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Mutagenicity tests with mebendazole in the mouse
Mutatio~z Research, 26 (I974) 427-43 ° ,¢, Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
427
M u t a g e n i c i t y tests with mebendazole in the mouse Mebendazole (methyl-5 benzoyl benzimidazole-2-carbamate) is a potent, orally active, broad spectrum anthelmintic. Studies in marts," and in various species of domestic animals4,~,13, z~ have shown mebendazole to be effective against numerous species of nematodes and cestodes. Toxicologic studies showed mebendazole to be safe in a variety of laboratory and domestic animals °. In view of its potentially widespread use for medical purposes it appeared important to test mebendazole for possible mutagenicity in mammals. The present paper deals with the results of mutagenicity tests in the mouse. Methods The induction of chromosome changes in the mouse was assayed after acute oral administration of mieronized mebendazole to male mice using the dominant lethality test, the spermatocyte test 7 and the Fz translocatioa t e s t t zo randomly bred Albino Swiss male mice of 12 weeks age were used per experimental group. They received r6o, 320 or 640 mg/kg mebendazole, 16o or 2io mg/kg cyclophosplaamide or the aqueous vehicle only by intubation of o.I ml solution. The compounds were suspended in a mixture prepared from 4 drops of Tween 80 (Atlas Company) and 2.5 ml tartaric acid (5 ml for the highest dose of mebendazole) complemented with water to IO ml. Immediately after treatment, each male was caged for 7 days with 4 untreated virgin females from the same strain. During 8 consecutive weeks the females were exchanged each week for fresh ones in order to test the dominant lethality induced in different stages of the male germ cells. On the ihth day after introduction to the male, the females were autopsied and the number of living and dead embryos recorded. Dominant lethality was evaluated directly by calculation of pre- and post-implantation losses. After their use for 8 weeks in the genetical test, 5 males from each group, either dosed with mebendazole at 26o, 320 or 640 mg/kg, or with cyclophosphamide at 21o mg/kg, or with the aqueous vehicle only, were sacrificed and the spermatocyte test was performed ~. The F I translocation test was done on 30 males sired in the first 3 weeks after treatment, by animals given mebendazole in a single I6o mg/kg dose. 200 spermatocytes at the diakinesis-first metaphase stage of meiosis were examined for each animal in the spermatocyte test and 25 in the F I translocation test. Results The results of the genetical tests are summarized in Table I and in Fig. I. In agreement with previous findings~, 10 single doses of eyclophosphamide have been found to induce dominant lethals with an increase of post-implantation deaths during the first 3 weeks of the experiment, indicating that spermatids and testicular as well as epididymal spermatozoa were affected. There is no evidence that mebendazole at single doses up to 640 m g / k g induces an increase of prenatal losses throughout the 8-week observation period. It can be inferred that mebendazole does not produce dominant lethals in the mouse male germ cells. The spermatocyte test as well as the Fz translocation test gave negative results in the controls and in each of the treated groups. Similar results have already been reportedwith cyclophosphamides,lz, 1=. It is
428 TABLE
SHORT COMMUNICATIONS I
RESULTS OF DISSECTIONS
Treatment
Week after Total treatment females
% of pregnant females b
Implantations per female a
Posti'mplantation loss b ( % of total implants)
Solvent
I 2 3 4 5 6 7 8
39 4° 4° 4° 4° 36 4° 4°
77 73 75 85 78 92 80 83
8.4 9.4 8.3 9.4 io.i 9.6 9.1 9.4
9. x 7.7 4..8 6.3 ,[.8 8.5 I2. 7 9.0
Cyclophosphamide 16o m g / k g
i 2
20 20
3 oa 60
3
2o
65
4 5 6 7 8
20 20 2o :20 2o
60 75 8o 80 7°
9.2 9,5 8.4 I0,2 9.9 9.6 9.9 8. 7
25.5 d 28,9e x 7.40 I2. 3 6.8 4.6 5.7 7.,I.
I 2 3 4 5 6 7 8
20 20 I6 16 16 I6 16 12
7° 81 4.3d 56 620 68 75
7.4
5.4
io.o 8.9
ii.8 3.8
x 2
4.0 39
65 6I 77 77 82 85 82 82
8.6 9,9 Io.I 9.4 9.2 9.2 Io. 5 9.6
9.8 9,7 7.0 6,8
13.9 8.I 7.9 6.6
12.3 9.2 5.6 8.9 8.3 3.9 Io. 7 8.2
Cyelophosphamlde 21o m g / k g
Mebendazole z6o m g / k g
Meb endazole 320 m g / k g
Meb endazole 640 m g / k g
8.I 5.70
37.00 48.80
7,8
25.5 o
8,6 5.90
8,3 I5.1 °
3
4°
4
4°
5
4°
6 7 8
4° 4° 4.o
i 2 3 4 5 6 7 8
4° 38 4° 4° 4° 4° 4° 4°
60 65 65 80 80 75 85
I o. 2 8. 9 9.3 9.9 9.6 9.6 IO.3 9.4
I 2 3 4 5 6 7 8
4° 4° 4° 4° 4° 4° 4° 4°
57 85 82 85 87 9o 85 80
9.5 8. 3 8. 7 8.6 8.9 8.6 8.8 9.9
a Mann-Whitney U test (two-tailed probability). b Z2 t e s t ( t w o - t a i l e d p r o b a b i l i t y ) . o p < 0.05. d p < o,oi. e p < o.ooi,
,1.5cl
77
,5,0
6. 4 6. 3 6.6
7.5 6,2 4.7 8.8
SHORT COMMUNICATIONS
429
%
°]o 100-
100
s0-" 6a-
40: 2o
0:
80 60
LL 12345678 I 160mg/kg
2 I
4
~0
2O a,,,.,,, 6
t
B I
210rag /kg
C ¥C L O P H O SPI.'IA M I D E
6
e
I CONTROL I
2
~, 6
lt,..h
s
kgl t160mgt ~
6
~'20
s
mg tkg I
,2 ~.
6
o swe~'ks...
6i 4 0- m9 1 kg i
MEBENDAZOLE
Fig. I. Number of postimplantation deaths expressed as percentage of total implants.
concluded that mebendazole does not induce cytologically detectable chromosome damage in pre- and post-meiotic germ ceils of the mouse but the negative findings in the reported tests do not preclude the possibility of the occurrence of genetic effects of a different nature. The authors wish to thank Mr. M. SWANNET for his valuable technical assistance. * Laboratory of Geuelics, Deflartme~¢tof Radiobiology, C.E.N./S.C.K., B-24oo, Mol and ** Janssen Pharmaceutic~, D@artment of Toxicology, B-234o, Beerse (Belgium)
A. L~ONARD* R. VANDESTEENE** 1~. MARSBOOM**
I BATEMAN, A. J. AND S. S. EPSTEIN, Dominant lethal mutations in mammals in A. HOLLAENDER (Ed.), Chemical Mutagens, Principles and Meehodsfor their Detection, Vol. 2, Plenum, New York, ~97I, pp. 541-558. 2 BI~ITTINGE~, D., Die mutagene Wirkung yon Endoxan bei der Maus, Humagenellk, 3 (I956) I56-~65. ] BRUGMANS, J, P., :D, TI-IIENPONT, I. VAN WIJNGAAI~DEN, 0. F. VANPARIJS, V. L. SCtlUERMANS AND H. L. LAUWERS, Mebendazole in enterobiasis : radiochemical and pilot clinical study in 1,278 subjects, J, Am. Med. Ass., 2I 7 (z97r) 313-315. 4 CALLEAR, J. F. F., AND R. M. S. NEAV~, The clinical use of the anthehnintic mebendazole, Brit. Vet. J., 127 (I97 I) xli. 5 DE VI~igs, J. P., Een smakelijk wormmiddel veer paarden, Tijdschr. Diergeneesk., 95 (IO) (I97Z), 693. 6 FI~RLAFUN, E., L'oxyurose: traitement aneien et actuel, Bruxelles-Med., 51 (9) (z97z), 6o56o8. 7 LI~ONARD, A., Observations on meiotic chromosomes of the male mouse as a test of the potential mutagenieity of chemicals in mammals, in A. ttOLLAENDEI~ (Ed.), Chemical Mutageus, Principles c~ndMethods for their Detection, Vol. 3, Plenum, New York, x973, PP. 21-56. 8 LI~ONARD, A. AND G, LINDEN, Observation of dividing spermatocytes for chromosome aberrations induced in mouse spermatogonia by chemical mutagens, Mutation Res., 16 (I972) 297~OO. 9 MARSBOOM, R., Toxicologic studies on mebendazole, Toxicol Appl. Pharmaeol, 24 (1972) 371-377. ZO I{61-IRBORN, G., The activity of alkylating agents, I. Sensitive mutable stages in spermatogenesis and oogenesis, in F. ¥OGI~:LA~D G. RSHI~BOr~N(Eds.), Chemical Mutagenesis in Mammals and ~/an, Springer, Berlin, 197 o, pp. i48-i52, 294-316. Ii SCnLEIEIeMACI{ER, E., Ober den Einfluss von Trenimon und Endoxan auf die Meiose der mi~nnlichen Maus, Humangenetik, 3 (I965) 134-I55.
430
SHORT COMMUNICATIONS
I2 SCHLEIERMACHER, E., The activity of alkylating agents, II. Histological and cytogenetic findings in spermatogenesis, in F. VOGEL AND G. RGHRBORN (Eds.), Chemical 3l~tagenesis in Mammals and Man, Springer, Berlin, 197 ° pp. 3~7-34 I. 13 THIENPONT, D., O. F. VANPARIJS AND L. C. HERMANS, Mebendazole, a new p o t e n t drug against s y n g a m o u s t r a c h e a in turkeys, poultry, Science, 52 (1973) 1712-1714 • 14 VANPARIJS, O. F., AND D. THIENPOINT, Anthelminthische W i r k u n g des Mebendazols gegen h e m a t o d e n u n d zestoden bei Hunden, De~t. Tierarzt. Wschrift, 8o (1973) 32o 322. Received January
2 3 r d , 1974
427
M u t a g e n i c i t y tests with mebendazole in the mouse Mebendazole (methyl-5 benzoyl benzimidazole-2-carbamate) is a potent, orally active, broad spectrum anthelmintic. Studies in marts," and in various species of domestic animals4,~,13, z~ have shown mebendazole to be effective against numerous species of nematodes and cestodes. Toxicologic studies showed mebendazole to be safe in a variety of laboratory and domestic animals °. In view of its potentially widespread use for medical purposes it appeared important to test mebendazole for possible mutagenicity in mammals. The present paper deals with the results of mutagenicity tests in the mouse. Methods The induction of chromosome changes in the mouse was assayed after acute oral administration of mieronized mebendazole to male mice using the dominant lethality test, the spermatocyte test 7 and the Fz translocatioa t e s t t zo randomly bred Albino Swiss male mice of 12 weeks age were used per experimental group. They received r6o, 320 or 640 mg/kg mebendazole, 16o or 2io mg/kg cyclophosplaamide or the aqueous vehicle only by intubation of o.I ml solution. The compounds were suspended in a mixture prepared from 4 drops of Tween 80 (Atlas Company) and 2.5 ml tartaric acid (5 ml for the highest dose of mebendazole) complemented with water to IO ml. Immediately after treatment, each male was caged for 7 days with 4 untreated virgin females from the same strain. During 8 consecutive weeks the females were exchanged each week for fresh ones in order to test the dominant lethality induced in different stages of the male germ cells. On the ihth day after introduction to the male, the females were autopsied and the number of living and dead embryos recorded. Dominant lethality was evaluated directly by calculation of pre- and post-implantation losses. After their use for 8 weeks in the genetical test, 5 males from each group, either dosed with mebendazole at 26o, 320 or 640 mg/kg, or with cyclophosphamide at 21o mg/kg, or with the aqueous vehicle only, were sacrificed and the spermatocyte test was performed ~. The F I translocation test was done on 30 males sired in the first 3 weeks after treatment, by animals given mebendazole in a single I6o mg/kg dose. 200 spermatocytes at the diakinesis-first metaphase stage of meiosis were examined for each animal in the spermatocyte test and 25 in the F I translocation test. Results The results of the genetical tests are summarized in Table I and in Fig. I. In agreement with previous findings~, 10 single doses of eyclophosphamide have been found to induce dominant lethals with an increase of post-implantation deaths during the first 3 weeks of the experiment, indicating that spermatids and testicular as well as epididymal spermatozoa were affected. There is no evidence that mebendazole at single doses up to 640 m g / k g induces an increase of prenatal losses throughout the 8-week observation period. It can be inferred that mebendazole does not produce dominant lethals in the mouse male germ cells. The spermatocyte test as well as the Fz translocation test gave negative results in the controls and in each of the treated groups. Similar results have already been reportedwith cyclophosphamides,lz, 1=. It is
428 TABLE
SHORT COMMUNICATIONS I
RESULTS OF DISSECTIONS
Treatment
Week after Total treatment females
% of pregnant females b
Implantations per female a
Posti'mplantation loss b ( % of total implants)
Solvent
I 2 3 4 5 6 7 8
39 4° 4° 4° 4° 36 4° 4°
77 73 75 85 78 92 80 83
8.4 9.4 8.3 9.4 io.i 9.6 9.1 9.4
9. x 7.7 4..8 6.3 ,[.8 8.5 I2. 7 9.0
Cyclophosphamide 16o m g / k g
i 2
20 20
3 oa 60
3
2o
65
4 5 6 7 8
20 20 2o :20 2o
60 75 8o 80 7°
9.2 9,5 8.4 I0,2 9.9 9.6 9.9 8. 7
25.5 d 28,9e x 7.40 I2. 3 6.8 4.6 5.7 7.,I.
I 2 3 4 5 6 7 8
20 20 I6 16 16 I6 16 12
7° 81 4.3d 56 620 68 75
7.4
5.4
io.o 8.9
ii.8 3.8
x 2
4.0 39
65 6I 77 77 82 85 82 82
8.6 9,9 Io.I 9.4 9.2 9.2 Io. 5 9.6
9.8 9,7 7.0 6,8
13.9 8.I 7.9 6.6
12.3 9.2 5.6 8.9 8.3 3.9 Io. 7 8.2
Cyelophosphamlde 21o m g / k g
Mebendazole z6o m g / k g
Meb endazole 320 m g / k g
Meb endazole 640 m g / k g
8.I 5.70
37.00 48.80
7,8
25.5 o
8,6 5.90
8,3 I5.1 °
3
4°
4
4°
5
4°
6 7 8
4° 4° 4.o
i 2 3 4 5 6 7 8
4° 38 4° 4° 4° 4° 4° 4°
60 65 65 80 80 75 85
I o. 2 8. 9 9.3 9.9 9.6 9.6 IO.3 9.4
I 2 3 4 5 6 7 8
4° 4° 4° 4° 4° 4° 4° 4°
57 85 82 85 87 9o 85 80
9.5 8. 3 8. 7 8.6 8.9 8.6 8.8 9.9
a Mann-Whitney U test (two-tailed probability). b Z2 t e s t ( t w o - t a i l e d p r o b a b i l i t y ) . o p < 0.05. d p < o,oi. e p < o.ooi,
,1.5cl
77
,5,0
6. 4 6. 3 6.6
7.5 6,2 4.7 8.8
SHORT COMMUNICATIONS
429
%
°]o 100-
100
s0-" 6a-
40: 2o
0:
80 60
LL 12345678 I 160mg/kg
2 I
4
~0
2O a,,,.,,, 6
t
B I
210rag /kg
C ¥C L O P H O SPI.'IA M I D E
6
e
I CONTROL I
2
~, 6
lt,..h
s
kgl t160mgt ~
6
~'20
s
mg tkg I
,2 ~.
6
o swe~'ks...
6i 4 0- m9 1 kg i
MEBENDAZOLE
Fig. I. Number of postimplantation deaths expressed as percentage of total implants.
concluded that mebendazole does not induce cytologically detectable chromosome damage in pre- and post-meiotic germ ceils of the mouse but the negative findings in the reported tests do not preclude the possibility of the occurrence of genetic effects of a different nature. The authors wish to thank Mr. M. SWANNET for his valuable technical assistance. * Laboratory of Geuelics, Deflartme~¢tof Radiobiology, C.E.N./S.C.K., B-24oo, Mol and ** Janssen Pharmaceutic~, D@artment of Toxicology, B-234o, Beerse (Belgium)
A. L~ONARD* R. VANDESTEENE** 1~. MARSBOOM**
I BATEMAN, A. J. AND S. S. EPSTEIN, Dominant lethal mutations in mammals in A. HOLLAENDER (Ed.), Chemical Mutagens, Principles and Meehodsfor their Detection, Vol. 2, Plenum, New York, ~97I, pp. 541-558. 2 BI~ITTINGE~, D., Die mutagene Wirkung yon Endoxan bei der Maus, Humagenellk, 3 (I956) I56-~65. ] BRUGMANS, J, P., :D, TI-IIENPONT, I. VAN WIJNGAAI~DEN, 0. F. VANPARIJS, V. L. SCtlUERMANS AND H. L. LAUWERS, Mebendazole in enterobiasis : radiochemical and pilot clinical study in 1,278 subjects, J, Am. Med. Ass., 2I 7 (z97r) 313-315. 4 CALLEAR, J. F. F., AND R. M. S. NEAV~, The clinical use of the anthehnintic mebendazole, Brit. Vet. J., 127 (I97 I) xli. 5 DE VI~igs, J. P., Een smakelijk wormmiddel veer paarden, Tijdschr. Diergeneesk., 95 (IO) (I97Z), 693. 6 FI~RLAFUN, E., L'oxyurose: traitement aneien et actuel, Bruxelles-Med., 51 (9) (z97z), 6o56o8. 7 LI~ONARD, A., Observations on meiotic chromosomes of the male mouse as a test of the potential mutagenieity of chemicals in mammals, in A. ttOLLAENDEI~ (Ed.), Chemical Mutageus, Principles c~ndMethods for their Detection, Vol. 3, Plenum, New York, x973, PP. 21-56. 8 LI~ONARD, A. AND G, LINDEN, Observation of dividing spermatocytes for chromosome aberrations induced in mouse spermatogonia by chemical mutagens, Mutation Res., 16 (I972) 297~OO. 9 MARSBOOM, R., Toxicologic studies on mebendazole, Toxicol Appl. Pharmaeol, 24 (1972) 371-377. ZO I{61-IRBORN, G., The activity of alkylating agents, I. Sensitive mutable stages in spermatogenesis and oogenesis, in F. ¥OGI~:LA~D G. RSHI~BOr~N(Eds.), Chemical Mutagenesis in Mammals and ~/an, Springer, Berlin, 197 o, pp. i48-i52, 294-316. Ii SCnLEIEIeMACI{ER, E., Ober den Einfluss von Trenimon und Endoxan auf die Meiose der mi~nnlichen Maus, Humangenetik, 3 (I965) 134-I55.
430
SHORT COMMUNICATIONS
I2 SCHLEIERMACHER, E., The activity of alkylating agents, II. Histological and cytogenetic findings in spermatogenesis, in F. VOGEL AND G. RGHRBORN (Eds.), Chemical 3l~tagenesis in Mammals and Man, Springer, Berlin, 197 ° pp. 3~7-34 I. 13 THIENPONT, D., O. F. VANPARIJS AND L. C. HERMANS, Mebendazole, a new p o t e n t drug against s y n g a m o u s t r a c h e a in turkeys, poultry, Science, 52 (1973) 1712-1714 • 14 VANPARIJS, O. F., AND D. THIENPOINT, Anthelminthische W i r k u n g des Mebendazols gegen h e m a t o d e n u n d zestoden bei Hunden, De~t. Tierarzt. Wschrift, 8o (1973) 32o 322. Received January
2 3 r d , 1974