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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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
(adopted 1997)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
6,6',6''-(1,3,5-triazine-2,4,6-triyltriimino)trihexanoic acid
EC Number:
279-505-5
EC Name:
6,6',6''-(1,3,5-triazine-2,4,6-triyltriimino)trihexanoic acid
Cas Number:
80584-91-4
Molecular formula:
C21H36N6O6
IUPAC Name:
6,6',6''-(1,3,5-triazine-2,4,6-triyltriimino)trihexanoic acid
Details on test material:
- Physical state: solid
- Storage conditions: in the dark at ambient room temperature

Test animals

Species:
mouse
Strain:
other: Hsd:ICR (CD-1)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Italy S.r.l., San Pietro al Natisone (UD)
- Age at study initiation: 5-6 weeks
- Weight at arrival: 22-30 g
- Housing: 5 animals per cage in clear polycarbonate cages
- Diet: commercially available laboratory rodent diet (Altromin MT, Altromin, D-32770 Lage, Postfach 1120, Germany), ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 10
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: CMC (carboxymethyl cellulose, 0.5%)
- Concentration of test material in vehicle: 200 mg/mL; 100 mg/mL; 50 mg/mL
- Amount of vehicle: 10 mL/kg
- Batch No. (from BDH): 1072067
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Solutions of the test item, as received, were prepared immediately before use in carboxymethylcellulose 0.5%. Solutions were prepared on a weight/volume basis without connection for the displacement due to the volume of the test item.
Duration of treatment / exposure:
Not applicable.
Frequency of treatment:
Single treatment.
Post exposure period:
Control group: 24 and 48 h
500 mg/kg bw: 24 h
1000 mg/kg bw: 24 h
2000 mg/kg bw: 24 and 48 h
Positive control: 24 h
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Control group: 10 per sex
500 mg/kg bw: 5 per sex
1000 mg/kg bw: 5 per sex
2000 mg/kg bw: 10 per sex
Positive control: 5 per sex
Control animals:
yes, concurrent vehicle
Positive control(s):
Mitomycin-C (batch no. 31K2504; SIGMA)
- Route of administration: oral, gavage
- Doses / concentrations: 3 mg/kg bw

Examinations

Tissues and cell types examined:
Erythrocytes of femur bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
A toxicity test was performed to aid in the selection of dose-levels. Groups of two male and two female mice were dosed by oral gavage with the test item at 2000, 1000 and 500 mg/kg bodyweight. The animals were observed for 48 hours and sacrificed. Scoring was performed on slides prepared from the femurs of animals.

DETAILS OF SLIDE PREPARATION:
Animals were sacrificed at appropriate sampling times and the femurs of animals were removed and bone marrow cells obtained by flushing with fetal calf serum. The cells were centrifuged and a concentrated suspension was prepared to make smears on slides. These slides were air-dried and then stained with May-Gruenwald and Giemsa, and mounted with Eukitt. Three slides were made from each animal.

METHOD OF ANALYSIS:
The slides were randomly coded by a person not involved in the subsequent microscope scoring. The slides were examined under low power (x 16 objective) and one slide from each animal was selected according to staining and quality of smears. Where no depression of polychromatic erythrocytes was observed at least 2000 polychromatic cells per animal were examined for the presence of micronuclei at high power (x 100 objective, oil immersion). At the same time the numbers of normal and micronucleated normochromatic erythrocytes were also recorded.
Evaluation criteria:
The test item is considered to induce micronuclei if a statistically significant increase in the micronucleus incidence in polychromatic erythrocytes (at P<0.05) is observed in any treatment group, in the pooled data for both sexes, or for either sex considered separately. Where increases in the incidence of micronucleated PCE's are observed which are statistically significant, but fall within the range of negative control values within this laboratory, then concurrent and historical control data are used to demonstrate that these increases do not have biological significance.
Statistics:
Only counts obtained from polychromatic cells were subjected to statistical analysis. Using the original observations (and not the micronucleus frequencies per 1000 cells), a modified Chi-squared calculation was employed to compare treated and control groups.
The degree of heterogeneity within each group was first calculated and where this was significant it was taken into account in the comparison between groups. Variance ratios or Chi-squared values are taken to show the significance of any difference between each treated group and the controls.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
Reduced activity was observed in female animals on the day of the treatment and the day after. No other adverse reactions to treatment were noted. Treatment with the test item had no adverse effect on the proliferation of bone-marrow erythropoietic cells. Based on the above results, dose-levels of 500, 1000 and 2000 mg/kg bodyweight were selected for the Main assay.

