EPA-560/6-77-022
TOXICITY STUDIES OF SELECTED CHEMICALS
TASK II:
THE DEVELOPMENTAL TOXICITY OF VINYLIDENE CHLORIDE INHALED
BY RATS AND MICE DURING GESTATION
FEBRUARY 1977
FINAL REPORT
ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF TOXIC SUBSTANCES
WASHINGTON, D.C. 20460
EPA 560/6-77-022
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EPA-560/6-77-022
TOXICITY STUDIES OF SELECTED CHEMICALS
TASK II:
THE DEVELOPMENTAL TOXICITY OF VINYLIDENE CHLORIDE INHALED
BY RATS AND MICE DURING GESTATION
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, D.C. 20460
February 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. Joseph Seifter is
the contract monitor for the project.
This work was conducted in the Biological Sciences Division under
the direction Dr. William B. House between October 1, 1975 and November 1,
1976. The experimental work was supervised directly by Dr. Cheng-Chun Lee,
Assistant Director, Biological Sciences Division for Pharmacology and
Toxicology; assisted by Dr. Robert D. Short Jr., Mr. Jan L. Minor, Dr. Paul
Peters, and Dr. Joseph M. Winston, Associate Toxicologists, with the tech-
nical assistance of Mr. Brett Ferguson, Mr. Timothy Unger and Miss Mary
Sawyer.
Approved for:
MIDWEST RESEARCH INSTITUTE
W. B. House, Director
Biological Sciences Division
ii
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TABLE OF CONTENTS
Summary 1
I. Introduction 2
II. Methods 3
A. Experimental Design 3
B. Exposure 4
C. Animals 6
D. Protocol for Morphological Evaluation 6
E. Protocol for Behavioral Evaluation 7
F. Statistics 9
III. Results 9
A. Chamber Concentrations of VDC 9
B. Morphological Studies on Mice and Rats 11
C. Behavioral Studies on Rats 40
IV. Discussion 54
A. Morphological Study 54
B. Behavioral Study 59
C. General Summary 60
References 61
LIST OF FIGURES
Figure Title Pag€
1 Diagram of Activity Maze 10
2 Cumulative Percent of Rats, Sexes Combined, Reaching a
Criterion of 1 Sec or Less for Righting on a Flat
Surface 47
3 Amount of Time Spent Pivoting During a 3-Min Test Period,
Sexes Combined 48
4 First a Complete Startle Response Was Observed for Various
Groups of Rats, Sexes Combined 49
iii
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TABLE OF CONTENTS (Continued)
LIST OF FIGURES (Concluded)
Figure Title Page
5 Cumulative Percent of Rats, Sexes Combined, Reaching a
Criterion of 15 Sec on the Bar-Holding Apparatus 50
6 First Day of Appearance for Righting in Air for Groups of
Rats, Sexes Combined. 51
7 First Day for Score of 2, 3, or 4 in the Swimming Test,
Sexes Combined 52
8 First Day of Appearance of Physical Parameters for Groups
of Rats, Sexes Combined 53
9 Average Body Weight of All Rat Pups 55
10 Continuous Activity of Groups of Three Rats (same sex) in
a Residential Maze, Both Sexes Combined 56
LIST OF TABLES
Table Title Page
1 Chamber Concentrations of Vinylidene Chloride During
Various Trials 12
2 Effect of Vinylidene Chloride Exposure on Gestational Days
6 to 16 on Maternal Welfare and Reproduction in Mice. . . 13
3 Effect of Vinylidene Chloride Exposure on Gestational Days
6 to 16 on Maternal Welfare and Reproduction in Rats. . . 14
4 Effect of Vinylidene Chloride Exposure on Gestational Days
6 to 16 on the Incidence of Soft Tissue Anomalies in
Mice 16
5 Effect of Vinylidene Chloride on Gestational Days 6 to 16
on the Incidence of Soft Tissue Anomalies in Rats .... 17
6 Effect of Vinylidene Chloride Exposure on Gestational Days
6 to 16 on the Incidence of Skeletal Anomalies in Mice. . 18
iv
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TABLE OF CONTENTS (Continued)
LIST OF TABLES (Continued)
Table Title Page
7 Effect of Vinylidene Chloride on Gestational Days 6 to 16
on the Incidence of Skeletal Anomalies in Rats 19
8 Effect of Vinylidene Chloride Exposure on Gestational Days
8 to 15 on Maternal Welfare and Reproduction in Mice. . . 20
9 Effect of Vinylidene Chloride Exposure at 41 ppm on Maternal
Welfare and Reproduction in Mice 22
10 Effect of Vinylidene Chloride (VDC) Exposure at 54 ppm on
Maternal Welfare and Reproduction in Mice 23
11 Effect of Vinylidene Chloride Exposure on the Incidence of
Gross Anomalies in Mice 24
12 Effect of Vinylidene Chloride (VDC) Exposure at 41 ppm on
the Incidence of Soft Tissue Anomalies in Mice 25
13 Effect of Vinylidene Chloride (VDC) Exposure at 54 ppm on
the Incidence of Soft Tissue Anomalies in Mice 26
14 Effect of Vinylidene Chloride (VDC) Exposure at 41 ppm on
the Incidence of Skeletal Anomalies in Mice 28
15 Effect of Vinylidene Chloride (VDC) Exposure at 54 ppm on
the Incidence of Skeletal Anomalies in Mice 29
16 Effect of Vinylidene Chloride Exposure on Gestational Days
6 to 9 on Maternal Welfare and Reproduction in Mice ... 30
17 Effect of Vinylidene Chloride Exposure on Gestational Days
9 to 12 on Maternal Welfare and Reproduction in Mice. . . 31
18 Effect of Vinylidene Chloride Exposure on Gestational Days
12 to 15 on Maternal Welfare and Reproduction in Mice . . 33
19 Effect of Vinylidene Chloride Exposure on Gestational Days
15 to 17 on Maternal Welfare and Reproduction in Mice . . 34
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TABLE OF CONTENTS (Concluded)
LIST OF TABLES (Concluded)
Table Title Page
20 Effect of Vinylidene Chloride (VDC) Exposure on the
Incidence of Gross Anomalies in Mice 35
21 Effect of Vinylidene Chloride Exposure on Gestational Days
6 to 9 on the Incidence of Soft Tissue Anomalies in Mice. 36
22 Effect of Vinylidene Chloride Exposure on Gestational Days
9 to 12 on the Incidence of Soft Tissue Anomalies in
Mice 37
23 Effect of Vinylidene Chloride Exposure on Gestational Days
12 to 15 on the Incidence of Soft Tissue Anomalies in
Mice 38
24 Effect of Vinylidene Chloride Exposure on Gestational Days
15 to 17 on the Incidence of Soft Tissue Anomalies in
Mice 39
25 Effect of Vinylidene Chloride Exposure on Gestational Days
6 to 9 on the Incidence of Skeletal Anomalies in Mice . . 41
26 Effect of Vinylidene Chloride Exposure on Gestational Days
9 to 12 on the Incidence of Skeletal Anomalies in Mice. . 42
27 Effect of Vinylidene Chloride Exposure on Gestational Days
12 to 15 on the Incidence of Skeletal Anomalies in Mice . 43
28 Effect of Vinylidene Chloride Exposure on Gestational Days
15 to 17 on the Incidence of Skeletal Anomalies in Mice . 44
29 Effect of Vinylidene Chloride Exposure on Gestational Days
8 to 20 on Maternal Welfare and Reproduction in Rats. . . 45
vi
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NOTICE
This document is a preliminary draft. It has
not been formally released by EPA and should
not at this stage be construed to represent
Agency policy. It is being circulated for
comment on its technical accuracy and policy
implications.
SUMMARY
Vinylidene chloride (VDC) was administered at various concentra-
tions by inhalation to two experimental animal species for 23 hr/day
(allowing 1 hr for servicing the animals and chambers) for various in-
tervals during gestation. Charles River CD rats and CD-I mice were used
in these tests. The development of these animals was observed using mor-
phological and behavioral parameters.
The results of this study indicate that (1) VDC is more toxic
in adult mice than adult rats, (2) adverse effects on maternal welfare,
as measured 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 VDC, these effects were observed at con-
centrations that also affected maternal welfare, (4) no problems with
neural development, as measured by behavioral parameters, were observed
in rats exposed to VDC, and (5) VDC was judged to be only a weak teratogen
with little primary effect on development.
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I. INTRODUCTION
Vinylidene chloride (1,1-dichloroethylene, VDC) and vinyl chloride
(1-chloroethylene, VCM) are monomeric intermediates used in the production
of plastics. These plastics are employed in food packaging, pipe construc-
tion, and upholstery manufacturing. The 1974 production of VCM in the U.S.
was 5.6 billion pounds.— Production figures for VDC are difficult to
obtain; however, the current output may be estimated at about 1 billion
pounds. Although VCM was associated with hepatic angiosarcoma in humansr/
there is relatively little information concerning the human toxicity of VDC,
a structurally related compound. However, there are studies in animals con-
cerning the toxicity of VDC.
A continuous 90-day inhalation exposure to VDC at a dose of 189 mg/
O
m (48 ppm) produced deaths in monkeys and guinea pigs, but not in dogs and
rats.—' A depression in growth occurred in all of the species tested. Mor-
phological changes occurred in livers from monkeys, dogs, and rats, and
kidneys from rats. Hepatic lesions consisted of fatty metamorphosis, focal
necrosis, hemosiderin deposition, lymphocytic infiltration, bile duct pro-
liferation, fibrosis, and pseudo-locule formation. The primary renal
lesion was nuclear hypertrophy of the tubular epithelium. VDC and VCM both
produced hepatic injury; however, VDC was a more potent hepatoxin.r.' In
addition, the hepatic injury following VDC exposure differed from that pro-
duced by VCM. Exposure to VDC, in contrast to VCM, produced changes in the
plasma membrane, mitochondria, and chromatin while sparing the endoplasmic
reticulum. Biochemical signs of toxicity included increased activity of
hepatic alkaline phosphatase and tyrosine transaminase; elevated plasma
alanine transaminase and alkaline phosphatase activity; and reduced hepatic
non-protein sulfhydryl groups and glucose-6-phosphatase activity.—2— The
toxicity of VDC was influenced by the nutritional status of exposed animals—
and the time of day the animals were exposed.—' The increased sensitivity
of these animals to VDC exposure was correlated with a reduction in the
hepatic non-protein sulfhydryl concentration.—'
Although the teratogenic potential of VDC has not been determined,
studies on the effect of VCM exposure during development may be relevant.
Mice, rats, and rabbits were exposed to 500 ppm of VCM during organogenesis.—'
Mice were more susceptible to VCM than either of the other two species.
Maternal deaths (six deaths per 30 exposed) and a slightly reduced weight
gain occurred in mice. In addition, there was a trend towards an increased
incidence of resorptions, reduced pregnancy rate, and reduced fetal body
weights. However, the incidence of external, soft tissue, and skeletal
malformations was equivalent in control and exposed mice. The addition of
15% ethanol to the drinking water increased the toxicity of VCM in mice. In
these mice, the maternal weight gain and pregnancy rate were further decreased
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while the incidence of cleft palate and certain skeletal anomalies was
significantly increased. VCM was judged, in this study, not to be tera-
togenic in mice, rats, and rabbits after daily 7-hr exposures at 500 ppm
during organogenesis.
In summary, VDC and VCM are toxic in the adult. Although the
effects of these compounds were similar, with the liver and kidney being
the primary sites of toxicity, there were some differences in the types
of lesions produced and the doses required to produce them. Although VCM
was judged not to be a teratogen, there is no information available to
evaluate the teratogenic potential of VDC.
The purpose of this study was to determine the effect of VDC
exposure on development. The two parameters of development examined were
the morphological development of rats and mice exposed to VDC in utero and
the behavioral development of rats similarly exposed.
II. METHODS
A. Experimental Design
In this study, rats and mice were exposed to various concentrations
of VDC in utero and their development was evaluated morphologically or be-
haviorally. The exposures were conducted in the trials listed below. In
addition to the VDC-exposed groups, each trial also contained two control
groups which were not exposed to VDC. One control group was given free
access to feed while feed was restricted in the other control group. These
two groups were included to provide a measure of normal development as well
as development influenced by malnutrition. For the purpose of this report,
the results are presented in the four parts indicated below. The morphologi-
cal studies are contained in Part 1 (Trials I, II and III), Part 2 (Trial IV),
and Part 3 (Trial V). The behavioral study is reported in Part 4 (Trial V).
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Trial
I
II
III
IV
Animal
Mice
Rats
Mice
Rats
Mice
Rats
Mice
Mice
•Rats
Concentration
VDC (ppm)
0, 15 and 300
0, 15 and 300
0, 57
0, 57
0, 30 and 144
0, 449
0 - 54 -
0, 41, 54 and 74
- 41, 54 -
- - 54 -
0, 56, 81 and 112
0, 56, 81 and 112
0, 56, 81 and 112
0, 56, 81 and 112
0, 56 and 283
Gestational
Days Exposed
6 to 16
6 to 16
6 to 16
6 to 16
6 to 16
6 to 16
6 to 15
8 to 15
10 to 15
12 to 15
6 to 9
9 to 12
12 to 15
15 to 17
8 to 20
B. Exposure
1. Chamber design; Five stainless steel cubical type animal
u— o
exposure chambers were used in these studies. Three chambers were of 3.5 m
volume and two chambers were 4.5 nH in volume. The contaminants entered the
inlet air stream and were mixed in a plenum at the top of each chamber. Air
was exhausted at the bottom of the chamber.
2. Chamber air supply and flow rate: The air supply to the
chambers was drawn from a stack on the roof of the building. Chamber air
passed through a coarse filter and then over coils for heating, cooling and
dehumidifying. Chamber temperature was maintained at 75°F ± 5° and humidity
at 50% ± 10%.
Initially, air flow rates were measured at the inlet site of the
chamber with pitot tubes connected to a magnehelic gauge. In later studies,
air flow rates were measured at the exhaust side of the chambers with ori-
fice plates connected to magnehelic gauges. Chamber air flows were calibrated
using an Autotronics 100-ssx air flow transducer. Air flow rates were ad-
justed to maintain 10 to 15 air changes per hour in each of the chambers.
3. Chamber safety; All chambers were operated at a slight nega-
tive pressure (0.1-0.2 in. of water) to prevent escape of the contaminant
into the room atmosphere. All air from the chambers traveled under negative
pressure to an incinerator operated at a temperature of 1700° to 1800°F.
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4. Generation of VDC vapor; Vinylidene chloride with a purity
of 99% was obtained from the Aldrich Company. Two methods of generating
VDC vapor were utilized. In Trials I to III, VDC was generated by heating
to 37°C. Since VDC has a boiling point of 32°C, all gas lines and rotometers
were heated to 40°C to prevent condensation. In Trials IV and V, the VDC
vapor was generated by bubbling nitrogen into VDC contained in a glass flask.
A stream of inlet air was directed through the flask and carried the VDC vapor
into the plenum of the chamber. The rate of VDC generation was determined by
the flow of nitrogen into the VDC flask. The nitrogen flow was controlled by
the use of rotometers.
5. Chamber atmosphere monitoring^; VDC was quantified with a
Varian 2700 gas chromatograph equipped with a flame ionization detector and
a stainless steel column (6 ft x 1/8 in.) packed with 0.4% Carbowax 1500 on
Carbopak A. The injector, column, and detector temperatures were 135°C,
95°C and 170°C, respectively.
VDC standards were prepared by serial dilution (weight/volume) of
VDC in carbon tetrachloride. The desired final concentrations in a 4 /^l
injection were determined by the following procedure:
mg/liter (/Ltg/ml) = ppm x mol wt
24450
The amount of VDC (mol wt = 96.94) in a 1-ml sample from chambers
that contain 10, 50 and 100 ppm VDC is 0.04, 0.20 and 0.40 ^g, respectively.
For 1 /xl of standard, these same amounts would be obtained from solutions
of 40, 200 and 400 mg/liter; but since 4 /nl of standard were injected, the
final dilutions of VDC in carbon tetrachloride were 10, 50 and 100 mg/liter.
Each point on the calibration curve was the mean of three determinations.
Each chamber was equipped with 10 sampling ports on two sides of
the chamber. Preliminary studies were conducted to determine the uniformity
of the concentration of the test material at different points in the chamber.
The concentration at the various sampling points was compared to a reference
point in the center of the chamber. It was found that the average concen-
tration at the various points was + 3% of the desired chamber concentration.
The concentration at the reference point was 98.4% to 100.4% of the average
chamber concentration. Therefore, the chambers were routinely sampled at
the reference point for determination of chamber concentrations. The
chamber samples were drawn through polyethylene sample lines into a gas
tight syringe. One milliliter samples of chamber air were injected into
the gas chromatograph for determination of chamber concentration.
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In Trials I to III, the chamber concentrations were determined
by measurement of peak heights on a recorder connected to the gas chromato-
graph. The chamber concentration was determined by extrapolation from a
standard curve prepared at the beginning of each day using VDC standards.
In Trials IV and V, a Varian CDS-111 chromatography data system was used
to determine chamber concentrations. The CDS-111 calculates chamber con-
centration by integration of peak areas. The CDS-111 was calibrated with
VDC standards at the beginning of each day and provided a direct printout
of chamber concentration in parts per million.
C. Animals
CD rats and CD-I mice (Charles River Breeding Laboratories, North
Wilmington, Massachusetts) were housed in our animal quarters for at least
7 days prior to use. These quarters were maintained at 24 + 2°C with a
relative humidity of 50 ± 10% and a 7 AM to 7 PM photoperiod. Rats were
generally housed two per cage and mice six per cage. Animals were given
free access to rodent chow (Wayne Lab-Blox, Allied Mills, Inc., Chicago,
Illinois) and tap water before, during, and after exposure, except where
indicated. During the exposure period, animals were housed in stainless
steel cages with wire mesh bottoms and their feed and water were changed
daily. When feed consumption was determined, the animals were given
powdered rodent chow in stainless steel feeders (Hoeltge, Cincinnati, Ohio)
which were designed to minimize spillage. Animals in the feed-restricted
groups were given their feed in round glass jars in order to provide better
access to their limited amount of feed. Initially, attempts were made to
feed these animals the same amount of feed consumed by the group exposed to
the high concentration of VDC. Since we did not achieve a high degree of
accuracy in this regard, the group has been termed a feed-restricted group
rather than a pair-fed group. The weight gain and, in most cases, the feed
consumption for this group is presented in the appropriate table.
D. Protocol for Morphological Evaluation
1. Mating: Sexually mature virgin female mice were housed over-
night with a proven male breeder. The next morning the females were examined
for evidence of copulation as demonstrated by the presence of a vaginal plug.
Sexually mature virgin female rats were examined by vaginal lavage late in
the afternoon for signs of proestrus (75 to 90% nucleated epithelial cells).
Females in proestrus were placed overnight with an experienced male. The
following morning, females were examined for the presence of sperm. The day
on which evidence of mating was discovered was identified as being day 0 of
gestation.
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2. Exposure; Groups of plug-positive mice and sperm-positive
rats were exposed to various concentrations of VDC as indicated in the above
section on experimental design. The treatments were administered for 22 to
23 hr a day. The exposures were terminated for 1 to 2 hr in the morning so
that animals could be added to or removed from the chamber and their feed and
water could be changed.
3. Examination: Mice and rats were sacrificed by CC>2 anesthesia on
gestational days 17 and 20, respectively. A laparotomy was performed and the
number and position of live, dead, and resorbed fetuses were recorded. The
umbilical cord was clamped and severed distally in order to prevent blood
loss. Fetuses were removed, weighed, and examined for gross anomalies.127
One-half of the fetuses from each litter were fixed in Bouins solution and
examined for soft tissue anomalies using a free hand slicing technique.±P_'
The remaining fetuses from each litter were processed for skeletal examina-
tion. Fetuses were fixed in 70% alcohol, eviscerated, and stored in 1% KOH
for 2 days. Afterwards, they were stained with alizarin red.il/ The skeletons
were examined for anomalies after differential decolorization.
E. Protocol for Behavioral Evaluation
1. Mating: Rats were mated as described above.
2. Exposure; Groups of sperm-positive rats were exposed to 0,
56 and 283 ppm of VDC from gestational days 8 to 20, as indicated above.
3. Examination: Pregnant females, exposed to 0 ppm (control),
0 ppm feed restricted, 56 ppm or 283 ppm of VDC from gestational days 8 to
20, were examined for litters at 8 AM, noon, 5 PM and 10 PM. Those litters
born between 10 PM and 9 AM, and those born between 8 AM and noon were
tested on the behavioral tasks starting on the morning following their birth
(day 1). Those born between noon and 5 PM were tested first on the afternoon
of the following day (day 1). Those litters born between 5 PM and 10 PM were
not used for preweaning tests. All groups, first tested in the morning, were
tested in the morning from then on. The same procedure was used for the
afternoon groups.
On day 1, the pups were sexed and weighed and each litter given a
coded number. Occasionally, pups were transferred to other litters within
age and treatment groups to balance the sexes* All litters were culled to
eight pups with equal numbers of each sex. Body weights were obtained on
alternate days until weaning at 21 days of age.
From each litter, one male and one female were marked by tail
clipping for inclusion into the preweaning behavioral testing. All testing
was done with the experimenter ignorant of the treatment group to which the
pup belonged.
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a. Preweaning tests:
(1) Surface righting ;l» This test started on day 1.
The pup was placed on its back and the time required for the pup to turn
over onto all four feet was recorded. The test was conducted three times
and the average recorded as the score for the day. Testing continued until
three daily average times were 1 sec or better. An upper limit of 30 sec
was applied.
(2) Pivoting ; Testing started on day 4. Young
animals frequently move in circles with one rear foot held in place; this
is called pivoting. The total time spent pivoting during a 3-min test was
recorded. Daily testing continued until the pups did not show any pivoting
during 3 consecutive days.
12 1 "\l
(3) Auditory startle; ""^ Testing started on day 10
and continued until a good response was observed on 3 consecutive days.
The pups were placed in a sound attenuating box and then a sharp noise was
produced over the animal by snapping a device constructed from a mouse trap.
The startle response consisted of sudden flexion and head turning of the pup
following the noise.
-i o/
(4) Bar holding :— ' This test was started on the 7th
day. The pup was placed on a 1/4-in. horizontal aluminum bar. If the pup
held onto the bar, the time it hung on was recorded in seconds up to a maxi-
mum of 15 sec. If the pup did not hang on the bar it fell onto a pile of
foam rubber. Pups that fell off immediately were given two other trials.
Testing continued until three consecutive daily scores of 15 sec were re-
corded, or until the pup was able to negotiate the bar and climb off.
12 I"}/
(5) Righting in air; ' ' This test was started on
day 12 and concluded upon three consecutive daily rightings. When held with
its stomach up and dropped, a mature rat will right itself in air and land
on its feet. Pups were dropped from a height of 50 cm onto a thick, soft
bed of foam rubber.
1 o I o /
(6) Visual placing ;•»•*• >•*••?/ This test started on day 12
and concluded when placing was present on 3 consecutive days. The test
consisted of grasping the pup by the flank and moving it toward the edge of
a table. A mature rat will reach out for the edge as its body approaches it.
(7) Swimming ; ^ > -* ' Swimming tests started on day 7
and continued until swimming ability was judged to be mature. Testing was
conducted in a 20-gal aquarium containing approximately 15 gal of water at
20°C. Judgements were based on a rating scale of 0 to 4 which reflected
the extent to which the animal kept its nose and ears out of the water.
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Pluses and minuses were also used to score the use of the forefeet or hind
feet in locomotion. An immature rat swims with its nose and ears submerged
and paddles with its front feet. A mature rat keeps it nose and ears out
of the water and kicks primarily with its hind feet only.
(8) Physical maturation: In addition to the preweaning
behavioral tests, indicies of physical maturation were recorded daily. These
included detachment of the pinnae (from day 1); incisor eruption (from day 5);
and opening of the eyes (from day 7). Also, body weights of the eight pups
in each litter were obtained on alternate days.
b. Activity test: At about 3 weeks of age, rats within
dose and sex were weaned into groups of three. They were used for measuring
activity levels, starting when they were 95 + 25 days of age. The apparatus
was a resident mazeis/ (Figure 1) which contained six infrared sensors. Five
of these mazes were used simultaneously. Groups of three rats were placed into
the maze and left there for 70 hr. Each time a rat crossed an infrared beam
a count was registered on a counter. Every 2 hr, a timer caused the counter
to print the total number of counts accumulated and then reset to zero.
In all, there were seven groups of males and seven groups of
females for each dose level. They were from 14 to 20 different litters.
Testing was in a counterbalanced order for both groups and apparatuses. The
sexes were tested on alternate days. Due to equipment malfunction, the data
for the last group of males was lost, leaving seven groups (21 rats) of
females and six groups (18 rats) of males for the data analysis.
F. Statistics
All quantitative data were reported as the mean + S.E. and analyzed
for significance by either parametric or nonparametric tests. The parametric
tests used in this study were the analysis of variance followed by Tukey's
omega procedureiZ/ for the morphological data and Duncan's multiple range
test—' for the behavioral data. The level of significance was chosen as
p <. 0.05. The non-parametric test used in this study was the two sample
rank test!§/ with a level of significance selected as p < 0.05 (one tail
test). Enumeration data were analyzed with the Fisher's exact probability
test.Ii/
III. RESULTS
A. Chamber Concentrations of VDC
The standard curve for measuring VDC concentrations was initially
prepared by injecting varying volumes of a VDC standard, prepared in chloro-
form, into the gas chromatograph. This procedure produced a non-linear response
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WATER
I NEST
I BOX
10
cm
Figure 1 - Diagram of Activity Maze. The sides and nest box are
constructed of galvanized steel and rests on an
expanded aluminum floor. The runway walls are 10 cm
high and the nest box walls are 25 cm high. Crossbars
indicate locations of infrared beams. The runways are
covered by a sheet of clear plexiglas with a raised
dome in the center.—'
10
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which prevented the accurate measurement of chamber concentrations. This
problem was corrected by injecting a constant volume of standards prepared
in carbon tetrachloride.
The chamber concentrations of VDC used during the various trials
are presented in Table 1. As the result of our early problems in obtaining
a linear standard curve it was not possible to accurately measure chamber
concentrations in Trial I by gas chromatography. Therefore, the concentra-
tions for this trial were calculated from mass balance data (i.e., the
number of air changes per hour and the amount of VDC consumed per day). In
subsequent trials, the chamber concentrations were measured using gas chro-
matography. For Trials II to IV, the concentrations were measured, on the
average, four to five times a day from 8 AM to 5 PM. In the evening, the
flow meters were monitored in an effort to control the chamber concentration.
During Trial V the VDC concentrations were measured, on the average, 10 to 11
times a day at 2-hr intervals. For Trials II to V, the chamber concentration
for a given day was determined by averaging the individual values determined
during that day.
B. Morphological Studies on Mice and Rats
1. Mice and rats exposed from gestational days 6 to 16 (Part 1):
a. General observations: Mice were exposed to 0, 15, 30,
57, 144 and 300 ppm of VDC from gestational day 6 to 16. The effects of
this treatment on maternal welfare and reproduction are presented in Table 2.
A reduced ratio of pregnant to exposed mice occurred in the group exposed to
30 and 57 ppm of VDC. Increased mortality in pregnant mice was observed at
VDC concentrations of 114 ppm and above. In addition, increased mortality
was observed in pregnant mice in the feed restricted group. Increased mor-
tality also occurred in non-pregnant mice exposed to 144 and 300 ppm of VDC.
Feed consumption and weight gain during exposure were reduced in the groups
exposed to 30 and 57 ppm of VDC as well as the feed restricted group. The
group exposed to 57 ppm of VDC had an increased number of dams with complete
resorptions. Viable fetuses were only produced in the control group, the
group exposed to 15 ppm of VDC, and the feed restricted group. Fetal body
weights were reduced only in the feed restricted group.
Rats were exposed to 0, 15, 57, 300 and 449 ppm of VDC from
gestational days 6 to 16. The effects of this treatment on maternal welfare
and reproduction are shown in Table 3. A reduced ratio of pregnant to ex-
posed rats occurred in the group exposed to 300 ppm of VDC. Deaths occurred
in pregnant rats exposed to VDC concentrations of 57 ppm and above. The
weight gain during exposure was reduced in all the groups exposed to VDC.
11
-------
TABLE 1
CHAMBER CONCENTRATIONS OF VINYLIDENE
CHLORIDE DURING VARIOUS TRIALS
Vinylidene Chloride (ppm)
Trial Mean Ranged/
300
II 57+2 (14)^ (51-81)
III 30+1 (10) (25-38)
144 + 12 (3) (131-168)
449+5 (14) (415-478)
IV 41+1 (18) (34-52)
54+2 (17) (46-61)
74 +2 (18) (51-86)
V 56+1 (18) (52-68)
81+1 (15) (74-91)
112 + 2 (13) (97-120)
283 + 4 (18) (228-303)
aj Mean calculated from mass balance data.
b_/ Mean + S.E. for the indicated number of daily
values determined by gas chromatography.
£/ Range of daily values.
12
-------
EFFECT
TABLE 2
OF VINYLIDENE CHLORIDE EXPOSURE ON GESTATIONAL DAYS 6 TO 16 ON
MATERNAL WELFARE AND REPRODUCTION IN MICE
Number Exposed
Pregnant
Alive
Non-Pregnant
Alive
al
Body Weight 'Change—
During exposure
After exposure
Feed Consumption".'
u>
Pregnant Surviviors
Implants /Dam
Viable fetuses (%)
Dead fetuses (%)
Early resorptions (7,)
Late resorptions (7o)
Dams with complete resorptions
Live Litters
Fetuses/Dam
Males (7o)
Fetal Weight (gm)
0£/
65
38
38
27
27
11.7 + 1.5 11
6.2 + 0.7 7
5.3 + 0.2 6
38
11.1 + 0.4 10
82 + 5
1 + 0
11 + 4
3 + 1
3
35
9.8 + 0.4 10
52 + 4
1.2 + 0.0 1
Vinylidene Chloride (ppm)
15
23
16
16
7
7
.0 + 1.0
.3 + 1.2
.0 + 0.9 4.
16
.7 + 0.6
87 + 6
2+1
8 + 6
5+2
0
16
.3 + 0.4
54 + 3
.3 + 0.1
30 57
19 21
2*.! 5l/
2 4
17 15
17 14
1 10.0 + 2. o!/
1 7.0 + 1.7
2 + O.ll/ 3.3 + 0.3^
2 4
4.0 6.8 + 2.3
0 Ol/
0 0
100 lOO^/
0 0
2 4l/
0 0
__
— — — _
-, _ _ _
144 300 O^/
18 15 20
889
0£/ 0£/ 7£/
10 7 11
Oi/ 0£/ 11
5.0 + 2.
5.1 + 1.
3.6l/
007
8.6 + 1.
42 + 18
0
46 + 19
12 + 12
3£/
0-0 4
9.0 + 2.
54+7
0.9 + 0.
oi/
7
7
f/
3
Ifi/
a_/ Gm/animal/interval for pregnant rats.
b/ Gm/animal/day during exposure for pregnant rats.
£/ Control group.
d/ Feed restricted group.
£/ Significantly different from
f/ Significantly different from
£/ Significantly different from
control (Fisher's exact probability
control (two sample
rank test) .
test).
control (Tukey's omega procedure).
-------
TABLE
MATERNAL WELFARE AND REPRODUCTION IN RATS
Vinylidene Chloride (ppm)
Number Exposed
Pregnant
Alive
Non-pregnant
Alive
Body Weight Change3.'
During exposure
After exposure
Feed Consumption—'
Pregnant Survivors
Implants/Dam
Viable fetuses (%)
Dead fetuses (%)
Early resorptions (%)
Late resorptions (70)
Dams with complete resorptions
Live Litters
Fetuses/Dam
Males (%)
Fetal weight (gm)
0£/
58
50
50
8
8
44 + 23
52+4
19.0 + 0.8
50
13.3 + 0.3
98 + 1
0
2+1
0.4 +0.2
0
50
13.0 + 0.4
51+2
3.6 + 0.1
15
18
14
13
4
3
57
20
16
13-
4
4
-28 + 8^ -64 + 6l/
91 + 6-7
15.3 + 3.6 7
13
12.5 + 0.5 12
95+2
0
5+2
0
0
13
11.9 + 0.6 12
53 + 4
3.4 + 0.1 3
80 + lOi/
.0 + l.l-/
13
.3 + 1.0
51 + 14-7
0
49 + 14^/
0
6£/
7
.9 + 0.9
46 + 5
.1 + 0.1^
300
18
9
6-'
9
8
-83 + 13l/
81 + 10-/
4.8 + 0.9i/
6
9.8 + 1.9
93 + 4
0
7 + 4
0
0
6 f
9.2 + 1.9-
49+11
3.0 + 0.1^
449 0*'
18 18
15 14
8-^ 14
4 4
3 4
-62 + 19l/ -61 + sl/
49+6 91 + 8l/
5.6 + O.sl/ 3. sl/
8 14
12.9 + 1.8 13.0 + 0.3
23 + l^ 98 + 1
0 0
64 + IS^/ 2+1
13+13 0
6^ 0
2 14
13.5 + 0.5 12.8 + 0.3
51+13 53 + 3
2.7 + 0.2I/ 3.0 + O.l^/
a/ Gm/animal/interval for pregnant rats.
b/ Gm/animal/day during exposure for pregnant rats.
c/ Control group.
d_/ Feed restricted group.
e/ Significantly different from
f/ Significantly different from
control (Fisher1
control (Tukey's
s exact probability
omega procedure).
test) .
-------
The weight gain after treatment was increased in all of the VDC exposed
groups except the group exposed to 449 ppm. Feed consumption was reduced
in the groups exposed to VDC concentrations of 57 ppm and above. Feed
consumption was also reduced in the feed restricted group. The percent of
implants with viable fetuses was reduced in the groups exposed to 57 and
449 ppm of VDC. In addition, the ratio of dams with complete resorptions
to pregnant survivors was increased in these groups. The resorptions which
occurred in these two groups were classified as being early resorptions.
Fetal body weights were reduced in the groups exposed to 57, 300 and 449 ppm
of VDC as well as the feed restricted group. The number of fetuses/dam was
reduced in the group treated with 300 ppm of VDC.
b. Anomalies: There was neither a significant increase in
gross anomalies nor a pattern of gross anomalies in either mice or rats
following exposure to VDC.
The soft tissue anomalies in mice and rats following exposure
to VDC are presented in Tables 4 and 5, respectively. There was no signifi-
cant increase in anomalies from mice exposed to 15 ppm of VDC. However, in
rats there was an increase in the incidence of lateral ventricle hydrocephalus
in rats exposed to 15 and 57 ppm of VDC. A similar incidence of this anomaly
also occurred in the group exposed to 300 ppm; however, this level was not
statistically significant.
Skeletal anomalies observed in mice and rats exposed to VDC
are reported in Tables 6 and 7, respectively. Ossification problems with
the incus and sternebrae occurred in mice exposed to 15 ppm of VDC. A
similar incidence of unossified incus also occurred in the feed restricted
group. Rats from the groups exposed to 15, 57 and 300 ppm of VDC had ossi-
fication problems with the sternebrae.
2. Mice exposed for various intervals ending on gestational day 15
(Part 2):
a. General observations: Mice were exposed to 0, 41, 54 and
74 ppm of VDC for 3 to 9 day intervals that ended on gestational day 15. The
effects of these concentrations of VDC on maternal welfare and reproduction
following a 7-day exposure, which started on gestational day 8, are presented
in Table 8. Significant mortality occurred in pregnant mice exposed to 74
ppm of VDC. A reduced weight gain during exposure occurred both in the VDC
treated groups and the feed restricted group. However, this effect was not as
dramatic in the latter group. At the end of the exposure period, the weight
change was significantly increased for all of these groups with the exception
of the group exposed to 74 ppm of VDC. In addition, dams in this group had a
significantly reduced number of implants and percent of these implants with
viable fetuses. All of the implants in the surviving dams were characterized
as being early resorptions.
15
-------
TABLE
EFFECT OF VINYLIDENE CHLORIDE EXPOSURE ON GESTATIONAL
DAYS 6 TO 16 ON THE INCIDENCE OF SOFT TISSUE ANOMALIES IN MICE
Vinylidene Chloride (p
Number of
Litters inspected
Fetuses inspected
Soft Tissue Anomalies
Hydrocephalus: lateral ventricle
lateral ventricle slight
Nasal passage occluded
Microphthalmia
Palate: cleft
Liver: small
Kidney: hydronephrosis
OS/
7
36
15
15
73
ok/
3
13
o+o0-/
0+0
0+0
0+0
0+0
0+0
0+0
2.
2.
1.
3.
1.
1.
5.
6+1.8
3+1.6
9+1.9
6+2.4
7+1.7
7+1.7
8+2.6
0+0
0+0
0+0
0+0
6.7+6.7
0+0
0+0
a/ Control group.
b/ Feed restricted group.
£/ Mean + S.E. of the percent of fetuses with the indicated anomaly cal-
culated on a per litter basis.
16
-------
TABLE
EFFECT OF VINYLIDENE CHLORIDE ON GESTATIONAL DAYS 6 TO 16 ON
THE INCIDENCE OF SOFT TISSUE ANOMALIES IN RATS
Vinylidene Chloride
Number of
Litters inspected
Fetuses inspected
Soft tissue anomalies
Hydrocephalus : lateral ventricle
lateral ventricle, slight
third ventricle
fourth ventricle
Nasopharyngeal canal occluded
Nasal passage occluded
Palate: short
Lung: agenesis
small
deflated
Small bronchus opening
Liver: small
Kidney: hydronephrosis
small
ectopic
Distended urinary bladder
oi'
50
211
2.5+2. 1-/
0.3+0.3
0.4+0.4
2.5+2.5
0.3+0.3
1.0+0.6
0+0
0.3+0.3
0+0
1.1+0.7
0.3+0.3
0+0
2.6+1.1
0+0
0.3+0.3
2.5+1.5
15
13
75
7.3+2. 3-/
1.3+1.3
0+0
0+0
0+0
0+0
2.4+1.6
0+0
0+0
0+0
0+0
0+0
5.5+2.5
1.5+1.5
0+0
0+0
57
7
41
15 . 1+6 . 3-/
0+0
0+0
0+0
0+0
0+0
0+0
0+0
4.9+3.2
0+0
0+0
0+0
0+0
0+0
0+0
0+0
(ppm)
300
6
27
33.3+21.1
0+0
0+0
0+0
0+0
0+0
0+0
0+0
0+0
0+0
0+0
8.3+8.3
25.0+17.1
6.1+3.9
0+0
0+0
QlD/
14
85
0+0
2.4+1.6
0+0
0+0
0+0
0+0
0+0
0+0
0+0
0+0
0+0
0+0
2.0+2.0
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 litter basis.
d_/ Significantly different from control in respective trial (two-sample rank test).
-------
TABLE
EFFECT OF VINYLIDENE CHLORIDE EXPOSURE ON GESTATIONAL
DAYS 6 TO 16 ON THE INCIDENCE OF SKELETAL
ANOMALIES IN MICE
Number of
Litters inspected
Fetuses inspected
Skeletal anomalies
Skull collapsed: marked
Nasal bones elevated
Premaxillary process: incompletely ossified
Supraoccipital: incompletely ossified
extra ossification
Incus: unossified
Mandible: short
incompletely ossified
Sternebrae: ossified normally
unossified
incompletely ossified
split
malaligned
extra ossification
Lateral curvature of spine
Centri: ossified normally
Ribs: extra
Paws: unossified
Vinylidene Chloride (ppm)
1
38
15
15
76
3
15
0+0
2.8+2.8
0+0
6.4+4.2
0+0
0+0
0+0
0+0
35.0+9.1
0+0
0+0
19.6+7.5
29.9+9.7
8.6+8.6
0+0
85 . 7+14
20.7+1.07
2.8+2.8
3.2+1.7
4.5+2.6
0+0
12.2+6.2
2.3+1.6
21. 4+8. O^/
1.1+1.1
4.1+2.8
37.3+6.2
1.7+1.7
13. 6+4. 4^
28.1+7.6
25.6+4.8
12.4+7.2
1+1
100+0
31.1+9.1
1.1+1.1
0+0
0+0
5.6+5.6
57.2+20.3
0+0
86 . 1+7 . 3-/
0+0
0+0
19.4+10.0
22.2+22.2
45.0+23.0
44.4+29.4
38.3+7.3
0+0
0+0
100+0
83.3+16.7
0+0
a/ Control group.
b_/ Feed restricted group.
c_/ Mean + S.E. of the percent of fetuses with the indicated anomaly calculated
on a per litter basis.
d/ Significantly different from control (two-sample rank test).
18
-------
TABLE 7
DAYS
EFFECT OF VINYLIDENE CHLORIDE ON GESTATIONAL
6 TO 16 ON THE INCIDENCE OF SKELETAL ANOMALIES IN RATS
Number
Number of
Lltcers inspected
Fetuses inspected
Skeletal anomalies
Skull collapsed
slight
marked
Nasal bones: elevated
Premaxillary: malaligned
Occipital fontanel: enlarged
Parietals: incompletely ossified
Interparietals: incompletely ossified
Supraoccipital: incompletely ossified
Squamosal: split
Mandibles: shortened
incompletely ossified
malaligned
Hyoid bone: unossified
incompletely ossified
malaligned
Sternebrae: ossified normally
unossified
incompletely ossified
split
malaligned
Lateral curvature to spine
Centri: ossified normally
lobed
split
Ribs: unossified
extra
Pelvis: unossified
incompletely ossified
Paws: incompletely ossified
Vinylidene Chloride (ppm)
OS.'
48
323
15
13
79
57
7
48
300
6
27
<£'
14
94
1.8+0.7-'
0.4+0.4'
0.3+0.3
0.3+0.3
4.4+2.1
1.2+0.8
0.3+0.3
1.6+1.0
0+0
1.0+1.0
0.8+0.5
0+0
6.4+2.1
2.7+1.0
0.3+0.3
25.4+3.8
25.7+5.0
44.8+3.5
5.0+2.2
5.7+1.9
0+0
69.9+4.0
25.3+3.6
5.9+1.6
0.34-0.3
9.3+2.4
1.7+1.7
1.7+1.7
0+0
5.9+4.5
4.4+3.2
0+0
0+0
0+0
0+0
1.0+1.0
1.3+1.3
0+0
0+0
0+0
1.0+1.0
0+0
0+0
0+0
10. 5+5. 3d./
51.4+7.7
50 .1+7.5
28.1+9.7
18. 3+3. &
0+0
72.9+8.2
24.6+7.3
3.7+1.9
0+0
0+0
0+0
1.3+1.3
2.2+2.2
4.2+2.7
3.8+2.5
0+0
0+0
0+0
2.4+2.4
0+0
7.1+5.0
4.4+2.9
0+0
0+0
0+0
21.4+9.5
3.6+3.6
0+0
4.2+2.7-'
72. 4+7. &
60.5+8.1
6.1+6.1
6.3+3.1
0+0
71.8+10.8
25.9+8.9
2.4+2.4
0+0
4.4+2.9
0+0
0+0
0+0
20.0+16.3
0+0
0+0
0+0
0+0
0+0
0+0
0+0
8.3+8.3
0+0
0+0
0+0
0+0
0+0
0+0
o+pi/
68.2+12.5
59.8+16.6
19.4+13.9
30.7+15.8
11.1+8.2
51.8+15.1
48.2+15.1
0+0
0+0
0+0
0+0
0+0
0+0
0+0
0+0
0+0
0+0
0+0
0+0
0+0
0+0
0+0
0+0
0+0
0+0
0+0
0+0
0+0
21.3+5.2
50.1+9.3
50.3+5.8
15.0+5.7
11.2+3.4
0+0
71.6+6.4
25.2+5.2
4.3+2.4
0+0
12.4+4.7
0+0
0+0
0+0
a/ Control group.
b/ Feed restricted group.
I/ Mean + S.E. of the percent of fetuses with the indicated anomaly calculated on a per litter basis.
d/ Significantly different from control in respective trial (two sample rank test).
19
-------
N5
O
TABLE 8
EFFECT OF VINYLIDENE CHLORIDE EXPOSURE ON GESTATIONAL DAYS 8 TO 15 ON
MATERNAL WELFARE AND REPRODUCTION IN MICE
Vinylidene Chloride
Number Exposed
Pregnant
Alive
Non-pregnant
Alive
a/
Body Weight Change—
During exposure
After exposure
Pregnant Survivors
Implants/dam
Viable fetuses (%)
Dead fetuses (%)
Early resorptions (%)
Late resorptions (%)
Dams with complete resorptions
Live Litters
Fetuses/dam
Males (%)
Fetal weight (gin)
a/ Cm/ animal/ interval for pregnant
b/ Control group.
£/ Feed restricted group.
0°'
24
17
17
7
7
12.5 +0.4
4.4 + 0.9
17
10.8 + 0.4
92+2
0
4 + 1
4 + 1
0
17
10.1 + 0.5
52+5
1.13 + 0.3
mice .
d/ Significantly different from control (Fishe
41
22
2 1-/
21
1
1
2.8 + l.l-/
11.4 + 1.1-/
21
10.0 + 0.7
75+8
0.3 + 0.3
21+9
4 + 1
4
17
10.5 + 0.3
43+4
1.08 + 0.05
sr's exact probab
54
20
12
12
8
8
1.3 + 1.5-/
12.2 + 1.2-'
12
10.3 + 0.9
83+8
1.5 + 1.5
11+8
5+2
1
11
9.7 + 0.8
44 + 4
1.06 + 0.03
ility test).
