EPA-560/6-77-028
TOXICITY STUDIES OF SELECTED CHEMICALS
TASK IV:
THE DEVELOPMENTAL TOXICITY OF ETHYLENE DIBROMIDE INHALED
BY RATS AND MICE DURING ORGANOGENESIS
JUNE 1977
FINAL REPORT
ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF TOXIC SUBSTANCES
WASHINGTON, D.C. 20460
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EPA-560/6-77-028
TOXICITY STUDIES OF SELECTED CHEMICALS
TASK IV:
THE DEVELOPMENTAL TOXICITY OF ETHYLENE DIBROMIDE INHALED
BY RATS AND MICE DURING ORGANOGENESIS
Final Report
Prepared by
Robert D. Short, Jr.
Jan L. Minor
Joseph M. Winston
Brett Ferguson
Timothy Unger
Cheng-Chun Lee
Contract No. 68-01-3242
Joseph Seifter
Project Officer
Office of Toxic Substances
U.S. Environmental Protection Agency
Washington, DC 20460
June 1977
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PREFACE
This report was prepared at Midwest Research Institute, 425 Volker
Boulevard, Kansas City, Missouri 64110, under Environmental Protection
Agency Contract No. 68-01-3242, MRI Project No. 4128-B, "Toxicity Studies
of Selected Chemicals." The work was supported by the Office of Toxic
Substances of the Environmental Protection Agency. Dr. William Marcus
and Dr. Joseph Seifter are consecutive contract monitors for this project.
This work was conducted in the Biological Sciences Division under
the direction of Dr. William B. House between April 1, 1977 and June 1, 1977.
The experimental work was supervised directly by Dr. Cheng-Chun Lee, Assis-
tant Director, Biological Sciences Division for Pharmacology and Toxicology;
assisted by Dr. Robert D. Short, Jr. (Senior Toxicologist), Dr. Joseph M.
Winston (Associate Toxicologist), Mr. Jan L. Minor (Associate Toxicologist),
with the technical assistance of Mr. Brett Ferguson and Mr. Timothy Unger.
Approved for:
MIDWEST RESEARCH INSTITUTE
W. B. House, Director
Biological Sciences Division
ii
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TABLE OF CONTENTS
Page
Summary 1
I. Introduction 2
II. Methods 3
A. Animals 3
B. Animal Exposure 3
C. Experimental Protocol 4
D. Statistical Methods 4
III. Results 4
A. Chamber Concentration 4
B. Maternal Welfare and Reproduction 5
C. Anomalies 5
References 14
LIST OF TABLES
Table Title
1 Effect of Ethylene Dibromide Exposure During Organo-
genesis on Maternal Welfare and Reproduction in Rats . . 6
2 Effect of Ethylene Dibromide Exposure During Organo-
genesis on Maternal Welfare and Reproduction in Mice . . 7
3 Effect of Ethylene Dibromide Exposure During Organo-
genesis on the Incidence of Soft Tissue Anomalies
in Rats 8
4 Effect of Ethylene Dibromide Exposure During Organo-
genesis on the Incidence of Skeletal Anomalies in Rats . 9
5 Effect of Ethylene Dibromide Exposure During Organogenesis
on the Incidence of Soft Tissue Anomalies in Mice. ... 11
6 Effect of Ethylene Dibromide Exposure During Organogenesis
on the Incidence of Skeletal Anomalies in Mice 12
iii
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SUMMARY
Ethylene dibromide (EDB) was administered at 20, 38, and 80 ppm by
inhalation to pregnant Charles River CD rats and CD-I mice for 23 hr/day.
The exposures started on day 6 of gestation and lasted for a total of 10
days. Observations were made on maternal welfare and fetal development.
The results of this study indicate that (1) EDB is more toxic in
adult mice than adult rats, (2) adverse effects on maternal welfare, as mea-
sured by weight gain, feed consumption, and survival were observed in both
mice and rats, (3) although morphological changes were observed in fetuses
from dams exposed to EDB, these effects were observed at concentrations that
also affected maternal welfare, and (4) EDB was judged to be only a weak
teratogen with little primary effect on development.
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I. INTRODUCTION
Ethylene dibromide (1,2-dibromethane, EDB), is used as a scavenger
in gasoline, a fumigant and a chemical intermediate. The estimated produc-
tion of EDB was about 315 million pounds in 1972 and 331 million pounds in
1973.
The inhalation of EDB produced toxicity in experimental animals .i/
Rats exposed to a concentration of EDB in excess of 200 ppm died within 24
hr from respiratory or cardiovascular collapse. Death at concentrations
less than 200 ppm were delayed and occurred sometimes as long as 12 days
after treatment. During this time, rats lost weight, appeared rough and
unkept, became irritable and produced a bloody nasal discharge. Chronic in-
halation studies (7 hr/day for 5 days/week for 6 months) indicated that rats,
guinea pigs, rabbits and monkeys generally tolerated EDB at levels of 25 ppm.
The results of these studies were used to establish a threshold limit value
of 20 ppm for EDB by the American Conference of Governmental Industrial Hy-
gienists.'
EDB exposure also produced a more insidious type of toxicity. A
carcinogenic response was demonstrated by administering EDB orally to rats
(40 and 80 mg/kg/day) and mice (60 and 120 mg/kg/day) five times a week.'
This treatment produced a high incidence of gastric squamous cell carcinomas
in both species as early as 10 weeks after treatment started. The tumors,
which were originally located in the forestomach, metastasized throughout the
abdominal cavity. In some animals the carcinoma migrated to the lungs and
other tissues.' EDB was also shown to be mutagenic in bacteria^/ and
Drosophila melanogaster.' EDB also affected spermatogenesis and the matura-
tion of sperm in bulls,-/-' damaged spermatogenic cells in ratsii' and reduced
the egg weight of laying hens.'
The teratogenic potential of inhaled EDB was studied in Charles
River CD rats and CD-I mice.' Animals were exposed to 32 ppm of EDB for
23 hr a day from days 6 through 15 of gestation. In addition to the con-
trol, a third group of animals was given a reduced diet, but no EDB expo-
sure. In mice, it was observed that exposed animals consumed less feed and
gained less weight than controls. Litter sizes were somewhat reduced as
were weights, and a variety of skeletal anomalies involving incomplete os-
sification was noted. Because increased occurrence of similar phenomena
was displayed by the reduced-diet, unexposed rats, it was determined that
the defects were most likely attributable to malnourishment rather than to
EDB exposure, per se. In rats, a similar reduction in feed consumption and
weight gain was seen. Similarly, litter size was somewhat reduced, but fetal
weights were near normal. As was noted in mice, many of the observed defects
could well be attributable to malnourishment rather than to EDB exposure.
per se. However, an increase in fourth-ventricular hydrocephaly, a reduction
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in the occurrence of the fourteenth rib, and an increase in the frequency of
wavy ribs appear to be correlated to EDB exposure in this species. The re-
sults section and tables for the above study are included in the present re-
port as Appendix I.
The present study was undertaken to extend the teratology observa-
tions to additional concentrations of EDB. The inhalation route of exposure
was selected for these studies because EDB is found in the atmosphere. The
occurence of congenital defects was used as a measure of toxicity because de-
velopment is a finely regulated process which is sensitive to disruption by
many agents. These agents include both carcinogens and mutagens. In addi-
tion, these tests may be performed in a short period of time and are useful
in identifying agents that may produce birth defects in humans.
II. METHODS
A. Animals
Charles River CD rats and CD-I mice (Charles River Breeding Labo-
ratories, North Wilmington, Massachusetts) were housed in our animal quarters
for at least 7 days prior to use. The quarters were maintained at 72 °C with
a relative humidity of 50 + 5% and a 7 AM to 7 PM photoperiod. Animals were
given free access to powdered rodent chow (Wayne Lab-Blox, Allied Mills, Inc.,
Chicago, Illinois), and tap water except where indicated in the experimental
protocol. During the treatment period, feed was changed daily in order to
prevent possible accumulation of EDB in the feed.
B. Animal Exposure
Rochester type stainless steel chambers with a volume of about 3.5
m^ were used in this study. Clean air at a flow rate of 10 to 12 changes per
hr entered at the top of the chamber. EDB vapor was generated by bubbling
nitrogen into a glass vessel which was maintained at 30°C. EDB entered the
air stream upstream from the chamber. Mixing was initiated in a plenum at
the top of the chamber and completed by two squirrel cage fans and a dif-
fusion plate.
The EDB concentration in the chamber was monitored using gas chro-
matography and a flame ionization detector. EDB was resolved using a stain-
less steel column packed with 5% didecyl phthalate on 80/100 chromosorb and
nitrogen (80 ml/min) as the carrier gas. The injection, column and detector
temperatures were 160°C, 145°C and 170°C, respectively. Standards were pre-
pared by serial dilutions of an EDB stock solution prepared in carbon tetra-
chloride.
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C. Experimental Protocol
Female rats and mice were exposed overnight to proven male breeders.
Successful mating was identified the next morning (day 0 of gestation) by
the presence of sperm in vaginal smears from rats and copulation plugs in
mice. Mated animals were divided into control and treated groups. The con-
trol group was subdivided into animals given free access fo feed and animals
whose feed was restricted. The EDB treated group was subdivided into animals
exposed to 20, 38, or 80 ppm of EDB. Animals were housed in the inhalation
chambers for 10 days starting on day 6 of gestation. During this time the
EDB treated group was exposed to EDB for 23 hr a day. The control animals
were housed under similar conditions; however, the EDB exposure was omitted.
Rats and mice were sacrificed on gestational day 20 or 18, respec-
tively. A laparotomy was performed and the uterine horns were exposed. The
umbilical cord was clamped and severed distally in order to prevent blood loss.
Fetuses were removed, weighed and examined for external anomalies.il/ One-
half of the fetuses from each litter was fixed in Bouin's solution and ex-
amined for soft-tissue anomalies by a free-hand slicing method.I The re-
maining fetuses were fixed in 70% alcohol, eviscerated, stored in 1% KOH and
stained with alizarin red.!2-' After differential decolorization, the skele-
tons were examined for anomalies.
D. Statsitical Methods
Quantitative data, reported as the mean + standard error, were
initially analyzed by Bartlett's test for homogeneity.11' The test of sig-
nificance for homogeneous data was Dunnett's procedure.!!/ In contrast,
heterogeneous data were analyzed by the two-sample rank test.JA' Enumera-
tion data were analyzed with the Fisher's exact probability test.l^/ For
all tests the 0.05 level of significance was chosen except where indicated.
The liter was considered to be the unit of observation.!^/ All statis-
tical tests, therefore, were based on the litter as the experimental unit.
III. RESULTS
A. Chamber Concentration
Rats and mice inhaled EDB 23 hr a day for 10 days starting at day
6 of gestation. During this time, the EDB concentration was measured by gas
chromatography generally every 2 hr. However, as the result of a mechanical
malfunction, the concentration was calculated by mass balance on one of the
exposure days. The values determined during the day were averaged to yield a
time weighed average concentration for that day. The average (range) of
these values for each chamber during the 17 day duration of the study were
20 (15 to 22), 38 (32 to 42), and 80 (71 to 84).
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B. Maternal Welfare and Reproduction
1. Rats; Adverse effects on maternal welfare and reproduction
were observed in rats exposed to EDB (Table 1). Deaths occurred only at
the high concentration. A weight loss was evident in rats exposed to 38
and 80 ppm of EDB. In addition, the weight gain at the end of exposure was
reduced at the high concentration. Feed consumption was reduced in rats
exposed to all concentrations of EDB and remained depressed in the high con-
centration group when the exposure was terminated. Dams exposed to 80 ppm
of EDB had a reduced number of implants and evidence of embryotoxicity,
as measured by increased resorptions. The body weight of fetuses from dams
exposed to 38 ppm was reduced. In the feed restricted group, feed con-
sumption and weight gain were reduced during organogenesis. When these rats
were given free access to feed a compensatory weight gain occurred. Al-
though fetal body weights were reduced, there was no evidence of embryo-
lethality.
2. Mice; In mice, death occurred in groups exposed to 38 and 80
ppm of EDB as well as the group with restricted feed consumption (Table 2).
In the last group deaths were due to canibalism. The weight change was re-
duced in the 20 and 38 ppm EDB exposed groups and the feed restricted group.
The weight change after the exposures were terminated was normal in all groups
except the one previously exposed to 38 ppm of EDB. In the group exposed to
20 ppm of EDB the percent of late resorptions was increased and fetal body
weights were reduced. In the group exposed to 38 ppm of EDB, the percent of
viable fetuses was reduced, the incidence of resorptions was increased, fetal
body weights were reduced, and the percent of male fetuses was increased. In
the feed restricted group the percent of viable fetuses was reduced and the
incidence of resorptions was increased. Although only one dam produced viable
fetuses, these fetuses had a reduced body weight and a reduced percent of males.
C. Anomalies
1. Rats; Hematoma were the most common external anomaly that
occurred in rats. They occurred in a variety of regions and were present
in all of the groups. The incidence (fetuses affected/fetuses inspected)
of umbilical hernia and clubbed feet in the group exposed to 38 ppm of EDB
were 1/184, and 2/184, respectively. The soft tissue anomalies observed
in rats are presented in Table 3. None of these anomalies was present in
any of the treated groups at an incidence that reached a level of statis-
tical significance.
The skeletal anomalies observed in rats are reported in Table.4.
A reduced percent of fetuses with normally ossified centri occurred in the
group exposed to 20, but not 38 ppm of EDB. In contrast this parameter was
increased in the feed restricted group. In general, EDB exposure did not
dramatically alter the incidence of skeletal anomalies.
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TABLE 1
EFFECT OF ETHYLENE DIBROMIDE EXPOSURE DURING ORGANOGENESIS
Number Exposed
Pregnant
Alive
Non-pregnant
Alive
Body Weight Change^/
During exposure
After exposure
Feed Consumption^./
During exposure
After exposure
Pregnant Survivors
Implants/dam
Viable fetuses (%)
Dead fetuses (?)
Early resorptions (7o)
Late resorptions
Live Litters
Fetuses/dam
Males (7o)
Fetal weight (gm)
ON MATERNAL WELFARE AND REPRODUCTION IN RATS
Ethylene Dibromide (ppm)
0£/
17
17
17
0
0
39+4
59+3
19+1
22+1
17
14.5 + 0.3
96+2
0+0
:%) 4+2
;) o + o
isorptions 0
17
13.9 + 0.3
45+3
4.0 +0.1
20
15
11
11
4
4
28+7
69+7
15 + 2l/
21+1
11
13.7 + 0.8
98+2
0+0
2+2
0+0
0
11
13.4 + 0.8
53 + 4
3.9 + 0.0
38_
16
15
15
1
1
-30 + 4l/
67 + 6
9 + ll/
22+1
15
12.7 + 1.0
98+2
0+0
2+2
0+0
0
15
12.5 + 1.0
43+3
3.6 + O.L&/
80i
16
16
.&£/
0
0
-77 + 5l/
11 + 6l/
3 + Ol/
7 + 2l/
8
11.3 + 1.3l/
0 + Ol/
13 + 13
88 + 13
0+0
7£/
0
--
__
--
£
16
15
15
1
1
-47 +
92 +
4 +
25 +
15
13.1 +
97 +
0 +
3 +
0 +
0
15
12.5 +
48 +
3.4 +
y
4l/
3l/
ll/
1
1.0
2
0
2
0
0.9
5
O.LS/
aj Gm/rat/interval for pregnant rats.
b^/ Gin/rat/day for pregnant rats.
£/ Control group.
d/ Feed restricted to the amount indicated on this table.
e/ Significantly different from control (Fisher exact probability test).
^/ Significantly different from control (two sample rank test).
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TABLE 2
EFFECT OF ETHYLENE DIBROMIDE EXPOSURE DURING ORGANOGENESIS
Number Exposed
Pregnant
Alive
Non- pregnant
Alive
Body Weight Change^/
During exposure
After exposure
Feed Consumptions./
During exposure
After exposure
Pregnant Survivors
Implants/dam
Viable fetuses (%)
Dead fetuses (%)
Early resorptions
Late resorptions (70)
Live Litters
Fetuses/dam
Males (5)
Fetal weight (gm)
ON MATERNAL WELFARE
0£/
18
18
18
0
0
16.2 + 0.6
5.6 + 0.3
6.3 + 0.4
6.4 + 0.1
18
12.3 + 0.3
94+2
0+0
.) 4+2
2+1
iorptions 0
18
11.5 + 0.4
53+4
1.35 + 0.03
AND REPRODUCTION IN MICE
20
20
19
19
1
1
7.5 + 0
6.5 + 0
3.9 + 0
5.6 + 0
19
12.8 + 0
90+2
0+0
4+1
6+2
0
19
11.5 + 0
58+3
1.09 + 0
Ethylene Dibromide
38
20
17
iol/
3
3
.8£/ 3.8 + 1.6
.7 0.5 + O.LS./
.3£/ 2.3 + 0.1&/ 0
.2 2.9 + 0.6£/
10
.4 12.3 + 0.7
35 + 14£/
1+1
51 + 16
I/ 13 + 10
6l/
4
.4 4.3 + i.aa/
21 + 9£7
.04£/ 0.25 + 0.1LS/
(ppm)
80
22
19
oi/
3
0
--
.8 + O.lS-'
--
--
--
--
__ .
--
--
_ _
--
--
--
Oi/
18«/
9
&
4
4
8.8 + 0.
4.6 + 0.
' 0.9 + 0.
5.8 + 1.
6
9.2 + 1.
2 + 2S
0+0
88 + 13
10 + 10
5l/
1
0.2 + 0.
17 + 17
0.16 + 0.
7&/
8
3&/
6
5
./
SJ
2Z./
&/
16&/
aj Gm/mouse/interval for pregnant mice.
b_/ Gm/mouse/day for pregnant mice.
£/ Control group.
^/ Feed restricted to the amount indicated on this table.
e/ Gestational status of five dead females was not determined.
j!/ Significantly different from control group (Fisher exact probability test),
g/ Significantly different from control group (two sample rank test).
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oo
TABLE 3
EFFECT OF ETHYLENE DIBROMIDE EXPOSURE DURING ORGANOGENESIS
ON THE INCIDENCE OF SOFT TISSUE ANOMALIES IN RATS
Ethylene Dibromide (ppm)
Number of
Litters inspected
Fetuses inspected
Soft Tissue Anomalies
Nasal passage occluded
Inferior vena cava hemorrhage
Hydronephrosis
Kidney cortex solidified
Distended urinary bladder
Blunt snout
0£/
17
112
6.0 + 3. DC./
0+0
13.0 + 4.0
0+0
1.0 + 1.0
0+0
20
11
71
1.0 + 1.0
0 + 0
12.0 + 4.0
1.5 + 1.5
0+0
0+0
38
15
88
6.0 + 3.0
0+0
14.0 + 7.0
0+0
0 + 0
0.8 + 0.8
oi/
15
88
3.0 + 2.0
3.2 + 1.7
0+0
0 + 0
0+0
0+0
a/ Control group.
b_/ Feed restricted group.
£/ Mean + S.E. of the percent of fetuses with the indicated anomaly calculated on a
per liter basis.
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TABLE 4
EFFECT OF ETHYLENE DIBROMIDE EXPOSURE DURING ORGANOGENESIS
ON THE INCIDENCE OF SKELETAL ANOMALIES IN RATS
Ethylene Dibromide (ppm)
O/' 20 38 <£/
Number of
Litters inspected 16 11 15 15
Fetuses inspected 115 75 96 97
Skeletal Anomalies
Skull collapsed: slight 6.0 + 2.8£/ 3.0+3.0 4.5 + 2.0 7.1+3.0
marked 1.0+1.0 0+3 1.1+1.1 1.1+1.1
Occipital fontanel enlarged 0+0 0+0 1.9 + 1.3 0.8 + 0.8
Parietals incompletely ossified 0+0 0 + 0 2.2+2.2 0+0
Interparietals: incompletely ossified 0+0 0+0 3.6 + 2.0 0+0
curved medially ° ± 0 0+0 4.8+3.3 0+0
Supraoccipital: incompletely ossified .0+0 0+0 0.8+0.8 0+0
Squamosal: split 0+0 2.5+1.7 1.7+1.2 0+0
Hyoid bone: unossified 0+0 0+0 1.0+1.0 0+0
split 0+0 0+0 1.0+1.0 0+0
Sternebrae: ossified normally 56.1+7.5 58.1+7.4 35.2+7.9 37.0+9.5
unossified 11.3 + 4.0 12.3 + 4.6 22.8 + 6.3 16.8 + 4.7
incompletely ossified 30.8 + 5.0 32.1+7.4 49.2+6.7 52.7 + 8.0
split 0+0 0+0 0+0 2.1+1.4
malaligned 5.0+2.7 4.1+2.1 7.7+2.4 4.2+2.5
Centri: ossified normally 69.3+5.3 47.4 + 10.0^54.8 + 8.8 84.7 + 4.51/
lobed 26.0+4.7 49.7 + 9.9 32.8+7.2 14.4+4.6
split 5.0+1.7 8.5+3.2 15.1+4.6 1.9+1.3
Ribs: extra 5.6+2.7 0+0 0+0 3.6+2.1
wavy 0+0 0 + 0 0.8+0.8 0+0
Radius absent 0+0 0+0 0+0 1.1 +.1.1
Paws: phalanges unossified 0 + 0 0 + 0 0.9 +0.9 0+0
a/ Control group.
b_/ Feed restricted group.
£/ Mean + S.E. of the percent of fetuses with the indicated anomally calculated on
a per litter basis.
d/ Significantly different from control group (two sample rank test p <0.10).
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2. Mice; Few external Anomalies were observed in mice. Exen-
cephally occurred in three of 218 fetuses examined from the group exposed
to 20 ppm of EDB. Hematomas occurred in a few of the fetuses from groups
exposed to 0 and 20 ppm of EDB.
The soft tissue anomalies observed in mice are presented in Table
5. There was a high percent of runts among the fetuses from the group ex-
posed to 38 ppm of EDB. This may be a reflection of the reduced fetal body
weight which in turn may be a reflection of the reduced feed consumption and
weight gain of the dams. None of the remaining anomalies listed occurred at
statistically significant rate in either of the groups exposed to EDB.
The skeletal anomalies observed in mice are reported in Table 6.
EDB exposure was associated with a statistically significant increase in some
of these anomalies. Of these anomalies, incomplete ossification of supraoc-
cipitals, unossified incus, and sternebrae anomalies (unossified and incom-
pletely ossified) were previously reported in mice exposed to 31.6 ppm of
EDfiiQ.' (Appendix I Table 4). Unfortunately, there were no fetal mice in
the present study to evaluate the effect of feed restriction on development.
However, the results of the previous study suggested that the incidence of
sternebrae anomalies was increased by restricting the availability of feed.
IV. DISCUSSION
Pregnant rats and mice were exposed to 0, 20, 38, or 80 ppm of
EDB 23 hr a day for 10 days starting on day 6 of gestation. EDB produced
effects on the dam, as measured either by weight change or feed consumption,
in both species at all of the doses tested. In addition, a significant in-
crease in adult mortality occurred in rats exposed to 80 ppm of EDB and in
mice exposed to 38 and 80 ppm of EDB. Fetal mortality, as measured by the
incidence of resorptions, was increased in rats exposed to 80 ppm of EDB
and in mice exposed to 38 ppm of EDB. Fetal mortality and reduced fetal
body weights may be due either to EDB exposure or to a decrease in feed con-
sumption during organogenesis.
In a previous study' rats similarly exposed to 31.6 ppm of
EDB, had a reduced litter size as measured by implants/dam and fetuses/
dam. In the present study the litter size was normal in rats exposed to 20
ppm of EDB. However, in rats exposed to 80 ppm there was a reduced number
of implants/dam and none of these implants was associated with viable fetuses.
Since the exposures started on gestational day 6, it is possible that the
treatment may have interfered with the process of implantation. In ad-
dition, it was also reported^' that exposure to 31.6 ppm of EDB produced
an increase in 4th ventricle hydrocephaly, reduction in the occurence of
a 14th rib and an increase in the frequency of wavy ribs. These findings
were not confirmed in the present study with EDB concentrations of 20, 38,
and 80 ppm.
10
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TABLE 5
EFFECT OF ETHYLENE DIBROMIDE EXPOSURE DURING ORGANOGENESIS
ON THE INCIDENCE OF SOFT TISSUE ANOMALIES
Number of
Litters inspected
Fetuses inspected
Soft Tissue Anomalies
Cerebrum, aplasia of
Hydrocephalus: lateral ventricle
Nasal cavity: immature
occluded
Nasopharyngeal canal occluded
Small nasal sinus
Nasal passage occluded
Nasopharypgeal canal absent
Palate: cleft
high
Hydronephrosis
Small kidney
Kidney cortex solidified
Exencephalus
Runt
Ethylene
OS/
18
98
0+0^
0.8 ± 0.8 2
0+0
0.8 + 0.8 0
0 + 0 1
0+0
4.0+_2.0 4
0+0 1
0 + 0 1
0+0 1
0.9 + 0.9 2
0+0 1
9.0 + 3.0 2
0+0 3
0+0 1
IN MICE
Dibromide
20
19
103
0+0
.5 + 1.5
0 + 0
.9 + 0.9
.1 + 1.1
0+0
.0+ 2.0
.8 +_ 1.2
.1 + 1.1
.8 + 1.2
.2+ 1.5
.9 + 1.3
.0 + 1.0
.1 + 1.7
.0 + 1.0
(ppo)
38
4
20
5.0 + 5.0
5.0 + 5.0
6.3 + 6.3
0+0
0+0
4.2 + 4.2
18.0 + 12.0
0 + 0
0+0
0+0
0+0
6.3 + 6.3
0+0
0 + 0
75.0 + 25.0!
a^l Control group.
W Mean + S.E. of the percent of fetuses with the indicated anomaly calculated
on a per litter basis.
c/ Significantly different from control group (two sample rank test p <0.05).
11
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TABLE 6
EFFECT OF ETHYLENE DIBROMIDE EXPOSURE DURING ORGANOGENESIS
ON THE INCIDENCE OF SKELETAL ANOMALIES IK MICE
Etjhylene Dlbromide (ppm)
IF 20 38_
Number of
Litters inspected 18 19 4
Fetuses inspected " 110 115 24
Skeletal Anomalies
Skull collapsed: slight 2.6 £ 2.0b-/ 9.8 + 4.7 5.0+5.0
marked 0+0 0.8 + 0.8 0+0
Nasal bones: elevated 0+0 0+0 11.3 + 7.0
curved medially 1.9+1.9 16.1+6.4 20.8+12.5
Premaxillary process incompletely
ossified 0+0 3.7 + 2.8 3.6 + 3.6
Occipital fontanel enlarged 0+0 15.7 + 6.9 88.1 + 4.0S./
Supraoccipital: unossified 0+0 1.8 + 1.8 41.4 + 20.1
incompletely ossified 0+0 15.7 + 6. ll/ 54.8 + 18.8l/
Tympanic annulus: incompletely ossified 0 + 0 0+0 3.6+3.6
Incus unossified 1.9 + 1.3 28.5 + 7.3d./ 95.8 + 4.2S./
Maxillary process: unossified 0+0 0.8+0.8 0+0
incompletely ossified 0+0 0+0 4.2+4.2
Maxillary: incompletely ossified 0 + 0 0+0 3.6+3.6
Mandible: incompletely ossified 0+0 3.7+2.8 0+0
Hyoid bone: unossified 0+0 4.6 + 3.6 35.5 + 16.&
incompletely ossified 0+0 0.8 + 0.8 21.7 + 10.4l/
Sternebrae: ossified normally 75.2 + 5.3 64.5 +7.6 0 + 0£/
unossified 4.7+2.2 7.5+4.8 91.7+ 4.8£/
Incompletely ossified 80.0+3.1 19.3+5.0 54.3 + 2.5£/
split 0+0 13.0+5.5 36.3 + 17.3d/
extra ossification between 1.9+1.9 0+0 0+0
Centri: ossified normally 100 +0 100+0 100+0
Ribs: extra 13.9+5.2 10.3+3.5 12.5+12.5
Pelvis: incompletely ossified 0+0 2.4+1.7 3.6+3.6
Paws: unossified 0+0 0+0 25.0 + 25.0
incompletely ossified 0+0 0+0 3.6 + 3.6
phalanges unossified 0+0 11.4 + 5.8 70.8 + 23.9^
Tibia: bent medially 0+0 0.8+0.8 0+0
a/ Control group.
b/ Mean + S.E. of the percent of fetuses with the indicated anomally calculated on
a per litter basis.
£/ Significantly different from control group (two sample rank test p <0.01).
d/ Significantly different from control group (two sample rank test p <0.05).
e/ Significantly different from control group (two sample rank test p <0.10).
12
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Although mice similarly exposed to 31.6 ppm of EDB had a variety
of skeletal anomalies, these defects were attributed to malnourishment rather
than to EDB exposure, per ^e_.' Some of these previously reported anoma-
lies (i.e., incompletely ossified supraoccipital, unossified incus, unossi-
fied sternabrae, incompletely ossified sternabrae, and split sternebrae) were
observed in the present study. Since an insufficient number of litters sur-
vived in the feed restricted group, it is not possible to attribute these
effects to EDB. Nevertheless the previous study suggests that malnourish-
ment contributes to an increased incidence of these anomalies.
In summary, the results of this study indicate that (1) EDB is
more toxic in pregnant mice than pregnant rats; (2) adverse effects on
maternal welfare, as measured by weight gain, feed consumption and survival
were observed in both rats and mice; (3) although morphological changes
were observed in fetuses from dams exposed to EDB, these effects were ob-
served at concentrations that also affected maternal welfare, and (4) EDB
was judged to be only a weak teratogen with little primary effect on devel-
opment .
13
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