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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:
18 September - 20 October 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Product name: Antiblaze V6
Appearance: clear pale yellow liquid

Test animals

Species:
mouse
Strain:
Swiss
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Iffa Credo, l'Arbresle, France
- Age at study initiation: approximately 6 weeks
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing: polycarbonate cages
- Diet: A04 C pelleted maintenance diet
- Water: Filtered , ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +- 2
- Humidity (%): 30 - 70%
- Air changes (per hr): at least 12 cycles/hour
- Photoperiod (hrs dark/hrs light): 12h/12h

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: Olive oil
- Amount of vehicle: 10 mL/kg
- Batch no: 010K6021 (Sigma, France)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was suspended in the vehicle in oerder to achieve concentrations of 50, 100, 200 mg/mL for males and 43, 75, 87.5 and 175 mg/mL for females. The target doses were 500, 1000 and 2000 mg/kg bw for males and 437.5, 875 and 1750 mg/kg for females.
The dosing solutions were prepared immediately before use.
Volume of administration: 10 ml/kg bw.


Frequency of treatment:
Two treatments, seperated by 24 hours
Post exposure period:
24 hours after last treatment (sampling time)
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
500, 1000, 2000 mg/kg/day (males)
Basis:
other: Target dose level
Remarks:
Doses / Concentrations:
437.5, 875, 1750 mg/kg/day (females)
Basis:
other: Target dose level
No. of animals per sex per dose:
5 males and 5 females for controls and all dose levels, except for high dose level where 8 males and 8 females were used
Control animals:
yes, concurrent vehicle
Positive control(s):
- Positive control: Cyclophosphamide (CPA)
- Route of administration: oral, dissolved in distilled water
- Concentration: 5 mg/mL
-Dose: 50 mg/kg single dose

Examinations

Tissues and cell types examined:
Bone marrow, erythrocytes
Details of tissue and slide preparation:
Stain used: Giemsa
Evaluation criteria:
- Polychromatic erythrocytes (PE), normochromatic erythrocytes (NE) ratio established by scoring total of 1000 erythrocytes
- Number of micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes
- Results considered positive if there is a statistically significant increase in the frequency of MPE compared to the concurrent vehicle control group
Statistics:
- no significant within-group heterogeneity MPE values: Chi-square test
- significant within-group heterogeneity MPE values: Mann-Whitney test
- PE/NE ratio comparison: student "t"-test

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Top dose-level determined in preliminary toxicity test
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In preliminary toxicity test, the test item was administered at 1500, 1750 and 2000 mg/kg/day (females) and 2000 mg/kg/day (males), to select the top dose level for the definitive test. For males no toxic effect was observed at 2000 mg/kg/day, this was selected as highest dose-level for the main test. For females 1/3 animals was found dead one h following a single dose of 2000 mg/kg bw. Clinical signs (half-closed eyes or hypoactivity) were observed after the first dose of 1750 mg/kg bw and 1750 and 1500 mg/kg/day in females and 1750 mg/kg was selected as highest dose level.

In the main test 1/5 of animals were found dead 24 hours after the first treatment in highest dose-level (2000 and 1750 mg/kg/day for males resp. females).

At the lower dose groups no clinical signs and mortality were observed. For both males and females, there were no significant differences between the MPE values of the treated groups compared to the control group.

In the males of the highest dose group a significant decrease in the PE/NE ratio was observed indicating that the bone marrow was reached. In otherdose levels the PE/NE ratio was not different from controls.

Any other information on results incl. tables

 

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
No statistically significant difference in the number of micronucleated polychromatic erythroxcytes was observed between treated and control animals. The test substance does not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two oral administrations at a 24-hour interval.
Executive summary:

In a Swiss Ico mouse bone marrow micronucleus assay, 5 males and 5 females per dose (8 animals for highest dose level) were treated orally with 2,2-bis(chloromethyl)trimethylene bis(bis(2-chloroethyl)phosphate) at doses of 0, 500, 1000 and 2000 mg/kg/day (for males) and 0, 437.5, 875 and 1750 mg/kg/day (for females).  The test item was administered twice, treatments seperated by 24 hours. Bone marrow cells were harvested 24 hours after last treatment. The vehicle used was olive oil.

 

There were signs of toxicity during the study, at the highest dose-level (mortality of 1/5 of animals). The PE/NE ratio was reduced in high dose male animals indicating that the bone marrow was reached. The test substance was tested at an adequate dose based on a preliminary toxicity test, where the highest dose-level for the main test was determined. The positive control (CPA) induced the appropriate response. No significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow was observed after any treatment time at any dose level.

This GLP study is classified as accepatble, it satisfies the requirements for OECD Test Guideline 474 for in vivo cytogenetic mutagenicity data.