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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:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study with acceptable restrictions (lack of data on test substance)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
(lack of data on test substance)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, St. Constant, Quebec, Canada
- Age at study initiation: approximately 9 weeks
- Weight at study initiation: 32.8 - 40.1 g (males); 27.1 - 31.8 g (females)
- Assigned to test groups randomly: yes, by a computer-generated body weight sorting program
- Housing: the animals were housed individually in suspended stainless steel and wire mesh cages with absorbent paper below the cages.
- Diet: Certified Rodent Diet # 5002 (pellets) (PMI Feeds Inc.), ad libitum
- Water: tap water, ad libitum
- Acclimation period: approximately 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 40 - 70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
From: 18 Sep 1995
To: 21 Sep 1995

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle/solvent used: peanut oil
- Justification for choice of solvent/vehicle: The test substance was soluble in peanut oil at the concentrations required for this study.
- Amount of vehicle: ≤ 1 mL/100 g bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was thoroughly mixed with the vehicle.
Duration of treatment / exposure:
three applications
Frequency of treatment:
approximately every 24 h
Post exposure period:
24 h after the last treatment
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:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: oral by gavage
- Dose: 20 mg/kg bw

Examinations

Tissues and cell types examined:
Tissue: bone marrow of the femur
Cell type: bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: range finding study performed to find the maximum tolerated dose

DETAILS OF SLIDE PREPARATION: 2 slides per animal were prepared. The slides were stained with acridine orange and wet mounted.

METHOD OF ANALYSIS: 2000 polychromatic erythrocytes (PCEs) from each animal were examined for the presence of micronuclei.
Evaluation criteria:
The test substance may be considered positive in this test system if at least one of the following criteria is met:
- The mean number of the micronucleated PCEs of at least one dose point is statistically different from the mean number of the micronucleated PCEs of the vehicle control. This value also must be outside the normal range of the micronucleated PCEs of the vehicle control. Additionally, a dose related statistical increase in the mean number of micronucleated PCEs must be observed.
- The mean number of the micronucleated PCEs of at least two dose points is statistically different from the mean number of the micronucleated PCEs of the vehicle control. This values also must be outside the normal range of the micronucleated PCEs of the vehicle control.
Statistics:
The statistical analysis included means and standard deviations of the micronuclei data and a test of equality of group means performed by a standard one-way analysis of variance (ANOVA).
Residuals from the ANOVA were analyzed for normality by either Wilk's Criterion or the Kolomogorov-Smirnov Statistic. If the residuals were not normally distributed (at 0.01 level of significance) in more than 25% of the analyses then nonparametric analyses were performed. The nonparametric analyses included the Kruskal-Wallis one-way analysis of variance followed by Dunn's Summed Rank Test if differences were indicated. Dose response was evaluated by Jonkheere's test of Ordered Response.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 500 - 2000 mg/kg bw
- Clinical signs of toxicity in test animals: No toxicity was observed up to the highest dose tested.
- Harvest times: 24 h

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: Treatment of the animals with the test substance did not lead to a dose-dependent and statistically significant increase in micronuclei formation.
- Cytotoxicity: No cytotoxicity was observed, since there were no statistically significant decreases in the percentage of PCEs of the treated animals in comparison with the vehicle control.

Any other information on results incl. tables

Table 1: Results of the in-vivo micronucleus assay in male animals

 

%PCE

at sampling time

Total micronuclei per 1000 PCEs at sampling time

Exp group

Number

of animals

Dose [mg/kg]

24 h

24 h

Vehicle control

(peanut oil)

5

0

54.14 ± 6.00

0.6 ± 0.7

Positive control

(cyclophosphamide)

5

20

40.24 ± 11.18**

18.7 ± 7.5**

Test substance

5

500

54.76 ± 4.81

0.9 ± 0.4

Test substance

5

1000

51.80 ± 7.46

1.2 ± 0.8

Test substance

5

2000

51.84 ± 8.85

0.8 ± 0.6

**statistically significant (p<0.01)

Table 2: Results of the in-vivo micronucleus assay in female animals

 

%PCE

at sampling time

Total micronuclei per 1000 PCEs at sampling time

Exp group

Number

of animals

Dose [mg/kg]

24 h

24 h

Vehicle control

(peanut oil)

5

0

55.30 ± 4.72

1.5 ± 0.4

Positive control

(cyclophosphamide)

5

20

44.54 ± 5.17**

10.5 ± 3.4**

Test substance

5

500

56.26 ± 5.89

0.6 ± 0.4*

Test substance

5

1000

60.68 ± 3.63

0.8 ± 0.7*

Test substance

5

2000

59.90 ± 2.7

0.4 ± 0.4*

*statistically significant (p<0.05);**statistically significant (p<0.01)

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative