United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 2771 1
Ja
600 1-80-008
ar/ 1 980
Research and Development
The Effect of Imida
Administered to
Pregnant Rats
-------
RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ENVIRONMENTAL HEALTH EFFECTS RE-
SEARCH series. This series describes projects and studies relating to the toler-
ances of man for unhealthful substances or conditions. This work is generally
assessed from a medical viewpoint, including physiological or psychological
studies. In addition to toxicology and other medical specialities, study areas in-
clude biomedical instrumentation and health research techniques utilizing ani-
mals — but always with intended application to human health measures.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
-------
EPA-600/1-8J-003
January 1980
THE EFFECT OF IMIDM ADMINISTERED TO PREGNANT RATS
by
Robert D. Short, Jan L. Minor, Timothy M. Unger,
Bradley Breeden, and Dan Van Goethem
Midwest Research Institute
425 Volker Boulevard
Kansas City, Missouri 64110
Contract No. 68-02-2746
Project Officer
Ronald L. Baron
Environmental Toxicology Division
Health Effects Research Laboratory
Research Triangle Park, North Carolina 27711
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND>:DEVELOPMENT
HEALTH EFFECTS RESEARCH LABORATORY
RESEARCH TRIANGLE PARK, NORTH CAROLINA 27711
-------
DISCLAIMER
This report has been reviewed by the Health Effects Research Laboratory,
U. S. Environmental Protection Agency, and approved for publication. Approval
does not signify that the contents necessarily reflect the views and policies
of the U. S. Environmental Protection Agency, nor does mention of trade names
or commercial products constitute endorsement or recommendation for the use.
1.1
-------
FOREWARD
The many benefits of our modern, developing, industrial society
are accompanied by certain hazards. Careful assessment of the relative
risk of existing and new man-made environmental hazards is necessary
for the establishment of sound regulatory policy. These regulations
serve to enhance the quality of our environment in order to promote the
public health and welfare and the productive capacity of our Nation's
population.
The Health Effects Research Laboratory, Research Triangle Park,
conducts a coordinated environmental health research program in toxicology,
epidemiology, and clinical studies using human volunteer subjects. These
studies address problems in air pollution, non-ionizing radiation, environ-
mental carcinogenesis and the toxicology of pesticides as well as other
chemical pollutants. The Laboratory participates in the development and
revision of air quality criteria documents on pollutants for which national
ambient air quality standards exist or are proposed, provides the data for
registration of new pesticides or proposed suspension of those already in
use, conducts research on hazardous and toxic materials, and is primarily
responsible for providing the health basis for non-ionizing radiation
standards. Direct support to the regulatory function of the Agency is
provided in the form of expert testimony and preparation of affidavits as
well as expert advice to the Administrator to assure the adequacy of health
care and surveillance of persons having suffered imminent and substantial
endangertnent of their health.
This report evaluates the teratogenic potential in Wistar rats.
F. G. Hueter, Ph.D.
Di rector
Health Effects Research Laboratory
iii
-------
ABSTRACT
The purpose of this study was to evaluate the teratogenic po-
tential of Imidan in Wistar rats. Accordingly, groups of pregnant
Wistar rats received Imidan by either a single or multiple dose protocol
and their fetuses were examined for gross, soft tissue, and skeletal
defects. In the single dose protocol, 30 mg/kg of Imidan.was administered
on gestational day 8 or 12. In the multiple dose protocol 0.06, 1.5, or
30 mg/kg of Imidan was administered every other day during gestation for
a total of nine doses. No mortality which was attributed to Imidan was
observed. Morbidity, as measured by reduced food consumption and weight
gain was observed in dams that received 30 mg/kg of Imidan by the single
and multiple dose protocol. None of the observed anomalies were increased
to a statistically significant degree. Therefore, it was concluded that
Imidan was not a teratogen in this study.
IV
-------
TABLE OF CONTENTS
Page
Forward ill
Abstract iv
List of Tables vi
I. Introduction 1
II. Methods . 1
A. Animals 1
B. Dose 2
C. Teratology Study 2
D. Interpretation of Data 3
III. Results A
A. Single Dose Protocol 4
B. Multiple Dose Protocol 8
IV. Discussion 8
References 14
-------
LIST OF TABLES
Number Title Page
Effect of Imidan Administered as a Single Dose During
Gestation on Maternal Welfare and Reproduction in Rats
Soft Tissue Anomalies in Rats Treated with a Single Dose
of Imidan During Gestation
Skeletal Anomalies in Rats Treated with a Single Dose
of Imidan During Gestation
Effect of Imidan Administered Every Other Day During Ges-
tation on Maternal Welfare and Reproduction in Rats . . 9
Gross Anomalies in Rats Treated Every Other Day During
Gestation with Imidan 10
Soft Tissue Anomalies in Rats Treated Every Other Day
During Gestation with Imidan 12
Skeletal Anomalies in Rats Treated Every Other Day During
Gestation with Imidan 13
vx
-------
I. INTRODUCTION
Imidan (phosmet, phosphorodithioic acid S-[(1,3-dihydro-l,3-
dioxo-2H-isoindol-2-yl)methyl], 0,0-dimethyl ester) is a widely used
organophosphate insecticide. The developmental toxicity of this pesti-
cide was the subject of a cooperative study between the United States
and the Soviet Union. In the United States study,!/ CD rats, which were
sperm positive on day 1 of gestation, received daily oral doses of 5,
10, 20, 25, or 30 mg/kg of Imidan on days 6 through 15 of gestation.
The dams were sacrificed on day 21 of gestation and their fetuses examined
for defects. Significant maternal mortality was observed in dams that
received 25 and 30 mg/kg/day of Imidan and food consumption was reduced
in dams that received 10 mg/kg/day and above of Imidan. On the other hand,
the incidence of fetal mortality, stunted fetuses, and malformations was
not significantly increased even at dose levels that produced obvious
maternal effects.
In the Russian study,J/ Wistar rats, which were sperm positive
on day 1 of gestation, received either multiple or single doses of
Imidan. For the multiple dose protocol, 0.06 or 1.5 mg/kg/day of Imidan
was given every other day during pregnancy and the dams were sacrificed
on day 19 of gestation. No adverse effects were observed at the low
dose; however, 1.5 mg/kg/day of Imidan increased postimplantation mor-
tality of the embryos. In addition, hydrocephaly and subcutaneous
hemorrhages were reported in this group. For the single dose protocol,
30 mg/kg of Imidan was given on day 9 or 13 of gestation. Abnormalities
which were reported with these treatments included hypognathia, general
edema, dislocation of the extremities, and hydrocepahly.
The United States and Russian studies differ not only in their
protocol but also their conclusions concerning the safety of Imidan. The
purpose of the present study was to evaluate the teratogenic potential
of Imidan by a protocol similar to the protocol used by the Russians.
Accordingly, pregnant Wistar rats were treated with multiple or single
doses of Imidan and their fetuses were examined for birth defects.
II. METHODS
A. Animals
Wistar rats were obtained from the Charles River Breeding
Laboratory (Wilmington, Massachusetts) and housed in our animal quarters
for at least 7 days prior to use. These quarters are maintained at 22 +
4°C with a relative humidity of 40 to 60% and a 7 AM to 7 PM photoperiod.
-------
Animals were given free access to rodent chow (Wayne Lab-Blox, Allied
Mills, Inc., Chicago, Illinois) and tap water.
B. Dose
1. Source of Imidan: A technical grade of Imidan (Stauffer
Chemical Company, Richmond, California) was received at the Midwest
Research Institute on March 29, 1979. The label contained the following
information: "Imidan, Composite, CGH-2,402,4921-31-3, milled 3/23/79."
2. Preparation; Imidan was administered to rats as a sus-
pension in a vehicle of 0.5% methylcellulose (MC, Methocel K4M Premium,
The Dow Chemical Company, Midland, Michigan). The ingredients for the
doses are listed below:
Dose Imidan
(mg/kg) (mg)
0 0 50 50
1.5 15 50 50
30.0 150 25 25
These doses were prepared daily by (a) adding Imidan to water; (b)
mixing for 15 to 20 sec with a Polytron (Brinkmann Instruments, Westbury,
New York); (c) adding 1% methylcullulose, gently mixing on Polytron, and
dipping tip of Polytron in suspension to wash off any material that adhered
to the blade. A suspension to deliver a dose of 0.06 mg/kg was prepared
by adding 2 ml of the above 0.15 mg/ml suspension of Imidan to 48 ml of
0.5% methylcellulose.
3. Administration; All doses were administered by oral intuba-
tion in a volume of 10 ml/kg. The body weight at the time of dosing was
used for calculating all doses.
C. Teratology Study
1. Mating; Sexually mature virgin Wistar rats were housed
overnight with a proven male breeder and examined for sperm-positive
vaginal smears the next morning. The morning evidence of mating was ob-
tained was identified as day 0 of gestation.
-------
2. Treatment
a. Single dose: Two groups each consisting of 25 mated
rats received 30 rag/kg of Imidan on day 8 or 12 of gestation. An addi-
tional group similar in size received only the vehicle on gestational days
8 and 12 and served as the control.
b. Multiple doses: Four groups each consisting of 25
mated rats received 0, 0.06, 1.5 or 30 mg/kg on alternate days of gesta-
tion starting on day 0 and ending on day 16. Therefore, rats in these
groups received a total of nine doses.
3. Maternal observations: Dams were observed for toxicological
responses. In addition, their body weight and food consumption was
monitored during gestation.
4. Fetal observations; Dams were sacrificed on gestational
day 21. A laparotomy was performed and the uterine horns exposed. The
number and position of live, dead, and resorbed fetuses was recorded.
