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Mutagenicity tests with a surfactant formulation, toximul MP-8, in Drosophila and mouse bone marrow cells
Letters, 8 (1981) 235-239
Toxicology
Elsevier/North-Holland
235
Biomedical
Press
MUTAGENICITY TESTS WITH A SURFACTANT FORMULATION, TOXIMUL MP-8, IN DROSOPHILA AND MOUSE BONE MARROW CELLS
O.P.
KAMRA
Laboratory
and B.B. GOLLAPUDI*
of Radiation Biology, Biology Department,
Dalhousie University, Halifax, N.S. (Canada
B3H 451) (Received
January
19th, 1981)
(Accepted
January
21st, 1981)
SUMMARY
Toximul
MP-8, a commercial
Drosophila inducing mouse
melanogaster
sex-linked micronucleus
emulsifier
sperm
recessive
and
formulation,
mouse
lethal mutations
bone
was evaluated marrow.
for possible
The emulsifier
in Drosophila but produced
mutagenic
was weakly no noticeable
activity in effective
effects
in
in the
test.
INTRODUCTION
Pesticides have attracted a great deal of attention in genetic toxicology testing because of their widespread use and the resulting degree of human exposure. A number of these chemicals have been identified as genetically active [l]. However, little attention has been directed to the genetic safety of the solvents and emulsifiers used to facilitate the spraying of water-insoluble ingredients and carriers. Toximul MP-8, a petroleum-based commercial emulsifier formulation, was extensively used some years ago for the aerial spraying of the organophosphorous insecticides, fenitrothion and phosphomidon, to control the spruce budworm infestation in the forests of Eastern Canada and North-Eastern United States. Cracker et al. [2] implicated Toximul MP-8 as the agent causing signs in mice similar to those of Rey’s Syndrome, an often fatal childrens’ disease prevalent in
* Present
address:
Institutes
of Health,
Abbreviations:
Laboratory
MN,
Bethesda,
of Cellular
and Molecular
Biology,
National
Cancer
micronuclei;
NCE,
normochromatic
erythrocytes;
erythrocytes.
0378-4274/81/OOOC-0000/$02.50
Institute,
National
MD 20014 (U.S.A.).
0 Elsevier/North-Holland
Biomedical
Press
PCE,
polychromatic
236
heavily
sprayed
areas.
Preliminary
highly toxic and clastogenic Tests have therefore
MATERIALS
to human
been undertaken
in two widely used and recessive lethal mutation micronucleus
experiments peripheral
indicated
that Toximul
blood lymphocytes
on the mutagenic
potential
MP-8 was
in culture
of Toximul
[3]. IMP-8
very sensitive in vivo test systems, i.e., the sex-linked assay in the mature sperm of D. melanogaster and the
test in mouse
bone marrow.
AND METHODS
Toximul MP-8 (Chas. Tenant and Co. Canada Ltd., Toronto, Ontario) was supplied by Dr. J.F.S. Cracker (I.W.K. Hospital for Children in Halifax, N.S.). (a) Sex-linked
recessive lethal
mutation
test in Drosophila
7-day-old males, genetic constitution X’2yB/y + Y, were either injected with Toximul solutions (0.2 ~1) prepared in 0.7% NaCl or fed for 24 h on the emulsifier in 5% sucrose solution. The feeding technique described by Traiit [4] was used; males were starved for 24 h prior to the start of feeding. The treated males were individually mated to four virgin females, genetic constitution ysc”In49sc*;bw;stpP, and the treated sperm was sampled in one 2-day brood. The FI progeny was allowed to mass-mate and the inseminated females (each representing one treated paternal X-chromosome) were individually placed in a culture vial. Sex-linked recessive lethal mutations were detected in the F2 from the absence of bar-eye males in any vial with at least 10 normal (round-eye) males. All other suspected lethals were retested and verified in the FJ. Statistical comparisons were made by the contingency chi-square test. (6) Micronucleus
test in the mouse
Out-bred ICT-Swiss female mice, aged IO-12 weeks, were injected (0.5 ~1) i-p., with two doses (24 h apart) of freshly prepared (in 0.9% NaCl) Toximul MP-8 solutions. The animals were killed at various intervals after the second injection (see Table II). Bone marrow smears were prepared by the method of Schmid [5] and stained in Giemsa [6]. A minimum of 2000 PCE and a corresponding number of NCE were scored from each animal for the presence of MN. The frequencies of micronucleated cells were expressed per 100 PCE scored. The percentage PCE was calculated as a ratio of (PCE x lOO)/(PCE + NCE). Statistical comparisons of the frequencies of total micronucleated cells between control and treated groups were performed by the two-sample t-test.
237
RESULTS
AND DISCUSSION
The survival
of the treated
Drosophila
males in both the injection
series ranged from approx. 90% (at the lowest concentration) concentration). The flies could tolerate higher concentrations
and feeding
to 25% (at the highest of the emulsifier when
fed than when injected. The mutagenicity tests in Drosophila are summarized in Table I. Although the controls of these experiments had a zero mutation frequency, the accumulated spontaneous recessive lethal frequency for this stock (historical controls) was 0.08%; the treatments were compared to this control value. A weak mutagenic response was noticed in some of the treated groups. However, there was no apparent dose-effect relationship. In feeding experiments, significant increases in the mutation frequency were noticed at 0.125% and 0.5%, but not at 0.25%. When the totals treated in injection and feeding series were compared with the controls, a significant increase in the mutation frequency (P
TABLE
I
EFFECT
OF TOXIMUL
MUTATIONS Toximul
MP-8
ON THE
IN THE DROSOPHILA
MP-8
INCIDENCE
OF SEX-LINKED
MELANOGASTER
Lethals/total
chromosomes
LETHAL
% Lethals
tested
(To) Injection
(i.p.)
0.005
O/610
0
0.01
2/438
0.46
0.02
O/389
0
0.04
31389
0.77”
0.05
2/602
0.33
0. IO
o/533
0
7/2961
0.24
0.125
6/686
0.87h
0.25
2/592
0.34
0.50
3/616
0.49a
11/1894
0.58”
6/7947
0.08
Total
RECESSIVE
SPERM
injected
Feeding
Total
fed
Controls’ a P
b P
c Total accumulated
controls
includes
O/403 in the injection
series and O/386 in the feeding experiment.
238
Ames test (a bacterial mutagenicity assay capable of detecting carcinogens as mutagens (8)). The emulsifier proved to be cytotoxic as assessed from the dosedependent reduction in the proportion of PCE in the bone marrow preparations made 30 h after the first injection (Table II). However, there was no effect of the treatment on the incidence of the micronucleated cells in these animals. Maier and Schmid [9] showed that clastogens, which are also cytotoxic, increase MN in the bone marrow only after the recovery of the celis from the mitotic delays induced by the cytostatic action of these agents. In order to explore this possibility, animals injected with Toximul (0.4 &‘kg body weight) were killed at various intervals after treatment and the bone marrow smears screened for micronucleated cells. The proportion of PCE, in the treated animals, gradually reached the control levels by 54 h indicating a complete recovery. Although the statistical analysis yielded a significant increase of micronucleated cells at 48 h, the data presented in Table II does not support the effect of the emulsifier on the incidence of micronucleated cells. Thus, Toximul MP-8 may be classified as weakly positive in the Drosophila test system and negative in the micronucleus test. Petropolis and Kamra [3] have observed that treatment of human peripheral blood lymphocytes from the start of the culture period induced chromosomal damage. We have also noticed ~hromosomal fragmentation and pulverization in human lymphocyte cultures treated for 2 h in the middle of their DNA synthetic period (unpublished). The potent clastogenic effect noticed in this in vitro system was, thus, not evident in any of the two in vivo systems reported in the present study. No clear explanation can be offered for this differential activity of Toximul MP-8. Differences in the metabolic fate of the genetically active component of the emulsifier in different biological systems may be considered as one of the causative factors for this differential activity. Toximul MP-8 is a complex mixture of several non-ionic-anionic blends of dodecyl benzene sulphates and polyoxyethylene ethers (Dr. Safe, University of It would be worthwhile to identify the Guelph, personal communication). mutagenic constituent(s) in this complex mixture. TABLE
II
EFFECT
OF TOXIMUL
Toximul
Number
MP-8 ON THE MOUSE --..h after Total
MP-8 (ml/kg
of
I St
body WI .)
animals
injection
BONE MARROW Vo PCE
PCE
CELLS
% PCE-
%NCE-
MN
MN
Total
Saline
3
30
10,914
54.8
0.19
0.05
0.24
0.2
3
30
X,277
47.4
0.27
0.13
0.40
0.4
3
30
7,756
39.8
0.23
0.17
0.40
0.8 0.4
2 6
30 42
5,317 13,663
35.9 41,7
0.15 0.12
0.23 0.18
0.38 0.31
3
48
7,118
52.5
0.24
0.18
0.42;1
3
54
9,897 ___.-__I___
53.7 _...__-
0.18
0.07
0.25 .._~
a P
MN
239
ACKNOWLEDGEMENTS
This study was supported in part through research grant A-4892 from the National Science and Engineering Research Council. Technical assistance of Mrs. Janet Wilson is gratefully acknowledged. REFERENCES S.S. Epstein J.F.S.
interaction P.N.
and MS.
Cracker,
K.R.
Rozee,
The Mutagenicity R.L.
Ozere,
SC.
as a cause of fatty visceral changes
Petropolis
culture:
Legator,
and O.P.
A preliminary
H. Tram,
Method
Kamra,
Cytotoxic Mutation
chemical
and
MIT Press, Cambridge, 0.
Hutzinger,
and encephalopathy
investigation,
of feeding
of Pesticides, Digout
effects
and viral
Lancet,
II (1974) 22.
in the mouse,
of Toximui
MP-8
1971.
Insecticide
in human
lymphocytes
in
Res., 58 (1978) 287.
mutagens
to adult Drosophila,
Drosophila
Inform.
Serv., 52
(1977) 168. W. Schmid, B. Gollapudi test, Mutation
The micronucleus and O.P.
Kamra,
test, Mutation Application
Res., 31 (1975) 9. of a simple giemsa staining
method
in the micronucleus
Res., 64 (1979) 45.
P. Maier and W. Schmid,
Ten model mutagens
evaluated
by the micronucleus
test, Mutation
Res., 40
(1976) 325. D. Jenssen and C. Ramel, for the prediction
The micronucleus
of carcinogenicity
B.N.Ames, J. McCann ~ff~~o~e~/a/rnarnrnalian
and E. Yamasaki, microsome
test as a part of a short-term
evaluated
by 143 agents
Methods
mutagenicity
tested,
for detecting
test, Mutation
mutagenicity
mutation carcinogens
test program
Res., 75 (1980) 191. and mutagens
Res., 31 (1975) 347.
with
Toxicology
Elsevier/North-Holland
235
Biomedical
Press
MUTAGENICITY TESTS WITH A SURFACTANT FORMULATION, TOXIMUL MP-8, IN DROSOPHILA AND MOUSE BONE MARROW CELLS
O.P.
KAMRA
Laboratory
and B.B. GOLLAPUDI*
of Radiation Biology, Biology Department,
Dalhousie University, Halifax, N.S. (Canada
B3H 451) (Received
January
19th, 1981)
(Accepted
January
21st, 1981)
SUMMARY
Toximul
MP-8, a commercial
Drosophila inducing mouse
melanogaster
sex-linked micronucleus
emulsifier
sperm
recessive
and
formulation,
mouse
lethal mutations
bone
was evaluated marrow.
for possible
The emulsifier
in Drosophila but produced
mutagenic
was weakly no noticeable
activity in effective
effects
in
in the
test.
INTRODUCTION
Pesticides have attracted a great deal of attention in genetic toxicology testing because of their widespread use and the resulting degree of human exposure. A number of these chemicals have been identified as genetically active [l]. However, little attention has been directed to the genetic safety of the solvents and emulsifiers used to facilitate the spraying of water-insoluble ingredients and carriers. Toximul MP-8, a petroleum-based commercial emulsifier formulation, was extensively used some years ago for the aerial spraying of the organophosphorous insecticides, fenitrothion and phosphomidon, to control the spruce budworm infestation in the forests of Eastern Canada and North-Eastern United States. Cracker et al. [2] implicated Toximul MP-8 as the agent causing signs in mice similar to those of Rey’s Syndrome, an often fatal childrens’ disease prevalent in
* Present
address:
Institutes
of Health,
Abbreviations:
Laboratory
MN,
Bethesda,
of Cellular
and Molecular
Biology,
National
Cancer
micronuclei;
NCE,
normochromatic
erythrocytes;
erythrocytes.
0378-4274/81/OOOC-0000/$02.50
Institute,
National
MD 20014 (U.S.A.).
0 Elsevier/North-Holland
Biomedical
Press
PCE,
polychromatic
236
heavily
sprayed
areas.
Preliminary
highly toxic and clastogenic Tests have therefore
MATERIALS
to human
been undertaken
in two widely used and recessive lethal mutation micronucleus
experiments peripheral
indicated
that Toximul
blood lymphocytes
on the mutagenic
potential
MP-8 was
in culture
of Toximul
[3]. IMP-8
very sensitive in vivo test systems, i.e., the sex-linked assay in the mature sperm of D. melanogaster and the
test in mouse
bone marrow.
AND METHODS
Toximul MP-8 (Chas. Tenant and Co. Canada Ltd., Toronto, Ontario) was supplied by Dr. J.F.S. Cracker (I.W.K. Hospital for Children in Halifax, N.S.). (a) Sex-linked
recessive lethal
mutation
test in Drosophila
7-day-old males, genetic constitution X’2yB/y + Y, were either injected with Toximul solutions (0.2 ~1) prepared in 0.7% NaCl or fed for 24 h on the emulsifier in 5% sucrose solution. The feeding technique described by Traiit [4] was used; males were starved for 24 h prior to the start of feeding. The treated males were individually mated to four virgin females, genetic constitution ysc”In49sc*;bw;stpP, and the treated sperm was sampled in one 2-day brood. The FI progeny was allowed to mass-mate and the inseminated females (each representing one treated paternal X-chromosome) were individually placed in a culture vial. Sex-linked recessive lethal mutations were detected in the F2 from the absence of bar-eye males in any vial with at least 10 normal (round-eye) males. All other suspected lethals were retested and verified in the FJ. Statistical comparisons were made by the contingency chi-square test. (6) Micronucleus
test in the mouse
Out-bred ICT-Swiss female mice, aged IO-12 weeks, were injected (0.5 ~1) i-p., with two doses (24 h apart) of freshly prepared (in 0.9% NaCl) Toximul MP-8 solutions. The animals were killed at various intervals after the second injection (see Table II). Bone marrow smears were prepared by the method of Schmid [5] and stained in Giemsa [6]. A minimum of 2000 PCE and a corresponding number of NCE were scored from each animal for the presence of MN. The frequencies of micronucleated cells were expressed per 100 PCE scored. The percentage PCE was calculated as a ratio of (PCE x lOO)/(PCE + NCE). Statistical comparisons of the frequencies of total micronucleated cells between control and treated groups were performed by the two-sample t-test.
237
RESULTS
AND DISCUSSION
The survival
of the treated
Drosophila
males in both the injection
series ranged from approx. 90% (at the lowest concentration) concentration). The flies could tolerate higher concentrations
and feeding
to 25% (at the highest of the emulsifier when
fed than when injected. The mutagenicity tests in Drosophila are summarized in Table I. Although the controls of these experiments had a zero mutation frequency, the accumulated spontaneous recessive lethal frequency for this stock (historical controls) was 0.08%; the treatments were compared to this control value. A weak mutagenic response was noticed in some of the treated groups. However, there was no apparent dose-effect relationship. In feeding experiments, significant increases in the mutation frequency were noticed at 0.125% and 0.5%, but not at 0.25%. When the totals treated in injection and feeding series were compared with the controls, a significant increase in the mutation frequency (P
TABLE
I
EFFECT
OF TOXIMUL
MUTATIONS Toximul
MP-8
ON THE
IN THE DROSOPHILA
MP-8
INCIDENCE
OF SEX-LINKED
MELANOGASTER
Lethals/total
chromosomes
LETHAL
% Lethals
tested
(To) Injection
(i.p.)
0.005
O/610
0
0.01
2/438
0.46
0.02
O/389
0
0.04
31389
0.77”
0.05
2/602
0.33
0. IO
o/533
0
7/2961
0.24
0.125
6/686
0.87h
0.25
2/592
0.34
0.50
3/616
0.49a
11/1894
0.58”
6/7947
0.08
Total
RECESSIVE
SPERM
injected
Feeding
Total
fed
Controls’ a P
b P
c Total accumulated
controls
includes
O/403 in the injection
series and O/386 in the feeding experiment.
238
Ames test (a bacterial mutagenicity assay capable of detecting carcinogens as mutagens (8)). The emulsifier proved to be cytotoxic as assessed from the dosedependent reduction in the proportion of PCE in the bone marrow preparations made 30 h after the first injection (Table II). However, there was no effect of the treatment on the incidence of the micronucleated cells in these animals. Maier and Schmid [9] showed that clastogens, which are also cytotoxic, increase MN in the bone marrow only after the recovery of the celis from the mitotic delays induced by the cytostatic action of these agents. In order to explore this possibility, animals injected with Toximul (0.4 &‘kg body weight) were killed at various intervals after treatment and the bone marrow smears screened for micronucleated cells. The proportion of PCE, in the treated animals, gradually reached the control levels by 54 h indicating a complete recovery. Although the statistical analysis yielded a significant increase of micronucleated cells at 48 h, the data presented in Table II does not support the effect of the emulsifier on the incidence of micronucleated cells. Thus, Toximul MP-8 may be classified as weakly positive in the Drosophila test system and negative in the micronucleus test. Petropolis and Kamra [3] have observed that treatment of human peripheral blood lymphocytes from the start of the culture period induced chromosomal damage. We have also noticed ~hromosomal fragmentation and pulverization in human lymphocyte cultures treated for 2 h in the middle of their DNA synthetic period (unpublished). The potent clastogenic effect noticed in this in vitro system was, thus, not evident in any of the two in vivo systems reported in the present study. No clear explanation can be offered for this differential activity of Toximul MP-8. Differences in the metabolic fate of the genetically active component of the emulsifier in different biological systems may be considered as one of the causative factors for this differential activity. Toximul MP-8 is a complex mixture of several non-ionic-anionic blends of dodecyl benzene sulphates and polyoxyethylene ethers (Dr. Safe, University of It would be worthwhile to identify the Guelph, personal communication). mutagenic constituent(s) in this complex mixture. TABLE
II
EFFECT
OF TOXIMUL
Toximul
Number
MP-8 ON THE MOUSE --..h after Total
MP-8 (ml/kg
of
I St
body WI .)
animals
injection
BONE MARROW Vo PCE
PCE
CELLS
% PCE-
%NCE-
MN
MN
Total
Saline
3
30
10,914
54.8
0.19
0.05
0.24
0.2
3
30
X,277
47.4
0.27
0.13
0.40
0.4
3
30
7,756
39.8
0.23
0.17
0.40
0.8 0.4
2 6
30 42
5,317 13,663
35.9 41,7
0.15 0.12
0.23 0.18
0.38 0.31
3
48
7,118
52.5
0.24
0.18
0.42;1
3
54
9,897 ___.-__I___
53.7 _...__-
0.18
0.07
0.25 .._~
a P
MN
239
ACKNOWLEDGEMENTS
This study was supported in part through research grant A-4892 from the National Science and Engineering Research Council. Technical assistance of Mrs. Janet Wilson is gratefully acknowledged. REFERENCES S.S. Epstein J.F.S.
interaction P.N.
and MS.
Cracker,
K.R.
Rozee,
The Mutagenicity R.L.
Ozere,
SC.
as a cause of fatty visceral changes
Petropolis
culture:
Legator,
and O.P.
A preliminary
H. Tram,
Method
Kamra,
Cytotoxic Mutation
chemical
and
MIT Press, Cambridge, 0.
Hutzinger,
and encephalopathy
investigation,
of feeding
of Pesticides, Digout
effects
and viral
Lancet,
II (1974) 22.
in the mouse,
of Toximui
MP-8
1971.
Insecticide
in human
lymphocytes
in
Res., 58 (1978) 287.
mutagens
to adult Drosophila,
Drosophila
Inform.
Serv., 52
(1977) 168. W. Schmid, B. Gollapudi test, Mutation
The micronucleus and O.P.
Kamra,
test, Mutation Application
Res., 31 (1975) 9. of a simple giemsa staining
method
in the micronucleus
Res., 64 (1979) 45.
P. Maier and W. Schmid,
Ten model mutagens
evaluated
by the micronucleus
test, Mutation
Res., 40
(1976) 325. D. Jenssen and C. Ramel, for the prediction
The micronucleus
of carcinogenicity
B.N.Ames, J. McCann ~ff~~o~e~/a/rnarnrnalian
and E. Yamasaki, microsome
test as a part of a short-term
evaluated
by 143 agents
Methods
mutagenicity
tested,
for detecting
test, Mutation
mutagenicity
mutation carcinogens
test program
Res., 75 (1980) 191. and mutagens
Res., 31 (1975) 347.
with