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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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Test method similar to OECD guideline 474, but no data on GLP.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1995

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
not specified
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): trichloroacetic acid (TCA)
- Analytical purity: 99.5 %

Test animals

Species:
mouse
Strain:
other: C57BL/6JfBL10/Alpk
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Barriered Animal Breeding Unit, Alderley Park, Cheshire.
- Age at study initiation: 6-7 weeks old
- Housing: They were housed up to 10 per cage
- Diet (e.g. ad libitum): Throughout the study, food (Porton Combined Diet (PCD)) was supplied ad libitum.
- Water (e.g. ad libitum): Throughout the study, water was supplied ad libitum.
- Acclimation period: At least 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23 ºC
- Humidity (%): 40-70 %
- Photoperiod (hrs dark / hrs light): cycled at 12 h intervals

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: water
Duration of treatment / exposure:
2 days
Frequency of treatment:
Daily
Post exposure period:
Animals were sacrificed 6 or 24 hours after the second injection.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
337, 675 and 1080 mg/kg
Basis:
nominal conc.
In male mice
Remarks:
Doses / Concentrations:
405, 810 and 1300 mg/kg
Basis:
nominal conc.
In female mice
No. of animals per sex per dose:
10 males and 10 females per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: i.p.
- Doses / concentrations: 25 mg/kg

Examinations

Tissues and cell types examined:
Bone marrow samples were taken 6 and 24 h after the second dose and the chromosomal damage assessed by analysis of the bone marrow for micronuclei.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
A median lethal dose (MLD) was determined for each sex by administering neutralized trichloroacetic acid to groups of two males and to groups of five males and five females via the intraperitoneal (i.p.) route at a dosing volume of 10 ml/kg. Each mouse was given two equal doses of neutralized trichloroacetic acid 24-h apart and was observed at least twice a day for a period of 4 days. Any clinical observations and deaths were recorded. The MLD for each sex was estimated from the levels of lethality observed by linear logarithmic interpolation (female data) or logistic regression (male data). Un-neutralized trichloroacetic acid could not be used owing to animal welfare constraints and ethical considerations.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Animals were given two i.p. doses 24 h apart of sterilized double deionized water or neutralized trichloroacetic acid at dose levels representing 25, 50 and 80% of the MLD in each sex using a dosing volume of 10 ml/kg. Animals were killed by asphyxiation in a rising concentration of CO2 followed by cervical dislocation 6 and 24 hours after receiving their second dose.

DETAILS OF SLIDE PREPARATION:
One femur was removed from each animal and stripped clean of muscle. Paint brush smears were prepared on clean, labelled glass microscope slides. The slides were air dried and stained with polychrome methylene blue and eosin using an Ames Hema-Tek staining machine. Slides were coded and analysed in ascending numerical slide code order.

METHOD OF ANALYSIS:
One thousand polychromatic erythrocytes were examined per animal using a x100 oil immersion objective lens and x10 or x12.5 eyepieces. The slides were assessed starting at the beginning of a smear and proceeding to the leading edge and the number of cells containing micronuclei recorded.
Evaluation criteria:
The criteria for the identification of a micronucleus were as described by Schmid (1976). The slides were also examined for evidence of cytotoxicity, which may be manifest as alterations in the ratio of different cell types in the bone marrow. This was assessed by counting the number of polychromatic and normochromatic cells in 1000 erythrocytes.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

No statistically or biologically significant increases in the incidence of micronucleated polychromatic erythrocytes compared with the solvent control dosed animals were observed in either sex at the 6 h sampling time or in the females at the 24 hr sampling time. A small but statistically significant increase in micronucleated polychromatic erythrocytes was observed in male mice 24 hr after a dose of 675 mg/kg (50% MLD). Since no increases were noted at the 25 or 80% MLD, and the levels recorded are within the range of the concurrent solvent control values, the small increase observed in the males at the 50% MLD is considered not to be biologically significant. No other increases in the micronuclei were observed.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
No statistically or biologically significant increases in the incidence of micronucleated polychromatic erythrocytes compared with the solvent control dosed animals were observed in either sex at the 6 h sampling time or in the females at the 24 hr sampling time. A small but statistically significant increase in micronucleated polychromatic erythrocytes was observed in male mice 24 hr after a dose of 675 mg/kg (50% MLD). Since no increases were noted at the 25 or 80% MLD, and the levels recorded are within the range of the concurrent solvent control values, the small increase observed in the males at the 50% MLD is considered not to be biologically significant. No other increases in the micronuclei were observed.
Executive summary:

In the mouse micronucleus test, neutralized trichloroacetic acid was administered in two equal intraperitoneal doses 24 h apart to C57BL/6JfBL10/Alpk mice (337, 675 and 1080 mg/kg in males; 405, 810 and 1300 mg/kg in females). These dose levels represent 25%, 50% and 80% of the median lethal dose (MLD) in this strain of mouse. Bone marrow samples were taken 6 and 24 h after the second dose and the chromosomal damage assessed by analysis of the bone marrow for micronuclei. No statistically or biologically significant increases in the incidence of micronucleated polychromatic erythrocytes compared with the solvent control dosed animals were observed in either sex at the 6 h sampling time or in the females at the 24 hr sampling time. A small but statistically significant increase in micronucleated polychromatic erythrocytes was observed in male mice 24 hr after a dose of 675 mg/kg (50% MLD). Since no increases were noted at the 25 or 80% MLD, and the levels recorded are within the range of the concurrent solvent control values, the small increase observed in the males at the 50% MLD is considered not to be biologically significant. No other increases in the micronuclei were observed.