Registration Dossier

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: Comparable to guideline study with acceptable restrictions: testing in single sex, limited documentation.

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
DNA damage in brain cells and behavioral deficits in mice after treatment with high doses of amantadine.
Author:
Kaefer V, Semedo JG, Silva Kahl VF, Von Borowsky RG, Gianesini J, Ledur Kist TB, Pereira P, Picada JN
Year:
2010
Bibliographic source:
J Appl Toxicol.; 30(8):745-53
Reference Type:
publication
Title:
In vivo rodent erythrocyte micronucleus assay.
Author:
Hayashi M, Tice RR, Macgregor JT, Anderson D, Blakey DH, Kirsch-Volders M, Oleson FB Jr, Pacchierotti F, Romagna F, Shimada H, Sutou S, Vannier B
Year:
1994
Bibliographic source:
Mutat. Res. 312: 293–304.

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
only male mice were used as test animals, limited documentation
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): Amantadine (hydrochloride-1-adamantanamine)
- Analytical purity: no data, purchased from Acros Organics (Belgium).

Test animals

Species:
mouse
Strain:
CF-1
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Lutheran University of Brazil (ULBRA)
- Weight at study initiation: 38–47 g
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: physiol. saline;
- Amount: 10 mL/kg bw
Duration of treatment / exposure:
3 days
Frequency of treatment:
daily
Post exposure period:
On the fourth day the animals were sacrificed.
Doses / concentrations
Remarks:
Doses / Concentrations:
15, 30 or 60 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
7 males
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide: 25 mg/kg bw

Examinations

Tissues and cell types examined:
Bone marrow from both femurs
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The highest dose tested (60 mg/kg bw) was previously established as the maximum tolerated dose (MTD), following the recommendations described by Mavournin et al., 1990 (Mutat. Res. 239: 29–80) and Hartmann et al., 2003 (Mutagenesis 18: 45–51), and the other doses used were 50 and 25% of MTD. All solutions were prepared immediately prior to administration.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): Male mice were treated i.p. once a day with saline solution, or the test item (15, 30 or 60 mg/kg bw) for three consecutive days. The genotoxic activities were evaluated using seven animals per group. On the fourth day the animals were sacrificed and for the micronucleus assay, samples of the bone marrow were collected from the femurs.
A positive control group with N = 5 animals was treated with 25 mg/kg bw cyclophosphamide and sacrificed after 24 h, to evaluate mutagenic activity.

DETAILS OF SLIDE PREPARATION: Bone marrow from both femurs was suspended in fetal calf serum and smears on clean glass slides were prepared. Slides were air-dried, fixed in methanol, stained in 10% Giemsa and coded for a blind analysis.

METHOD OF ANALYSIS: Polychromatic erythrocytes: normochromatic erythrocytes (PCE/NCE) ratio was scored in 1000 cells. The incidence of micronuclei (MN) was observed in 2000 PCE per animal.

Statistics:
The statistical evaluation of data from micronucleus assay was carried out using Tukey’s test.
In all comparisons, P ≤ 0.05 was considered as indicating statistical significance.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes

Any other information on results incl. tables

Table 3: Results of the in vivo micronucleus assay.

Treatment group

Dose

[mg/kg]

PCE/NCE ratio

Micronucleated polychromatic erythrocytes in 2000 PCE per animal

Vehicle control

0

2.79 ± 0.86

1.14± 0.37

Test substance

15

2.16 ± 0.84

1.28± 0.48

 

30

3.50 ± 1.13

1.43±0.53

 

60

2.58 ± 0.95

1.71± 0.76

Positive control (Cyclophosphamide)

25

1.16 ± 0.80*

11.6± 2.07**

Significant difference: *P0.05; **P0.01 (ANOVA, Tukey’s test) in comparison with the saline group.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative