EPA-560/6-76-018
TOXICITY STUDIES OF SELECTED CHEMICALS
TASK I:
THE DEVELOPMENTAL TOXICITY OF ETHYLENE DIBROMIDE INHALED
BY RATS AND MICE DURING ORGANOGENESIS
APRIL 1976
FINAL REPORT
ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF TOXIC SUBSTANCES
WASHINGTON, D.C. 20460
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EPA-560/6-76-018
TOXICITY STUDIES OF SELECTED CHEMICALS
TASK I:
THE DEVELOPMENTAL TOXICITY OF ETHYLENE DIBROMIDE INHALED
BY RATS AND MICE DURING ORGANOGENESIS
Final Report
Prepared by
Robert D.. Short,; Jr.
Jan L. Minor
Brett Ferguson
Timothy Unger
Cheng-Chun Lee
Contract No. 68-01-3242
William L- Marcus, Ph.D.
William A. Coniglio
Project'officers
Of.fice o'f Toxic Substances
U.S. Environmental Protection Agency
Washington, D.C. 20460
'April 1976
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NOTICE
' This report has been reviewed by the Office of Toxic Substances, .
EPA, and approved for publication. Approval does not signify that the
contents necessarily reflect the views and policies of the Environmental
Protection Agency, nor does' mention .of trade names or commercial products
constitute endorsement or recommendation'for use.
11
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SUMMARY
Ethylene dibromide (EDB) was administered at 32 ppm by inhala-
tion to two experimental animal species for 23 hr/day (allowing 1 hr for
servicing the .animals and chambers) during organogenesis (day 6 through
15 of gestation). Charles River CD rats and CD-I mice were used in these
tests. In addition to the control, a third group of animals was used.
These were given a reduced diet, but no EDB exposure.
' i . •
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 ossification were' 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 mal-
nourishment rather than to EDB exposure, per se.
I ,
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,
reduction in the -occurrence of fourteenth rib, and increase in the
frequency of wavy ribs appear to be correlated to EDB exposure in this
species.
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I. INTRODUCTION
Ethylene dibromide (1,2-dibromoethane, EDB), is us°ed as a scavenger
in gasoline, a fumigant and a chemical intermediate. The estimated production
of EDB was about 315 million pounds in 1972 and 331 million pounds in 1973.
i
The inhalation of.EDB produced toxicity in experimental animals.—
Rats exposed to a concentration of1 EDB in excess of 200' ppm died within 24
hr from respiratory or cardiovascular collapse. Mortality 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
unkempt, became irritable and,produced a bloody nasal discharge. Chronic
inhalation 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
rr 2/ : . ' . • '
Industrial Hygienists.—'
i
i
EDB exposure also .produced a more insidious type of toxicity than
previously described. A carcinogenic response was demonstrated by adminis-
tering EDB orally to rats (40land 80 mg/kg/day) and mice (60 and 120 mg/kg/
3/
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, me-
tastasized 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 spermatogen-
esis and the maturation of sperm in bulls.— damaged spermatogenic cells in
rats—' and reduced the egg weight of laying hens.—' This type-of toxicity
is insidious because the continuous exposure to EDB can produce dramatic
effects after a symptom-free latency period.
The present study was undertaken to evaluate the ability of EDB
to produce this insidious type of toxicity. The inhalation route of exposure
was selected.because EDB is found in the atmosphere. The production of con-
genital defects was used as a measure of toxicity because development is a
finely regulated process which is sensitive to disruption by many agents.
Those agents include both carcinogens and mutagens. In addition, these tests
r.i.-iy be performed in a short period of time rind arc useful in identify ing agents
that may "produce birth defects in humans.
2.
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II. METHODS
A. Animals
Charles River CD rats and CD-I mice (Charles River Breeding Labo-
rnLories, 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
n relative humidity of 50 i 5% arid a 7 AM to 7 PM photoperiod. Animals were
given Tree access to powdered rodent chow (Wayne Lab-Blox,. Allied Mills, Inc.,
Chicago, Illionis), 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 in were us.ed 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 stainless-steel vessel which was maintained at 30°C. EDB
entered the air stream .upstream from the chamber. Mixing was initiated in
n plenum at the top of the chamber and completed by two squirrel cage fans
and a diffusion plate.
The EDB concentration in the chamber was monitored using gas chro-
matography and a flame ionizatlon detector. EDB was resolved using a stainless
steel column packed with 5% didecyl phthalate on 80/100 chromosbrb and i
ni.trogen (80 ml/min) as the carrier gas. The injection, column and detector
temperatures were 160°C, 145°C and 170°C, respectively. Standards were
prepared by serial dilutions of an EDB stock solution prepared in carbon
tetrachloride.
C. Experimental Protocol
Female rats and mice' were exposed overnight to proven male breeders.
Successful mating (day 0 of gestation) was identified the next morning by
the presence of sperm in vaginal smears.from rats and"copulation plugs in
in.ice. Mated animals were divided into three groups: a control group, an
KEW tinted group arid a feed restricted group. .Animals were housed in the
i nlinL.it.ion chambers for 10 days starting on day 6 of gestation. During this
time the EDB treated group was exposed to 30 ppm .of EDB for 23 hr a day.
The remaining animals were housed under similar conditions; however, the EDB
exposure was omitted.
3 .
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Rats and mice were sacrificed on gestational day 20 or 18, re-
spectively. A laparotomy was performed and the uterine horns were exposed.
The umbilical cord was clamped and severed distaily in order to prevent blood
Loss. Fetuses were removed, weighed and examined for external anomalies.—'
One-half of the fetuses from each litter were fixed in Bouin's solution and
examined for soft-tissue anomalies by a free-hand slicing method.— The
remaining fetuses were fixed in 7070 alcohol, eviscerated, stored in 17, KOH
and stained with alizarin red.—' After differential decolorization, the
skeletons were examined for anomalies.
i). Statistical Methods
Quantitative data, 'reported as the mean ± standard error, were
: "127
initially analyzed by Bartlett's test for homogeneity.—' The test of sig-
1 *"*' /
nificancc for .homogeneous data was Dunnett's procedure.—' In contrast,
; 13/
luit(?r.op,cncous data were analyzed by the two-sample rank test.— For all
tests the 0.05 level of significance was chosen except where indicated.
The litter was considered to [be the unit of observation.-!^-/ All statis-
tical tests, therefore, wereibased on the litter as the experimental
unit.
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III. RESULTS
Rats and mice inhaled EDB 23 hours a day for 10 days starting on
day 6 of gestation. During this time, the average concentration of EDB
was 31.6 + 1.9 (mean + S.E. for 36 determinations).
EDB exposure did not produce significant mortality in either
rats or mice .(Table 1). During the 10 day treatment period, rats and
mice exposed to EDB consumed less feed and gained less weight than con-
trols. After treatment, feed consumption of these animals returned, to
normal and they regained weight.
The various parameters of reproduction which were determined in
this study are summarized in Table 2. EDB exposure was associated with a
reduced litter size in rats. .The fetal body weight was also reduced in mice
exposed to EDB and mice whose feed consumption was limited during the
treatment period. This effect on fetal .weight was probably due to malnourish-
ttient during development rather than a direct toxicity of EDB on develop-
ment . | .
i
i '
The anomalies observed in fetuses from .rats and mice are summarized
in tables 3 and 4, respectively. In rats, EDB exposure significantly in-
creased the incidence of hydrocephaly of the fourth ventricle and of minor
changes in rib development. In addition, one fetus had two shortened
limbs with one ending in only four digits. These anomalies were not ob-
served in fetuses from the rats with a restricted feed intake. Anomalies
observed following exposure of mice to EDB included hydrocephaly of the
third ventricle, hydrocephaly of the fourth ventricle, and 'variations
in the normal ossification of the supraoccipital, incus and sternabrae.
Thes'e anomalies were observed at a similar frequency in fetuses from
mice exposed to EDB and from mice on a restricted feed intake.
• 5
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TABLE 1
EFFECT OF ETHYLENE DIBROMIDE EXPOSURE DURING ORGANQGENESIS
ON NUMBER, BODY WEIGHT CHANGE AND FEED CONSUMPTION
OF RATS AND MICE
ETHYLENE DIBROMIDE (ppm)
Kn t s
Number
Pregnant
• A I ive
Non-pregnant
Alive
liody Wei glit ClinngeS/
IXiring treatment
After treatment
Feed Consumption"/
During treatment
After treatment
M i c i:
Number
Pregnant
Alive
Non-pregnant
AI ivc
Body Weight Change3-'
IXiring treatment
Aft.er treatment
Feed Consumption—' .
IXiring treatment
After treatment ,
OS/
18
18
0
0
49 i 5
61 i 5
21.1 ± 0.4
30 .,8 ±3.2
17
7
7
14 ± 1 '
5.0 ± 0.9
5.6 ± 0.2
6.4 ± 1.6
31. 6
18
18
. 10
10
-27 i t£/
. 80 ± 9
10,5 ± 0.61/
27.7. ± 1.2
13
13
15
14
• 1 ± I-1
6.2 ± 0.9
3.5 ± 0.21/
6.4 i 0.4
17
17
1
1
-46 ± 5^
99 ±
5.0 ±0.1-
29.7 ± 1.3
9
7
11
5 ± 2^
5.1 ± 1.7
3.6 ±0.1-
8.1 j- 0.2
;i/ Cm/animal/interval for pregnant animals.
b/ Cm/animal/day for pregnant animals.
£/ Control group..
i|/ l-'ced restricted to the amount indicated on this table.
£/ Significantly different from control (two-sample rank test).
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TABLE .2
F.FFFCT OF ETHYLENE DIBROMIDE EXPOSURE DURING ORGANOGENESIS
ON REPRODUCTION IN
RATS AND MICE
ETHYLENE DIBROMIDE
s
regnant survivors
Implants/Jam
Viable fetuses (70)
Dead fetuses (7»)
Knrly resorptions (%)
Late resorptions (70)
Dams with complete resorptions
15.4
99
i
] 1
i
i
i
Lve 1 ittcrs ;
Fetuses /dam
'Males (%>
Fetal wciglit (gm)
15.3
48
3.62
regnant survivors
j
Implants/dam
Viable fetuses (%)
Dead fetuses (7o)
F.arly. resorptions (%)
Late resorptions (?<,)
Dams- with complete resorptions
ve litters
Fetuses/ dam
Males (7o)
Fctnl weight (gm)
10.3
83
15
2
10.3
50
1.24
18.
±
• ±
0
±
0
0
18
±
i
*
17
±
±
0
±
±
2
15
±
±
±
0.3
1
1
0.3
3
0.04
0.6
8
8
1
0.4
4
0.03
31.6 , •
18
12.4 ± 0.9£/
. 92 ± 6 .
0
8 '± 6
• 1 ± 1
1
17
12.2 J- 0.9-/
59 ± 4
3.53 ± 0.10
13
9.5 ± 0.8
61 ± 11
0
32 x 11
7 ±5
3
'• 10
8.2 ± 0.7
. 52'± 6
0. 93 .i 0.06^-'
(ppm)
.ok/.
15.2
99
1
15.1
50
3.12
.8.6
42
46
12
9.0
54
0.95
17
±
±
0
i'
0
0
17
±
±
±
1
•±
±
0
i
±
3
4
±
±
±
0:3
0
0
0.3
3
0.06-/
1.7
18
.
:19
12
2.3
7
0.09-/
Control group.
Feed restricted to the amount indicated on Table 1.
Sigr-ificantly different from control (two-sample rank test).
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TABLE 3
EFFECT OK PTIIYLENE IK BROMIDE EXPOSURE DURING ORGANOGENESIS ON THE
INCIDENCE OF ANOMALIES IN FETAL RATS
Ethylene Dibromide
Inspected for
F.xt.crnal anomalies
Soft tissue anomalies
Skeletal anomalies
tlxicrti.il anomalies
I.iml) reduction
Sc>fi tissue anomalies
llydrocephaly; lateral ventricles
third ventricle
l
fourth ventricle
Hydroncphrosis . 1
Cliil> I'oot •
.Skeletal anomalies :
Supraoccipi. tal , incompletely ossified
I'nrictals, incompletely ossified
Fourteenth pair of ribs :
Wavy ribs '
Fused ribs
0
269
129
140
0.
0.
0.
6.
7.
0.
3.
14.
0.
0.
a/'
(18)^
(18)
(18)
o*/-
0
0
9
3
0
1
2
9
0
0
(9)
(6)
,
(2)'
(6)
(10)
i
31
207
99
108
0
0
1
17
9
2
11
2
0
.4
.9
.6
.8
.7
.3
12
.6
.4
4
.8
16.
(17)
(17)
(17)
(1)
(1)
(2) ,
(11)^
(7)
(2)
(5)
(7)
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TABLE 4
DK I'l'HYLKNK DIBROMIDE EXPOSURE' DURING ORGANOGEN'ESIS ON THE
INCIDENCE OF ANOMALIES IN FETAL MICE
Ethylene Dibromide
1 n n n_r c_ t_c^J _1 oj;
Y.x t c r i in 1 n n oma lies
ilofi tlnsuc anomalies
SVfllolal anomalies
Jxtnrn.'il anomalies
0^
155
75
80
(15)^
(15)
(15)
3_;
81
40
41
(10)
(10)
(10)
i
36
17
19
(4)
(4)
(A)
inly • I .
S o ( t . t I .*; sue anomalies ;.
llydrocep'naly: lateral ventricles
third ventricle '
fourth ventricle
S V rj_c t_nj anomalies I
Supr.iocc ipital, incompletely ossified
Incus, not. ossified
St-criKibrae: incompletely ossified
not ossified
split
0.7
'0.0
0.0
0
0
0
7
5
4
0
5
.0
.0
.0
.2
.4
.7
.0
.2
(3)
(3)
W .
(3)
0
12
5
52
56
22
46
30
.0
.0
.8
.2
.2
.3
.5
.0
(3)
(2)
(9)
(8)
£/
£/
-Q
(6)^
(7)
(5)
£1
16
16
16
33
41
25
41
..29
.7
.7
.1
.3
.7
.0
.5.
.1
(1)
(1)
(1)
(2)
(2)
(3?/
(3)£'
(3)
!>/
c/
f/
t.'oiilrol i;rqup.
Ki-L'i.1 restricted to the amount indicated in. Table .1.
focal number of fetuses (total number of litters).
IVrccntage of fetuses with the indicated anomaly calculated on a per'
litter basis. Number of litters affected is given in parenthesis.
Significantly different from control group (two-sample rank test),
(i1 < 0. 10). ' . . •
Significantly different from control group (two-sample rank test),
(!' ^_ 0.05). ' . .
Sl^ntficantly different from control group (two-sample rank test),
(F1 < 0.01).
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REFERENCES
I. Hovo, V. I. i-t:_al. "Toxicity of Ethyl en e Dibromide Determined on Experi-
oVninl Animals," AMA Arch, of Ind. Hyg. and Occup. Med., (6:158, 1952.
I
.'. . IXjc-umcMU.iC ion of the Threshold Limit Values, American Conference of
Governmental Industrial Hygienists, Cincinnati,'Ohio, 1974.
1. Ol;uM», c c nI. "Induction of Stomach Cancer in Rats and Mice with Halo-
R.-nnCcd Aliphatic Fumigants," J. Nat. Cancer Inst. , 5_1(6).: 1993, 1973o
Favors,.M. B., cc al. "Carcinogenicity of Ethylene Dibromide (EDB) and
1 .2-r>ibronio-3-Chlor6propane (DBCP) after Oral Administration in Rats
nnil Mice," Toxicol. Appl. Pharmacol., 33:171, 1975..
!
>, flrcro, II.,.A. B. Stein, and H. S. Rosenkranz, "The Mutagenicity and DNA-
.VoiUfying Effect of Haloalkanes," Cancer Research, _34:2576, 1974.
i
»i. Vof-el, K. , and J. L. R. Chandler, "Mutagenicity Testing of Cyclamate and
Some Pesticides in Drosophila Melanogaster," Experietia, 30:621, 1974.
7. Aslr, D., "Sites of Spermicidal Action-of Ethylene Dibromide in Bulls,"
J. Keprod. Pert. , .35^:519, 1973. •
tt. Wvnrd.i, K., et al. "Studies with Alkylating Agents II. A Chemical
Interpretation through Metabolic Studies of the Antifertility Effects
of Kthylcne Dimethanesulphonate and Ethylene Dibromide," Biochern.
I'liarm. . _L9: 1783, 1970.
9. Ikxuli , A., E. Olomucki, and.M. Calderon, "Problems Connected with Ethylene
Dibromide Fumigation of Cereals II. Feeding Experiments with Laying
• lions," J. Sci. Food Agric. . 6:600, 1955.
10. 'Wilson, J. G., "Methods for Administering Agents and Detecting Malfor-
ni.it ions in Experimental Animals," in Teratology—Principles and Tech-
niquos, J. G. Wilson and J. Warkany (eds.), University of Chicago
Tress, Chicago,.Illinois, pp. 262-277 (1965). . ' •
11. Stnplcs', R. C. and V. L. Schnell, "Refinements in Rapid Clearing Tech-
• . niqucs'in the KOH-Alizarin Red S Method for Fetal Bones," Strain
Tcchnol., _39:61-63,. 1964.
I.!. SioeL, R. G. D., and J. .H. Torrie, Principles and Procedures o-f Statistics,
Nc-Graw-Hill Book Company, New York, 1960.
10
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. H. B. . niul I). R. Whitney, "On a Test of Whether One of Two Random
Y,»rUbl
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lECHNICAL REPORT DATA
;/'/. .;vr'/rrt, .1. I.. Minoir, B. Ferguson, T. linger,
'
(ti.ii'. . MIU.ANI.'A | | or-. NAMi: AND ADDRESS
|( Kr.'search Institute
j'lVcv Uou 1 cvard
I City . MO Ml 10
Jl.M.i-ii. M.I ttC'f NAMl AND ADDRESS
I o! icxic Substances :
KHV i i iMiin.-nta 1 1'rol cc t-ion Agency
IK.!"". I). C.. 21V.60 :
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
68-01-3242,.Task #1 .
13. TYPE OF REPORT AND PERIOD COVERED
Final
14. SPONSORING AGENCY CODE
.'ik wns initialed to investigate the ability of Ethylene Dibromide to produce
ir. hv the inhalation route.
urpt'iK! of this study was to determine the teratogenic potential of Ethylene
t
»i
rent available to public through the
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ttfiold. Virginia 22151
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Unclassified
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