, staggering gait, prone position, flanks pinched in, coat bristling, panting, clonic convulsions, palpebral fissure narrow, palpebral fissure very narrow, no macroscopic findings were observed; mortality: 1/3 males, 3/3 females
800 mg/kg bw: spontaneous activity decreased, squatting posture, prone position, staggering gait, stilted gait, ataxic gait, panting, clonic convulsions, no macroscopic findings were observed; mortality: 3/3 males, 2/3 females
600 mg/kg bw: spontaneous activity decreased, increased activity when touched, squatting posture, unsteady gait, forward crawling, stilted gait, ataxic gait, prone position, flanks pinched in, marked flank respiration, tonic convulsions when touched, palpebral fissure narrow, lacrimation clear, colourless; macroscopic findings: hepatic tissue lightly coloured, distinctly patterned appearence of hepatic tissue; mortality: 2/3 males, 2/3 females
400 mg/kg bw: spontaneous activity decreased, increased activity when touched, stilted gait, forward crawling, unsteady gait, squatting posture, prone position, coat bristling, marked flank respiration, panting, palpebral fissure narrow, palpebral fissure very narrow, twitching; macroscopic findings: hepatic tissue lightly coloured, distinctly patterned appearence of hepatic tissue; mortality: 2/3 males, 1/3 females
200 mg/kg bw: spontaneous activity decreased, increased activity when touched; no macroscopic findings were observed; mortality: 0/6 animals

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): The incidence of micronucleated polychromatic and normochromatic erythrocytes in the dose groups was within the normal range of the negative control groups
- Ratio of PCE/NCE (for Micronucleus assay): The ratio of polychromatic erythrocytes to normocytes remained essentially unaffected by the test compound.
- Appropriateness of dose levels and route: In a preliminary dose range finding study, oral administration of 400 mg/kg bw test substance resulted in mortality in male and female mice. Therefore the highest sublethal dose of 200 mg/kg body weight was selected for the main study.
- Statistical evaluation: One-sided Wilcoxon-Test was performed for each treatment interval.

Any other information on results incl. tables

Table 1: Results of the in vivo micronucleus assay in male animals.

 

Mean PCEs / 1000 NCEs
at sampling time ± SD

Total micronuclei per 1000 PCEs at sampling time

Total micronuclei per 1000 NCEs at sampling time

Exp group

Number of animals

Dose [mg/kg bw]

12 h

24 h

48 h

12 h

24 h

48 h

12 h

24 h

48 h

Vehicle control (corn oil)

5

0

1.1±0.08

0.8±0.07

0.8±0.10

1±0.0

1.2±0.11

1.4±0.09

1.8±0.08

0.8±0.08

1.6±0.09

Positive control (Cyclophosphamide)

5

50

n.d

0.8±0.19

n.d.

n.d.

40±0.73

n.d.

n.d.

1.60±0.05

n.d.

Test substance

5

20

1±0.22

1.0±0.09

0.8±0.10

1±0.1

2±0.12

0.8±0.04

0.6±0.05

0.80±0.04

0.8±0.11

Test substance

5

100

0.8±0.09

0.9±0.07

0.8±0.07

1.2±0.11

1±0.07

0.8±0.08

1.4±0.09

0.8±0.08

1±0.10

Test substance

5

200

1.1±0.09

1.1±0.09

0.9±0.19

2.4±0.11

1.8±0.11

0.8±0.04

0.8±0.13

0.6±0.09

0.8±0.13

n.d. = not determined

 

Table 2: Results of the in vivo micronucleus assay in female animals.

 

Mean PCEs / 1000 NCEs
at sampling time ± SD

Total micronuclei per 1000 PCEs at sampling time

Total micronuclei per 1000 NCEs at sampling time

Exp group

Number of animals

Dose [mg/kg bw]

12 h

24 h

48 h

12 h

24 h

48 h

12 h

24 h

48 h

Vehicle control (corn oil)

5

0

1.1±0.13

1±0.10

1±0.16

2±0.17

1.2±0.11

0.8±0.08

0.4±0.05

1.2±0.13

0.4±0.09

Positive control (Cyclophosphamide)

5

50

n.d.

0.7±0.18

n.d.

n.d.

29.8±0.73

n.d.

n.d.

0.8±0.08

n.d.

Test substance

5

20

1.0±0.21

1.1±0.14

0.9±0.13

1.2±0.08

2.4±0.11

1±0.1

1±0.07

0.2±0.04

1±0.07

Test substance

5

100

0.8±0.10

0.8±0.20

0.9±0.17

1.8±0.08

1.2±0.11

1±0.17

0.6±0.09

0.6±0.05

0.6±0.09

Test substance

5

200

1.0±0.09

1.0±0.13

0.8±0.14

1.2±0.13

2.2±0.18

1.2±0.11

1±0.07

0.8±0.08

0.6±0.05

n.d. = not determined

 

Table 3: Results of the in vivo micronucleus assay.

Treatment group

Dose

[mg/kg]

Sampling time

[h]

PCE with MN ± SD

Mean frequency of PCE with MN

PCE/NCE ratio

Vehicle control

 

 

0

12

1.5±0.13

0.2

1.1±0.11

0

24

1.2±0.10

0.1

0.9±0.15

0

48

1.1±0.09

0.1

0.9±0.15

Test substance

 

 

200

12

1.8±0.13

0.2

1.1±0.13

200

24

2.0±0.14

0.2

1±0.12

200

48

1.0±0.08

0.1

0.9±0.16

Positive control (Cyclophosphamide)

40

24

34.9±0.87*

3.5

0.8±0.18

* statistically significant (p<0.05)

Conclusion: The test substance was not mutagenic (clastogenic) in this mouse bone marrow micronucleus test.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative