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DRUG AND CHEMICAL TOXICOLOGY, 14(4), 353-373 (1991)
TOXICITY AND MUTAGENICITY OF THE MOLLUSCICIDAL PLANT AMBROSIA MARITIMA L.
Alard, F a * , Stievenart. C . * , Vanparys, P h . * * , Thilemans, L.** and Gaerts, S.* * Institute of Tropical Medicine Veterinary Departement Nationalestraat, i 5 5 , B-2000 Antwerp, Belgium * * Janssen Pharmaceutica N.V. Department of Toxicology B-2340 Beerse, Belgium
ABSTRACT The acute and subchronic toxicity of the
mol-
luscicidal plant, Ambrosia maritima L., has been tested on rats.
No toxic signs could be detected neither af-
ter oral administration of 5 g/kg of dried leaves of the plant as a powder or as a methanoiic extract, nc3r after the incorporation of 50,300 ppm powdered leaves in the feed during 4 weeks. Using an aqueous extract of the plant material of A.maritima or using ambrosin, one of the active mo1.-
luscicidal components of the plant, no mutagenic acti-
353 Copyright 0 199 1 by Marcel Dekker, Inc.
354
ALARD ET AL.
vizy could be detected in the S.typhimurium strai:;s
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TA97, TA 98, TAl538, TAlOO and TA1535.
INTRODUCTION
The
molluscicidal
activity
of
k.maritima
L.
(Asteraceae) has been repeatedly proven in irhc labaratory'
and in the field'
'.
Doses of 35 to 7C myil of
the dried leaves of the plant were able t o kill m r e than 90 % of the snails in irrigation canals and drairis ?he secondary metabolites of t h c plant. tie
in Egypt'.
sesquiterpene lactones, are the active principliis ?resent in the leaves, flowers and seeds of A.maritima. Ambrosin and damsin are the two most important
ses-
quiterpene lactones' whose molluscicidal activity has I
been proven
.
The toxicity of d.maritima has been eva-
luated in fish, crustacea and algae:. It was showr? thst the plant had a very low toxicity to aquatic ncn-target organisms and that it was not toxic when used at the molluscicidal concentrations of 35-70 mg/l. Up
to
however, on
now the
virtually mammalian
no
data
toxicity
were of
available, d.maritima.
Since these data are necessary before the plant can be used on a larger scale in the field, these experiments were set up in order to study the mutagenic potential and the acute and the subchronic toxicity on rats.
TOXICITY AND MUTAGENICITY OF AMBROSIA MARITIMA L.
MATERIAL-=
355
METHODS
A?nbro~*. mari tima. Drug and Chemical Toxicology Downloaded from informahealthcare.com by Cornell University on 11/18/14 For personal use only.
Seeds of A.maritima from Egyptian origin were sown in Senegal.
The plants were dried and used within the
first year after harvesting.
Acute toxicity tests were
carried out using a powder or an extract of A.maritima. The powder was made from the leaves of A.maritima : 150 mg was suspended in 1 mi of an aqueous solution of 1 % sodium carboxymethyl-celluiose. The extract of the plant was made by macerating the powder of the ieaves in methanol during 4 hours. Afterwards the suspension was filtered to separate the solxtion from the sediment.
This latter was percolated
during 14 hours in methanol.
The percolate and the
solution were than distilled at a temperature of ~ 4 0 ° C . The residue was dissolve2 in a mixture of 2 @ % polyethylene glycol water.
(PEGQOO) and 4 % DMSO in distilled
One ml of this solution contained the eqai-
valent of 0.6 g dried leaves. The content of sesquiterpene lactones of the methanolic extract of A.maritima was determined by column chromatography (silica gel), essentially as described by Bohlmann et al.'. Ambrosin as well as water extracts from A.maritima were tested
ir,
the Ames test.
Ambrosin was obtained as
3 56
ALARD ET AL.
a gift from Dr. J. Ziesche (Institute for Organic Chemistry, Technical University of Berlin, D - 1000 Berlin
12, West Germany) and was dissolved in ethanol.
To
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obtain the water extract, 2 g powder mixture from flowers, seeds and leaves of the plant were extracted with 14 ml distilled water for 48 h. fuging for
10 min. at
900 g
and for
After centri-
5 min. at 1000
g, the water fraction was transferred into a vial and tested directly.
In order to know the amount of plant
material dissolved in water, the pellet was evaporated at 12O'C during 72 h.
The residue was then weighed and
substracted from the initial amount of dry material.
Acute Toxicity Studies Albino Wistar rats, weighing about 200 g at the start of the experiment, were used for the toxicity studies.
Since it was evident from preliminary obser-
vationsic that
A.maritima
was
probably
very
little
toxic, only one dose (5 to 5.6 g/kg) was administered (Table 1). The absence of toxic signs at this dose level is generally considered as a guarantee for the toxicological acceptability of the test substance. Due to the large volume of the powder, it was divided in two parts, two thirds being administered first and the other third 24 hours later. administered as a
The extract, however, was
single dose.
The
control rats
357
TOXICITY AND MUTAGENICITY OF AMBROSIA MARITIMA L.
TABLE 1
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Acute and Subchronic Toxicity Tests of A.maritima in Rats Toxicity Test Acute Subchronic
....................................................... Ambrosia mari tima - Presentation - Dose(s)
powder
extract
5 g/kg
5.3
5.6 g/kg*
-
3,125 12,500 5 0 , 0 0 0 ppm
p.0.
p.0.
p.0. (feed)
Administration
No. of Rats Used 12 - Control + male + female 8 12 - Test + male 10 + female Duration of test (days1
13
powder
-
12
5
12 12
5 15 15
10 13
28 __._____
* extract, equivalent to
5 . 3 and 5.6 g/kg respectively
for the males and females
received the same volume of the solvent as the test rats in an aqueous solution of 1 % sodium carboxymethylcellulose or a mixture of 2 0 % PEG400
and 4 %
DMSO for the experiment using the powder or the extract of A.maritima respectively.
The body weights of
the rats were followed during the course of the experiment and autopsy was carried out 13 days after the administration of the plant material. (lungs,
spleen,
liver,
heart,
All the organs
pancreas,
358
ALARD ET AL.
kidneys, brain, thymus, adrenals, thyroids and gonads) were examined in detail and weighed.
Nineteen serum-
and 10 blood parameters were examined : sodium, potasDrug and Chemical Toxicology Downloaded from informahealthcare.com by Cornell University on 11/18/14 For personal use only.
sium, chloride, calcium, inorganic phosphate, total protein, albumin, haptoglobin, glucose, cholesterol, triglycerides, phospholipids,
blood
creatinine,
alkaline
total
bilirubin,
urea
nitrogen,
phosphatase.
aspartate aminotransaminase, alanine aminotransaminase, cholinesterase, haematocrit, haemoglobin, red
blood
cells, white blood cells, thrombocytes, normoblasts, mean ce'll volume, mean cell haemoglobin, mean cell haemoglobin concentration, differential count of white blood cells.
Subchronic Toxicity Tesi Forty rats weighing between 120 and 130 g were used for this test.
Powder of A.maritima leaves was
thoroughly mixed in the feed (meal) at doses of 3,125, 12,500 and 50,000 ppm mixer
(Table.1) using an industrial
(Collette MPH 150). Control rats received the
same feed without A.maritima. dually housed and fed
All rats were indivi-
ad libitum.
The animals were
regularly weighed and their food and water consumption was measured during the course of the experiment.
A
urine sample was taken on day 25 after the start of the experiment and examined for the presence of epithelial
TOXICITY AND MUTAGENICITY OF AMBROSIA MARITIMA L.
359
cells, casts, crystals, red and white blood cells and bacteria.
The following parameters were determined
:
creatinine, urobilinogen, pH, volume, specific gravity, Drug and Chemical Toxicology Downloaded from informahealthcare.com by Cornell University on 11/18/14 For personal use only.
proteins, glucose, acetone, bilirubin and occult blood. Autopsy was carried out on day 28.
The examination of
the organs and haematoloqical and serological parameters was similar as for the acute toxicity test.
Statical Analysis of Data Statistical analysis of the data of the toxicity studies was carried out using the Mann Whitney U test or the chi square test.
Salmonella Revertant Mutation Tests Bacterial strains The
S.typhimurium
strains TA97, TA98, TA1538,
TAlOO and TA1535 were kindly provided by Prof. B.N. Ames, Berkeley, University of California, U.S.A.
The
tester strain genotypes, as described by Ames et al.", were confirmed before the start of each study.
The
spontaneous reversion of the tester strains used in this study were within our historical control range. 2-nitrofluorene, sodium
azide
and
2-aminoanthracene
(Janssen Chimica, Belgium) were used as positive controls.
ALARD ET AL.
360
Metabolic activatior? system In all the tests, a rat liver homogenate (S9-mix) was used as metabolic activation system.
Adult male
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Wistar rats were injected once intraperitoneally with 200 mg/kg Aroclor 1254 (Monsanto, U.S.A.) in order to
induce drug-metabolizing enzymes.
The preparation of
the S9-mix was done according to the methods of Ames et 'C
al." Concentrations of S9 per plate were 20 and 50 p l .
Plate incorporation tests Two experiments were performed using the standard ? C
plate incorporation test described by Ames et al.'.. The
plate
incorporation test
with
ambrosin was
performed in triplicate using two S.typhimurium strains TA98 and TA100.
The test was done in the presence and
in the absence of a rat liver metabolic activation sysTop agar, 0.1 ml of bacteria, S9-mix (20 p l /
tem.
plate) if needed and 15.7, 31.3, 62.5, 125, 250, 500 and 1000 pq ambrosin/plate were sequentially added, shaken and poured onto agar plates. The plate incorporation test with the water fraction of plant material was performed using the five standard strains TA97, TA98, TA1535, TAlOO and TA1535 in the presence and in the absence of rat liver S9-mix. The test was performed in triplicate and was repeated once.
Top agar, 0.1 ml of bacteria, S9-mix (20 and 50
TOXICITY AND MUTAGENICITY OF AMBROSIA MARITIMA L.
361
ul/plate) if needed and the water fraction containing 78.1, 156.3, 312.5, 625, 1250 and 5000 pg piant ma-
terial per plate were sequentially added, shaken and
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poured onto agar plates. In both tests, the number of revertant colonies were counted after 48 h. of incubation in the dark at 37'C.
The results were considered as positive if there
was at least a twofold increase in the mean number of revertants with one of the strains TA37, TA38 or TAlOO or a threefold increase in the mean number of revertants with one of the strains TA1535 or TA1538.
The
increase in revertants must be dose related and reproduc i ble .
RESULTS
Toxicity Tests The sesquiterpene lactone content of the dried leaves of A.maritima, which were used in the toxicity tests, was determined by column chromatography (silica gel). It was shown to be 1.4 to 1.6 % of the dry weight of the plant.
No significant differences in weight gain were
present between treated and control animals in any of the experiments carried out (Tables 2 and 3 ) .
ALARD ET AL.
362
TABLE 2 Change in Eody Weights of Rats during the A c l i t s Toxicity Test using a Powder or an Extract of A.maritima
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Mean Body Weight Gain
(g) 2 5.D.
A . mciri tina
powder
extract
-__---------------------------------------------------Male Rats i'ontro 1 Test
6 6 . 3 2 13.;: 63.3 2 6 . 4 " '
8 2 . 6 z 212 8 ,!i : 30.3 2 13.J
Female Rats Control Test
36.3 2 9.5 31.0 2 6.9"
2 7 . 2 f 8.4: 32.3 z 10.0
_.___
*
S.'
___.
~
~
gain of weight from day 1 to day 12 not significantly different from the controi rats
TABLE 3 Change in Body Weights of Rats during the Subchronic Toxicity Test using A.maritima, incorporated iE t h e Feed
--------------Male Rats Contro 1 Treated 3 , 1 2 5 ppm 1 2 , 5 0 0 ppm 50,000 ppm Female Rats Contro 1 Treated 3 , 1 2 5 ppm 1 2 , 5 0 0 pprn 5 0 , 0 0 0 ppm l i
128.82 2.5
338.8221.8
200
k22.1
128.8 128.82 128.82
~6 . 7 5.9 5.6
323.2218.2 335.4-+22 302,8216
194.4217.2 206.6217.2' ' 1 7 4 216.1'
118.6t16.9
236.8528.6
118.2213.1
118.4216.7 118.8214 118.6213.9
240.2k33.8 240,8216.6 229.8212.1
1 2 1 . 8 k 2 0 . 1Y 122 216.7' 113.22 7.5"
Y :
:
'
not significantly different from the controls
S
TOXICITY AND MUTAGENICITY OF AMBROSIA MARITIMA L.
363
No adverse signs were observed during the course of any of the tests and no macroscopic lesions were present
iZ
the organs of the treated animals. The presence of sofr
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feces had been noted in 4 of the 30 treated rats used in the subchronic toxicity test.
