Amantadine hydrochloride

Genetic toxicity: in vivo

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

Reference

Reference 1

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 2

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

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

Test material information

Constituent 1

Reference substance name:

Amantadine hydrochloride

EC Number:

211-560-2

EC Name:

Amantadine hydrochloride

Cas Number:

665-66-7

Molecular formula:

C10H17N.ClH

IUPAC Name:

adamantan-1-amine hydrochloride

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 or test system 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 validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

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: *P≤0.05; **P≤0.01 (ANOVA, Tukey’s test) in comparison with the saline group.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative