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Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lesb20
Teratologic assessment of maleic hydrazide and daminozide, and formulations of ethoxyquin, thiabendazole and naled in rats a
a
a
K.S. Khera , C. Whalen , G. Trivett & G. Angers
a
a
Bureau of Chemical Safety , New Research Centre, Tunney's Pasture , K1A OL2, Canada Published online: 14 Nov 2008.
To cite this article: K.S. Khera , C. Whalen , G. Trivett & G. Angers (1979) Teratologic assessment of maleic hydrazide and daminozide, and formulations of ethoxyquin, thiabendazole and naled in rats, Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes, 14:6, 563-577, DOI: 10.1080/03601237909372151 To link to this article: http://dx.doi.org/10.1080/03601237909372151
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J. ENVIRON. SCI. HEALTH, B14(6), 563-577 (1979)
TERATOLOGIC ASSESSMENT OF MALEIC HYDRAZIDE AND DAMINOZIDE, AND FORMULATIONS OF ETHOXYQUIN,
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THIABENDAZOLE AND NALED IN RATS Key words:
Teratogenicity, embryotoxicity, maleic hydrazide, daminozide, ethoxyquin, thiabendazole, naled
K.S. Khera, C. Whalen, G. Trivett, and G. Angers Bureau of Chemical Safety, New Research Centre, Tunney's Pasture, Ottawa K1A OL2, Canada ABSTRACT Teratogenicity studies were conducted in rats
treated orally from days 6-15 of gestation with single daily doses of 400-1600 mg/kg of maleic hydrazide, 3001000 mg/kg daminozide, 125-500 mg/kg ethoxyquin or thiabendazole, or 25-100 mg/kg naled.
Dams were killed on
the 22nd day of gestation, and fetuses were evaluated
by routine teratologic methods.
No adverse effect was
related to any treatment other than an increased incidence of anomalous fetuses at the highest dose (500 mg/kg) of thiabendazole.
563 Copyright © 1979 by Marcel Dekker, Inc. All Rights Reserved. Neither this work nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher.
564
KHERA ET AL. INTRODUCTION Maleic hydrazide, a synthetic plant growth regu-
lator and a herbicide, is known to be carcinogenic, mutagenic and antimitotic 1 '2 . It has been shown to translocate to and accumulate in.onions 3 , beans H ,
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tomato, rice, peanuts , silver maple and American sycamore .
Maleic acid is allowed for usage on vege-
tables provided residue levels do not exceed SO ppm in potatoes, 30 ppm in beets, carrots and rutabagas, and 15 ppm in onions
. No teratogenic activity was ob-
served following its administration to rabbits during 7 pregnancy . Daminozide (Alar), a growth regulator, increases the firmness and yield of apples, bananas, pears, brussel sprouts and carrots.
It is stable in the soil
p
for four weeks , and has been detected on apples for 9 10 three weeks , cherries for seven weeks plants for variable time periods
, and other
. Residues of 2.12-
9.10 ppm have been detected in apples stored for seven months after harvesting from commercial orchards 12 sprayed with 0.2% A'lar . In the rat, Daminozide has produced leukopenia after dosaging at 5 mg/kg/day for two months and an acute oral LD
value of >10 g/kg
,
but little is known otherwise about its toxicity. Tolerances have been established under the U.S. Federal
TERATOLOGIC ASSESSMENTS IN RATS
565
Food, Drug and Cosmetic Act for daminozide residues of 14 up to 3 0 ppm on apples and 10 ppm on grapes Ethoxyquin, commonly added to commercial feeds to retard oxidation of carotenes, xanthophylls and vitamins, is also used on apples and Anjou pears to prevent Downloaded by [Virginia Tech Libraries] at 14:24 14 March 2015
scalds.
Maximum residue limits allowed in Canada are 3
ppm in apples, pears and poultry livers and 0.5 ppm in egg, meat and poultry meat
. A study on its terato-
genic activity and reproductive effects in rabbits
,
is of questionable value because 55% of offspring in
the control group died within two weeks post-partum. Thiabendazole, an anthelmintic agent, is used on
a variety of fruits and vegetables for controlling fungal diseases.
Thiabendazole has been reported to
degrade slowly in the soil
and translocate, after
absorption by roots, to leaves of pepper and tomato plants
, sugar beets
and potato tubers
.
Residues
as high as 11.8 ppm on lemons and 9.3 ppm on oranges 21 have been detected upon commercial application Thiabendazole occurred in an undegraded form for four
weeks in the peel after the oranges were dipped in a 22 0.1% suspension . Maximum tolerances allowed are 10 ppm in apples, citrus fruits and pears, and 4 ppm in potatoes
. Thiabendazole produced no teratogenic
activity in rabbits when administered orally in 100-800 mg/kg doses from the eighth to the 16th day of pregnancy 23
566
KHERA ET AL. Naled or dibrom, an acetylcholinesterase inhibi-
tor, is used on a large number of vegetables.
Its
residues (0.1 mg/kg) have been found in peas, cucumbers 24 2 5 and tomatoes 5-10 days after spraying ' . Naled has been incriminated for causing dermatitis in female Downloaded by [Virginia Tech Libraries] at 14:24 14 March 2015
workers exposed soon after its application on vegetation
.
Permitted residue levels are 3 ppm on
spinach, turnips and citrus fruits and 0.5 to 1 ppm on vegetables and cereals
.
Limited data are available
on the toxicity of naled. Human ingestion of the above pesticides or their metabolites is highly likely due to their occurrence in foods.
Since the teratologic safety of these pesti-
cides has not yet been established, studies were conducted and the results are herein reported. MATERIALS AND METHODS Chemicals Samples tested were:
maleic hydrazide containing
97% 3,6-dihydroxypyridazine, and daminozide, 99% succinic acid 2,2-dimethylhydrazide (Aldrich Chemical, Milwaukee, Wisconsin); an ethoxyquin commercial preparation containing 67% of 1,2-dihydro-6-ethoxy-2,2 ,4trimethylquinoline and 33% of unknown ingredients (Santoquin, mixture 6, lot 417, Monsanto, St. Louis, Missouri); a thiabendazole formulation containing 45% w,v of 2-(4-thiazolyl) benzimidazole and 55% of unknown
TERATOLOGIC ASSESSMENTS IN RATS
567
ingredients (Mertect, lot 2362-X, Merck Sharp S Dohme, Kirkland, Quebec); a naled (or dibrom) formulation containing 36% w/v of dimethyl 1,2-dibromo-2,2-dichloroethyl phosphate and 64% of unknown ingredients
("Fly
killer D", Chevron Chemical, Oakville, Ontario).
All
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samples on receipt from the suppliers were stored at 6°C till tested.
Animals Female Wistar rats, 175-225 g in body weight, were
paired overnight with proven males, and the morning that a positive vaginal smear was observed was counted as day 1 of gestation.
Twenty mated females, randomly
selected, were assigned to each experimental group.
The test doses, representing the levels of the composite samples and not the active ingredients, on a per kg basis were 400, 800, 1200 or 1600 mg of maleic
hydrazide; 300, 600 or 1000 mg of daminozide; 125, 250 or 500 mg of ethoxyquin or thiabendazole; and 25, 50 or 100 mg of naled.
Maleic hydrazide, ethoxyquin and
naled suspended in corn oil, and daminozide and thiabendazole in distilled water, were administered once daily by esophageal intubation from the sixth to the 15th day of gestation, inclusive.
The volume of
vehicle or vehicle plus chemical was 10 ml/kg body weight.
A control group was included for each test
568
KHERA ET AL.
chemical.
Females were weighed at frequent intervals
during gestation.
On the 22nd day of gestation, each
dam was killed, its uterine contents removed, its number of corpora lutea determined, and was then necropsied.
The fetuses were weighed and examined for via-
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bility and external malformations.
Early resorptions
or implantation sites and fetuses dying at a late stage in their development, were recorded.
Two-thirds of the
live fetuses from each litter were examined for skeletal development after alizarin red staining.
The
remainder, fixed in Bouin's fluid, were dissected to study visceral anomalies. Statistical Analysis To determine effects on maternal body weight, the mean and standard error were calculated for each experimental group, and values of t_ were obtained for 27 test and control group differences in means . Fetal values were analysed with the litter as the basic unit. The proportion (p_) of litter having a particular attribute was calculated and transformed to a normally distributed variable arc sin/p_ value
.
The mean and
standard error of these values for various.test groups, 29 were then derived
.
The t_ test was used for com-
parison of test and the respective control values. Differences were considered significant at £ < 0.05.
TERATOLOGIC ASSESSMENTS IN RATS
569
RESULTS No sign of toxicity or any adverse effect on maternal body weight was noticed during pregnancy, in any treatment or control group.
The number of females
pregnant in the different test groups varied within the
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control range (Tables 1 and 2) and was unrelated to treatment. Values for corpora lutea, total implants, resorp-
tions, dead fetuses, live fetuses and their male/female
ratio, and fetal weight from groups treated with maleic hydrazide, daminozide, and formulations of ethoxyquin, thiabendazole or naled, are listed in Tables 1 and 2.
All values listed for the treatment groups were comparable to the control values except the fetal weight
at the 125 mg/kg dose of thiabendazole formulation that
was significantly reduced (P^ = 0.05).
The numbers of
live fetuses at the 1200 mg/kg dose of maleic hydrazide, and total implants/pregnancy at the 100 mg/kg of
naled, were reduced; whereas, the resorption at the 125
mg/kg ethoxyquin formulation and 250 mg/kg thiabendazole formulation were increased.
However, none of them
were statistically different from control values. Incidences of anomalous fetuses, anomalous litters (each having at least one anomalous fetus), fetuses having individual anomalies for the different pesticide treated groups are given (Tables 3 and 4 ) . The in-
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TABLE 1 Prenatal data from rats dosed orally with maleic hydrazide or damlnozide on dayl 6 through 15 of gestation
Daminozide
Maleic hydrazide
Number of females pregnant/number inseminated
0
400 mg/kg
800 mg/kg
1200 mg/kg
1600 mg/kg
0
300 mg/kg
600 mg/kg
1000 mg/kg
19/20
20/20
IB/20
18/20
20/20
19/20
20/20
19/20
19/20
Number of corpora lutca, mean + SE
13.3+0.5
13.4*0.4
13.8*0.4
12.8*0.4
13.3*0.5
12.3+0.4
12.4.0.4
12.1*0.4
12.6*0.3
Implants per pregnancy, mean + SE
12.0*0.7
12.9+0.4
12.7*0.9
9.8*1.0
11.9*0.6
11.1*0.6
11.7*0.6
11.3*0.7
11.3*0.7
Number of females with resorptions
7
12
7
7
5
9
11
8
12
Resorptions, total number
11
13
11
7
9
14
15
11
17
Percent resorptions
4.9
5.1
4.9
4.0
3.8
6.6
6.4
5.1
7.9
Dead fetuses, total
1
1
1
0
0
0
0
0
0
99/116
126/118
104/112
81/89
94/134
94/104
130/89
118/86
108/89
3
0
1
0
0
0
2
11.4+0.6
10.4*0.6
11.0*0.7
10.7*0.7
10.4*0.6
Male/female-llve fetuses Runted fetuses, total number Live fetuses per dam, mean + SE Fetal weight (gm), mean + SE
a
0
0
11.3*0.7
12.2*0.4,
5.0*0.04
Number of resorptions/total implants x 100
5.4*0.04
12.0*0.9 5.3*0.04
9.4*1.0 5.1*0.04
5.0*0.05
5.3*0.04
5.1*0.03
5.1*0.04
5.4*0.05
a
TABLE 2
W
Prenatal data from rats dosed orally with formulations of ethoxyquin, thiabendazole, or Naled on days 6 through 15 of gestation
^
Number of females oregnant/number inseminated
Thiabendazole
Naled
H O
0
125 mg/kg
250 mg/kg
500 mg/kg
0
125 mg/kg
250 mg/kg
500 mg/kg
0
25 mg/kg
50 mg/kg
100 mg/kg
17/20
17/20
19/20
17/20
18/20
16/20
19/20
16/20
17/20
19/20
15/20
18/20
12.5.0.5
14.2*0.5
13.2*0.4
13.1*0.3
12.2.0.5
13.4*0.4
13.5*0.3
12.4.0.9
12.6.0.4
13.1*0.4
12.5*0.3
12.4*0.4
Implants per pregnancy, mean * SE
11.5*0.8
13.5*0.5
12.7*0.4
12.5*0.3
10.6*1.1
12.8*0.7
12.8.0.6
10.8.1.1
12.0.0.7
11.4*0.7
12.4.0.3
10.8*0.8
Number of females with resorptions
10
13
8
9
a
9
13
8
7
10
8
8
Resorptions, total number
12
27
10
17
10
12
17
15
14
13
14
9
Percent resorptions
6
12
4
8
5
5
7
9
7
6
8
5
Dead fetuses, total
0
1
0
0
0
0
0
2
0
1
0
0
102/31
101/100
116/115
106/90
82/99
101/118
117/109
76/80
98/92
103/100
86/86
96/90
Male/female-llve Runted fetuses, total number Live fetuses per dam, mean • SE Fetal weight(gm), mean
8
10.8*0.B 5.0.0.05
0
1
11.8*0.8 -
12.2.0.5 -
4.9*0.03 -
4.8.0.04 -
Number of resorptions/total implants x 100, b P < 0.05
1 11.5*0.5 _ 4.8*0.05 -
1
0
10.1.1.0 12.2.0.8 _ _ _ 4.7*0.04 _
0 11.9.0.7 _
1
2 9.B.I.2 _
4.4*0.10 b 4.7*0.04 4.6.0.05 _ _ _ _
0
0
11.2.0.6 _
10.7.0.7
5.0.0.03 -
4.9.0.03 -
0 11.5^0.4 — 5.2.0.04 -
1 10.3.0.8 5.0*0.05 -
M
RATS
Number of corpora lutea, mean_* SE
w ££j
ISMENTE
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O Ethoxyquin
572
KHERA ET AL.
TABLE 3 Incidence of anomalies in fetuses from rats treated orally with maleic hydrazEde or daminozide from day 6 to 15 of gestation
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Maleic hydrazlde
Daminozide
0
400 mg/kg
800 mg/kg
1200 mg/kg
1600 mg/kg
0
300 mg/kg
600 mg/kg
10 mg,
Number of anomalous fetuses/number examined
8/215
1/244
8/216
5/170
16/228
9/198
8/219
6/204
9/1
Number of litters with anomalous fetuses/ number examined
7/19
1/20
7/18
3/1B
8/20
7/19
5/20
4/19
6/
Extra ribs, uni- & bilateral
4
0
3
0
2
5
2
2
1
13th rib, rudimentary or missing
1
0
1
3
4
1
1
1
5
Fused ribs
0
0
0
0
1
0
0
0
C
Sternoschisis
0
0
0
0
2
0
0
0
0
Sternebrae fused or non-opposed
1
0
0
1
3
0
1
0
1
Sternebrae, delayed ossification
0
0
2
0
5
2
2
2
]
Calvarium, delayed ossification
0
0
0
0
1
0
0
0
(
Runted fetus
0
0
3
0
1
0
0
0
Miscellaneous
0
0
l"
0
2C
0
0
0
Anomalies (number of fetuses affected)
"a fetus may have more than one anomaly, b cleft palate, c one fetus each had a cleft upper lip and rudimentary tail
crease in total number of anomalous fetuses in the 2 50 mg/kg group of thiabendazole formulation was quite significant (£ = 0.005).
The incidences found to be of
borderline significance (£ = 0.05) were the total number of anomalous fetuses in the 2 50 mg/kg group of ethoxyquin formulation and the delayed ossification of sternebrae in the 2 5 and 50 mg/kg groups of naled. All
TABLE 4 Incidence of anomalies in fetuses from rats treated orally with formulations of ethoxyquin, thiabendazole or naled from day 6 to 15 of gestation
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Ethoxyquin
Thiabendazole >
Number of anomalous fetuses/number examined
0
125 mg/kg
250 mg/kg
500 mg/kg
13/183*
12/201
32/231b
22/196
8/17
7/17
13/19
9/17
Number of litters with anomalous fetuses/ number examined
3/181
2/18
125 mg/kg
250 mg/kg
500 mg/kg
10/217
8/226
9/156 b
6/18
6/19
8/14
25 mg/kg
50 mg/kg
100 mg/kg
9/190
16/203
9/172
11/186
6/17
8/19
7/15
6/18
a
I
Anomalies (number of fetuses affected)
CO
Wavy ribs, uni- & bilateral
0
0
0
1
4
2
Extra ribs, uni- & bilateral
6
5
15
12
0
13th rib, rudimentary or missing
1
0
1
0
Strenebraa fused or non-opposed
0
0
1
Sternebrae, delayed ossification
7
5
Calvarium, delayed ossification
2
0
0
0
0
1
0
0
2
1
0
3
2
2
3
0
0
0
0
4
5
2
0
1
0
0
1
0
0
1
1
0
8
12
3
7
5
7
1
5b
5b
6
1
1
0
0
0
0
0
3
1
3
Runted fetus
0
1
1
1
0
0
1
2
0
0
0
1
Kinky or short tail
0
0
2
0
0
0
0
0
1
0
0
0
Miscellaneous
lc
0
2d
0
0
0
le
0
0
0
l'
1«
8
a fetui may have more than one anomaly, b P < 0.05, c itemoschitli, d one fetui each had fused ribs and umbilical herina. generalized «ubcutaneoui edema, hydroureter, ^hydronephrosls
en en M en
574
KHERA ET AL.
other measures of teratogenicity in Tables 3 and 4 were within the control range. DISCUSSION In evaluating their teratogenic potential, 25 fetal and maternal parameters were measured at 3-4 dose
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levels of each of the five pesticides in the present study.
Only four of these parameters showed differences
between the test and control values at levels of P < 0.05.
Rather clear was an increase in the total number
of anomalous fetuses/number of fetuses examined in the 500 mg/kg group of thiabendazole formulation (P < 0.005), although no single anomaly was significantly increased in incidence.
The remaining three para-
meters, at the borderline of P = 0.05, were; a reduced fetal weight, at the 12 5 mg/kg of thiabendazole formulation; an increased total number of anomalous fetuses/ number of fetuses examined, at the 25 0 mg/kg ethoxyquin formulation; and a delayed sternebral ossification, at the 25 and 50 rng/kg naled formulation.
These findings
were inconsistent with the lack of effects at the higher dose levels.
It is possible that these effects
are unreal since among the 25 x 5 x 3 parameters presently examined, one would expect at least four values to be significantly different just by chance alone.
TERATOLOGIC ASSESSMENTS IN RATS
575
It is not known whether et_ seq. pesticides,.if chemically pure, or the unknown ingredients, on separate testing, would give negative responses as have been obtained with their combinations.
However, as it
is, our findings do not suggest making any changes in Downloaded by [Virginia Tech Libraries] at 14:24 14 March 2015
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John Wiley and Sons 1952,
p. 391. Received: March 30, 1979.
Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lesb20
Teratologic assessment of maleic hydrazide and daminozide, and formulations of ethoxyquin, thiabendazole and naled in rats a
a
a
K.S. Khera , C. Whalen , G. Trivett & G. Angers
a
a
Bureau of Chemical Safety , New Research Centre, Tunney's Pasture , K1A OL2, Canada Published online: 14 Nov 2008.
To cite this article: K.S. Khera , C. Whalen , G. Trivett & G. Angers (1979) Teratologic assessment of maleic hydrazide and daminozide, and formulations of ethoxyquin, thiabendazole and naled in rats, Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes, 14:6, 563-577, DOI: 10.1080/03601237909372151 To link to this article: http://dx.doi.org/10.1080/03601237909372151
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J. ENVIRON. SCI. HEALTH, B14(6), 563-577 (1979)
TERATOLOGIC ASSESSMENT OF MALEIC HYDRAZIDE AND DAMINOZIDE, AND FORMULATIONS OF ETHOXYQUIN,
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THIABENDAZOLE AND NALED IN RATS Key words:
Teratogenicity, embryotoxicity, maleic hydrazide, daminozide, ethoxyquin, thiabendazole, naled
K.S. Khera, C. Whalen, G. Trivett, and G. Angers Bureau of Chemical Safety, New Research Centre, Tunney's Pasture, Ottawa K1A OL2, Canada ABSTRACT Teratogenicity studies were conducted in rats
treated orally from days 6-15 of gestation with single daily doses of 400-1600 mg/kg of maleic hydrazide, 3001000 mg/kg daminozide, 125-500 mg/kg ethoxyquin or thiabendazole, or 25-100 mg/kg naled.
Dams were killed on
the 22nd day of gestation, and fetuses were evaluated
by routine teratologic methods.
No adverse effect was
related to any treatment other than an increased incidence of anomalous fetuses at the highest dose (500 mg/kg) of thiabendazole.
563 Copyright © 1979 by Marcel Dekker, Inc. All Rights Reserved. Neither this work nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher.
564
KHERA ET AL. INTRODUCTION Maleic hydrazide, a synthetic plant growth regu-
lator and a herbicide, is known to be carcinogenic, mutagenic and antimitotic 1 '2 . It has been shown to translocate to and accumulate in.onions 3 , beans H ,
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tomato, rice, peanuts , silver maple and American sycamore .
Maleic acid is allowed for usage on vege-
tables provided residue levels do not exceed SO ppm in potatoes, 30 ppm in beets, carrots and rutabagas, and 15 ppm in onions
. No teratogenic activity was ob-
served following its administration to rabbits during 7 pregnancy . Daminozide (Alar), a growth regulator, increases the firmness and yield of apples, bananas, pears, brussel sprouts and carrots.
It is stable in the soil
p
for four weeks , and has been detected on apples for 9 10 three weeks , cherries for seven weeks plants for variable time periods
, and other
. Residues of 2.12-
9.10 ppm have been detected in apples stored for seven months after harvesting from commercial orchards 12 sprayed with 0.2% A'lar . In the rat, Daminozide has produced leukopenia after dosaging at 5 mg/kg/day for two months and an acute oral LD
value of >10 g/kg
,
but little is known otherwise about its toxicity. Tolerances have been established under the U.S. Federal
TERATOLOGIC ASSESSMENTS IN RATS
565
Food, Drug and Cosmetic Act for daminozide residues of 14 up to 3 0 ppm on apples and 10 ppm on grapes Ethoxyquin, commonly added to commercial feeds to retard oxidation of carotenes, xanthophylls and vitamins, is also used on apples and Anjou pears to prevent Downloaded by [Virginia Tech Libraries] at 14:24 14 March 2015
scalds.
Maximum residue limits allowed in Canada are 3
ppm in apples, pears and poultry livers and 0.5 ppm in egg, meat and poultry meat
. A study on its terato-
genic activity and reproductive effects in rabbits
,
is of questionable value because 55% of offspring in
the control group died within two weeks post-partum. Thiabendazole, an anthelmintic agent, is used on
a variety of fruits and vegetables for controlling fungal diseases.
Thiabendazole has been reported to
degrade slowly in the soil
and translocate, after
absorption by roots, to leaves of pepper and tomato plants
, sugar beets
and potato tubers
.
Residues
as high as 11.8 ppm on lemons and 9.3 ppm on oranges 21 have been detected upon commercial application Thiabendazole occurred in an undegraded form for four
weeks in the peel after the oranges were dipped in a 22 0.1% suspension . Maximum tolerances allowed are 10 ppm in apples, citrus fruits and pears, and 4 ppm in potatoes
. Thiabendazole produced no teratogenic
activity in rabbits when administered orally in 100-800 mg/kg doses from the eighth to the 16th day of pregnancy 23
566
KHERA ET AL. Naled or dibrom, an acetylcholinesterase inhibi-
tor, is used on a large number of vegetables.