This sign was only
temporarily present and disa2peared after two days. The meaii body and organ weights of the rats treated with an extract of A.maritima in the acute toxicity test are shown in Table 4. In Table 5 only the weights of those organs are presented, where some differences between treated and control rats in the subchronic toxicity test were remarked. Almost
no
significant differences
in
haemato-
logical and serum chemistry values were observed in the acute and subchronic toxicity tests.
The serum para-
meters, for which some significant results were obtained, are summarized in Tables 6 and 7 .
The analysis
of the urine, taken on day 25 in the subchronic toxi-
city test, didnot show any
significant differences
between treated and control groups.
Salmoneila Revertant Mutation T e . s The results of the Salmonella revertant mutation tests with ambrosin are shown in Table 8. Ambrosin was not mutagenic to the strains TA98 and TAlOO when tested up to the concentration of 1 0 0 0 pg/plate.
The inclu-
ALARD ET AL.
364
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TABLE 4 Mean Body and Organ Weights of Rats, treated with an Extract of A.maritirna (Acute Toxicity Test) - ~ _ _ . . ___.. .-_ Male Rats Female Rats ...................................... Weight Control Treated Control Treated
....................................................... 311 2130 6898 Spleen 828 2656 Liver 13641 43760 Kidneys (mg) 2436 (mg/kg) 7849 Brain (mg) 1821 5877 (mg/kg) Thymus (mg) 459 (mg/kg) 1475 Gonads (mg) 2677 8642 (mg/kg) _____ significance computed by tailed) : * p < .05 (9)
Body Lungs
(mg) (mg/kq) (mg) (mg/kg) (mg) (mg/kg)
250 1980 7959 681 2719 986 1 37842 1762 7057 1773 7110 407 1632 135 540
298 2004 6722 749 2513 13278 44432 2312 7746 1833 6174 507 1700 2613 8795 ____
Mann-Whitney
U
240 1762 7381 649 2711 9724 40573 1735 7258 1756 7357 424 1771" 122 509 -
test
(two
sion of a rat liver metabolic activation system did not alter
the
positive
mutagenic
potential
of
ambrosin.
The
controls 2-nitrofluorene and sodium azide,
which are direct acting mutagens, caused a strong increase in the number of revertant colonies in the strains TA98 and TA100, respectively. The indirect mutagen 2-amino-anthracene, which is only mutagenic after metabolic activation, also caused a strong increase in the number of revertants in both strains. Ambrosin was found to be slightly toxic to the bacteria at the concentration level of 1000 pg/glate.
TOXICITY AND MUTAGENICITY OF AMBROSIA MARITIMA L.
365
TABLE 5
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Mean Body and selected Organ Weights of Rats which received A.maritima in the Feed during 28 says (Subchronic Toxicity Test) 5 . A . Male Rats ______.
A.maritima in Feed (ppmj
Weight
Control
3.125
12.500
50.009
329 738 2247 1948 5949 2474 7558
323 776 2393 1854 5757 2750 8540
335 784 2350 1980 591 1 2547 7602
333 703 2327 1838* 6086 2658 8764*
_____-___-----____-------------------_-_______------_Body (9) Spleen (mg) (mg/kg)
Brain
(mg) (mg/kg)
Gonads (mg) img/kg) ___
.______-.______
significance computed tailed) : * p ( .05
5.B.
by
Mann-Whitney
U
.~
test
(two
Female Rats A.maritima in Feed (ppm)
Weight
Contro 1
3.125
12.500
50.000
237 673 2847 1730 7337 155 649
240 678 2746 1841 7709 142 585
241 668 2752 1765 7335 138 572
230 553* 2413'* 1708 7447 140 611
--__-__-----------------------------------------------Body !gj Spleen (mg) (mg/kg) (mg) (mg/kg) Gonads (mg) (mg/kgj
Brain
significance computed by Mann-Whitney tailed) : * p < . 0 5 * * p < .01
U
test
(two
Table 9 shows the results of the Salmonella revertant mutation tests with the water fraction of the plant material.
No mutagenic activity was found with the
water fraction towards the strains TA97, TA98, TA1538,
3 66
ALARD ET AL.
TABLE 6
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Selected Haematological and Serum Chemistry Values of Rats, treated with an Extract of A.maritima (Acute Toxicity Test) Male Rats
Female
rats ......................................... Control Treated Control Treated -------------_-----------------------------------------
parameter
HCT HGB RBC WBC TOP
36.1 12.8 Cg/pl) . (lO,/mmJ) 7.32 (10'/mm') 8.3 (9%) 6.3 3.3 ALB (9%) 23.9 BUN (mg%) 329 ALP (u/l) -______ (%)
36.1
38.0 13.3
12.7
7.32 10.1 5.9** 3.2 23.9
267*
38.1 i3.4
7.76
7.75
9.0 6.6 3.6 22.6 154
8.6 6.5 ?.4*
lS.EC** 157 .-
~
HCT : haematocrit; HGB : haemoglobin; RBC : red blood cells; WBC : leucocytes; TOP : total protein; ALE : albumin; BUN : blood urea nitrogen; ALP : alcalic phosphatase significance computed by Mann-Whitney U test (two tailed) : * p < .05 * * p < .01
TAlOO and TA1535 in the presence and in the absence of a rat liver metabolic activation system (20 te).
111
S9/pla-
Higher concentrations of S9 (50 pl/plate) did not
induce an increase in the number of revertant colonies.
DISCUSSION
These results show that there are no signs of toxicity
after the administration of a single dose of
5 g Ambrosia maritima per kg bodyweight as a powder or
TOXICITY AND MUTAGENICITY OF AMBROSIA MARITIMA L.
TABLE
367
7
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Selected Haematological and Serum Chemistry Values of Rats in the Subchronic Toxicity Test using A.mar-itima in the Feed 7 . A . Male Rats
A.maritima in Feed (ppm)
Contro1
Parameter
3.125
12.500 50.000 ____------_________-----__------__--------------_-
HCT HGB RBC WBC TOP
ALB
40.0 14.0 7.35 9.2 6.3 3.6 16.4 388
(%)
Cg/pl) . (lO:/mmi j (10;/mm') (9%) (g%)
BUN (mg%)
ALP (u/l) -
_.__ -.--..I--.-._.
41.0 14.2 7.51 9.7 6.2 3.6 15.8 325
40.7 14.2 7.22 10.0 6.3 3.6 18.1 261*
-
...
40.4 14.2 7.43 7.9 6.5 3.7 15.8 273
__
___.
-.
HCT : haematocrit; HGB : haemoglobin: RBC : red blood cells; WBC : leucocytes; TOP : total protein: ALB : albumin: BUN : blood urea nitrogen; ALP : alkaline phosphatase significance computed by Mann-Whitney U test (two tailed) : * p < . 0 5 7 . B . Female Rats
A.maritima in Feed (pprn) 3.125 12.500 50.000 .__----________________________________ 42.4 41.2 40.8 41.8 14.7 14.5 14.4 14.8 7.85 7.54 7.50 7 72 11.2 8.7 8.5 9.5 6.1 6.3 6.3 6.1 3.6 3.4 3.6 3.5 16.5 15.5 13.7 12.8* 220 258 231 170
Control
I
_
-
~
~
-
~
_
_
_
~
-
-
_
I
_
-.
HCT : haematocrit; HGB : haemoglobin; RBC : red blood cells; WBC : leucocytes; TOP : total protein: ALE : albumin; BUN : blood urea nitrogen; ALP : alkaline phosphatase significance computed by Mann-Whitney U test (two tailed) : * p < .05
368
ALARD ET AL.
TABLE 8
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Salmonella Revertant Mutation Test Results Ambrosin using the Plate Incorporation Assay Dosage Group
Conc . pg/plate
with
Number of revertants/plate (mean of 3 plates 2 SD) TA98 TAlOO -----------------------,
-s9
+s9
-S9
+s9
212 4 302 3 25+ 7 27+ 5 20k 1 27+ 8
612 1 5 5 8 2 11 57.r 8 602 3 63k 12 662 8 532 4
812 7 78210 862 7 332 7 602 3 77+ 6 68+?i 44224
....................................................... Ambros in (ethanol)
0 15.7 31.3 67.5 125 250 500 1000
14k 132 152
162 142
172 18t 12k
3 2 4 5 5 2
4 5
142 3 82 3
332
2
Positive Controls 2-nitrofluorene 5
8032211
sodiumazide 587+142
1
2-aminoanthracene 1
259268
371260
-S9 : without metabolic activation +S9 : with metabolic activation
an extract.
Similarly no toxic effects could be ob-
served after the administration of 50,000 ppm of A . maritima in the feed during 2 8 days.
Although signi-
ficant differences have been observed between some organ weights and serum parameters of treated and control rats, these differences were rather small and they were never present simultaneously in male and female ani-
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ALARD ET A L . ,
mals.
Consequently they can be considered as toxi-
cologically insignificant. Based on a total feed consumption of 718 g to 812 g over the whole period this
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means that a total uptake of 35.9 to 40.6 g of A . maritima over a period of 4 weeks had no observable
adverse effect on these rats. These results confirm the preliminary data of Vassiliades et al.", who reported no adverse signs of toxicity in mice after the consumption of water, containing 1 g/l of A.maritima during one week. Intravenous injection of mice on the other hand with arnbrosin and damsin, dissolved in 4 -- 8 % alcohol saline at dose rates of respectively 40 and 80 mg/kg, killed 10 % of the animals".
Reports from the field some-
times mention that small or large ruminants eat the plant"!' whereas other authors observed that animals avoid eating the plant".
Up to now, however, no case
of intoxication of animals due to A.maritima has ever
been reported. Hymenovin, a sesquiterpene lactone, has been shown to be mutagenic to
S.typhimurium
strains TA98 and
TA100'6. In this study, another sesquiterpene lactone, ambrosin, has been tested for its mutagenic potential in S.typhimurium strains TA98 and TA100.
Ambrosin,
when tested up to the concentration of 1000 pgjplate, showed no mutagenic properties towards the S.typhi-
TOXICITY AND MUTAGENICITY OF AMBROSIA MARITIMA L.
murium
371
strains TA98 and TAlOO under the experimental
conditions stated.
As ambrosin is not the only active
molluscicidal substance of A.maritima, additional stu-
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dies were done with water extracts of plant material of A.maritima.
These studies showed that none of the wa-
ter soluble substances of the plant material are mutagenic towards the S.typhimurium strains
TA97, TA98,
TA1538, TAlOO and TA2535 under the experimental conditions stated.
It can be concluded that acute and subchronic toxicity as well as mutagenic effects of A.maritima for rats are virtually absent at the concentrations tested. Since the toxicity of the plant to aquatic nontarget organisms (fish, crustacea and algae) is also very low or absent', A.maritima can be considered as a molluscicidal plant, which is environmentally acceptable and which can be used on a larger scale in the field.
ACKNOWLEDGEMENTS
The authors wish to thank Dr. J. Belot (Dakar, Senegal) for providing the plant material, Prof. A.J. Vlietinck (BUCA, Antwerp) and his staff for preparing the extract of A.maritima, Dr. L. Triest (University of Brussels, VUB) for the phytochemical analyses and Dr.
372
ALARD ET AL.
J. Ziesche (Technical University, Berlin) for providing the ambrosin.
The skillful1 work of the technical
staff of the toxicology department of Jansscn Pharma-
Drug and Chemical Toxicology Downloaded from informahealthcare.com by Cornell University on 11/18/14 For personal use only.
ceutica and the veterinary department of the Institute of Tropical Medicine is gratefully acknowledged.
This
work was supported by a grant from the National Fund for Scientific Research (NFWO), Belgium (No.2.0030.88).
REFERENCES
1.
A.F. Sherif and M.F. El Sawy, Alex.Med.J., 8, 139 (1962) .
2.
M. Sidhom and S. Geerts, Rev.Elev.Med.V.6t.Pays Trop., 37, 442 (1984).
3. M.F. El Sawy, H.K. Bassiouny, A. Rashwan and A. I. El Magdoub, Bull.High Inst.Publ.Hlth.Alex., s, 307 (1978). 4. G. Vassiliades and O.T. Diaw, Rev.Elev.Med.V&t. Pays Trop., S, 179 (1982). 5.
M.F. El Sawy, J. Duncan, T.F. Marshall, M.A.R. Shehata and N. Brown, Trop.Med.Parasito1.,- 3 ,101 (1984).
6. J. Jakupovic, H. Sun, S. Geerts and F. Bohlmann, Planta Med. 52, 49 (1987). 7.
H.A. Shoeb and M.A. El Emam, Egypt.J.Bilh., 3, 157 (1976)
.
8.
F. Alard, S. Geerts & L. Triest. Toxicon 750 (1991).
9.
F.
B , 745-
Bohlman, C. Zdero , R.M. King & Robinson.Phytochemistry 23, 1979-1988 (1984).
H.
10.
J. Duncan, Pharmac.Ther., 27, 243 (1985).
11.
G. Vassiliades and O.T. Diaw, Rev.Elev.Med.VCt. Pays Trop., 33, 401 (1980).
TOXICITY AND MUTAGENICITY OF AMBROSIA MARITIMA L.
12.
373
H. Ah-Shady and T.O. Soine, J.Am.Fharm.Ass.,,L.4 384 (1953).
Drug and Chemical Toxicology Downloaded from informahealthcare.com by Cornell University on 11/18/14 For personal use only.
13.
M.F. El Sawy, Biological combat of schistosomiasis IDRC report (unpublished) 55 pp.
14. J. Xerharo and J.G. Adam, "La pharmacopee sCnegalaise traditionelle : alantes medicinales et toxiques" Vigot, Paris, 1974. 15. B.N. Ames, J . McCann and E. Yamasaki, Mutation R e s . , 2 , 347 ( 1 9 7 5 ) . 16. G . W . Ivie and D.A. Witzel in "Handbook of natural Vol. 1. Plant and fungal toxins, R.F. toxins" Keiler and A.T. Tu (Eds.), Marcel Dekker, Inc., New York and Basel, 543 (1983).
DRUG AND CHEMICAL TOXICOLOGY, 14(4), 353-373 (1991)
TOXICITY AND MUTAGENICITY OF THE MOLLUSCICIDAL PLANT AMBROSIA MARITIMA L.
Alard, F a * , Stievenart. C . * , Vanparys, P h . * * , Thilemans, L.** and Gaerts, S.* * Institute of Tropical Medicine Veterinary Departement Nationalestraat, i 5 5 , B-2000 Antwerp, Belgium * * Janssen Pharmaceutica N.V. Department of Toxicology B-2340 Beerse, Belgium
ABSTRACT The acute and subchronic toxicity of the
mol-
luscicidal plant, Ambrosia maritima L., has been tested on rats.
No toxic signs could be detected neither af-
ter oral administration of 5 g/kg of dried leaves of the plant as a powder or as a methanoiic extract, nc3r after the incorporation of 50,300 ppm powdered leaves in the feed during 4 weeks. Using an aqueous extract of the plant material of A.maritima or using ambrosin, one of the active mo1.-
luscicidal components of the plant, no mutagenic acti-
353 Copyright 0 199 1 by Marcel Dekker, Inc.
354
ALARD ET AL.
vizy could be detected in the S.typhimurium strai:;s
Drug and Chemical Toxicology Downloaded from informahealthcare.com by Cornell University on 11/18/14 For personal use only.
TA97, TA 98, TAl538, TAlOO and TA1535.
INTRODUCTION
The
molluscicidal
activity
of
k.maritima
L.
(Asteraceae) has been repeatedly proven in irhc labaratory'
and in the field'
'.
Doses of 35 to 7C myil of
the dried leaves of the plant were able t o kill m r e than 90 % of the snails in irrigation canals and drairis ?he secondary metabolites of t h c plant. tie
in Egypt'.
sesquiterpene lactones, are the active principliis ?resent in the leaves, flowers and seeds of A.maritima. Ambrosin and damsin are the two most important
ses-
quiterpene lactones' whose molluscicidal activity has I
been proven
.
The toxicity of d.maritima has been eva-
luated in fish, crustacea and algae:. It was showr? thst the plant had a very low toxicity to aquatic ncn-target organisms and that it was not toxic when used at the molluscicidal concentrations of 35-70 mg/l. Up
to
however, on
now the
virtually mammalian
no
data
toxicity
were of
available, d.maritima.
Since these data are necessary before the plant can be used on a larger scale in the field, these experiments were set up in order to study the mutagenic potential and the acute and the subchronic toxicity on rats.
TOXICITY AND MUTAGENICITY OF AMBROSIA MARITIMA L.
MATERIAL-=
355
METHODS
A?nbro~*. mari tima. Drug and Chemical Toxicology Downloaded from informahealthcare.com by Cornell University on 11/18/14 For personal use only.
Seeds of A.maritima from Egyptian origin were sown in Senegal.
The plants were dried and used within the
first year after harvesting.
Acute toxicity tests were
carried out using a powder or an extract of A.maritima. The powder was made from the leaves of A.maritima : 150 mg was suspended in 1 mi of an aqueous solution of 1 % sodium carboxymethyl-celluiose. The extract of the plant was made by macerating the powder of the ieaves in methanol during 4 hours. Afterwards the suspension was filtered to separate the solxtion from the sediment.
This latter was percolated
during 14 hours in methanol.
The percolate and the
solution were than distilled at a temperature of ~ 4 0 ° C . The residue was dissolve2 in a mixture of 2 @ % polyethylene glycol water.
(PEGQOO) and 4 % DMSO in distilled
One ml of this solution contained the eqai-
valent of 0.6 g dried leaves. The content of sesquiterpene lactones of the methanolic extract of A.maritima was determined by column chromatography (silica gel), essentially as described by Bohlmann et al.'. Ambrosin as well as water extracts from A.maritima were tested
ir,
the Ames test.
Ambrosin was obtained as
3 56
ALARD ET AL.
a gift from Dr. J. Ziesche (Institute for Organic Chemistry, Technical University of Berlin, D - 1000 Berlin
12, West Germany) and was dissolved in ethanol.
To
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obtain the water extract, 2 g powder mixture from flowers, seeds and leaves of the plant were extracted with 14 ml distilled water for 48 h. fuging for
10 min. at
900 g
and for
After centri-
5 min. at 1000
g, the water fraction was transferred into a vial and tested directly.
In order to know the amount of plant
material dissolved in water, the pellet was evaporated at 12O'C during 72 h.
The residue was then weighed and
substracted from the initial amount of dry material.
Acute Toxicity Studies Albino Wistar rats, weighing about 200 g at the start of the experiment, were used for the toxicity studies.
Since it was evident from preliminary obser-
vationsic that
A.maritima
was
probably
very
little
toxic, only one dose (5 to 5.6 g/kg) was administered (Table 1). The absence of toxic signs at this dose level is generally considered as a guarantee for the toxicological acceptability of the test substance. Due to the large volume of the powder, it was divided in two parts, two thirds being administered first and the other third 24 hours later. administered as a
The extract, however, was
single dose.
The
control rats
357
TOXICITY AND MUTAGENICITY OF AMBROSIA MARITIMA L.
TABLE 1
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Acute and Subchronic Toxicity Tests of A.maritima in Rats Toxicity Test Acute Subchronic
....................................................... Ambrosia mari tima - Presentation - Dose(s)
powder
extract
5 g/kg
5.3
5.6 g/kg*
-
3,125 12,500 5 0 , 0 0 0 ppm
p.0.
p.0.
p.0. (feed)
Administration
No. of Rats Used 12 - Control + male + female 8 12 - Test + male 10 + female Duration of test (days1
13
powder
-
12
5
12 12
5 15 15
10 13
28 __._____
* extract, equivalent to
5 . 3 and 5.6 g/kg respectively
for the males and females
received the same volume of the solvent as the test rats in an aqueous solution of 1 % sodium carboxymethylcellulose or a mixture of 2 0 % PEG400
and 4 %
DMSO for the experiment using the powder or the extract of A.maritima respectively.
The body weights of
the rats were followed during the course of the experiment and autopsy was carried out 13 days after the administration of the plant material. (lungs,
spleen,
liver,
heart,
All the organs
pancreas,
358
ALARD ET AL.
kidneys, brain, thymus, adrenals, thyroids and gonads) were examined in detail and weighed.
Nineteen serum-
and 10 blood parameters were examined : sodium, potasDrug and Chemical Toxicology Downloaded from informahealthcare.com by Cornell University on 11/18/14 For personal use only.
sium, chloride, calcium, inorganic phosphate, total protein, albumin, haptoglobin, glucose, cholesterol, triglycerides, phospholipids,
blood
creatinine,
alkaline
total
bilirubin,
urea
nitrogen,
phosphatase.
aspartate aminotransaminase, alanine aminotransaminase, cholinesterase, haematocrit, haemoglobin, red
blood
cells, white blood cells, thrombocytes, normoblasts, mean ce'll volume, mean cell haemoglobin, mean cell haemoglobin concentration, differential count of white blood cells.
Subchronic Toxicity Tesi Forty rats weighing between 120 and 130 g were used for this test.
Powder of A.maritima leaves was
thoroughly mixed in the feed (meal) at doses of 3,125, 12,500 and 50,000 ppm mixer
(Table.1) using an industrial
(Collette MPH 150). Control rats received the
same feed without A.maritima. dually housed and fed
All rats were indivi-
ad libitum.
The animals were
regularly weighed and their food and water consumption was measured during the course of the experiment.
A
urine sample was taken on day 25 after the start of the experiment and examined for the presence of epithelial
TOXICITY AND MUTAGENICITY OF AMBROSIA MARITIMA L.
359
cells, casts, crystals, red and white blood cells and bacteria.
The following parameters were determined
:
creatinine, urobilinogen, pH, volume, specific gravity, Drug and Chemical Toxicology Downloaded from informahealthcare.com by Cornell University on 11/18/14 For personal use only.
proteins, glucose, acetone, bilirubin and occult blood. Autopsy was carried out on day 28.
The examination of
the organs and haematoloqical and serological parameters was similar as for the acute toxicity test.
Statical Analysis of Data Statistical analysis of the data of the toxicity studies was carried out using the Mann Whitney U test or the chi square test.
Salmonella Revertant Mutation Tests Bacterial strains The
S.typhimurium
strains TA97, TA98, TA1538,
TAlOO and TA1535 were kindly provided by Prof. B.N. Ames, Berkeley, University of California, U.S.A.
The
tester strain genotypes, as described by Ames et al.", were confirmed before the start of each study.
The
spontaneous reversion of the tester strains used in this study were within our historical control range. 2-nitrofluorene, sodium
azide
and
2-aminoanthracene
(Janssen Chimica, Belgium) were used as positive controls.
ALARD ET AL.
360
Metabolic activatior? system In all the tests, a rat liver homogenate (S9-mix) was used as metabolic activation system.
Adult male
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Wistar rats were injected once intraperitoneally with 200 mg/kg Aroclor 1254 (Monsanto, U.S.A.) in order to
induce drug-metabolizing enzymes.
The preparation of
the S9-mix was done according to the methods of Ames et 'C
al." Concentrations of S9 per plate were 20 and 50 p l .
Plate incorporation tests Two experiments were performed using the standard ? C
plate incorporation test described by Ames et al.'.. The
plate
incorporation test
with
ambrosin was
performed in triplicate using two S.typhimurium strains TA98 and TA100.
The test was done in the presence and
in the absence of a rat liver metabolic activation sysTop agar, 0.1 ml of bacteria, S9-mix (20 p l /
tem.
plate) if needed and 15.7, 31.3, 62.5, 125, 250, 500 and 1000 pq ambrosin/plate were sequentially added, shaken and poured onto agar plates. The plate incorporation test with the water fraction of plant material was performed using the five standard strains TA97, TA98, TA1535, TAlOO and TA1535 in the presence and in the absence of rat liver S9-mix. The test was performed in triplicate and was repeated once.
Top agar, 0.1 ml of bacteria, S9-mix (20 and 50
TOXICITY AND MUTAGENICITY OF AMBROSIA MARITIMA L.
361
ul/plate) if needed and the water fraction containing 78.1, 156.3, 312.5, 625, 1250 and 5000 pg piant ma-
terial per plate were sequentially added, shaken and
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poured onto agar plates. In both tests, the number of revertant colonies were counted after 48 h. of incubation in the dark at 37'C.
The results were considered as positive if there
was at least a twofold increase in the mean number of revertants with one of the strains TA37, TA38 or TAlOO or a threefold increase in the mean number of revertants with one of the strains TA1535 or TA1538.
The
increase in revertants must be dose related and reproduc i ble .
RESULTS
Toxicity Tests The sesquiterpene lactone content of the dried leaves of A.maritima, which were used in the toxicity tests, was determined by column chromatography (silica gel). It was shown to be 1.4 to 1.6 % of the dry weight of the plant.
No significant differences in weight gain were
present between treated and control animals in any of the experiments carried out (Tables 2 and 3 ) .
ALARD ET AL.
362
TABLE 2 Change in Eody Weights of Rats during the A c l i t s Toxicity Test using a Powder or an Extract of A.maritima
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Mean Body Weight Gain
(g) 2 5.D.
A . mciri tina
powder
extract
-__---------------------------------------------------Male Rats i'ontro 1 Test
6 6 . 3 2 13.;: 63.3 2 6 . 4 " '
8 2 . 6 z 212 8 ,!i : 30.3 2 13.J
Female Rats Control Test
36.3 2 9.5 31.0 2 6.9"
2 7 . 2 f 8.4: 32.3 z 10.0
_.___
*
S.'
___.
~
~
gain of weight from day 1 to day 12 not significantly different from the controi rats
TABLE 3 Change in Body Weights of Rats during the Subchronic Toxicity Test using A.maritima, incorporated iE t h e Feed
--------------Male Rats Contro 1 Treated 3 , 1 2 5 ppm 1 2 , 5 0 0 ppm 50,000 ppm Female Rats Contro 1 Treated 3 , 1 2 5 ppm 1 2 , 5 0 0 pprn 5 0 , 0 0 0 ppm l i
128.82 2.5
338.8221.8
200
k22.1
128.8 128.82 128.82
~6 . 7 5.9 5.6
323.2218.2 335.4-+22 302,8216
194.4217.2 206.6217.2' ' 1 7 4 216.1'
118.6t16.9
236.8528.6
118.2213.1
118.4216.7 118.8214 118.6213.9
240.2k33.8 240,8216.6 229.8212.1
1 2 1 . 8 k 2 0 . 1Y 122 216.7' 113.22 7.5"
Y :
:
'
not significantly different from the controls
S
TOXICITY AND MUTAGENICITY OF AMBROSIA MARITIMA L.
363
No adverse signs were observed during the course of any of the tests and no macroscopic lesions were present
iZ
the organs of the treated animals. The presence of sofr
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feces had been noted in 4 of the 30 treated rats used in the subchronic toxicity test.
This sign was only
temporarily present and disa2peared after two days. The meaii body and organ weights of the rats treated with an extract of A.maritima in the acute toxicity test are shown in Table 4. In Table 5 only the weights of those organs are presented, where some differences between treated and control rats in the subchronic toxicity test were remarked. Almost
no
significant differences
in
haemato-
logical and serum chemistry values were observed in the acute and subchronic toxicity tests.
The serum para-
meters, for which some significant results were obtained, are summarized in Tables 6 and 7 .
The analysis
of the urine, taken on day 25 in the subchronic toxi-
city test, didnot show any
significant differences
between treated and control groups.
Salmoneila Revertant Mutation T e . s The results of the Salmonella revertant mutation tests with ambrosin are shown in Table 8. Ambrosin was not mutagenic to the strains TA98 and TAlOO when tested up to the concentration of 1 0 0 0 pg/plate.
The inclu-
ALARD ET AL.
364
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TABLE 4 Mean Body and Organ Weights of Rats, treated with an Extract of A.maritirna (Acute Toxicity Test) - ~ _ _ . . ___.. .-_ Male Rats Female Rats ...................................... Weight Control Treated Control Treated
....................................................... 311 2130 6898 Spleen 828 2656 Liver 13641 43760 Kidneys (mg) 2436 (mg/kg) 7849 Brain (mg) 1821 5877 (mg/kg) Thymus (mg) 459 (mg/kg) 1475 Gonads (mg) 2677 8642 (mg/kg) _____ significance computed by tailed) : * p < .05 (9)
Body Lungs
(mg) (mg/kq) (mg) (mg/kg) (mg) (mg/kg)
250 1980 7959 681 2719 986 1 37842 1762 7057 1773 7110 407 1632 135 540
298 2004 6722 749 2513 13278 44432 2312 7746 1833 6174 507 1700 2613 8795 ____
Mann-Whitney
U
240 1762 7381 649 2711 9724 40573 1735 7258 1756 7357 424 1771" 122 509 -
test
(two
sion of a rat liver metabolic activation system did not alter
the
positive
mutagenic
potential
of
ambrosin.
The
controls 2-nitrofluorene and sodium azide,
which are direct acting mutagens, caused a strong increase in the number of revertant colonies in the strains TA98 and TA100, respectively. The indirect mutagen 2-amino-anthracene, which is only mutagenic after metabolic activation, also caused a strong increase in the number of revertants in both strains. Ambrosin was found to be slightly toxic to the bacteria at the concentration level of 1000 pg/glate.
TOXICITY AND MUTAGENICITY OF AMBROSIA MARITIMA L.
365
TABLE 5
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Mean Body and selected Organ Weights of Rats which received A.maritima in the Feed during 28 says (Subchronic Toxicity Test) 5 . A . Male Rats ______.
A.maritima in Feed (ppmj
Weight
Control
3.125
12.500
50.009
329 738 2247 1948 5949 2474 7558
323 776 2393 1854 5757 2750 8540
335 784 2350 1980 591 1 2547 7602
333 703 2327 1838* 6086 2658 8764*
_____-___-----____-------------------_-_______------_Body (9) Spleen (mg) (mg/kg)
Brain
(mg) (mg/kg)
Gonads (mg) img/kg) ___
.______-.______
significance computed tailed) : * p ( .05
5.B.
by
Mann-Whitney
U
.~
test
(two
Female Rats A.maritima in Feed (ppm)
Weight
Contro 1
3.125
12.500
50.000
237 673 2847 1730 7337 155 649
240 678 2746 1841 7709 142 585
241 668 2752 1765 7335 138 572
230 553* 2413'* 1708 7447 140 611
--__-__-----------------------------------------------Body !gj Spleen (mg) (mg/kg) (mg) (mg/kg) Gonads (mg) (mg/kgj
Brain
significance computed by Mann-Whitney tailed) : * p < . 0 5 * * p < .01
U
test
(two
Table 9 shows the results of the Salmonella revertant mutation tests with the water fraction of the plant material.
No mutagenic activity was found with the
water fraction towards the strains TA97, TA98, TA1538,
3 66
ALARD ET AL.
TABLE 6
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Selected Haematological and Serum Chemistry Values of Rats, treated with an Extract of A.maritima (Acute Toxicity Test) Male Rats
Female
rats ......................................... Control Treated Control Treated -------------_-----------------------------------------
parameter
HCT HGB RBC WBC TOP
36.1 12.8 Cg/pl) . (lO,/mmJ) 7.32 (10'/mm') 8.3 (9%) 6.3 3.3 ALB (9%) 23.9 BUN (mg%) 329 ALP (u/l) -______ (%)
36.1
38.0 13.3
12.7
7.32 10.1 5.9** 3.2 23.9
267*
38.1 i3.4
7.76
7.75
9.0 6.6 3.6 22.6 154
8.6 6.5 ?.4*
lS.EC** 157 .-
~
HCT : haematocrit; HGB : haemoglobin; RBC : red blood cells; WBC : leucocytes; TOP : total protein; ALE : albumin; BUN : blood urea nitrogen; ALP : alcalic phosphatase significance computed by Mann-Whitney U test (two tailed) : * p < .05 * * p < .01
TAlOO and TA1535 in the presence and in the absence of a rat liver metabolic activation system (20 te).
111
S9/pla-
Higher concentrations of S9 (50 pl/plate) did not
induce an increase in the number of revertant colonies.
DISCUSSION
These results show that there are no signs of toxicity
after the administration of a single dose of
5 g Ambrosia maritima per kg bodyweight as a powder or
TOXICITY AND MUTAGENICITY OF AMBROSIA MARITIMA L.
TABLE
367
7
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Selected Haematological and Serum Chemistry Values of Rats in the Subchronic Toxicity Test using A.mar-itima in the Feed 7 . A . Male Rats
A.maritima in Feed (ppm)
Contro1
Parameter
3.125
12.500 50.000 ____------_________-----__------__--------------_-
HCT HGB RBC WBC TOP
ALB
40.0 14.0 7.35 9.2 6.3 3.6 16.4 388
(%)
Cg/pl) . (lO:/mmi j (10;/mm') (9%) (g%)
BUN (mg%)
ALP (u/l) -
_.__ -.--..I--.-._.
41.0 14.2 7.51 9.7 6.2 3.6 15.8 325
40.7 14.2 7.22 10.0 6.3 3.6 18.1 261*
-
...
40.4 14.2 7.43 7.9 6.5 3.7 15.8 273
__
___.
-.
HCT : haematocrit; HGB : haemoglobin: RBC : red blood cells; WBC : leucocytes; TOP : total protein: ALB : albumin: BUN : blood urea nitrogen; ALP : alkaline phosphatase significance computed by Mann-Whitney U test (two tailed) : * p < . 0 5 7 . B . Female Rats
A.maritima in Feed (pprn) 3.125 12.500 50.000 .__----________________________________ 42.4 41.2 40.8 41.8 14.7 14.5 14.4 14.8 7.85 7.54 7.50 7 72 11.2 8.7 8.5 9.5 6.1 6.3 6.3 6.1 3.6 3.4 3.6 3.5 16.5 15.5 13.7 12.8* 220 258 231 170
Control
I
_
-
~
~
-
~
_
_
_
~
-
-
_
I
_
-.
HCT : haematocrit; HGB : haemoglobin; RBC : red blood cells; WBC : leucocytes; TOP : total protein: ALE : albumin; BUN : blood urea nitrogen; ALP : alkaline phosphatase significance computed by Mann-Whitney U test (two tailed) : * p < .05
368
ALARD ET AL.
TABLE 8
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Salmonella Revertant Mutation Test Results Ambrosin using the Plate Incorporation Assay Dosage Group
Conc . pg/plate
with
Number of revertants/plate (mean of 3 plates 2 SD) TA98 TAlOO -----------------------,
-s9
+s9
-S9
+s9
212 4 302 3 25+ 7 27+ 5 20k 1 27+ 8
612 1 5 5 8 2 11 57.r 8 602 3 63k 12 662 8 532 4
812 7 78210 862 7 332 7 602 3 77+ 6 68+?i 44224
....................................................... Ambros in (ethanol)
0 15.7 31.3 67.5 125 250 500 1000
14k 132 152
162 142
172 18t 12k
3 2 4 5 5 2
4 5
142 3 82 3
332
2
Positive Controls 2-nitrofluorene 5
8032211
sodiumazide 587+142
1
2-aminoanthracene 1
259268
371260
-S9 : without metabolic activation +S9 : with metabolic activation
an extract.
Similarly no toxic effects could be ob-
served after the administration of 50,000 ppm of A . maritima in the feed during 2 8 days.
Although signi-
ficant differences have been observed between some organ weights and serum parameters of treated and control rats, these differences were rather small and they were never present simultaneously in male and female ani-
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3 3
.. ..
m m
Vlvl
I +
310
ALARD ET A L . ,
mals.
Consequently they can be considered as toxi-
cologically insignificant. Based on a total feed consumption of 718 g to 812 g over the whole period this
Drug and Chemical Toxicology Downloaded from informahealthcare.com by Cornell University on 11/18/14 For personal use only.
means that a total uptake of 35.9 to 40.6 g of A . maritima over a period of 4 weeks had no observable
adverse effect on these rats. These results confirm the preliminary data of Vassiliades et al.", who reported no adverse signs of toxicity in mice after the consumption of water, containing 1 g/l of A.maritima during one week. Intravenous injection of mice on the other hand with arnbrosin and damsin, dissolved in 4 -- 8 % alcohol saline at dose rates of respectively 40 and 80 mg/kg, killed 10 % of the animals".
Reports from the field some-
times mention that small or large ruminants eat the plant"!' whereas other authors observed that animals avoid eating the plant".
Up to now, however, no case
of intoxication of animals due to A.maritima has ever
been reported. Hymenovin, a sesquiterpene lactone, has been shown to be mutagenic to
S.typhimurium
strains TA98 and
TA100'6. In this study, another sesquiterpene lactone, ambrosin, has been tested for its mutagenic potential in S.typhimurium strains TA98 and TA100.
Ambrosin,
when tested up to the concentration of 1000 pgjplate, showed no mutagenic properties towards the S.typhi-
TOXICITY AND MUTAGENICITY OF AMBROSIA MARITIMA L.
murium
371
strains TA98 and TAlOO under the experimental
conditions stated.
As ambrosin is not the only active
molluscicidal substance of A.maritima, additional stu-
Drug and Chemical Toxicology Downloaded from informahealthcare.com by Cornell University on 11/18/14 For personal use only.
dies were done with water extracts of plant material of A.maritima.
These studies showed that none of the wa-
ter soluble substances of the plant material are mutagenic towards the S.typhimurium strains
TA97, TA98,
TA1538, TAlOO and TA2535 under the experimental conditions stated.
It can be concluded that acute and subchronic toxicity as well as mutagenic effects of A.maritima for rats are virtually absent at the concentrations tested. Since the toxicity of the plant to aquatic nontarget organisms (fish, crustacea and algae) is also very low or absent', A.maritima can be considered as a molluscicidal plant, which is environmentally acceptable and which can be used on a larger scale in the field.
ACKNOWLEDGEMENTS
The authors wish to thank Dr. J. Belot (Dakar, Senegal) for providing the plant material, Prof. A.J. Vlietinck (BUCA, Antwerp) and his staff for preparing the extract of A.maritima, Dr. L. Triest (University of Brussels, VUB) for the phytochemical analyses and Dr.
372
ALARD ET AL.
J. Ziesche (Technical University, Berlin) for providing the ambrosin.
The skillful1 work of the technical
staff of the toxicology department of Jansscn Pharma-
Drug and Chemical Toxicology Downloaded from informahealthcare.com by Cornell University on 11/18/14 For personal use only.
ceutica and the veterinary department of the Institute of Tropical Medicine is gratefully acknowledged.
This
work was supported by a grant from the National Fund for Scientific Research (NFWO), Belgium (No.2.0030.88).
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