Live fetuses were removed, weighed and immediately examined for external
anomalies as described by Wilson._3/
Approximately one-half of the viable fetuses from each litter
were dissected and examined for soft tissue anomalies by the free-hand
slicing method of Wilson.J./ Each fetus was fixed in 20 to 25 ml of
Bouins fluid for 2 weeks. The hardened fetuses were examined for external
anomalies and serially cut from the head through the trunk using a sharp
razor blade. No slices were made beyond the kidneys and the intestines
were carefully removed from the pelvic cavity. The cross-sections of
the fetuses and the genitourinary organs on the pelvic floor were care-
fully examined by experienced personnel. The remaining viable fetuses
from each litter were processed for skeletal examination. Fetuses were
fixed in 70% alcohol for 2 weeks and enviscerated. The fetuses were
stored in 1% KOH for 2 days and then stained with alizarin red .A/
After differential decolorization, the skeletons were examined by experi-
enced personnel for anomalies.
D. Interpretation of Data
1. Statistics; Quantitative data are reported as the mean +
standard error. These data were analyzed by Bartlett's test for homo-
geneity ._!/ Homogeneous data were analyzed by Tukey's omega procedure.2J
Heterogeneous data were analyzed by a nonparametric rank test.,6/ The
level of statistical significance was selected as p < 0.05 unless in-
dicated otherwise. The litter was considered the experimental unit.
The percent of fetuses with a given anomaly was calculated for each
-------
litter, and these values were averaged to provide a measure of the
affected fetuses per litter.
2. Ranking of anomalies: The various anomalies have been as-
signed a rank. The ranking system is based on our subjective feeling as
to the value of a particular anoamly in predicting the teratogenic poten-
tial of a compound. Accordingly, anomalies with a rank of one have little
value in such predictions while anomalies with a rank of four are more
valuable. The rank is indicated by each anomaly in the various tables.
In addition, the various groups of anomalies are summarized by rank at the
end of each table.
III. RESULTS
A. Single Dose Protocol
1. Maternal welfare and reproduction: A single dose of 30
mg/kg of Imidan on days 8 or 12 of gestation did not produce mortality
which was attributable to Imidan (Table 1). The one death that occurred
in nonpregnant rats treated on day 8 was due to a ruptured esophagus pro-
duced during dosing. The food consumption and body weight of dams
treated on day 12 were significantly reduced after dosing. In addition,
food consumption of dams treated on day 8 was also significantly reduced
after treatment; however, there was no significant effect on body weight.
The various parameters used to monitor reproduction were
normal in dams that received a single dose of Imidan (Table 1). The
litter sizes were normal and there was no evidence of fetal toxicity, as
monitored by percent viable fetuses and fetal body weight.
2. Evaluation of C-section time; Additional dams, which are
not included in Tables 1 through 3, were sacrificed on day 18 of gesta-
tion. Six dams were from the control group and six were from the group
treated on day 12 of gestation. The fetuses were very small and had
sticky skin. These fetuses were processed for soft tissue and skeletal
examination. As a result of the immaturity of these fetuses, it was con-
cluded that C-sections should be performed later in gestation. Accordingly,
all subsequent C-sections were performed on day 21 of gestation.
3. Gross anomalies; There were no gross anomalies to report.
4. Soft tissue anomalies; The soft tissue anomalies are pre-
sented and summarized by rank in Table 2. There was no statistically
significant increase in any of the observed anomalies. In addition,
there was no significant increase in the combined incidence of anomalies
with a given rank.
-------
TABLE 1
EFFECT OF IMIDAN ADMINISTERED AS A
SINGLE DOSE DURING GESTATION ON
MATERNAL WELFARE AND REPRODUCTION IN RATS
Imidan (rag /kg)
Day(s) Treated
Number Treated
Pregnant
Alive
Non-Pregnant
Alive
Body Weight (g/rat)
Day 0
8
9
12
13
20
Food Consumption (g/rat /day)
Days 8-12
12-16
8-16
8-20
Pregnant Survivors
Implants /Dam
Viable Fetuses (%)
Dead Fetuses (%)
Early Resorptions (%)
Late Resorptions (%)
0
8 and 12
25
23
23
2
2
223 + 3
254 + 3
254 + 3
271 + 3
278 + 3
353 + 5
25.4 + 0.3
27.0 + 0.6
26.2 + 0.4
27.1 + 0.4
22
12.5 + 0.6
98 + 1
0 + 0
2 + 1
0 + 0
Dams with Complete Resorptions 0
Live Liters
Fetuses /Dam
Males (%)
Fetal Weight
22
12.2 + 0.6
47 + 3
5.31 + 0.08
30
8
25
22
22
3
2
224 + 3
252 + 4
248 + 4
261 + 4
267 + 4
343 + 8
^.v ± v.u-
25.6 + 0.9
24.6 + 0.6
25.4 + 0.7
22
13.5 + 0.3
93 + 5
0 + 0
7 + 5
0 + 0
1
21
12.3 + 0.3
52 + 3
5.28 + 0.05
30
12
25
23
23
2
2
223 + 3
250 + 3
254 + 4
267 + 3
266 + 3^'
344 + 5
24.3 + 0.5
24.4 + 0.5^
24.4 + 0.4^.'
24.7 + Q.&
23
12.6 + 0.4
93 + 2
1 + 1
5 + 1
1 + 1
0
23
11.7 + 0.4
49 + 3
5.36 + 0.05
a/ Significantly different from control (Dunnett's procedure)
b/ Significantly different from control (two sample rank test)
-------
TABLE 2
SOFT TISSUE ANOMALIES IN RATS TREATED WITH A
SINGLE DOSE OF IMIDAN DURING GESTATION
Eiose (mg/kg/day) 0 30 30
Days Treated 8 and 12 8 12
Number of
Litters Affected/Examined (%) 10/23 (43) 4/21 (19) 4/23 (17)
Fetuses Affected/Examined (%) 15/137 (11) 6/133 (4) 8/132 (6)
Soft Tissue Anomalies (Rank)—
Micropthalmia (.4) 0 (O)^/ 0 CO) 0.7 (1)
Trachea Occluded (1) 1.7 (2) 0.8 (1) 2.2 (.2)
Right Ventricle Collapsed (2) 0 (0) 0.7 (1) 0 (0)
Hydronephrosis (3) 0.9 (1) 0 (0) 3.8 C2)
Slight (1) 4.5 (6) 2.8 (2) 0 (0)
Blood in Kidney (3) 0 (0) 0 (0) 1.1 (1)
Distended Urinary Bladder (2) 0 (0) 0 (0) 0.5 CD
Summary by Rank 1-4 10.2 (10) 4.3 C4) 6.7 (4)
2-4 4.0 (2) 0.7 CD 4.5 C3)
3-4 1.5 (2) 0 (0) 4.5 C3)
4 0.6 (1) 0 (0) 0.7 (1)
a/ Ranked by increasing value in predicting teratogenic potential.
b_/ Mean of the percent of fetuses with the indicated anomaly calculated on
a litter basis. The number in parenthesis is the number of affected
litters.
-------
TABLE 3
SKELETAL ANOMALIES IN RATS TREATED WITH A
SINGLET DOSE OF IMISAN 3UUING GESTATION
Oose (ing/kg/day) 0 30 30
Days Treated 3 and 12 3 12
Number of
Litters Affected/Examined (%) 23/23 (100) 21/21 (100) 23/23 (100)
Fetuses Affected/Examined (%) 127/148 (86) 105/145 (72) 102/137 (74)
Skeletal Anomalies (Rank)—^
Squamosal Split (1) 18.2 (12)£/ 27.9 (16) 17.4 (14)
Inc. Ossified (1) 7.9 (6) 7.9 (5) 4.0 (2)
Hyoid Bone Split (3) 1.8 (2) 0 (0) 0 (0)
Inc. Ossified (1) 21.8 (15) 26.2 (13) 18.5 (15)
Unossified (1) 0 (0) 1.5 (2) 2.6 (2)
Frontal Bones Inc.
Ossified (1) 0.6 (1) 2.3 (4) 2.5 (2)
Frontal Foncanel
Enlarged (2) 6.0 (5) 3.1 (5) 5.1 (3)
Occipital Fontanel
Enlarged (2) 1.8 (3) 0.7 (1) 0.7 (1)
Pariecals Inc. Ossified (1) 37.3 (19) 43.5 C17) 37.5 (17)
Interparietal Inc.
Ossified (1) 39.7 (19) 45.5 (18) 45.0 (21)
Supraoccipital Inc.
Ossified (1) 29.2 (14) 31.3 C16) 32.3 (16)
Ribs Extra (2) 0.7 (1) 0.6 CD 0.7 (1)
Wavy (2) 13.0 C9) 14.1 Cll) 17.9 CIO)
Short (2) 0.7 CD 0 CO) 0 (0)
Centra Ossified Normally 54.3 (23) 74.5 (21)-, 69.7 (23) .
Lobed (2) 43.9 (20) 23.0 (15)- 23.0 (16)~
Split (2) 3.3 (3) 3.3 (5) 7.1 (9)
Hemi-Cencra (4) 0 (0) 0 (0) 1.4 (2)
Vertebra Inc. Ossified (1) 0 (0) 0 CO) 0.7 (1)
aerni-Vertebra (4) 0 (0) 0 CO) 0.7 (1)
Stemebrae Ossified
Xoraally 55.2 C22) 70.6 (20) 70.2 (23)
Unossified (1) 4.5 (5) 2.3 (2) 2.5 C3)
Inc. Ossified (1) 7.9 (S) 7.5 C6) 10.7 C3)
Lobed (2) 15.3 (15) 7.9 (7) 7.3 C3)
Halalignmenc of Fusion (3) 17.2 CW) 11.6 0-4) 10.0 (10)
of Stemebrae
Summary by Rank 1-4 I 85.5 (23) 71.7 (21) 73.4 (23)
2-4 66.4 (23) 48.3 (20) 47.3 C.22)
3-4 17.9 (14) 11.3 C14) 10.3 (11)
4 0 (0) 0 CO) 1-4 (2)
a/ Ranked by increasing value in predicting caratogenic potential.
b_/ Mean of the percent of fetuses with the indicated anomaly calculated on a
litter basis. The number in parenthesis is the number of affected littars.
^ Significantly different from control (Tukey'* procedure).
-------
5. Skeletal anomalies; The skeletal anomalies are presented
and summarized by rank in Table 3. In the two groups treated with
Imidan, the incidence of normally ossified centra was significantly
increased and the incidence of lobed centra was significantly reduced.
Other than these observations, there was no significant increase in
either specific anomalies or anomalies that were grouped by rank.
S. Multiple Dose Protocol
1. Maternal welfare and reproduction: In this protocol, rats
were treated with 0.06, 1.5, or 30 mg/kg of Imidan every other day
during gestation for a total of nine doses. Mortality was not observed
in any of the treatment groups (Table 4). Morbidity, as measured by
reduced food consumption and weight gain, was consistently observed only
in dams from the high dose group.
The various parameters used to monitor reproduction were
normal in all groups treated with Imindan (Table 4). There was no
evidence of fetal toxicity as monitored by litter size, percent viable
fetuses and fetal body weight. Although the percent males was reduced
in the group treated with 1.5 mg/kg of Imidan, this effect did not occur
in other groups and, therefore, is probably a chance occurrence.
2. Gross anomalies: The gross anomalies observed in these
groups are present in Table 5. There was no significant increase of in-
dividual anomalies or anomalies summarized by rank.
3. Soft tissue anomalies; The soft tissue anomalies observed
during this protocol are presented in Table 6. There was no significant
increase of individual anomalies or summaries of anomalies by rank.
4. Skeletal anomalies; The skeletal anomalies observed
during this protocol are presented in Table 7. There was no significant
increase in individual anomalies or summaries of anomalies by rank.
IV. DISCUSSION
The purpose of the present study was to evaluate the teratogenic
potential of Imidan. This study was necessary because the literature con-
tains conflicting information concerning the safety of this insecticide
during pregnancy. The obvious differences between previous studies^-»2/
include strains of rats used, dosing protocol, and time of C-section. As
described in the introduction, the protocol that was adopted for the present
study was similar to one which reported to demonstrate the teratogenic
potential of Imidan.2J
8
-------
TABLE 4
EFFECT OF IMIDAN ADMINISTERED EVERY OTHER
DAY DURING GESTATION ON MATERNAL
WELFARE AND REPRODUCTION IN RATS
Number Treated
Pregnant
Alive
Non-Pregnant
Alive
Body Weight (g/rat)
Day 0
2
4
8
16
20
Food Consumption
(g/rat /day)
Days 0-4
4-8
8-16
16-20
Pregnant Survivors
Implants/Dam
Viable Fetuses (%)
Dead Fetuses (%)
Early Resorptions
0
25
21
21
4
4
247 + 4
253 + 4
256 + 4
268 + 4
309 + 5
364 + 6
23.9 + 0.4
23.5 + 0.6
26.1 + 0.6
29.2 + 0.8
19
12.8 +0.6
96 + 2
0 + 0
(%) 4 + 2
Late Resorptions (%) 0+0
Dams with Complete
Resorptions
Live Liters
Fetuses /Dam
Males (%)
Fetal Weight
0
19
12.5 + 0.6
55 + 3
5.21 + 0.07
Imidan
0.06
25
24
24
1
1
234 + 3£/
241 + 3
247 + 3
258 + 3
298 + 4
348 + 6
22.1 + 0.7^/
22.3 + 0.4
25.3 + 0.4
27.3 + 0.8
23
11.8 + 0.8
92 + 3
0 + 0
8 + 3
0 + 0
0
23
10.8 + 0.8
49 + 3
5.32 + 0.12
(mg/kg)
1.5
25
19
19
6
6
240 + 3
244 + 3
248 + 4
262 + 4
304 + 4
358 + 5
24.4 + 0.7
23.6 + 0.7
27.1 + 0.7
28.1 + 1.7
18
12.7 + 0.6
95 + 1
0 + 0
4 + 1
1 + 1
0
18
12.0 + 0.5
39 + 3S/
5.25 + 0.07
30
25
24
24
1
1
244 + 3
246 + 4
243 + kSJ
248 + 4a./
280 + 6^
331 + 9±/
20.1 + O.S^/
18.5 + 0.7^
21.4 + l.ok/
27.0 + O.S^/
23
12.7 + 0.7
91 + 4
0 + 0
9 + 4
0 + 0
1
22
12.0 + 0.7
44 + 4
5.08 + 0.07
&/ Significantly different from control (Dunnett's procedure)
b/ Significantly different from control (two sample rank test)
-------
TABLE 5
GROSS ANOMALIES IN RATS TREATED EVERY OTHER DAY
DURING GESTATION WITH IMIDAN
Imidan (mg/kg/day)
Number of
Litters Affected/Examined (%)
Fetuses Affected/Examined (%)
Gross Anomalies
(Rank)-/
Ear Pinna Reduced or Misplaced
Snout Reduced
Lower Jaw Reduced
Snout Upturned
Short Neck
Appendicular Reduction
Anomaly
Hindquarters Reduced
Rotund Fetus
Rank 1-4
2-4
3-4
4
0/21 (0)
0/255 (0)
(4)
(3)
(3)
(3)
(3)
(4)
(4)
(1)
0
0 (0)
o CO)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
o CO)
0 (0)
0 (0)
0 (0)
0.06
1.5
2/24 (8)
2/260 (1)
2.1 (1)
0 (0)
0 (0)
2.1 (1)
0 (0)
0 CO)
0.4 (1)
0 CO)
.5 C2)
.5 C2)
2.
2.
2.5
2.5
C2)
C2)
0/19 (0)
0/231 (0)
0 (0)
0 (0)
0 (0)
0 CO)
0 CO)
0 CO)
o CO)
0 (0)
0 (.0)
0 (0)
0 (0)
0 (0)
30.0
1/23 (4)
1/275 (1)
0.3 (1)
0.3 (1)
0.3 (1)
0 CO)
0.3 CD
0
0
0.3
0.3
0.3
0.3
0.3
3 (1)
(0)
(1)
(1)
CD
(1)
(1)
a/ Ranked by increasing value in predicting teratogenic potential.
b/ Mean of the percent of fetuses with the indicated anomaly calculated on a
litter basis. The number in parenthesis is the number of affected litters.
10
-------
No mortality was observed in Wistar rats that received up to 30
rag/kg every other day during gestation (Tables 1 and 4). This is 'contrast
to the observations in CD rats where continuous doses of 25 mg/kg/day of
Imidan produced death..!/ Morbidity, as measured by reduced weight gain
and reduced food consumption, was observed in Wistar rats that received
30 mg/kg/day of Imidan (Tables 1 and 4). Similar observations were ob-
served in CD rats that received 10 mg/kg/day and above of Imidan.I/ The
previous Imidan teratology study in Wistar rats does not provide the above
types of maternal observation.JV
We decided to perform C-sections on day 21 of gestation because
fetuses were too immature for meaningful examinations if C-sections were
performed on gestational day 18. A similar conclusion regarding the best
time to perform C-sections in Wistar rats was reacted by others.U This
is in contrast to the previous Wistar teratology study2/ in which it was
reported that dams were sacrificed at the earlier time. However, in
pictures, their fetuses appear to be older than the stated gestational age.
Dams treated with up to 30 mg/kg of Imidan by either the single
or multiple dose protocol did not produce fetuses with a statistically
significant increase in gross (Table 5) soft tissue (Tables 2 and 6), or
skeletal (Tables 3 and 7) anomalies.
In order to be classified as a teratogen an agent must alter the
structure or function of a statistically significant number of young..§/ An
agent is not classified as a teratogen if it only produced fetal death or
reduces fetal growth. In addition, an agent is not classified as a tera-
togen if the dose required to produce an effect in the embryo or fetuses
is overtly toxic to the dam. According to these criteria Imidan is not a
teratogen in Wistar rats.
11
-------
TABLE 6
SOFT TISSUE ANOMALIES IN RATS TREATED EVERY OTHER DAY
DURING GESTATION WITH
IMIDAN
Imidan (mg/kg/day)
Number of
Litters Affected/Examined
Fetuses Affected /Examined
(%)
(%)
a/
Soft Tissue Anomalies (Rank)—
Nasal Passage Occluded
Trachea Occluded
Bifurcated Esophagus
Right Ventricle Collapsed
Blood in Abdomen
Small Intestines
Hydronephrosis
Slight
Kidney Pelvis Collapsed
No Kidneys
Hydroureter
Distended Urinary Bladder
Summary by Rank l-r-4
2-4
3-4
4
CD
(1)
(4)
(2)
(2)
(3)
(3)
(1)
(3)
(4)
(2)
(2)
0
12/21 (57)
26/123 (21)
, /
0 (0)^'
2.9 (1)
0 (0)
0 (0)
0 (0)
0 (0)
0.8 (1)
1.5 (2)
0 (0)
0 (0)
5.5 (3)
1.6 (2)
21.5 (12)
15.6 (8)
8.5 (6)
0 (0)
0.06
5/24 (21)
10/125 (8)
2.1 (1)
1.7 (2)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
1.6 (1)
0 (0)
0 (0)
3.1 (2)
0 (0)
7.5 (5)
3.1 (2)
0 (0)
0 (0)
1.5
6/19 (32)
12/109 (11)
0 (0)
0 (0)
0 (0)
0 (0)
1.8 (1)
0 (0)
1.8 (1)
2.7 C3)
0 (0)
0 (0)
4.2 (3)
1.9 (2)
10.4 (6)
8.7 (5)
1.8 (1)
0 (0)
30.0
5/23 (22)
10/130 (8)
0 (0)
2.0 (2)
0.9 (1)
0.9 (1)
0.9 (1)
0.5 CD
0 CO)
0 (0)
0.5 (1)
0.5 (1)
1.7 (1)
2.2 (3).
8.6 (6)
7.1 (5)
1.4 (2)
1.4 (2)
a/ Ranked by increasing value in predicting teratogenic potential.
b/ Mean of the percent of fetuses with the indicated anomaly calculated on
a litter basis. The number in parenthesis is the number of affected
litters.
12
-------
TABLE 7
SKELETAL ANOMALIES IN RATS TREATED EVERY OTHER DAY
DURING GESTATION *ITH IMIDAN
tnidan (mg/kg/dav)
Number of
Litters Affected/Examined
Fetuses Affected/Examined
Skeletal Anomalies (Rank)-''
Tympanic Annulus Inc.
Ossified
Squamosal Split
Inc. Ossified
Hyoid Bone Split
Inc. Ossified
Unossified
Maxillary Process
Inc. Ossified
Frontal Bones
Inc. Ossified
Frontal Fontanel Enlarged
Occipital Fontanel
Enlarged
Parietals Inc. Ossified
Interparietals
Inc. Ossified
Supraoccipital
Inc. Ossified
Exoccipital Inc. Ossified
Ribs Extra
Wavy
Centra Ossified Normally
Lobed
Split
Sternebrae Ossified
Normally
Unossified
Inc. Ossified
Lobed
Malalignment of Fusion
of Sternebrae
Pubis Unossified
Ulna Reduced
Radius Reduced
Femur Reduced
Tibia Reduced
Fibula Reduced
Summary by Rank 1-4
2-4
3-4
4
0.06
20/21 (95)
99/132 (75)
L.5
30.0
23/24 (96) 19/19 (100) 22/23 (96)
112/135 (33) 103/122 (84) 122/143 (35)
(2)
(1)
(1)
(3)
(1)
(1)
(1)
(1)
(2)
(2)
(1)
(1)
(1)
(2)
(2)
(2)
(2)
(2)
(D
(1)
(2)
C3)
(3)
(3)
(4)
(3)
(3)
(3)
0 (0)—
19.1 (14)
8.5 (5)
0.8 (1)
23.6 (13)
0 (0)
0 (0)
4.9 (4)
3.6 (3)
0 (0)
32.7 (14)
41.2 (15)
24.4 (12)
0 (0)
1.4 (1)
14.9 (5)
71.7 (20)
23.6 (10)
4.6 (4)
67.1 (21)
4.3 (4)
3.8 (3)
15.4 (11)
9.5 (11)
0, (0)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
73.2 (20)
58.1 (19)
9.5 (11)
0 (0)
4.2 (1)
18.4 (13)
5.3 (4)
0 (0)
16.0 (12)
0.5 (1)
0 (0)
7.2 (6)
8.3 (5)
2.0 (3)
40.4 C20)
40.0 (19)
33.5 (17)
4.2 (1)
1.2 (2)
13.3 (15)
58.7 C23)
39.0 (20)
2.3 (3)
71.9 (22)
0.7 (1)
11.3 (6)
5.9 (7)
10.2 (9)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
80.3 (.23)
64.3 C23)
10.2 (9)
0 (0)
0 (0)
29.9 (14)
8.3 (6)
0 (0)
32.3 (16)
0 (0)
0.9 (1)
6.1 (3)
2.1 (2)
0 (0)
41.2 C15)
43.7 CIS)
37.9 (15)
0.9 (D
0 (0)
21.6 (12)
63.8 (IS)
30.7 CIS)
5.5 (5)
74.3 (19)
1.1 (1)
3.9 C3)
5.2 C5)
15.1 (13)
0 CO)
0 CO)
0 CO)
0 (0)
0 (0)
0 CO)
84.7 (19)
59.7 (19)
15.1 C13)
0 CO)
0 (0)
19.4 (14)
3.3 (3)
0 (0)
21.1 (13)
0.6 (1)
0 (0)
3.2 (4)
0.6 (D
3.7 (5)
35.7 (17)
44.2 (19)
25.5 (.15)
0 (0)
0 (0)
21.3 (1-)
62.5 (21)
35.9 (19)
3.0 Ci)
60.5 (23)
6.0 (7>
±5.5 < 10)
10.3 (ID
'.1 C)
0.5 (D
0.5 (D
0.5 CD
0.5 CD
0.5 (D
0.5 CD
83.3 (22)
61.2 (22)
9.5 :"8)
C.5 (1)
a/ Ranked by increasing value in predicting teratogenic potential.
b_/ Mean of the percent of fetuses with the indicated anomaly calculated on a
litter basis. The number in parenthesis is the r.utnber of affected liters.
