United States
Environmental Protection
Agency
Health Effects Research EPA-600 1-80-017
Laboratory February 1980
Research Triangle Park NC 2771 1
Research and Development
Teratology of a
Zineb Formulation
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
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This report has been assigned to the ENVIRONMENTAL HEALTH EFFECTS RE-
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This document is available to the public through the National Technical Informa-
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EPA-600/1-80-017
February 1980
TERATOLOGY OF A ZINEB FOMULATION
by
Robert D. Short, Jan L. Minor, Timothy M. Unger, Bradley Breeden,
Dan VanGoethem and Cheng-Chun Lee
Midwest Research Institute
425 Volker Boulevard
Kansas City, Missouri 64110
Contract No. 68-02-2982
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
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DISCLAIMER
This report has been reviewed by the Health Effects Research
Laboratory, U. S. Environmental Protection Agency, and approved for
publication. Mention of trade names or commercial products does not
constitute endorsement or recommendation for use.
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FOREWORD
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 toxi-
cology, epidemiology, and clinical studies using human volunteer subj-
ects. These studies address problems in air pollution, non-ionizing
radiation, environmental 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 pollu-
tants 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 provi-
ding 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 endanger-
ment of their health.
"Because the fungicide zineb is applied to the foliage of a variety
of fruits and vegetables, it is important to determine the hazard to
humans associated with its use. The present study evaluates the tera-
togenic potential of one zineb formation in both rats and mice."
F. G. Hueter, Ph.D.
Director
Health Effects Research Laboratory
111
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ABSTRACT
The purpose of the present study was to evaluate the teratogenic
potential of a zineb formulation. An initial toxicity study indicated
that oral doses of 1,000 or 2,000 mg/kg/day adversely affected the
weight gain of nonpregnant rats but not nonpregnant mice. In the tera-
tology study, pregnant rats and mice received daily oral doses of 0,
200, 632, or 2,000 mg/kg from day 6 of gestation until the day before
C-section. Maternal welfare, as monitored by body weight and food
consumption, was affected only in rats that received 2,000 mg/kg/day of
the formulation. Evidence of embryo or fetal lethality was not present
in rats or mice. However, fetuses from rats that received 2,000 mg/kg/
day of the formulation had a reduced body weight. Some anomalies were
significantly increased in rats that received 2,000 mg/kg/day of the
formulation. These anomalies included hydrocephalus, split centra,
incompletely ossified frontal bones, and enlarged occipital fontanel.
None of the anomalies observed in mice were increased to a statistically
significant level in any of the groups treated with the formulation.
This study indicated that the zineb formulation produced anomalies in
rats at doses which adversely affected maternal welfare. In addition,
there was no evidence of teratogenicity in mice treated with similar
doses.
IV
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CONTENTS
FOREWORD 111
ABSTRACT iv
TABLES vi
I. Introduction 1
II. Methods 1
A. Animals 1
B. Dose 1
C. Toxicity Study 2
D. Teratology Study 2
E. Interpretation of Data 3
III. Results 4
A. Analysis of Zineb Formulation 4
B. Toxicity Study 4
C. Teratology Study in Rats and Mice Treated with
a Zineb Formulation 4
IV. Discussion 16
References 18
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List of Tables
Number Title Page
1 Analysis of Dithane Z-78 Lot 0.170 5
2 Body Weight and Food Consumption of Rats and Mice ... 6
Treated With a Zineb Formulation
3 Effect of a Zineb Formulation Administered During . . .
Organogenesis on Maternal Welfare and Reproduction .
in Rats 7
4 Effect of a Zineb Formulation Administered During . . .
Organogenesis on Maternal Welfare and Reproduction .
in Mice : 8
5 Gross Anomalies in Rats Treated on Gestational Days
6-19 With Zineb 10
6 Gross Anomalies in Mice Treated on Gestational Days . .
6-17 With Zineb 11
7 Soft Tissue Anomalies in Rats Treated on Gestational .
Days 6-19 With Zineb 12
8 Soft Tissue Anomalies in Mice Treated on Gestational. .
Days 6-17 With Zineb 13
9 Skeletal Anomalies in Rats Treated on Gestational Days
6-19 With Zineb 14
10 Skeletal Anomalies in Mice Treated on Gestational Days
6-17 With Zineb 15
vi
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I. INTRODUCTION
Zineb (zinc ethylenebisdithiocarbamate) is a protective fungi-
cide used for foliage application on a variety of fruits and vegetables.
Since this fungicide may contaminate some foods consumed by humans, it is
imperative to understand the hazards associated with its use. Accordingly,
the purpose of the present study was to evaluate the teratogenic potential
of a zineb formulation in both rats and mice.
The present study was divided into two phases. During the first
phase, toxicity data were obtained for virgin rats and mice. This infor-
mation was used to identify doses for the second phase. During this phase,
pregnant rats and mice were treated with the formulation during organo-
genesis and their offspring were examined for birth defects.
II. METHODS
A. Animals
CD rats and CD-I mice 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 and analysis: A zineb femulation (Dithane Z-78,
Lot No. 0170) was used in this study. The formulation was received from
Rohm and Haas (Philadelphia, Pennsylvania) on November 15, 1978. The
formulation was repackaged in glass jars on December 6, 1978, wrapped in
aluminum foil and stored in the refrigerator. After repackaging samples
of the formulation were sent to Dr. Don Cowers (Research Division, Rohm
and Haas, Spring House, Pennsylvania) for analysis. In addition, at the
completion of both the toxicology and teratology studies, additional
samples of the formulation were sent to Dr. Cowers on March 20, 1979,
for analysis.