RESULTS OF DEFINITIVE STUDY
No increases in the numbers of micronucleated PCE's were observed in any treatment group at any sampling time. Pronounced increases in the frequency of micronucleated PCE's were observed in the positive control group, indicating the correct functioning of the test system.
The ratio of mature to immature erythrocytes and the proportion of immature erythrocytes among total erythrocytes were analyzed to evaluate the bone marrow cell toxicity. Based on these results, no inhibitory effect on erythropoietic cell division was observed in male or female animals at any sampling time.

Any other information on results incl. tables

Table 1: Incidence of micronucleated PCEs and ratio of PCE/NCE of the in-vivo bone marrow micronucleus study.

Treatment

Dose level (mg/kg bw)

Incidence of micronucleated PCEs

PCE/(PCE+NCE)

% over the mean negative control value

 

male/female

Mean

SE

range

 

24 hr sampling time

Vehicle

10 mL/kg

0.6

0.2

0

1.5

100

Test item

500

0.9

0.3

0

3.0

99

Test item

1000

0.5

0.2

0

1.5

101

Test item

2000

0.8

0.3

0

2.5

98

Mitomycin C

3

10.6***

1.4

6.0

17.0

102

 48 hr sampling time

Vehicle

10 mL/kg

0.6

0.2

0

1.5

100

Test item

2000

0.8

0.3

0

3.0

98

***: Incidence significantly greater than control value at p < 0.001

Applicant's summary and conclusion

Conclusions:
On the basis of the results obtained, it is concluded that the test article administered by oral gavage, does not induce micronuclei in the polychromatic erythrocytes of treated mice, under the reported experimental conditions.
Executive summary:

The test article's potential to cause chromosomal damage in vivo was investigated in a micronucleus test. Based on results obtained in a preliminary toxicity trial, dose-levels selected were 500, 1000 and 2000 mg/kg bodyweight for male and female animals. Hsd:ICR (CD-I) mice were dosed once by gavage with the vehicle alone (carboxymethylcellulose 0.5%), the test material in the vehicle at the selected dose-levels or with the positive control substance Mitomycin-C. Each group consisted of five male and five female animals with the exception of the control and high-dose group, which included an additional five animals of each sex per group. Five animals per sex from each group were sacrificed at the 24 hour sampling time. The additional animals were sacrificed at the 48 hour sampling time. Bone-marrow smear slides were made and stained with May-Gruenwald and Giemsa stains. At least 2000 polychromatic cells per animal were examined for the presence of micronuclei. The slides were coded prior to scoring. The results obtained at each sampling time were subjected to statistical analysis using a modified chi-squared test. Following treatment with the test substance, no statistically significant increase in the incidence of micronucleated PCE's over the control value was observed at any dose level, at any sampling time. Following treatment with the positive control Mitomycin-C, statistically significant increases in the incidence of micronucleated PCE's over the control values were observed, indicating the correct functioning of the test system. No clinical signs or depression of bone marrow erythropoietic cell division, were observed at any dose-level, at any sampling time. It is concluded that, under the reported experimental conditions, the test item administered by oral gavage at the selected dose-levels to male and female mice, does not induce micronuclei in the polychromatic erythrocytes.

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