13
-------
REFERENCES
1. Staples, R. E., R. G. Kellam, and J. K. Haseman, "Developmental
Toxicity in the Rat After Ingestion or Gavage of Organophosphate
Pesticides (Dipterex, Imidan) During Pregnancy," Environ. Health
Perspect.. 13:133-140 (1976).
2. Martson, L. V. and V. M. Voronina, "Experimental Study of the Effect
of a Series of Phosphoroorganic Pesticides (Dipterex and Imidan)
on Embryogenesis," Environ. Health Perspect. , 3.3:121-125 (1976).
3. Wilson, J. G., "Methods for Administering Agents and Detecting Malfor-
mations in Experimental Animals," In Teratology—Principles and
Techniques, J. G. Wilson and J. Workany (eds.), University of Chicago
Press, Chicago, Illinois, pp. 262-277 (1965).
4. Staples, R. C. and V. L. Schnell, "Refinements in Rapid Clearing Tech-
niques in the KOH-Alizarin Red S Method for Fetal Bones," Stain
Techno 1., _39_: 61-63 (1964).
5. Steel, R. G. D. and J. H. Torrie, Principles and Procedures of
Statistics, McGraw-Hill Book Co., New York (1960).
6. Mann, H. B. and D. R. Whitney, "On a Test of Whether One of Two
Random Variables is Stochastically Larger than the Other," Ann.
Math. Stat., 18:50-60 (1947).
7. Aliverti, V., L. Bonanomi, E. Giavini, V. G. Leone, and L. Mariani,
"The Extent of Fetal Ossification as an Index of Delayed Development
in Teratogenic Studies on Rats," Teratology, ^0:237-242 (1979).
8. Staples, R. E. and J. G. Wilson, Definition of teratogenesis and tera-
togen, in Methods for Detection of Environmental Agents that Produce
Confenital Defects (T. H. Shepard and J. R. Millers, eds.) pp. 25-26,
American Elsevier Publishing Company, New York (1975).
14
-------
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/1-80-008
2.
3. RECIPIENT'S ACC=SSICr»NC.
4. TITUS AIMO SUBTITLE
The Effect of Imidan Administered to Pregnant Rats
S. REPORT DATS
January 19.80
6. PERFORMING ORGANIZATION COOS
7. AUTHORIS)
Robert D. Short, Jan L. Minor, Timothy M. Unger
Bradley Breeden, and Dan Van Goethem
8. PERFORMING ORGA.NIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Midwest Research Institute
425 Volker Boulevard
Kansas City, Missouri 64110
10. PROGRAM ELEMENT NO.
1EA615
11. CONTRACT/GRANT NO.
68-02-2746
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory RTP,NC
U.S. Environmental Protection Agency
Office of Research and Development
Research Triangle Park, North Carolina 27711
13. TYPE OF REPORT AND PERIOD COVSRSO
14. SPONSORING AGENCY CODE
EPA 600/11
15. SUPPLEMENTARY NOTES
18. ABSTRACT
The purpose of this study was to evaluate the teratogenic potential of Imidan in
Wistar rats. Accordingly, groups of pregnant Wistar rats received Imidan by either a
single or multiple dose protocol and their fetuses were examined for gross, soft tis-
sue, and skeletal defects. In the single dose protocol, 30 mg/kg of Imidan was admin-
istered on gestational day 8 or 12. In the multiple dose protocol 0.06, 1.5, or 30
mg/kg of Imidan was administered every other day during gestation for a total of nine
doses. No mortality which was attributed to Imidan was observed. Morbidity, as mea-
sured by reduced food consumption and weight gain was observed in dams that received
30 mg/kg of Imidan by the single and multiple dose protocol. None of the observed
anomalies were increased to a statistically significant degree. Therefore, it was
concluded that Imidan was not a teratogen in this study.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lOENTIFIERS/CPS.N ENDED TERMS C. COSAT; Field/Groan
Insecticide
Pesticide
Organophosphate insecticide
Imidan
Teratology
Rat
06 T
13. 3lSTRI3UT:ON STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS iTha Report)
UNCLASSIFIED
21- >*O. OP rAGSS
21
20. SECURITY CLASS iThispagei
UNCLASSTFTFD
SPA Farm 2220-1 (9-73)
15
------- |