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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1999-11-23 to 1999-12-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline and GLP conform well documented scientific study report.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report date:
1999

Materials and methods

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

Test material

Constituent 1
Reference substance name:
921-522-3
EC Number:
921-522-3
IUPAC Name:
921-522-3
Constituent 2
Reference substance name:
Tetrapropylene succinic acid monoisobutylester
IUPAC Name:
Tetrapropylene succinic acid monoisobutylester
Test material form:
other: viscous liquid, light brown
Details on test material:
- Name of test material (as cited in study report): Hostacor 4323
- Chemical name: Tetrapropenyl succinic acid monoisobutylester
- Physical state: viscous liquid, light brown
- Density: 0.969 g/cm3 (at 20°C)
- Analytical purity: n.a.
- Lot/batch No.: 137304039
- Expiration date of the lot/batch: December 2000
- Storage condition of test material: darkness at approximately 20°C in a fume cupoard
- Solubility: Solution in sesame oil

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
solution in sesame oil
Duration of treatment / exposure:
overall: 2 days
Frequency of treatment:
The test substance was administered twice at an interval of 24 hours orally by gavage to the test animals.
Post exposure period:
24 hours after the second application to the test animals, the animals were killed by carbon dioxide asphyxiation
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
Test substance: 2000 mg/kg bw (dissolved in 20 % (w/v) sesame oil)
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
positive substance: 50 mg/kg bw (dissolved in 0.5 % (w/v) distilled water)
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
positive controls: cyclophosphamide

Examinations

Tissues and cell types examined:
bone marrow,
polychromatic erythrocytes,
erythrocytes
Details of tissue and slide preparation:
24 hours after dosing, animals were killed by carbon dioxide asphyxiation. Both femora were removed. After opening of the proximal end of the femora the bone marrow was flushed into a centrifuge tube containing 3 mL fetal bovine serum. The solution 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, identifed by project code number and air-dried for about 12 hours. The staining follows according to protocol.
Evaluation criteria:
2000 polycromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded, not the number of individual micronuclei. In addition the ratio for the polychromatic erythrocytes to 200 total erythrocytes was determined. Main parameter for statisitical analysis, i.e. validity assessment of the study and mutgenicity of the test substance, was the proportion of polychromatic erythrocytes with micronuclei out of 2000 counted erythrocytes. All bone marrow smears for evaluation were coded to ensure that the group from they were taken remained unknown to the investigator.

Criteria for a positive response:
Both biological and statistical significances were considered together for evaluation purposes. A substance is considered postive if there is a significant increase in the number of micronucleated polychromatic erythrocytes compared with the concurrent negative control group. A test substance producing no significant increase in hte number of micronucleated polychromatic erythrocytes is concsidered non-mutagenic in this system.
Statistics:
A one-sided Wilcoxon-Test was evaluated to check the validity of the study. The study was considered as valid in case the proportion of polychromatic erythrocytes with micronuclei in the positive control was significantly higher than the negative control (p =0.05).

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
The following signs of toxicity were observed: motor activity decreased, gait stilted, coat bristling, trembling, squatting posture, palpebral fissure narrow and eyelids adhering.
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Table: Summary of results of MNT assay with Hostacor 4323

Substance

Dose

 

Sex

 

Number of animals

Poly/animal

Poly/Ery

Poly with MN

Poly with MN

 

Mutagenic Index

 

mg/kg bw

pooled

(f + m)

counted

Mean

Mean

[%]

Mu. I.

controls

0

pooled

10

2000

0.48

1.40

0.1

1.0

Test item

2000

pooled

10

2000

0.44

1.60

0.1

1.1

Cyclo-phosphamide

50

pooled

10

2000

0.45

44.60*

2.2

31.9

Poly= polychromatic erythrocytes

Poly/Ery = polychromatic erythrocytes / erythrocytes

MN = Micronucleus; * = significantly different from control (p<0.05)

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Hostacor 4323 did not cause any significant increase of micronucleated polychromatic erythrocytes and is not mutagenic in the micronucleus test under the conditions described in this report.
Executive summary:

In order to study the induction of micronuclei in bone marrow cells Hostacor 4323 was tested in a micronucleus test in erythrocytes according to OECD 474 guideline performed in NMRI mice. The test substance was administered to ten mice (5/ sex) twice at an interval of 24 ours orally by gavage to the test animals at a dose of 2000 mg/kg bw. The vehicle, sesame oil, was administered in the same way to the negative control groups. Cyclophosphamide was used as positive substance and was administered once orally at a dose of 50 mg/kg bw.

The following signs of toxicity were observed: motor activity decreased, gait stilted, coat bristling, trembling, squatting posture, palpebral fissure narrow and eyelids adhering. According to test procedure all animals were killed 24 hours after administration and bone marrow were harvested from both femora of each animal. Smears were prepared from the bone marrow and further processed according to protocol. 2000 polychromatic erythrocytes were counted for each animal.

The number of polychromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic erythrocytes to total erythrocytes in both male and female animals remained unaffected by the treatment with Hostacor 4323 and was not less than 20% of control value. Cyclophosphamide induced a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the test system. The ratio of polychromatic erythrocytes to total erythrocytes was not changed to a significant extent.

Under the conditions of this study the results indicate that Hostacor 4323 is not mutagenic in the micronucleus test.

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