Its
residues (0.1 mg/kg) have been found in peas, cucumbers 24 2 5 and tomatoes 5-10 days after spraying ' . Naled has been incriminated for causing dermatitis in female Downloaded by [Virginia Tech Libraries] at 14:24 14 March 2015
workers exposed soon after its application on vegetation
.
Permitted residue levels are 3 ppm on
spinach, turnips and citrus fruits and 0.5 to 1 ppm on vegetables and cereals
.
Limited data are available
on the toxicity of naled. Human ingestion of the above pesticides or their metabolites is highly likely due to their occurrence in foods.
Since the teratologic safety of these pesti-
cides has not yet been established, studies were conducted and the results are herein reported. MATERIALS AND METHODS Chemicals Samples tested were:
maleic hydrazide containing
97% 3,6-dihydroxypyridazine, and daminozide, 99% succinic acid 2,2-dimethylhydrazide (Aldrich Chemical, Milwaukee, Wisconsin); an ethoxyquin commercial preparation containing 67% of 1,2-dihydro-6-ethoxy-2,2 ,4trimethylquinoline and 33% of unknown ingredients (Santoquin, mixture 6, lot 417, Monsanto, St. Louis, Missouri); a thiabendazole formulation containing 45% w,v of 2-(4-thiazolyl) benzimidazole and 55% of unknown
TERATOLOGIC ASSESSMENTS IN RATS
567
ingredients (Mertect, lot 2362-X, Merck Sharp S Dohme, Kirkland, Quebec); a naled (or dibrom) formulation containing 36% w/v of dimethyl 1,2-dibromo-2,2-dichloroethyl phosphate and 64% of unknown ingredients
("Fly
killer D", Chevron Chemical, Oakville, Ontario).
All
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samples on receipt from the suppliers were stored at 6°C till tested.
Animals Female Wistar rats, 175-225 g in body weight, were
paired overnight with proven males, and the morning that a positive vaginal smear was observed was counted as day 1 of gestation.
Twenty mated females, randomly
selected, were assigned to each experimental group.
The test doses, representing the levels of the composite samples and not the active ingredients, on a per kg basis were 400, 800, 1200 or 1600 mg of maleic
hydrazide; 300, 600 or 1000 mg of daminozide; 125, 250 or 500 mg of ethoxyquin or thiabendazole; and 25, 50 or 100 mg of naled.
Maleic hydrazide, ethoxyquin and
naled suspended in corn oil, and daminozide and thiabendazole in distilled water, were administered once daily by esophageal intubation from the sixth to the 15th day of gestation, inclusive.
The volume of
vehicle or vehicle plus chemical was 10 ml/kg body weight.
A control group was included for each test
568
KHERA ET AL.
chemical.
Females were weighed at frequent intervals
during gestation.
On the 22nd day of gestation, each
dam was killed, its uterine contents removed, its number of corpora lutea determined, and was then necropsied.
The fetuses were weighed and examined for via-
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bility and external malformations.
Early resorptions
or implantation sites and fetuses dying at a late stage in their development, were recorded.
Two-thirds of the
live fetuses from each litter were examined for skeletal development after alizarin red staining.
The
remainder, fixed in Bouin's fluid, were dissected to study visceral anomalies. Statistical Analysis To determine effects on maternal body weight, the mean and standard error were calculated for each experimental group, and values of t_ were obtained for 27 test and control group differences in means . Fetal values were analysed with the litter as the basic unit. The proportion (p_) of litter having a particular attribute was calculated and transformed to a normally distributed variable arc sin/p_ value
.
The mean and
standard error of these values for various.test groups, 29 were then derived
.
The t_ test was used for com-
parison of test and the respective control values. Differences were considered significant at £ < 0.05.
TERATOLOGIC ASSESSMENTS IN RATS
569
RESULTS No sign of toxicity or any adverse effect on maternal body weight was noticed during pregnancy, in any treatment or control group.
The number of females
pregnant in the different test groups varied within the
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control range (Tables 1 and 2) and was unrelated to treatment. Values for corpora lutea, total implants, resorp-
tions, dead fetuses, live fetuses and their male/female
ratio, and fetal weight from groups treated with maleic hydrazide, daminozide, and formulations of ethoxyquin, thiabendazole or naled, are listed in Tables 1 and 2.
All values listed for the treatment groups were comparable to the control values except the fetal weight
at the 125 mg/kg dose of thiabendazole formulation that
was significantly reduced (P^ = 0.05).
The numbers of
live fetuses at the 1200 mg/kg dose of maleic hydrazide, and total implants/pregnancy at the 100 mg/kg of
naled, were reduced; whereas, the resorption at the 125
mg/kg ethoxyquin formulation and 250 mg/kg thiabendazole formulation were increased.
However, none of them
were statistically different from control values. Incidences of anomalous fetuses, anomalous litters (each having at least one anomalous fetus), fetuses having individual anomalies for the different pesticide treated groups are given (Tables 3 and 4 ) . The in-
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TABLE 1 Prenatal data from rats dosed orally with maleic hydrazide or damlnozide on dayl 6 through 15 of gestation
Daminozide
Maleic hydrazide
Number of females pregnant/number inseminated
0
400 mg/kg
800 mg/kg
1200 mg/kg
1600 mg/kg
0
300 mg/kg
600 mg/kg
1000 mg/kg
19/20
20/20
IB/20
18/20
20/20
19/20
20/20
19/20
19/20
Number of corpora lutca, mean + SE
13.3+0.5
13.4*0.4
13.8*0.4
12.8*0.4
13.3*0.5
12.3+0.4
12.4.0.4
12.1*0.4
12.6*0.3
Implants per pregnancy, mean + SE
12.0*0.7
12.9+0.4
12.7*0.9
9.8*1.0
11.9*0.6
11.1*0.6
11.7*0.6
11.3*0.7
11.3*0.7
Number of females with resorptions
7
12
7
7
5
9
11
8
12
Resorptions, total number
11
13
11
7
9
14
15
11
17
Percent resorptions
4.9
5.1
4.9
4.0
3.8
6.6
6.4
5.1
7.9
Dead fetuses, total
1
1
1
0
0
0
0
0
0
99/116
126/118
104/112
81/89
94/134
94/104
130/89
118/86
108/89
3
0
1
0
0
0
2
11.4+0.6
10.4*0.6
11.0*0.7
10.7*0.7
10.4*0.6
Male/female-llve fetuses Runted fetuses, total number Live fetuses per dam, mean + SE Fetal weight (gm), mean + SE
a
0
0
11.3*0.7
12.2*0.4,
5.0*0.04
Number of resorptions/total implants x 100
5.4*0.04
12.0*0.9 5.3*0.04
9.4*1.0 5.1*0.04
5.0*0.05
5.3*0.04
5.1*0.03
5.1*0.04
5.4*0.05
a
TABLE 2
W
Prenatal data from rats dosed orally with formulations of ethoxyquin, thiabendazole, or Naled on days 6 through 15 of gestation
^
Number of females oregnant/number inseminated
Thiabendazole
Naled
H O
0
125 mg/kg
250 mg/kg
500 mg/kg
0
125 mg/kg
250 mg/kg
500 mg/kg
0
25 mg/kg
50 mg/kg
100 mg/kg
17/20
17/20
19/20
17/20
18/20
16/20
19/20
16/20
17/20
19/20
15/20
18/20
12.5.0.5
14.2*0.5
13.2*0.4
13.1*0.3
12.2.0.5
13.4*0.4
13.5*0.3
12.4.0.9
12.6.0.4
13.1*0.4
12.5*0.3
12.4*0.4
Implants per pregnancy, mean * SE
11.5*0.8
13.5*0.5
12.7*0.4
12.5*0.3
10.6*1.1
12.8*0.7
12.8.0.6
10.8.1.1
12.0.0.7
11.4*0.7
12.4.0.3
10.8*0.8
Number of females with resorptions
10
13
8
9
a
9
13
8
7
10
8
8
Resorptions, total number
12
27
10
17
10
12
17
15
14
13
14
9
Percent resorptions
6
12
4
8
5
5
7
9
7
6
8
5
Dead fetuses, total
0
1
0
0
0
0
0
2
0
1
0
0
102/31
101/100
116/115
106/90
82/99
101/118
117/109
76/80
98/92
103/100
86/86
96/90
Male/female-llve Runted fetuses, total number Live fetuses per dam, mean • SE Fetal weight(gm), mean
8
10.8*0.B 5.0.0.05
0
1
11.8*0.8 -
12.2.0.5 -
4.9*0.03 -
4.8.0.04 -
Number of resorptions/total implants x 100, b P < 0.05
1 11.5*0.5 _ 4.8*0.05 -
1
0
10.1.1.0 12.2.0.8 _ _ _ 4.7*0.04 _
0 11.9.0.7 _
1
2 9.B.I.2 _
4.4*0.10 b 4.7*0.04 4.6.0.05 _ _ _ _
0
0
11.2.0.6 _
10.7.0.7
5.0.0.03 -
4.9.0.03 -
0 11.5^0.4 — 5.2.0.04 -
1 10.3.0.8 5.0*0.05 -
M
RATS
Number of corpora lutea, mean_* SE
w ££j
ISMENTE
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O Ethoxyquin
572
KHERA ET AL.
TABLE 3 Incidence of anomalies in fetuses from rats treated orally with maleic hydrazEde or daminozide from day 6 to 15 of gestation
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Maleic hydrazlde
Daminozide
0
400 mg/kg
800 mg/kg
1200 mg/kg
1600 mg/kg
0
300 mg/kg
600 mg/kg
10 mg,
Number of anomalous fetuses/number examined
8/215
1/244
8/216
5/170
16/228
9/198
8/219
6/204
9/1
Number of litters with anomalous fetuses/ number examined
7/19
1/20
7/18
3/1B
8/20
7/19
5/20
4/19
6/
Extra ribs, uni- & bilateral
4
0
3
0
2
5
2
2
1
13th rib, rudimentary or missing
1
0
1
3
4
1
1
1
5
Fused ribs
0
0
0
0
1
0
0
0
C
Sternoschisis
0
0
0
0
2
0
0
0
0
Sternebrae fused or non-opposed
1
0
0
1
3
0
1
0
1
Sternebrae, delayed ossification
0
0
2
0
5
2
2
2
]
Calvarium, delayed ossification
0
0
0
0
1
0
0
0
(
Runted fetus
0
0
3
0
1
0
0
0
Miscellaneous
0
0
l"
0
2C
0
0
0
Anomalies (number of fetuses affected)
"a fetus may have more than one anomaly, b cleft palate, c one fetus each had a cleft upper lip and rudimentary tail
crease in total number of anomalous fetuses in the 2 50 mg/kg group of thiabendazole formulation was quite significant (£ = 0.005).
The incidences found to be of
borderline significance (£ = 0.05) were the total number of anomalous fetuses in the 2 50 mg/kg group of ethoxyquin formulation and the delayed ossification of sternebrae in the 2 5 and 50 mg/kg groups of naled. All
TABLE 4 Incidence of anomalies in fetuses from rats treated orally with formulations of ethoxyquin, thiabendazole or naled from day 6 to 15 of gestation
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Ethoxyquin
Thiabendazole >
Number of anomalous fetuses/number examined
0
125 mg/kg
250 mg/kg
500 mg/kg
13/183*
12/201
32/231b
22/196
8/17
7/17
13/19
9/17
Number of litters with anomalous fetuses/ number examined
3/181
2/18
125 mg/kg
250 mg/kg
500 mg/kg
10/217
8/226
9/156 b
6/18
6/19
8/14
25 mg/kg
50 mg/kg
100 mg/kg
9/190
16/203
9/172
11/186
6/17
8/19
7/15
6/18
a
I
Anomalies (number of fetuses affected)
CO
Wavy ribs, uni- & bilateral
0
0
0
1
4
2
Extra ribs, uni- & bilateral
6
5
15
12
0
13th rib, rudimentary or missing
1
0
1
0
Strenebraa fused or non-opposed
0
0
1
Sternebrae, delayed ossification
7
5
Calvarium, delayed ossification
2
0
0
0
0
1
0
0
2
1
0
3
2
2
3
0
0
0
0
4
5
2
0
1
0
0
1
0
0
1
1
0
8
12
3
7
5
7
1
5b
5b
6
1
1
0
0
0
0
0
3
1
3
Runted fetus
0
1
1
1
0
0
1
2
0
0
0
1
Kinky or short tail
0
0
2
0
0
0
0
0
1
0
0
0
Miscellaneous
lc
0
2d
0
0
0
le
0
0
0
l'
1«
8
a fetui may have more than one anomaly, b P < 0.05, c itemoschitli, d one fetui each had fused ribs and umbilical herina. generalized «ubcutaneoui edema, hydroureter, ^hydronephrosls
en en M en
574
KHERA ET AL.
other measures of teratogenicity in Tables 3 and 4 were within the control range. DISCUSSION In evaluating their teratogenic potential, 25 fetal and maternal parameters were measured at 3-4 dose
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levels of each of the five pesticides in the present study.
Only four of these parameters showed differences
between the test and control values at levels of P < 0.05.
Rather clear was an increase in the total number
of anomalous fetuses/number of fetuses examined in the 500 mg/kg group of thiabendazole formulation (P < 0.005), although no single anomaly was significantly increased in incidence.
The remaining three para-
meters, at the borderline of P = 0.05, were; a reduced fetal weight, at the 12 5 mg/kg of thiabendazole formulation; an increased total number of anomalous fetuses/ number of fetuses examined, at the 25 0 mg/kg ethoxyquin formulation; and a delayed sternebral ossification, at the 25 and 50 rng/kg naled formulation.
These findings
were inconsistent with the lack of effects at the higher dose levels.
It is possible that these effects
are unreal since among the 25 x 5 x 3 parameters presently examined, one would expect at least four values to be significantly different just by chance alone.
TERATOLOGIC ASSESSMENTS IN RATS
575
It is not known whether et_ seq. pesticides,.if chemically pure, or the unknown ingredients, on separate testing, would give negative responses as have been obtained with their combinations.
However, as it
is, our findings do not suggest making any changes in Downloaded by [Virginia Tech Libraries] at 14:24 14 March 2015
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