2. Preparation: Corn oil was selected as the vehicle for
dosing since zineb was reported to be more stable in a nonaqueous medium.
In order to prepare the suspension, the formulation was added to a
weighed amount of cold pressed corn oil which was reported to be free of
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preservatives (Hain Pure Food Compnay, Los Angeles, California) and
mixed for 5 to 10 sec with the aid of a polytron (Brinkmann Instruments,
Westbury, New York). Suspensions were prepared every 2 to 3 days and
stored at refrigerator temperature in glass bottles wrapped with aluminum
foil. The actual weight of corn oil and zineb formulation used to
prepare 50 ml of the various dosing solutions is shown below. For the
calculation of these values it was determined that 0.5 g of zineb formu-
lation occupies a volume equivalent to 1 ml of corn oil and that the
density of our corn oil was 0.92 g/tnl. All doses are expressed in terms
of the formulation.
Weight (g) of
Dose (mg/kg) Zineb Formulation Corn Oil
200 2.00 45.1
250 2.50 44.8
500 5.00 43.7
632 6.32 43.1
1,000 10.00 41.4
2,000 20.00 36.8
3. Administration: All doses, which are expressed in terms
of the zineb formulation, were administered by oral intubation in a
volume of 5 ml/kg.
C. Toxicity Study
Five groups each consisting of 10 virgin female CD rats and 10
virgin female CD-I mice received 0, 250, 500, 1,000, and 2,000 rag/kg/day
of the zineb formulation. The treatment period was 14 days for rats and
11 days for mice. Body weights were determined at the start and end of
treatment, and food consumption was measured during treatment.
D. Teratology Study
1. Mating; Sexually mature virgin female CD rats and CD-I
mice were housed overnight with a proven male breeder. In the morning
rats were examined for sperm-positive vaginal smears and mice were ob-
served for vaginal copulating plugs. The morning evidence of mating was
obtained and identified as day 0 of gestation.
2. Treatment; Four groups, each consisting of at least 60
mated rats and mice, received by oral intubation 0, 200, 632, or 2,000
mg/kg/day of the zineb formulation. The treatments were administered in
daily doses from day 6 of gestation until the day before C-section.
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3. Maternal observations; Dams were observed for toxicological
responses. Their body weight was determined at the beginning and end of
treatment and at the time of C-section. In addition, food consumption was
determined during and after treatment.
4. Fetal observations; Pregnant rats and mice were sacrificed
on gestational days 20 and 18, respectively. A laparotomy was performed
and the uterine horns exposed. The number and position of live, dead, and
resorbed fetuses were recorded. Live fetuses were removed, weighed and
immediately examined for external anomalies as described by Wilson.-^
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.I/ 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 carefully 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 .2J After differential
decolorization, the skeletons were examined by experienced personnel for
anomalies.
E. 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 ..3/ Homogeneous'data were analyzed by Dunnett's procedure or Tukey's
omega procedure.JL/ Heterogeneous data were analyzed by a nonparametric
rank test.A/ The level of statistical significance was selected as p <
0.05 unless indicated otherwise. The litter was considered the experimental
unit. The percentage 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 anomaly in predicting the teratogenic
potential of a compound. Accordingly, anomalies with a rank of 1 have
little value in such predictions while anomalies with a rank of 4 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.
3
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III. RESULTS
A. Analysis of Zineb Formulation
The chemical analyses of the zineb formulation (Dithane Z-78,
Lot 0170) used in this study is presented in Table 1. From these data,
Dr. Cowers concluded in a letter to Dr. Ron Baron (Environmental Protec-
tion Agency, Research Triangle Park, North Carolina) dated May 2, 1979,
that there was no evidence of progressive degradation of the product, or
of build-up of ETU. The overall averages for all the determinations were
85.5% EBDC + S.D. 0.8% (n = 12) and 0.35% ETU + S.D. 0.16% (n = 10).
B. Toxicity Study
Five groups each consisting of 10 rats and 10 mice received
daily doses of 0, 250, 500, 1,000 or 2,000 mg/kg of the zineb formula-
tion. The treatment period lasted for 14 days in rats and 11 days in
mice.- No deaths occurred in either rats or mice which were attributed
to the formulation. Two deaths occurred in rats as the result of esophageal
perforation during dosing. The body weight and food consumption of rats
and mice are presented in Table 2. The daily weight gain was reduced in
rats that received 1,000 or 2,000 mg/kg/day of the formulation. The
weight change of mice was not significantly affected at any of the doses
studied. In addition, food consumption was not reduced at any of the
doses studied.
C. Teratology Study in Rats and Mice Treated With a Zineb Formulation
1. Maternal welfare and reproduction
a. Rats; None of the parameters used to monitor maternal
welfare and reproduction were significantly affected in rats treated with
200 or 632 mg/kg/day of a zineb formulation (Table 3). In contrast, rats
treated with 2,000 mg/kg/day of the formulation had a reduced body weight
and consumed less feed than did the controls. In addition, fetuses from
these rats weighed less than from the controls.
b. Mice; None of the parameters used to monitor maternal
welfare and reproduction were significantly affected in mice treated with
200, 632, or 2,000 mg/kg/day of a zineb formulation (Table 4).
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TABLE 1
ANALYSIS OF DITHANE Z-78 LOT 0.170
Original MRI Before Study MRI After Study
Bag EBDC % ETU % EBDC % ETU % EBDC % ETU %
A 84.5 0.29 86.4 0.40 84.9 0.34
85.0 0.27 86.3 - 85.2 0.34
B 84.5 0.27 86.3 0.80 85.2 0.28
84.9 0.28 86.9 - 85.5 0.27
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TABLE 2
BODY WEIGHT AND FOOD CONSUMPTION OF RATS
AND MICE TREATED WITH A
ZINEB FORMULATION
Dosea./
(mg/kg/day)
Rats
0
250
500
1,000
2,000
Mice
0
250
500
1,000
2,.000
Initial
Body Weight (g)
Final
214 + 3k/
210 + 4
209 + 3
210 + 4
211 + 4
242 + 3
232 + 6
234 + 6
226 + 5
227 + 6
g/day
1.99
1.56
1.80
1.17S/
23.8 + 0.4
23.4 + 0.4
24.7 + 0.5
23.9 + 0.4
24.1 + 0.4
23.9 + 0.5
24.4 + 0.4
25.0 + 0.4
23.3 + 0.4
24.5 + 0.2
0.01
0.09
0.03
-0.05
0.04
Food Consumption
(g/animal/day)
15.8 + 0.5
14.8 + 0.4
16.4 + 0.6
14.8 + 0.5
14.3 + 1.0
3.8 + 0.1
4.1 + 0.2
3.8 + 0.1
3.6 + 0.1
4.3 + 0.1£/
a/ Dose of zineb formulation administered to virgin rats and mice for
14 and 11 .days, respectively.
b/ Mean + SE or mean.
£/ Significantly different from control (Dunnett's test).
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TABLE 3
EFFECT OF A ZINEB FORMULATION ADMINISTERED DURING ORGANOGENESIS
ON MATERNAL WELFARE AND REPRODUCTION IN RATS
Zineb Formulation (mg/kg/day)
Number Treated
Pregnant
Alive
Non-pregnant
Alive
Body Weight (g/rat)
Day 0
6
9
12
15
20
Food Consumption (g/rat/day)
Days 6-9
9-12
12-15
15-20
Pregnant Survivors
Implants/Dam
Viable fetuses (Z)
Dead fetuses (Z)
Early resorptions (Z)
Late resorptions (Z)
Dams with complete resorptions
Live Liters
Fetuses/Dam
Males (Z)
Fetal weight (g)
0
26
25
25
1
1
225 t 3
250 + 3
259 + 3
273 ± 4
285 + 4
340 + 6
12.4 t 2.1
20.1 + 1.4
17.9 ± 1.1
21.7 +0.5
25
12.9 ±. 0.6
85 ± 4
0 i 0
9 + 3
6 + 3
0
25
11.0 + 0.8
58 ± 4
3.91 ± 0.07
200
27
26
26
1
1
225 i 3
249 + 4
258 + 4
271 + 4
282 ± 5
337 ± 6
10.9 ± 1.8
20.3 ± 0.6
19.7 ± 0.6
22.2 + 0.5
26
12.5 = 0.5
76 + 6
0 + 0
9 ± 3
15 + 5
2
24
10.2 ± 0.7
46 t 4
3.84 + 0.07
632
26
22
22
4
.3
228 + 3
251 + 3
264 + 6
270 + 4
283 + 5
331 ± 8
11.0 + 1.7
17.6 + 1.1
18.1 i 0.6
20.8 t 0.6
22
12.5 ± 0.7
80 ± 7
0 + 0
4 + 2
16 + 7
2
20
11.3 + 0.9
55 ± 5
3.81 ± 0.06
2,000
26
24
24
2
2
226 : 4
248 i 4
245 +. 3
258 ± 4*-'
267 + l&!
318 ± 7
6.9 ± 1.3^
16.2 ± 0.7^
16.0 + 0.7^
18.3 * 0.7-
24
13.0 ± 0.6
85 + 6
0 ± 0
9 ± 4
5 ± 4
:
22
12.4 + 0.5
48 ± 2
3.58 + 0.08*-'
aj ' Significantly different from control (Dunnett's procedure).
b/ Significantly different from control (two-sample rank test).
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TABLE 4
EFFECT OF A ZINEB FORMULATION ADMINISTERED DURING ORGANOGENESIS
ON MATERNAL WELFARE AND REPRODUCTION IN MICE
Zigeb Formulation (mg/kg/day)
Number Treated
Pregnant
Alive
Non-pregnant
Alive
Body Weight (g/mouse)
Day 0
6
9
12
15
IS
Food Consumption (g/mouse/day)
Days 6-9
9-12 •
12-15
15-18
Pregnant Survivors
Imp lane /Dam
Viable fetuses (X)
Dead fetuses (%)
Early resorptions (Z)
Lace resorptions (%)
Dams with complete resorpcions
Live Litters
£
28
26
26
2
2
28.4 ± 0.4
30.8 * 0.3
32.6 i 0.3
37.3 ± 0.4
43.6 * 0.5
51.6 ± 0.6
4.9 ± 0,2
5.3 ± 0.2
5.7 ± 0.2
5.9 ± 0.2
26
12.3 t 0.4
88 t 4
0 ± 0
6 ± 1
3*1
0
26
200
28
23
23
5
5
28.5 * 0.4-
30.6 ± 0.4
32.4 ± 0.4
36.9 + 0.5
43.5 ± 0.7
52.0 t 1.2
4.9 ± 0.1
5.2 ± 0.1
5.9 t 0.2
6.3 ± 0.2
22
11.6 ±0.5
93 ± 2
1 + 1
5 ± 2
1*1
0
22
632
28
26
26
2
2
28.5 ± 0.4
31.0 ± 0.5
32.5 * 0.5
37.3 ±0.7
43.5 ± 0.8
51.7 ± 1.0
4.9 ± 0.2
5.3 * 0.1
5.5 ± 0.2
6.4 ± 0.2
26
11.9 ± 0.4
92 ± 2
0 - 0
6 ± 2
2*1
0
26
2.000
28
24
22
4
3
28.4 * 0.4
30.5 ± 0.4
32.5 i 0.4
36.6 ± 0.5
42.8 ± 0.7
50.9 * 1.0
4.4 * 0.4
5.2 * 0.1
5.4 ± 0.2
6.4 * 0.1
22
11.6 ± 0.4
91 ± 2
0*0
6 ± 2
2*1
0
22
Fetuses/Dam
Males 0!)
Fetal weight (g)
10.8.± 0.6 10.9 * 0.5
75 £ 17 45 * 4
1.25 = 0.02 1.30 t 0.03
10.9 ± 0.5 10.6 * 0.5
46 ± 3 43 ± 3
1.28 * 0.02 1.29 ± 0.03
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2. Gross anomalies
a. Rats (Table 5): External hematomas were observed in
all dose levels, including the control group. Short tail and kinked tail
were found in the 632 and 2,000 mg/kg/day dose levels. Raised craniums
were found in the 200 and 2,000 mg/kg/day levels. One runt was found in
one litter in the 632 mg/kg/day dose. In addition, the 200 mg/kg/day dose
w
level had single incidences of mottled skin, upturned snout, and edetnatous
fetus, along with two incidences of short neck and short limbs.
b. Mice (Table 6): External hematomas and kinked tails
were found in all dose levels except the control group. Cleft lip and
small fetuses were observed in the 200 and 632 mg/kg/day dose groups.
Single incidences of abnormally placed eye bulges occurred in the 632
and 2,000 mg/kg/day dose groups. Also, there were single incidences of
immature skin and malformed rear legs in the 632 mg/kg/day group and mottled
skin in the 200 mg/kg/day group. The only external anomalies observed in
the control group were two cases of exencephaly in one litter.
3. Soft tissue anomalies
a. Rats (Table 7): The occurrence of distended esophagus
was significantly reduced in both the 200 and 2,000 mg/kg/day dose levels.
Also, the incidence of lateral hydrocephalus in the 2,000 mg/kg/day group
was significantly increased relative to the other three groups. The sum-
mary of anomalies by rank was significantly increased in the group treated
with 2,000 mg/kg/day of the formulation.
b. Mice (Table 8): Zineb did not significantly increase,
in a dose-related fashion, any of the soft tissue anomalies observed in
mice or the summaries by rank.
4. Skeletal anomalies
a. Rats (Table 9): Significant increases were found in the
2,000 mg/kg/day dose level in the incidence of enlarged frontal fontanel,
enlarged occipital fontanel and split centra. When the anomalies were
summarized by rank this group had a significant increase in the incidence
of anomalies with a rank of 1 to 4.
b. Mice (Table 10): Zineb did not significantly increase,
in a dose-related fashion, any of the soft tissue anomalies observed in
mice.
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TABLE 5
GROSS ANOMALIES IN RATS TREATED ON GESTATIONAL
DAYS 6-19 WITH ZINEB
Zineb (mg/kg/day)
Dumber of
Litters Affected/Examined (7,)
Fetuses Affected/Examined (%)
Gross Anomalies (Rank)—'
Hematoma
Mottled Skin
Raised Cranium
Snout Upturned
Short Neck
Short Limbs
Short Tail
Tail Kinked
Runt, Small or Dwarf
Rotund Fetus
Edematous Fetus
Summary by Rank 1-4
2-4
3-4
5/25 (20)
6/274 C2)
(2)
(2)
(3)
(3)
(3)
(2)
(3)
(3)
(1)
(1)
(2)
1.9 (5)-
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
1.9 (5)
1.9 (5)
0 (0)
0 (0)
200
632
4/24 (17)
6/246 (2)
4/20 (20)
4/226 (2)
2.000
9/22 (41)
55/272 (20)
2.0 (3)
1.4 (1)
0.8 (1)
0.3 (1)
0.8 (1)
0.8 (1)
0 (0)
0 (0)
0 (0)
0.8 (1)
0.3 (1)
3.1 (4)
3.4 (4)
1.1 (2)
0 (0)
0.8 (2)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
0.4 (1)
0.4 (1)
0.8 (1)
0 (0)
0 (0)
2.0 (4)
1.2 (3)
0 (0)
0 (0)
1.3 (3)
0 (0)
2.8 (3)
0 (0)
0 (0)
0 (0)
10.9 (5)
16.2 (6)
0 (0)
0 (0)
0 (0)
18.5 (9)
18.5 (9) .
17.2 (7)-
0 (0)
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 parentheses is the number of affected litters.
£/ Significant from control (two-sample rank'test).
10
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TABLE 6
GROSS ANOMALIES IS MICE TREATED ON GESTATIONAL
DAYS 6-17 WITH ZINEB
Zineb (mg/kg/day)
Number of
Litters Affected/Examined
Fetuses Affected/Examined
Gross Anomalies (Rank)—
Immature Skin
Hematoma
Mottled Skin
Exencephalocele
Eye Bulges' Abnormally
Placed or Misshaped
Cleft Palate, Lip or Face
Forepaws Malformed
Tail Kinked
Runt, Small or Dwarf
Summary by Rank 1-4
2-4
3-4
4
(Z)
(t)
(1)
(2)
(2)
(4)
(3)
(4)
(4)
(3)
(1)
0
1/26 (4)
2/290 (1)
0 (0)-'
0 (0)
0 (0)
0.6 (1)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
0.6 (1)
0.6 (1)
0.6 (1)
0.6 (1)
200
6/23 (26)
7/251 (3)
0 (0)
0.4 (1)
0.4 (1)
0 (0)
0 (0)
0.3 (1)
0 (0)
1.2 (2)
0.8 (2)
2.7 (6)
1.9 (4)
1.5 (3)
0.3 (1)
632
5/26 (19)
11/283 (4)
0.3 (1)
0.3 (1)
0 (0)
0 (0)
0.3 (1)
0.6 (2)
0.3 (1)
1.6 (1)
0.3 (1)
3.1 (5)
2.9 (4)
2.9 (4)
2.5 (4)
2,000
3/22 (14)
3/233 (1)
0 (0)
0.9 (2)
0 (0)
0 (0)
0.4 (1)
0 (0)
0 (0)
0.4 (1)
0 (0)
1.3 (3)
1-3 (3)
0.8 (2)
0 (0)
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 parentheses is the number of affected litters.
-------
TABLE 7
SOFT TISSUE ANOMALIES IN RATS TREATED ON
GESTATIONAL DAYS 6-19 WITH ZINEB
Zlneb (mg/kg/day)
Number of
Litters Affected/Examined C)
Fetuses Affected/Examined (Z)
Soft Tissue Anomalies (Rank)-
Hydrocephalus Lateral
Slight
Hydrocephalus 3rd Ventricle
Slight
Hydrocephalus 4th Ventricle
Slight
Trachea Occluded
Distended Esophagus
Agenesis of Lungs
Aortic Valve Medially Displaced'
Heart Malformed
ECtopic Heart
Hemorrhage in Liver
Hemorrhage in Abdomen
ECtopic Stomach
Hydronephrosis
Slight Enlargement of Kidney
Pelvis
Pelvis of Kidney Collapsed
Ectopic Kidney
Small Kidney
Kidney Pelvis Asymetrical
Urinary Bladder Absent
Distended
Hemorrhage of Ductus Venosis
Crypcorchid Testicle
Malplaced Testicle
Short Seek
Summary by Sank 1-4
2-4
3-4
200
14/25 (56)
31/132 (97)
632
2.000
15/24 (62) 10/20 (50) 18/22 (82)
24/118 (20) 18/110 (16) 75/133 (56)
(4)
(U
(4)
(1)
(1)
0 CO)
0.8 (1)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
0.8 (1)
0 (0)
0 (0)
0 (0)
26.3 (12)-'
18.3
3.9 (4)
1.3 (2)
1.3 (1)
(1)
(1)
(4)
(4)
(4)
(4)
(3)
(2)
(3)
(3)
(1)
0 (0)
7.6 (8)
0 (0)
0 (0)
0 (0)
0 (0)
0.6 (1)
0 (0)
1.8 (2)
0.8 (1)
0.6 (1)
2.4 (2)
0.7 (!)£'
0.6 (1)
0 (0)
0.6 (1)
1.0 (1)
0 (1)
0 (0)
0 (0)
1.4 (1)
0.6 (1)
' 0 (0)
2.3 (3)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
0.7 (1)
0 (0)
0 (0)
1.4 (1)
0.6 (1)
0 (0)^'
0 (0)
0.6 (1)
0 (0)
0.6 (1)
0.6 (1)
0 (0)
0 (0)
2.4 (3)
3.9 (2)
(3)
(3)
(3)
(2)
(4)
(2)
(3)
(4)
(2)
(3)
0 (0)
0.5 (1)
0 (0)
0 (0)
0 (0)
4.7 (4)
0 (0)
0 (0)
0 (0)
0 (0)
22.9 (14)
8.8 (7)
5.1 (6)
0 (0)
2.8 (3)
1.4 (1)
0 (0)
0 (0)
0 (0)
0 (0)
0.6 (1)
0 (0)
0.7 (1)
0.8 (1)
21.3 (14)
6.2 (6)
5.6 (5)
1.6 (2)
1.7 (2)
0.8 (1)
0 (0)
0 (0)
0 (0)
1.5 (1)
0 (0)
0 (0)
0 (0)
0 (0)
15.7 (10)
4.8 (4)
2.5 (3)
0 (0)
0.8 (1)
1.8 (2)
3.2 (2)
0.9 (1)
0.6 (1)
0 (0)
0 (0)
0.9 (1)
2.3 (3)
0 (0)
54.5 (18
34.6 (15
32.6 (15
27.7 (14
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 parentheses is the number of affected litters.
£/ Significant from control (two-sample rank test).
12
-------
TABLE 3
SOFT TISSUE ANOMALIES IN MICE TREATED ON
GESTATIONAL
DAYS 6-17 WITH ZINEB
Zincb (mg/kg/day)
Number of
Litters Affected/Examined (Z)
Fetuses Affected/Examined (Z)
Soft Tissue Anomalies (Rank)—
Hydrocephalus Lateral Slight
(1)
Hydrocephalus 4th Ventricle Slight(l)
Exencephaly
Blood in Tissue by Nasal Passage
Micropthalaia
Eye Intraorbital Hemorrhage
Cleft Palate
Trachea Occluded
Short Neck
Enlarged Bronchi
Hemorrhage in Pericardium
Small Heart
Stomach Distended
Hemorrhage in Abdomen
Duodenum Enlarged
Small Kidney
Urinary Bladder Distended
Summary by Hank 1-4
2-4
3-4
4
(4)
(3)
(4)
(3)
(4)
(1)
(3)
(1)
(3)
(2)
(1)
(2)
(3)
(3)
(2)
0
13/26 (50)
21/139 (15)
0.8 (1)-'
0 (0)
1.5 (1)
0 (0)
0.8 (1)
0.8 (1)
0 (0)
6.5 (7)
0 (0)
0 (0)
a. 5 (i)
0.5 (1)
1.6 (1)
0 (0)
0 (0)
0 (0)
4.2 (6)
13.8 (13)
6.3 (7)
2.6 (2)
1.5 (1)
200
13/23 (56)
17/119 (14)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
0.6 (1)
5.4 (5)
0 (0)
1.4 (1)
0 (0)
0 (0)
3.6 (4)
0.7 (1)
0.9 (1)
0.6 (1)
1.7 (2)
13.4 (13)
3.1 (4)
1.5 (2)
• 0.6 (1)
632
7/26 (27)
16/136 (12)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
1.1 (2)
3.3 (3)
0.5 (1)
0 (0)
0.5 (1)
0 (0)
0 (0)
0.5 (1)
0 (0)
0.5 (1)
3.8 (2)
9.4 (7)
6.fi (5)
2.2 (4)
1.1 (2)
2,000
9/22 (41)
13/112 (12)
0 (0)
0.8 (1)
0 (0)
0.8 (1)
0 (0)
0 (0)
0 (0)
3.8 (4)
0 (0)
0 (0)
0 (0)
0 (0)
2.6 (2)
0 (0)
0.9 (1)
0 (0)
3.4 (3)
10.5 (9)
5.0 (5)
1.7 (2)
0 (0)
£/ Ranked by increasing value in predicting teracogenic potential.
t>/ Mean of the percent of fetuses vith the indicated anomaly calculated on a. litter
basis. The number in parentheses is the number of affected litters.
13
-------
TABLE 9
SKELETAL ANOMALIES IK RATS TREATED OK GESTATIONAL
DAYS 6-19 WITH ZINEB
Zineb (mg/kg/dav)
Number of
Litters Affected/Examined (%)
Fetuses Affected/Examined (%)
Skeletal Anomalies (Rank)—
Basioccipital Inc. Ossified (2)
Squamosal Split (1)
Squamosal Inc. Ossified (2)
Jugal Inc. Ossified (1)
Hyoid Bone Unossified (1)
Inc. Ossified (1)
Nasal Bones Inc. Ossified (1)
Frontal Bones Inc. Ossified (1)
Frontal Fontanel Enlarged (2)
Occipital Fontanel Enlarged (2)
Parietals Inc. Ossified (1)
Interparietals Inc. Ossified(l)
Curved Medially 3
Supraoccipital Inc. Ossified(l)
Unossified (2)
Extra Ribs (2)
Ribs Inc. Ossified (3)
Ribs Fused (4)
Centra Ossified Normally
Lobed (2)
Split (2)
Unossified (1)
Sternebrae Ossified Normally
Unossified (1)
Inc. Ossified (1)
Malalignment of Fusion (3)
Lobed (2)
Ischium Unossified (3)
Pub is Unossified (3)
Inc. Ossified (2)
Paws Inc. Ossified (1)
Phalanges of Paws Unosslfied(l)
Femur Broken and Dislocated
Summary by Rank 1-4
2-4
3-4
4
0
23/25 (92)
103/142 (72)
0 (0)-/
0 (0)
0.9 (1)
0.4 (1)
4.8 (2)
6.6 (5)
0.4 (1)
0.4 (1)
0 (0)
0 (0)
3.7 (2)
12.1 (5)
0 (0)
5.5 (3)
0 (0)
2.3 (2)
0 (0)
0 (0)
79.3 (24)
17.4 (11)
3.3 (4)
0 (0)
40.2 (18)
31.0 (15)
28.3 (17)
2.3 (4)
6.0 (4)
0.4 (1)
2.4 (2)
2.1 (2)
2.0 (10
0 (0)
0.6 (1)
70.5 (23)
28.6 (14)
2.9 (2)
0 (0)
200
23/24 (96)
83/128 (65)
1.0 (1)
0.6 (1)
0 (0)
0 (0)
3.8 (4)
4.6 (3)
0 (0)
0 (0)
11.6 (5)
2.0 (2)
0.6 (1)
5.2 (4)
0 (0)
7.1 (4)
0 (0)
1.9 (3)
0.6 (1)
0 (0)
72.4 (23)
22.3 (15)
5.9 (6)
0 (0)
47.5 (22)
26.2 (14)
25.2 (16)
2.1 (3)
3.0 (3)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
65.2 (23)
35.5 (18)
0.6 (1)
0 (0)
632
19/19 (100)
78/116 (67)
0 (0)
0.8 (1)
0 (0)
0.8 (1)
4.2 (4)
0.7 (1)
0 (0)
0 (0)
2.0 (1)
0 (0)
0 (0)
2.1 (1)
0.8 (1)
1.5 (2)
0 (0)
1.3 (1)
0 (0)
0 (0)
77.8 (19)
18.5 (12)
7.0 (4)
0 (0)
54.9 (17)
21.4 (11)
23.6 (11)
8.9 (6)
2.2 (2)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
65.5 (19)
34.4 (15)
0.8 (1)
0 (0)
2.000
22/22 (100)
128/137 (93)
6.9 (3)
0.5 (1)
0 (0)
0 (0)
4.3 (4)
2.7 (1)
0 (0)
0 (0)
34.4 (12)-y
17.4 (8)^'
0.6 (1)
11.5 (7)
0.8 (1)
20.8 (10)
0.6 (1)
3.0 (2)
0.8 (1)
0.9 (1)
31.9 (17)
42.3 (21)
39.6 (20)-'
0.9 (1)
28.0 (7)
43.0 (17)
23.0 (17)
9.6 (8)
11.3 (10)
0 (0)
0 (0)
1.6 (2)
0 (0)
0 (0)
0 (0)
94.3 (22)£'
81.9 (21)
5.8 (7)
0.9 (1)
b/ Mean of the percent of fetuses with the indicated anomaly calculated on a litter
basis. The number in parentheses is the number of affected litters.
c/ Significant from control (two-sample rank test).
14
-------
TABLE 10
SKELETAL ANOMALIES IN HICE TREATED ON GESTATIONAL
DAYS 6-17 WITH ZIKEB
Zineb (ma/kg/dav)
200
632
2.000
Number of
Litters Affected/Examined (I)
Fetuses Affected/Examined (J)
21/26 (81) 20/23 (87) 22/26 (85) 14/22 (64)
53/151 (35) 53/132 (40) 63/147 (43) 26/121 (21)
Skeletal Anomalies (Rank)!/
Short Ribs
Extra Ribs
Centra Ossified Normally
Steraebrae Ossified Nor-
mally
Onossified
Inc. Ossified
Lobed
Malalignment of Fusion
$UOfllATY bv Rank 1**4
2-4
(2)
(2)
(1)
(1)
(2)
(3)
k /
0.6 (l)i/
5.5 (4)
99.4 (26)
75.0 (26)
0.6 (1)
.3.8 (5)
19.5 (13)
8.8 (7)
36.2 (21)
33.0 (20)
0 (0)
6.4 (6)
100.0 (23)
68.8 (23)
0 (0)
1.6 (2)
17.7 (11)
16.5 (12)
39.6 (20)
38.0 (19)
0 (0)
8.9 (5)
100.0 (26)
66.2 (22)
0 (0)
0.5 (1)
25.6 (12)
7.6 (7)
42.1 (22)
41.5 (21)
0 (0)
6.9 (6)
100.0 (22)
84.4 (22)
0 (0)
3.0 (2)
7.4 (5)
5.1 (6)
21.0 (14)
18.8 (13)
a/ Ranked by increasing value in predicting teratogenlc potential.
b_/ Mean of the percent of fetuses with the indicated anomaly calculated on a
litter basis. The number in parentheses Is the number of affected litters.
15
-------
IV. DISCUSSION
Adult virgin rats were more sensitive to the zineb formulation
than were virgin mice (Table 2). For example, the daily weight gain was
reduced in rats that received 1,000 or 2,000 mg/kg/day of the formulation.
In contrast, there was no significant effect on weight gain in mice that
received up to 2,000 mg/kg/day of the formulation. Mortality was not ob-
served in either rats or mice that received up to 2,000 ng/kg/day of the
formulation.
Likewise, pregnant rats were more sensitive to the zineb formula-
tion than were pregnant mice (Tables 3 and 4). Maternal welfare, as mon-
itored by body weight and food consumption, was significantly affected in
rats but not mice that received 2,000 mg/kg/day. No adverse effects on
maternal welfare were observed in rats that received 200 or 632 mg/kg/day
of the formulation or in mice at any of the doses tested.
Embryo or fetal lethality, as monitored by the status of the im-
plants, was not significantly affected in rats or mice at any of the doses
tested (Tables 3 and 4). Embryo or fetal toxicity, as monitored by fetal
weight, occurred only in rats that received 2,000 mg/kg/day of the formu-
lation .
None of the gross anomalies observed in rats or mice were in-
creased to a statistically significant level at any of the doses tested
(Tables 5 and 6). However, fetuses from rats that received 2,000 mg/kg/day
of the formulation appeared to have a higher incidence of short tails and
kinky tails. In addition, when the anomalies were summarized by rank this
dose group had a higher incidence of anomalies which were more predictive
of teratogenic potency.
None of the soft tissue anomalies observed in rats or mice was
increased in a clear dose-related manner (Tables 7 and 8). However, in
fetuses from rats that received 2,000 mg/kg/day there was a statistically
significant increase in lateral hydrocephalus and the various summaries of
anomalies by rank. In addition, fetuses from this group also appeared to
have a higher incidence of hydrocephalus of the third ventricle. In mice,
there did not appear to be an increase in any of the observed anomalies.
None of the skeletal anomalies observed in rats or mice were in-
creased in a clear dose-related manner (Tables 9 and 10). However, in
fetuses from rats that received 2,000 mg/kg/day of the formulation there
was a statistically significant increase in enlarged frontal fontanel,
enlarged occipital fontanel, split centra, and anomalies with a rank of
1 to 4. In addition, fetuses from this group appeared to have a higher in-
cidence of incompletely ossified supraoccipital. In mice, there did not
appear to be an increase in any of the observed anomalies.
16
-------
In another study ,J/ maneb or zineb was given orally to rats in
doses of 2,000 to 4,000 mg/kg on days 11 or 13 of gestation and a high
incidence of neural tube closure defects, cleft lip and palate and skeletal
defects resulted. The teratogenic potential of ferbam and thiram was also
evaluated in rats and mice.JL/ Ferbam administered to rats during organo-
genesis by gavage at doses of 114 mg/kg reduced the maternal weight gain,
litter size, and fetal body weight. Mice survived ferbam treatment during
gestation at doses of 228 mg/kg without adverse effects on these parameters.
Thiram treatment during organogenesis reduced maternal weight gain and
fetal body weight in rats given 40 mg/kg or more. Litter size was, de-
creased in rats given thiram at doses of 136 mg/kg or more. Some mice
died at thiram doses of 300 mg/kg; however, development was not affected
in the survivors. These fungicides were judged to have little teratogenic
activity.
Ethylenethiourea (ETU) arises from the degradation of ethylene-
bisdithiocarbamate fungicides and is a potent teratogen in rats.Z' ETU
wast' administered during organogenesis at doses ranging from 5 to 80 mg/kg/
day. ETU at doses of 10 mg/kg and above produced neural tube closure de-
fects, hydrocephalus and other malformations of the brain, kinky tails,
and limb defects.
Chemical analysis of the zineb formulation used in the present
teratology study indicated that it contained 85.5% zineb and 0.35% ETU
(Table 2). Consequently, animals that were dosed with 2,000 mg/kg/day
of the formulation received 1,710 mg/kg/day of zineb and 7 mg/kg/day of
ETU.
In conclusion, the present study indicates that (a) rats were
more sensitive than mice to adverse effects of the formulation; (b) no
adverse effects on maternal welfare or embryonic and fetal- development
were observed in rats that received 200 or 632 mg/kg/day of the formulation
or in mice that received 200, 632, or 2,000 mg/kg/day of the formulation;
and (c) maternal welfare and embryonic and fetal development was adversely
affected in rats that received 2,000 mg/kg/day. These adverse affects
on development may have been related to the maternal effects or ETU con-
tamination of the formulation. Therefore, the present study indicates
that the zineb formulation was not teratogenic in mice but did produce
anomalies in rats at doses that adversely affected maternal welfare.
17
-------
REFERENCES
1. 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).
2. Staples, R. C. and V. L. Schnell, "Refinements in Rapid Clearing Tech-
niques in the KOH-Alizarin Red S Method for Fetal Bones," Stain
Technol.. 39.: 61-63 (1964).
3. Steel, R. G. D. and J. H. Torrie, Principles and Procedures of
Statistics, McGraw-Hill Book Co., New York (1960).
4. 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., 1.8:50-60 (1947).
5. Petrovoa-vergieva, T. and L. Ivanova-tchemishanska, "Assessment of the
Teratogenic Activity of Dithiocarbamate Fungicides," Food Cosmet.
Toxicol., 11:239-244 (1973).
6. Short, R. D., J. Q. Russel, J. L. Minor and C. C. Lee, "Development
Toxicity of Ferric Dimethyldithiocarbamate and bis(Dimethylthio-
carbamoyl)disulfide in Rats and Mice," Toxicol. Appl. Pharmacol.,
35_: 83-94 (1976).
7. Khera, K. S., "Ethylenethiourea: Teratogenicity Study in Rats and
Rabbits," Teratology, 7:243-252 (1973).
18
-------
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing/
RE=OST NO,
EPA-600/1-80-017
3. RECIPIENT'S ACCESSIOf*NO.
4. TITLE AND SUBTITLE
5. REPORT DATE
February 1980
Teratology of a Zineb Formulation
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Robert D. Short, Jan L. Minor, Timothy M. Unger, Bradley
Breeden, Dan VanGoethem and Cheng-Chun Lee
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Midwest Research Institute
425 Volker Boulevard
Kansas City, Missouri 64110
TO. PROGRAM ELEMENT NO.
1EA615
11. CONTRACT/GRANY NO.
68-02-2982
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory RTP,NC
U.S. Environmental Protection Agency
Office of Research and Development
Research Trianele Park. North Carolina 27711
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
EPA 600/11
15. SUPPLEMENTARY NOTES
16. ABSTRACT
The purpose of the present study was to evaluate the teratogenic potential of a
zineb formulation. An initial toxicity study indicated that oral doses of 1,000 or
2,000 mg/kg/day adversely affected the weight gain of nonpregnant rats but not non-
pregnant mice. In the teratology study pregnant rats and mice received daily oral
doses of 0, 200, 632, or 2,000 mg/kg from day 6 of gestation until the day before C-
section. Maternal welfare, as monitored by body weight and food consumption, was af-
fected only in rats that received 2,000 mg/kg/day of the formulation. Evidence of
embryo or fetal lethality was not present in rats or mice. However, fetuses from rats
that received 2,000 mg/kg/day of the formulation had a reduced body weight. Some
anomalies were significantly increased in rats that received 2,000 mg/kg/day of the
formulation. These anomalies included hydrocephalus, split centra, incompletely
ossified frontal bones, and enlarged occipital fontanel. None of the anomalies ob-
served in mice were increased to a statistically significant level in any of the groups
treated with the formulation. This study indicated that the zineb formulation pro-
duced anomalies in rats at doses which adversely affected maternal welfare. In addi-
tion, there was no evidence of teratogenicity in mice treated with similar doses.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFlERS/OPEN ENDED TERMS
COSATI Field/Group
Toxicology
Teratology
Fungicide
Zineb formulation
Rats
Mice
06F,T
3. jlSTPiSUT'GN STATEMENT
Release to public
19. SECURITY CLASS 'This Report I
JflJCLASSIFIED
21. NO. OF PAGES
19
20. SECURITY CLASS lThispage>
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
19
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