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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1990

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- State of aggregation: liquid

Test animals

Species:
mouse
Strain:
other: Tif: MAGf (SPF)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: animal farm CIBA-GEIGY
- Age at study initiation: Young adult mice
- Weight at study initiation: Females: 23-30 g; Males: 28-39 g
- Housing: group-caging
- Diet (e.g. ad libitum): standard diet of NAFAG No. 890 ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: at least 3 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-23
- Humidity (%): 50-65
- Photoperiod (hrs dark / hrs light): 12 hours/12 hours

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
Arachis oil
Details on exposure:
A solubility test was performed to determine the highest applicable dose level (solution/suspension) of the test substance for the tolerability test (up to a top dose level of 5000 mg/kg body weight). Arachis oil was found to be the best suited vehicle, yielding the highest applicable dose level of 5000 mg/kg.
A tolerability test was performed to determine the maximum tolerated dose level of the test substance, which is the highest causing no death in a group of four animals for the observation period of three consecutive days; that is the interval between administration and sacrifice of the animals in the micronucleus test, plus one day. The tolerability test was performed with three groups of four mice (two females and two males) receiving one single application. One group received the highest applicable dose (5000 mg/kg) and the other two groups received the doses of 1/5 and 1/25 of that amount respectively.
The animals were treated once with the highest tolerated dose of the test substance, 5000 mg/kg, and the appropriate treatment groups were sacrificed 16, 24 and 48 hours thereafter. Subsequently their femoral bone marrow erythrocytes were scored for micronuclei.
Duration of treatment / exposure:
one administration and then sacrificed after a period of 16, 24 and 48 hours
Frequency of treatment:
once
Post exposure period:
16, 24 and 48 hours
Doses / concentrations
Dose / conc.:
5 000 mg/kg bw/day
No. of animals per sex per dose:
Five animals were evaluated out of 8 treated per group
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide (CPA, 64 mg/kg) was administered by oral gavage, at a volume of 20 ml/kg.

Examinations

Tissues and cell types examined:
bone marrow: micronuclei in polychromatic erythrocytes
Details of tissue and slide preparation:
Preparation of bone marrow and preparation of the slides:
The animals were sacrificed by dislocation of the cervical vertebrae. Bone marrow was harvested from the shafts of both femurs with fetal calf serum and prepared on slides. After air-drying, the slides were stained with May-Gruenwald/Giemsa solution and mounted.
Evaluation criteria:
Criteria for scoring micronuclei:
Micronuclei are uniform, darkly stained, more or less round bodies in the cytoplasm of PCEs. Inclusions in PCEs which are reflective, improperly shaped or stained, or which are not in the focal plain of the cell are judged to be artifacts and are not scored as micronuclei. Cells containing more than one micronucleus are only counted once.
Prior to analysis the slides were coded. The slides of five animals/ sex/dose, showing good differentiation between mature and polychromatic erythrocytes, were selected for scoring. From each animal 1000 polychromatic erythrocytes were scored for micronuclei. The ratio of polychromatic to normochromatic erythrocytes was determined for each animal by counting a total of 1000 erythrocytes.

Assay evaluation criteria:
A test substance is considered to be positive in this test system if a statistically significant increase in the number of micronuclei in polychromatic erythrocytes occurs in comparison with the negative control at any dose and sampling time respectively. If ecjuivocal results are obtained, the final decision has to be based on scientific judgement.

Assay acceptance criteria:
1. The result of the experiments should not be influenced by a significant technical error or a recognized artifact.
2. The high dose of the test substance applied should be the maximum tolerated dose causing no death in a group of four animals in the range finding-test. In case of missing toxicity the high dose should be up to a maximum of 5000 mg/kg body weight or the highest applicable dose due to the limited solubility of the test substance.
3. The (juality of the slides must allow a clear differentiation between polychromatic and normochromatic erythrocytes.
4. At least five female and five male animals per dose and control group should be evaluated.
5. The positive control should fulfil the criteria for a positive substance.
Statistics:
The significance of differences was assessed by the Chi-Square-test (p < 0.05).

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
In the first step of the tolerability test the maximum dose of TK 13 282 (CGL 123) (5000 mg/kg) and the doses of 1000 and 200 mg/kg caused no death in a group of four animals.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
The tolerability test was performed with three groups of four mice (two females and two males) receiving one single application. One group received the highest applicable dose (5000 mg/kg) and the other two groups received the doses of 1/5 and 1/25 of that amount respectively.
- Solubility: Arachis oil was found to be the best suited vehicle, yielding the highest applicable dose level of 5000 mg/kg.

Any other information on results incl. tables

EXPERIMENTAL RESULTS

Number of polychromatic erythrocytes with micronuclei and ratio of polychromatic to normochromatic (p/n) erythrocytes, arithmetic mean per sex and group.
Sacrifice Dose Sex polychromatic erythrocytes normochromatic
erythrocytes
ratio of
p / n erythrocytes
number of
polychromatic
erythrocytes
with micronuclei
% of
polychromatic
erythrocytes
with micronuclei
16h Control (Arachis oil) f 524 476 1.1 0.6 0.06
m 498 502 1 0.2 0.02
Test substance 5000 mg/kg f 541 459 1.2 1 0.1
m 505 495 1 0.4 0.04
24h Control (Arachis oil) f 514 486 1.1 1 0.1
m 504 496 1 0.6 0.06
Test substance 5000 mg/kg f 519 481 1.1 0.4 0.04
m 525 475 1.1 0.4 0.04
48h Control (Arachis oil) f 517 483 1.1 0.8 0.08
m 481 519 0.9 0.4 0.04
Test substance 5000 mg/kg f 506 494 1 0.4 0.04
m 486 514 0.9 0.4 0.04
CONTROLS
24h Control (Arachis oil) f 514 486 1.1 1 0.1
m 504 496 1 0.6 0.06
Cyclophosphamid (64 mg/kg) f 512 488 1 16.6 1.66
m 470 530 0.9 12.8 1.28

Applicant's summary and conclusion

Conclusions:
Under the given experimental conditions no evidence for clastogenic or aneugenic effects was obtained in mice treated with the test item.