(ppm)
74
17
9
3d/
8
4
-8.7 + l.S^
-0.3 + 0.7-/
3
6.3 + 1.9-/
0-'
0
100 + 0£/
0
3-
0
-
-
—
(£'
17
13
13
4'
4
10.2 + 0.4-^
7.5 + 0.4-
13
10.6 + 0.5
90 + 2
0.6 + 0.6
5+2
3+1
0
13
9.6 + 0.5
53+4
1.23 + 0.03
e_/ Significantly different from control (two sample rank test).
-------
The effects of 41 ppm of VDC on maternal welfare and repro-
duction, following both a 7- and 5-day exposure ending on gestational day
15, are presented in Table 9. The control and VDC exposed group from
Table 8 are included for comparison. The weight gain during exposure was
reduced for both VDC exposed groups relative to the appropriate control
values. However, the weight change after exposure was increased only in
the group with the more prolonged exposure. Dams in the group exposed to
VDC from gestational days 10 to 15 had a reduced percent of viable fetuses
and an increase both in the percent of early resorptions and the number of
dams with complete resorptions. In addition, the number of fetuses/dam was
also reduced in this group.
The effects of 54 ppm of VDC on maternal welfare and repro-
duction following exposure periods of 9, 7, 5 and 3 days ending on gestational
day 15, are presented in Table 10. A significant increase in mortality
occurred in the group whose exposure started on gestational day 6. The
weight gain during exposure was reduced for all of the VDC treated groups
relative to the appropriate control values. An increased weight gain after
exposure occurred only in the group of mice whose treatment period started
on gestational day 8. All of the surviving dams exposed from gestational
days 6 to 15 had complete resorptions. Dams treated for 5 days starting
on gestational day 10 had a reduced percent of viable fetuses, an increased
percent of both early and late resorptions, and an increased number of dams
with complete resorptions. In addition, fetuses/dam and fetal body weight
were reduced in this group. Similar effects were observed in dams treated
for 3 days starting on gestational day 12; however, there was no effect on
the incidence of early resorptions.
b. Anomalies; The gross anomalies observed in fetuses
from dams exposed to 41 and 54 ppm of VDC are presented in Table 11. Imma-
ture skin, which was defined as being a sticky, poorly developed integument,
hematoma, and exencepaly were observed. Immature skin and hematoma occurred
in both the control and VDC exposed groups and did not increase in a dose
related manner. Exencepaly, in contrast, occurred only in the group exposed
to 81 ppm of VDC.
The effects of exposing pregnant mice to 41 and 54 ppm of VDC
on the incidence of soft tissue anomalies are presented in Tables 12 and 13,
respectively. Although anomalies were observed in mice exposed to 41 ppm
of VDC from gestational days 8 to 15 and 10 to 15 there was no significant
increase in the incidence of these anomalies relative to the control group.
However, an increased incidence of cleft palate occurred in mice exposed to
54 ppm of VDC from gestational days 10 to 15 and 12 to 15 but not 8 to 15.
21
-------
TABLE 9
EFFECT OF VINYLIDENE CHLORIDE EXPOSURE AT 41 PPM ON
MATERNAL WELFARE AND
VDC (ppm)
Days Exposed
Number Exposed
Pregnant
Alive
Non-pregnant
Alive
Body Weight Change-/
During exposure
After exposure
Pregnant Survivors
Implants/dam
Viable fetuses (%)
Dead fetuses (%)
Early resorptions (%)
Late resorptions (%)
Dams with complete resorptions
Live Litters
Fetuses/dam
Males (%)
Fetal weight (gm)
REPRODUCTION
8 to 15
24
17
17
7
7
12.5 + 0.4
4.4 + 0.9
17
10.8 + 0.4
92+2
0
4 + 1
4+1
0
17
10.1 + 0.5
52+5
1.13 + 0.03
IN MICE
41
8 to 15
22
21
21
1
1
2.8 + I. I-1
11.4 + 1.1-
21
10.0 + 0.7
75+8
0
21 + 9
4 + 4
4
17
10.5 + 0.3
43+4
1.08 + 0.05
41
10 to 15
20
18
17
2
2
-4.0 + 0.7-/
5.7 + 1.0
17
9.3 + 0.9
29 + 8-/
1 + 1
64 + 9-^
6+3
8-/
9
5.2 + 1.QS-'
45 + 11
1.20 + 0.20
a_/ Gm/animal/interval for pregnant mice.
b/ Control group.
c_/ Significantly different from control (two sample rank test).
d/ Significantly different from control (Fisher's exact probability test).
22
-------
NJ
LO
TABLE 10
EFFECT OF VINYLIDENE CHLORIDE (VDC) EXPOSURE AT 54 PPM ON
MATERNAL WELFARE
VDC (ppm)
Days Exposed
Number Exposed
Pregnant
Alive
Non-pregnant
Alive
a/
Body Weight Change—
During exposure
After exposure
Pregnant Survivors
Implants/dam
Viable fetuses (%)
Dead fetuses (%)
Early resorptions (%)
Late resorptions (%)
Dams with complete resorptions
Live Litters
Fetuses/dam
Males (%)
Fetal weight (gm)
a/ Gm/animal/ interval for pregnant
b/ Control group.
6 to 15
24
17
17
7
7
12.5 + 0.4
4.4 +0.9
17
10.8 + 0.4
92+2
0
4 + 1
4 + 1
0
17
10.1 + 0.5
52 + 5
1.13 + 0.03
mice.
£/ Significantly different from control (Fisher1
d/ Significantly different from control (Tukey's
AND REPRODUCTION IN MICE
54
6 to
22
7
3-
15
11
-4.7 +
1.3 +
3
3.3 +
0
0
100 +
0
3
0
-
-
-
s exact
omega p
54
15 8 to 15
20
12
L/ 12
8
8
0.9-/ 1.3 + 1.5^/
0.7 12.2 + 1.2-'
12
0.9^' 10.3 + 0.9
83+8
2+2
Oi/ 11 + 8
5+2
1
11
9.7 + 0.8
44 + 4
1.06 + 0.03
probability test).
>rocedure) .
54
10 to 15
24
17
12
7
6
-3.6 + 0.8^
6.2 + 0.5
12
10.5 + 0.8
23 + 9-
o • —
23 + 8^
54 + 13^/
6-'
6
5.2 + 1.81/
51 + 13
0.93 + 0.05-/
54
12 to 15
13
11
10
2
1
-8.1 + 0.
2.7 + 0.
10
10.2 + 0.
32 + 9-
10 + 10
9 + 5
49 + 12
4£/
6
5.0 + 0.
47 + 6
0.81 + 0.
7^
5
8
/
£./
6*'
06^
e_/ Significantly different from control (two sample rank test).
-------
TABLE 11
EFFECT OF VINYLIDENE CHLORIDE EXPOSURE ON THE
INCIDENCE OF GROSS ANOMALIES IN MICE
VDC
(ppm)
Oa/
41
54
Qb/
Gestational
Days
Exposed
8 to 15
8 to 15
8 to 15
10 to 15
12 to 15
8 to 15
Gross Anomalies
Immature
Skin
1.2+1.2^
18.7+9.4
6.5+4.6
0+0
0+0
1.5+1.5
Hematoma
4.4+1.6
2.3+1.1
0+0
0+0
0+0
1.5+1.5
Exencephaly
0+0
0+0
5.3+3.2
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 litter basis.
24
-------
TABLE 12
EFFECT OF VINYLIDENE CHLORIDE (VDC) EXPOSURE AT 41 PPM ON THE
INCIDENCE OF SOFT TISSUE ANOMALIES IN MICE
Number of
Litters inspected
Fetuses inspected
a/
VDC (ppm) 0-
Days Exposed 6 to 15
17
32
41
8 to 15
17
86
41
10 to 15
9
22
<£'
8 to
13
62
t
15
Soft tissue anomalies
Hydrocephalus: lateral ventricle 0+0-' 9.8+6.4 11.1+11.1 0+0
third ventricle 0+0 12.6+6.6 0+0 0+0
fourth ventricle 5.3+3.2 6.4+3.3 0+0 1.9+1.9
Olfactory bulb with depression 0+0 1.5+1.5 0+0 0+0
Nasal cavity occluded 1.0+1.0 0+0 11.1+11.1 2.6+1.7
Nasopharyngeal canal occluded 2.0+1.3 2.5+1.7 0+0 0+0
Nasal passage occluded 4.5+3.6 5.3+2.5 8.3+6.0 14.4+5.0
Cleft palate 0+0 1.5+1.5 10.2+7.6 0+0
Deflated lung 1.2+1.2 2.4+1.6 0+0 0+0
Kidney: hydronephrosis 1.2+1.2 2.8+2.8 2.9+2.0 1.3+1.3
small 0+0 0+0 16.7+11.8 0+0
cortex solidified 0+0 6.3+2.5 5.6+5.6 5.8+3.0
£/ Control group.
b_/ Feed restricted group.
c_/ Mean + S.E. of the percent of fetuses with the indicated anomaly calculated
on a per litter basis
25
-------
TABLE 13
EFFECT OF VINYLIDENE CHLORIDE (VDC) EXPOSURE AT 54 PPM ON THE
INCIDENCE OF SOFT TISSUE ANOMALIES IN MICE
VDC (ppm)
Days Exposed
Number of
Litters inspected
Fetuses inspected
Soft tissue anomalies
Hydrocephalus : lateral ventricle
third ventricle
fourth ventricle
Temporal artery hemorrhage
Nasal cavity occluded
Nasopharyngeal canal occluded
Nasal passage occluded
Micropthalmia
Ectopic eye
Cleft palate
Small thymus
Foramen ovale enlarged
Atrio-ventricular communis
Atrio-ventricular valve enlarged
Deflated lung
Kidney: hydronephrosis
small
cortex solidified
Distended urinary bladder
f
6 to 15
17
82
u /
0+0^
0+0
5.3+3.2
0+0
1.0+1.0
2.0+1.3
4.5+3.6
0+0
0+0
0+0
0+0
0+0
0+0
0+0
1.2+1.2
1.2+1.2
0+0
0+0
0+0
54
8 to 15
11
54
5.3+3.6
4.5+3.0
14.5+4.9
3.8+2.6
1.5+1.5
1.3+1.3
14.5+7.7
7.6+6.1
2.3+2.3
7.1+3.8
0+0
1.5+1.5
3.0+3.0
1.5+1.5
4.8+3.4
0+0
0+0
2.3+2.3
0+0
54
10 to 15
6
16
0+0
0+0
11.7+8.3
0+0
0+0
16.7+16.7
0+0
0+0
0+0
31. 7+16. l&l
16 . 7+16 . 7
3.3+3.3
0+0
0+0
0+0
0+0
3.3+3.3
0+0
8.3+8.3
«y
12 to 15
6
13
0+0
0+0
5.6+5.6
0+0
8.3+8.3
0+0
16.7+16.7
0+0
0+0
44.4+20.5£/
0+0
0+0
0+0
0+0
0+0
0+0
0+0
0+0
0+0
a/ Control group.
W Mean + S.E. of the percent of fetuses with the indicated anomaly calculated
on a per litter basis.
£/ Significantly different from control (two sample rank test).
26
-------
The skeletal anomalies observed in fetuses from mice exposed
to 41 and 54 ppm of VDC are reported in Tables 14 and 15. An increased
incidence of unossified incus occurred in the groups exposed to 41 ppm of
VDC from gestational days 8 to 15 and 10 to 15. In addition, an increased
. incidence of unossified sternebrae occurred in the groups exposed from
days 10 to 15. Ossification problems with the incus also occurred in mice
exposed to 54 ppm of VDC from gestational days 8 to 15 and 12 to 15. Anoma-
lies of the sternebrae occurred in the groups exposed to 54 ppm of VDC from
gestational days 8 to 15 and 12 to 15. Incompletely ossified supraoccipitals
occurred, at an increased frequency, in the groups exposed to VDC from gesta-
tional days 10 to 15 and 12 to 15. In addition, the normal ossification of
centri was reduced in the group exposed from gestational days 8 to 15.
3. Mice exposed for 2- to 3-day intervals during gestation (Part 3) :
a. General observations: Mice were exposed to 0, 56, 81 and
112 ppm of VDC for 2- to 3-day intervals beginning and ending at various times
during gestation. In Part 3, one control group was used for all the treatment
periods. However, each treatment period had its own feed restricted group.
Mice in these groups were not given any feed during the appropriate intervals.
At the end of the exposure period, all groups were given free access to feed.
The effects of a 3-day exposure, which started on gestational
day 6, on maternal welfare and reproduction are presented in Table 16. A
reduced ratio of pregnant to exposed mice occurred in all the VDC treated
groups and the feed restricted group. Significant mortality occurred in the
group exposed to 112 ppm of VDC and there were no pregnant survivors. Complete
resorptions occurred in the one surviving dam from the group exposed to 81 ppm
of VDC and the two surviving dams from the feed restricted group. Other than
a significant reduction in the ratio of pregnant to exposed mice, there was
no effect of 56 ppm VDC exposure on the parameters of maternal welfare and
reproduction presented in Table 16.
The effects of a 3-day exposure, which started on gestational
day 9, on maternal welfare and reproduction are presented in Table 17. A
reduced ratio of pregnant to treated mice was observed in the group exposed
to 56 ppm of VDC. Mortality occurred in the dams exposed to 112 ppm and
there were no survivors. The weight gain during treatment was signifi-
cantly reduced both in the group exposed to 81 ppm of VDC and the group
starved during exposure. The weight gain of the latter group increased
after exposure; however, this increase may reflect the greater weight loss
during treatment. The group exposed to 81 ppm of VDC had a reduced percent
viable fetuses, an increased percent of early resorptions, a reduced number
of fetuses/dam, and a reduced fetal body weight. These effects did not reach
a level of significance in the feed restricted group.
27
-------
TABLE 14
EFFECT OF VINYLIDENE CHLORIDE (VDC) EXPOSURE AT 41 PPM ON THE
INCIDENCE OF
VDC (ppm)
Days Exposed
pected
epcted
SKELETAL ANOMALIES
O*/
8 to 15
17
168
IN MICE
41
8 to 15
17
93
41
10 to 15
9
26
ok/
8 to 15
13
118
Number of
Skeletal anomalies
Skull collapsed: slight O+O-/ 2.2+1.5 14.8+11.3 5.8+3.2
marked 0+0 0+0 0+0 7.3+4.3
Nasal bones: incompletely ossified 0+0 11.8+8.1 5.6+5.6 0+0
elevated 0+0 5.9+5.9 10.0+6.7 0+0
Occipital fontanel enlarged 1.0+1.0 17.8+8.5 0+0 0+0
Interparietals curved medially 0+0 1.0+1.0 5.9+4.1 0+0
Supraoccipital: unossified 2.4+2.3 15.3+8.5 0+0 0+0
. incompletely ossified 4.3+2.6 25.3+6.6^/ 8.3+5.9, 3.8+3.8
Incus unossified 23.4+7.7 54.9+9.4- 58.3+14 ."5-/ 11.5+6.4
Hyoid bone: unossified 7.8+6.1 17.6+9.5 0+0 1.3+1.3
incompletely ossified 0+0 0+0 0+0 1.3+1.3
Sternebrae: ossified normally 61.4+7.8 54.0+8.4 42.0+12.5 67.6+7.2
unossified 7.8+6.1 19.7+9.4 26.9+11.I-/ 0+0
incompletely ossified 7.1+3.3 14.7+6.8 29.8+11.7 2.6+1.7
split 6.4+3.0 12.2+4.0 28.1+11.4 13.1+7.1
fused 1.2+1.2 0+0 0+0 0+0
malaligned 14.4+4.4 14.9+4.3 3.7+3.7 21.9+5.1
Centri: ossified normally 100+0 99.0+1.0 100+0 100+0
Vertebrae: hemi-vertebra 0+0 1.0+1.0 0+0 0+0
Ribs: vertically or malfused 0+0 1.0+1.0 . 0+0 0+0
extra 22.8+6.5 13.1+4.9 40.6+14.1 33.8+8.4
wavy 0+0 1.0+1.0 0+0 1.9+1.9
a/ Control group.
b_/ Feed restricted group.
c_/ Mean + S.E. of the percent of fetuses with the indicated anomaly calculated on a per
litter basis.
d/ Significantly different from control (two sample rank test).
28
-------
TABLE 15
EFFECT OF VINYLIDENE CHLORIDE (VDC) EXPOSURE AT 54 PPM ON THE
INCIDENCE OF SKELETAL ANOMALIES IN MICE
VDC (ppm)
Days Exposed
Number of
Litters inspected
Fetuses inspected
Skeletal anomalies
Skull collapsed: slight
Nasal bones: incompletely ossified
elevated
Occipital fontanel enlarged
Interparietals curved medially
Supraoccipital : unossified
incompletely ossified
Incus unossified
Palatine bones incompletely ossified
Mandibles shortened
Hyoid bone: unossified
incompletely ossified
split
Sternebrae: ossified normally
unossified
incompletely ossified
split
fused
malaligned
Centri: ossified normally
fused vertically
malaligned
Vertebral: fused vertically
Ribs: vertically or malfixed
extra
wavy
Paws: incompletely ossified
o*/
6 to 15
17
168
0+0 ~'
0+0
0+0
1.0+1.0
0+0
2.4+2.3
4.3+2.6
23.4+7.7
0+0
0+0
7.8+6.1
0+0
0+0
61.4+7.8
7.8+6.1
7.1+3.3
6.4+3.0
1.2+1.2
14.4+4.4
100+0
0+0
0+0
0+0
0+0
22.8+6.5
0+0
1.2+1.2
54
8 to 15
11
55
3.6+3.6
0+0
4.9+3.7
11.4+6.2
4.9+3.7
0+0
21.7+9.0
65.0+8.6-'
0+0
0+0
0+0
1.8+1.8
2.6+2.6
38.1+7.01/
7.3+7.3
23.0+8.3
23. 9+7. Q&
0+0
30.6+5.7-'
86.6+6.3^
4.3+2.2
1.3+1.3
5.8+3.3
8.7+4.5
25.3+8.2
0+0
4.4+4.4
54
10 to 15
4
15
0+0
0+0
0+0
33.3+23.6
0+0
0+0
57.5+21.7^'
95.0+5.0^'
0+0
0+0
0+0
0+0
0+0
40.0+24.5
41.7+25.0
18.3+10.7
37.5+23.9
0+0
0+0
100+0
0+0
0+0
0+0
0+0
45.0+16.6
25.0+25.0
0+0
54b/
12 to 15
6
17
0+0
16.7+16.7
0+0
61.1+20.0
0+0
18.1+12.6
76. 4+12. 3-'
100+0^'
16 . 7+16 . 7
16.7+16.7
8.3+8.3
0+0
0+0
0+0d/
88. 9+7. 2-1
23.6+12.3
25.0+12.0
0+0
8.3+8.3
100+0
0+0
0+0
0+0
0+0
11.1+7.0
0+0
0+0
a/ Control group.
b/ Feed restricted group.
c_/ Mean + S.E. of the percent of fetuses with the indicated anomaly calculated on
a litter basis.
d_/ Significantly different from control (two sample rank test).
29
-------
TABLE 16
EFFECT OF VINYLIDENE CHLORIDE EXPOSURE ON GESTATIONAL DAYS 6 TO 9 ON
MATERNAL WELFARE AND REPRODUCTION IN MICE
Vinylidene Chloride (ppm)
Number Exposed
Pregnant
Alive
Non-pregnant
Alive
a/
Body Weight Change—
During exposure
After exposure
Feed Consumption—
During exposure
After exposure •
Pregnant Survivors
Implants /dam
Viable fetuses (%)
Dead fetuses (%)
Early resorptions (%)
Late resorptions (%)
Dams with complete resorptions
Live Litters
Fetuses/dam
Males (70)
Fetal weight (gm)
oE/
12
10
10
2
2
2.1 + 0.3
15.4 + 0.7
5.4 + 0.2
5.8 + 0.4
10
10.7 + 0.7
91+4
0
3 + 1
5 + 3
0
10
9.6 + 0.5
53 + 3
1.15 + 0.04
56
16
5-
5
11
11
0.9 + 0.8
20.0 + 2.2
1.7+0.5 1-1
5.2 + 0.3 3.7
5
10.4 + 0.9
97+3
0
2+2 1
2+2
0
5
10.0 + 0.8
64 + 6
•1.12 + 0.04
81 112
16 14
I-' 6^
I 0-
15 8
15 7
-4.0
-2.2
+ 0.2
+ 0.6
1 0
12.0
0
0
ooS/
0
1
0 0
-
-
— —
(£'
17
3^
2
14
14
-7.5 + 0.5-7
4.2 + 0 . 8—
0
5.3 + 0.5
2
6.5 + 2.5
0
0
100 + Q%/
0
2
0
-
-
—
a/ Gm/animal/interval for pregnant mice.
b/ Gm/animal/day for pregnant mice.
c_/ Control group.
d/ Feed restricted group.
e/ Significantly different from
f/ Significantly different from
£/ Significantly different from
control ,!(Fisher
control (Tukey1
's exact probability test).
s omega procedure).
control (two sample rank test).
,
-------
TABLE 17
EFFECT OF VINYLIDENE CHLORIDE EXPOSURE ON GESTATIONAL DAYS 9 TO 12 ON
MATERNAL WELFARE AND REPRODUCTION IN MICE
Number Exposed
Pregnant
Alive
Non-pregnant
Alive
3. /
Body Weight Change—
During exposure
After exposure
Feed Consumption—
During exposure
After exposure
Pregnant Survivors
Implants/dam
Viable fetuses (%)
Dead fetuses (70)
Early resorptions (%)
Late resorptions (%)
Dams with complete resorptions
Live Litters
Fetuses /dam
Males (%)
Fetal weight (gm)
0£/
12
10
10
2
2
3.9 + 0.3
11.5 + 0.6
5.2 + 0.4
6.4 + 0.2
10
10.7 + 0.7
91 +4
0
3 + 1
5 + 3
0
10
9.6 + 0.5
53 + 3
1.15 + 0.04
Vinylidene Chloride (ppm)
56 81
16 19
6- 12
5 12
10 7
10 7
-0.4 + 2.2 -4.6 + l.O-/
19.6 + 1.5&' 14.8 + 1.2
1.9 + 0.2 1.5 + 0.2
5.0 + 0.2 4.6 + 0.3
5 12
11.2 + 0.4 11.2 + 0.4
93+3 65 + ~lU
2.0 + 2.0 0
2 + 2 22 + 8^
4 + 4 14 + 3
0 1
5 11
10.4 + 0.6 7.8 + 0.4^
41 + 2 54 + 3
1.16 + 0.07 0.88 + 0.04S/
112 O-/
12 14
7 7
0— 7
5 7
4 7
-8.0 + 0.7|/
19.5 + 1.4fi
0
7.0+2.5
0 7
11.6 + 0.8
78 + 11
0
4 + 3
19+9
0
0 7
- • 9.1 + 1.6
54 + 6
1.01 + 0.04
a/ Gm/animal/interval for pregnant mice.
b_/ Gm/animal/day for pregnant mice.
c_/ Control group.
d_/ Feed restricted group.
e/ Significantly different from
fj Significantly different from
g/ Significantly different from
control (Fisher1
s exact probability test).
control (two sample rank test).
control (Tukey's
omega procedure) .
-------
The effects of a 3-day exposure, which started on gestational
day 12, on maternal welfare and reproduction are presented in Table 18. A
reduced ratio of pregnant to exposed mice occurred in the group treated with
81 ppm of VDC. Deaths occurred in the dams exposed to both 81 and 112 ppm
of VDC. The weight gain during treatment was reduced in the groups exposed
to 81 and 112 ppm VDC and the feed restricted group. The weight gain after
treatment was normal for all of the groups except the group exposed to 112
ppm of VDC. A significant increase in early resorptions occurred in all of
the VDC exposed groups and the feed restricted group. In addition, there
was a significant increase in the ratio of dams with complete resorptions
to pregnant survivors in the 112 ppm VDC exposure group and the feed re-
stricted group. The number of fetuses/dam and the fetal body weight was
reduced only in the feed restricted group.
The effect of a 2-day exposure, which started on gestational
day 15, on maternal welfare and reproduction is presented in Table 19. A
significant reduced ratio of pregnant to exposed mice occurred in the 81 ppm
VDC treated group. There was no significant increase in mortality in any
of the groups. The weight gain during treatment was reduced for all of the
VDC exposed groups and the feed restricted group. After exposure, the weight
gain after treatment was normal for both the 56 and 81 ppm VDC exposed groups.
The percent viable fetuses was reduced for all of the VDC exposed groups and
the feed restricted group. None of the dams in the groups exposed to 112
ppm of VDC produced any viable fetuses. At the time of examination, the
implants in these dams were classified as being a mixture of early and late
resorptions. Increased resorptions also occurred in the feed restricted
group. Fetal body weights were reduced in the 56 and 81 ppm exposed groups
as well as the feed restricted group.
b. Anomalies: Gross anomalies observed in fetuses from dams
exposed to 56 and 81 ppm of VDC are presented in Table 20. Immature skin
and hematoma were the most frequent anomalies observed. The incidence of
these anomalies was increased on the group exposed to 81 ppm of VDC from
gestational days 15 to 17. In addition, this group also had an increased
incidence of runting among the fetuses. Hematomas were also observed in the
feed restricted group.
Soft tissue anomalies observed in fetal mice from dams exposed
to various concentrations of VDC for 2- to 3-day intervals during gestation
are presented in Tables 21 to 24. A significant increase in hydrocephalus
of the lateral ventricles occurred in mice from the groups exposed to 56 and
81 ppm of VDC from gestational days 15 to 17 (Table 24). However, this
anomaly occurred at a similar, although not statistically significant, fre-
quency in the feed restricted group. Small kidneys were more frequent in
mice from the group exposed to 81 ppm of VDC from gestational days 15 to 17.
Although some soft tissue anomalies were observed in all of the groups, none
of these anomalies, other than those described above, occurred at a signifi-
cantly greater frequency in the VDC-treated groups relative to the control
groups.
32
-------
TABLE 18
EFFECT OF VINYLIDENE CHLORIDE EXPOSURE ON GESTATIONAL DAYS 12 TO 15 ON
CO
MATERNAL WELFARE AND REPRODUCTION IN MICE
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 (70)
Early resorptions (%)
Late resorptions (%)
Dams with complete resorptions
Live Litters
Fetuses /dam
Males (%)
Fetal weight (gm)
0£/
12
56
7
10
10
2
2
4.9
6.6
5.6
6.7
+ 0.5
+ 0.6
+ 0.4
+ 0.6
0.4
6.2
1.7
4.5
10
10.7
91
3
5
+ 0.7
± *
0
+ 1
+ 3
0
8.2
71
22
8
10
9.6
53
1.15
+ 0.5
± 3
+ 0.04
6.3
61
1.17
5
5
2
2
+
+
+
+
5
+
+
0
+
+
1
4
+
+
+
Vinylidene
Chloride (ppm)
81
24
20
20
2.0 -6.0
1.3 6.3
0.5 1.7
0.5 7.7
2.3 8.5
19 42
20^ 58
8
2.1
13
0.16
+
+
+
+
2
+
+
0
+
0
1
1
10
50
0
3.01/
2.8
0.1
1.8
3.5
42
.«£/
.83
112
8
6
#/
2
0
-9.0 + 2.0-/
-2.3 + 0.6&/
1.0 + 0.0
1.2 + 0.0
3
6.7 + 2.7
0
0 '
100 + 0^
0 .
3-
0
-
_
-
<£/
15
-6.3
7.7
6.1
10.6
22
55
23
10
10
5
5
+
+
0
+
10
+
+
0
+
+
"~o
0.4^/
1.3
0.6
0.9
gl/
12f/
}°
4-
4.2
51
0.86
6
+
+
+
1.4^/
14
0.06^
a/ Gm/animal/ interval for pregnant mice.
b_/ Gm/animal/day for pregnant mice.
c/ Control group.
df Feed restricted group.
e_/ Significantly different from
f/ Significantly different from
g_/ Significantly different from
control
control
control
(Fisher
1 s exact
probability test).
(two sample rank test).
(Tukey1
s omega
procedure).
-------
OJ
TABLE 19
EFFECT OF VINYLIDENE CHLORIDE EXPOSURE ON GESTATIONAL DAYS 15 TO 17 ON
MATERNAL WELFARE AND REPRODUCTION
IN MICE
, Vinylidene Chloride (ppm)
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 (%)
Late resorptions (%)
Dams with complete resorptions
Liver Litters
Fetuses/dam
Males (%)
Fetal weight (gm)
a/ Gm/animal/interval for pregnant
b/ Gin/animal/day for pregnant mice
£/ Control group.
d/ Feed restricted group.
°-
12
10
10
2
2
6.2 + 0.7
0.4 + 0.7
5.4 + 0.6
5.5 + 2.0
10
10.7 + 0.7
91 + 4
0
3 + 1
5+3
0
10
9.6 + 0.5
53+3
1.15 + 0.04
mice.
.
56
8
7
7
1
1
-5.1 + 0.3-A
0.3 + 0.7
2.2 + 0.4
3.3 + 1.2
7
11.0 + 0.6
69 + 8^/
0
17 + 7
14 + 7
0
7
7.7 + 1.1
55+6
0.66 + 0.03&
81
24
11-'
11
13
13
-5.5 + Q.I-1
0.7 + 0.9
1.8 + 0.2
2.4 + 0.5
11
12.5 + 0.3
47 + 12l/
0
29 + 12
24 + 9
3
8
8.3 + 1.4
54 + 9
0.55 + 0.02^y
112
7
6
4
1
0
-4.0 + 1.1-/
-5.5 + 2.1&I
0.3 + 0.2
1.2 + 0.5
4
12.3 + 0.8
ol/
0
75 + 22-/
25 + 22
4-
0
-
/
f
°-
16
9
9
7
7
-9.3 +
6.6 +
0
5.2 +
9
10.7 +
48 +
0
38 +
14 +
3
6
7.0 +
38 +
0.81 +
\l
<*.£'
0.5&/
2.7
0.7,
14f/
12^/
8
1.1
5
0.03&/
e_/ Significantly different from control (Fisher's exact probability test).
fj Significantly different from control (two sample rank test).
g_/ Significantly different from control (Tukey's omega procedure).
-------
TABLE 20
EFFECT OF VINYLIDENE CHLORIDE (VDC) EXPOSURE ON
THE INCIDENCE OF GROSS ANOMALIES IN MICE
VDC (ppm)
<*/
56
81
b/
OT
Gestational
Days
Exposed
6-17
6-9
9-12
12-15
15-17
9-12
12-15
15-17
9-12
12-15
15-17
Gross
Immature
Skin
3.S+2.4-7
0+0
4.4+4.4
2.3+2.3
0+0
18.7+9.5
0
18. 7+6. 0-
0+0
5.5+5.5
0+0
Anomalies
Hematoma
0.8+0.8
4.0+4.0
5.5+5.5
0+0
1.3+1.3
5.6+2.6
0
9.0+3. 4-^
6.1+4.7
33.7+15.1^
10. 5+4. 3-'
Runt ing
0+0
0+0
0+0
0+0
0+0
0+0
0
71.4+18
, °±°
0+0
0+0
£/ Control group.
Jb/ Feed restricted group.
£/ Mean + S.E. of the percent of fetuses with the indicated anomaly
calculated on a per litter basis.
d/ Significantly different from control (two sample rank test) .
35
-------
TABLE 21
EFFECT OF VINYLIDENE CHLORIDE EXPOSURE ON GESTATIONAL DAYS 6 TO 9
ON THE INCIDENCE OF SOFT TISSUE ANOMALIES IN MICE
Vinylldene Chloride (ppm)
0*' 56
Number of
Litters inspected 10 5
Fetuses inspected 46 24
Soft tissue anomalies
Hydrocephalus: lateral ventricle 0±0-/ 5.0±5.0
fourth ventricle 5.0±3.3 4.0±4.0
Nasopharyngeal canal occluded 1.7±1.7 0±0
Nasal passage occluded 7.3±4.9 25.3±12.7
Deflated lung 2.5±2.5 0±0
Kidney: hydronephrosis 1.7±1.7 0±0
small 0±0 4.0±4.0
cortex solidified 0±0 3.3±3.3
a/ Control group.
b/ Mean ± S.E. of the percent of fetuses with the indicated anomaly
calculated on a per litter basis.
36
-------
TA.BLE 22
EFFECT OF VINYLIDENE CHLORIDE EXPOSURE ON GESTA.TIONAL DAYS 9 TO 12
ON THE INCIDENCE OF SOFT TISSUE ANOMALIES IN MICE
Vinylidene Chloride (ppnp
Q$J 56 81 O
Number of
Litters inspected 10 5 11 7
Fetuses inspected 46 24 40 29
Soft tissue anomalies
Hydrocephalus: third ventricle OiO9- 0±0 3.0±3.0 0±0
fourth ventricle 5.0±3.3 0±0 6.8±4.9 2.9±2.9
Nasopharyngeal canal occluded 1.7±1.7 0±0 0±0 0±0
Nasal passage occluded 7.3±4.9 0±0 10.6±6.4 7.1±4.6
Micropthalmia 0±0 0±0 2.3±2.3 0±0
ECtopic eye 0±0 0±0 2.3±2.3 0±0
Cleft palate 0±0 0±0 9.1±9.1 25.0±14.4
Deflated lung 2.5±2.5 0±0 9.8±4.2 14.3±14.3
Kidney: hydronephrosis 1.7±1.7 4.0±4.0 0±0 0±0
small 0±0 0±0 4.5±4.5 0±0
cortex solidified 0±0 0±0 3.0±3.0 0±0
a/ Control group.
jb/ Feed restricted group.
c/ Mean ± S.E. of the percent of fetuses with the indicated anomaly calculated
on a litter basis.
37
-------
TA.BLE 23
EFFECT OF VINYLIDENE CHLORIDE EXPOSURE ON GESTA.TIONAL DAYS 12 TO 15
ON THE INCIDENCE OF SOFT TISSUE ANOMALIES IN MICE
Vinylidene Chloride (ppm)
Q*l 56 81 0-2'
Number of
Litters inspected 10 416
Fetuses inspected 46 12 5 13
Soft tissue anomalies
Hydrocephalus: lateral ventricle 0±0-£/ 0±0 60 25.0±15.8
fourth ventricle 5.0±3.3 0±0 0 0±0
Nasopharyngeal canal occluded 1.7±1.7 12.5±12.5 0 0±0
Nasal passage occluded 7.3±4.9 0±0 20 12.5±8.5
Cleft palate 0±0 16.3±9.9 20 25.0±14.4
Deflated lung 2.5±2.5 0±0 20 4.2±4.2
Kidney: hydronephrosis 1.7±1.7 0±0 0 8.3±8.3
small 0±0 0±0 0 4.2i4.2
cortex solidified 0±0 0±0 20 4.2±4.2
Distended urinary bladder 0±0 25.0±25.0 0 0±0
a/ Control group.
b/ Feed restricted group.
c/ Mean ± S.E. or mean of the percent of fetuses with the indicated anomaly
calculated on a litter basis.
38
-------
TA.BLE 24
°-'
10
46
56
7
25
81
8
34
°-7
6
20
EFFECT OF VINYLIDENE CHLORIDE EXPOSURE ON GESTATIONAL DAYS 15 TO 17
ON THE INCIDENCE OF SOFT TISSUE ANOMALIES IN MICE
Vinylidene Chloride (ppm)
Number of
Litters inspected
Fetuses inspected
Soft tissue anomalies
Hydrocephalus: lateral ventricle
third ventricle
fourth ventricle
Subarachnoidal space enlarged
Nasopharyngeal canal occluded
Nasal passage occluded
Micropthalmia
Cleft palate
Deflated lung
Ductus venosis hemorrhage
Kidney: hydronephrosis
small
cortex solidified
Distended urinary bladder
Misplaced testes
Misplaced ovary
OiO^/
OiO
5 . 0±3 . 3
OiO
1 . 7±1 . 7
7.3±4.9
0±0
OiO
2.5±2.5
OdbO
1 . 7±1 . 7
0±0
0±0
0±0
OiO
0±0
27.1il3.5-d-/
0±0
OiO
0±0
0±0
4.8±4.8
0±0
0±0
2.9i2.9
7.1i7.1
OiO
10.0±7.2
2.8±2.8
7.1±7.1
0±0
0±0
26.3±11.8^/
11.3±5.9
13 . 3±5 . 3
5.0±5.0
0±0
10.4±8.3
2.5±2.5
20.8±12.5
8.3±6.3
0±0
0±0
20.8i8.2-/
0±0
0±0
2.1±2.1
2.1i2.1
27.8±12.7
15.8±8.2
0±0
0±0
0±0
0±0
OiO
8.3±8.3
4.2±4.2
OiO
OiO
7.5i4.8
OiO
OiO
OiO
OiO
a/ Control group.
b/ Feed restricted group.
cl Mean ± S.E. of the percent of fetuses with the indicated anomaly calculated on
a litter basis.
d/ Significantly different from control group (two sample rank test).
39
-------
Skeletal anomalies observed in fetal mice from dams exposed
to various concentrations of VDC for 2- to 3-day intervals during gestation
are presented in Tables 25 to 28. No increase in skeletal anomalies was
produced in the group of mice exposed to 56 ppm of VDC from gestational
days 6 to 9 (Table 25). In addition, no increase in skeletal anomalies
was observed when mice were exposed to this concentraton of VDC from
gestational days 9 to 12 (Table 26) or 12 to 15 (Table 27). However, if
mice were exposed to 56 ppm of VDC from gestational days 15 to 17 then there
was an increase in unossified supraoccipitals, hyoid bones, and sternabrae
(Table 28). In addition, there was a reduction in the number of normally
ossified sternabrae and a corresponding reduction in the incidence of mal-
aligned sternabrae. If the concentration of VDC was increased to 81 ppm
then anomalies of the sternebrae and centri were observed in the group
exposed from gestational days 9 to 12 (Table 26). However, these anomalies
also occurred at a similar frequency in the feed restricted group. There
were not enough litters in the group exposed to 81 ppm of VDC from gesta-
tional days 12 to 15 to determine if exposure during this period produced
skeletal anomalies. If mice were exposed to 81 ppm of VDC from gestational
days 15 to 17 then there was an increased incidence of both slight and marked
collapsed skull (Table 28). This type of anomaly did not occur in the feed
restricted group. In addition, there were also unossified supraoccipitals,
sternabrae, and paws as well as malaligned sternabrae in the group exposed
to 81 ppm of VDC.
C. Behavioral Studies on Rats
1. General observations; General observations, which summarize
the effects of VDC exposure on maternal welfare and reproduction, are pre-
sented in Table 29. No deaths occurred during exposure. A weight loss
occurred during the initial portion of the exposure period in the VDC
exposed groups as well as the feed restricted group. During the latter
portion of the exposure period, only the high dose VDC group lost weight.
The total weight gain during the exposure period was reduced in both VDC
exposed groups and the feed restricted group. The body weight of day old
pups in the normalized litters was also reduced in these groups. There was
a trend towards a reduced number of viable pups in the high dose VDC group.
In conjunction with this observation, there was a significant reduction in
the number of pups/implants in this group. The number of implants were
determined at weaning when the dam was sacrificed and the uterus examined
for implantation sites.
2. Behavioral observations: Although there were significant
effects due to sex on some of the tests, in no case was there a significant
sex times treatment interaction. Since we are only interested in effects
due to treatments, and these effects were not differentially affected by
sex, the data from both sexes were combined for presentation.
40
-------
TABLE 25
EFFECT OF VINYLIDENE CHLORIDE EXPOSURE ON GESTATIONAL
DAYS 6 TO 9 ON THE INCIDENCE OF SKELETAL ANOMALIES IN MICE
Vinylldene Chloride (ppm)
^
Number of
Litters inspected
Fetuses inspected
Skeletal anomalies
Skull collapsed: slight
Nasal bones: curved, medially
Occipital fontanel enlarged
Supraoccipital: unossified
incompletely ossified
Incus unossified
Sternebrae: ossified normally
unossified
incompletely ossified
split
malaligned
extra
Centri: ossified normally
Paws: unossified
phalanges unossified
10
50,
4.0+2. 7
7.7+6.0
4.0+2.7
2.0+2.0
9.3+5.0
28.0+9.3
44.3+8.2
10.0+8.0
26.3+5.1
9.3+4.0
25.3+5.9
2.0+2.0
100+0
4.0+2.7
13.3+6.4
5
26
5.0+5.0
11.4+11.4
16.0+11.7
0+0
17.0+7.7
26.7+14.6
40.1+16.9
17.0+11.8
28.0+13.6
0+0
18.9+9.0
0+0
100+0
0+0
29.0+10.0
a/ Control group.
b/ Mean + SE of the percent of fetuses with the indicated anomaly calculated on a
per litter basis.
41
-------
TABLE 26
EFFECT OF VINYLIDENE CHLORIDE EXPOSURE ON GESTATIONAL
DAYS 9 TO 12 ON THE INCIDENCE
OF SKELETAL
ANOMALIES
Vinyl idene
Number of
Litters inspected
Fetuses inspected
Skeletal anomalies
Skull collapsed: slight
marked
Nasal bones: curved medially
Occipital fontanel enlarged
Supraoccipital: unossified
incompletely ossified
Incus unossified
Hyoid bone unossified
Sternebrae: ossified normally
unossified
incompletely ossified
lobed
split
maligned
extra
Centri: ossified normally
lobed
incompletely ossified
Paws: unossified
phalanges unossified
OS/
10
50
4.0+2.7-'
0+0
7.7+6.0
4.0+2.7
2.0+2.0
9.3+5.0
28.0+9.3
0+0
44.3+8.2
10.0+8.0
26.3+5.1
0+0
9.3+4.0
25.3+5.9
2.0+2.0
100+0
0+0
0+0
4.0+2.7
13.3+6.4
56
5
28
8.0+4.9
0+0
14.7+9.0
4.0+4.0
0+0
17.3+9.2
13 .3+9.7
0+0
63.3+17.0
4.0+4.0
18.7+12.2
0+0
10.0+6.7
7.3+4.5
3.3+3.3
100+0
0+0
0+0
0+0
8.0+8.0
IN MICE
Chloride (ppm)
81
11
47
7.1+3. 8
6.8+4.9
14.1+6.1
2.3+2.3
2.3+2.3
6.4+4.2
47.3+13.6
6.8+6.8
25. 6+6. ^
22.9+7.1
26.7+8.0
0+0
54. 1+10. l&!
17.1+4.4
0+0
76. 1+7. t&!
16.7+7.9
5.5+2.8
0+0
62.3+11.1
Ob/
7
34
2.9+2.9
0+0
11.3+9.4
0+0
0+0
24.2+8.2
52 . 9+17 . 8
0+0
29.6+13.0
9.9+3.6
27.3+13.6
1.8+1.8
40.8+13.3^
21.1+6.3
0+0
95.2+4.8
4.8+4.8
0+0
0+0
21.1+8.2
a/ Control group.
b/ Feed restricted group.
£/ Mean + SE of the percent of fetuses with the indicated anomaly calculated
on a per litter basis.
d/ Significantly different from control (two sample rank test).
42
-------
TABLE 27
EFFECT OF VINYLIDENE CHLORIDE EXPOSURE ON GESTATIONAL
DAYS 12 TO 15 ON THE INCIDENCE
Number of
Litters inspected
Fetuses inspected
Skeletal anomalies
Skull collapsed: slight
Nasal bones: .curved medially
Occipital fontanel enlarged
Supraoccipital: unossified
incompletely ossified
Incus unossified
Sternebrae: ossified normally
unossified
incompletely ossified
split
malaligned
extra
Centri: ossified normally
Paws; unossified
phalanges unossified
OF SKELETAL ANOMALIES
0^
10
50
4.0+2.7-
7.7+6.0
4.0+2.7
2.0+2.0
9.3+5.0
28.0+9.3
44.3+8.2
10.0+8.0
26.3+5.1
9.3+4.0
25.3+5.9
2.0+2.0
100+0
4.0+2.7
13.3+6.4
Vinylidene
56
3
13
/
1 16.7+16.7
0+0
0+0
11.1+11.1
50.0+9.6
22.2+22.2
30.6+19.4
41.7+30.0
30.6+2.8
33.3+33.3
8.3+8.3
0+0
100+0
5.6+5.6
5.6+5.6
IN MICE
Chloride
81
1
5
80
80
0
0
100
100
0
100
40
100
20
0
100
0
100
(ppm)
Qb/
4
12
25.0+25.0
0+0
0+0
5.0+5.0
56.3+21.3
25.0+25.0
5.0+5.01/
67 . 5+19 . 7^'
27.5+16.0
47. 5+20. 6^'
23.8+10.3
0+0
100+0
0+0
61.3+17.1
a/ Control group.
b/ Feed restricted group.
£/ Mean + SE or individual value of the percent of fetuses with the indicated
anomaly calculated on a per litter basis.
d/ Significantly different from control (two sample rank test).
43
-------
TABLE 28
EFFECT OF VINYLIDENE CHLORIDE EXPOSURE ON GESTATIONAL
DAYS 15 TO 17 ON THE INCIDENCE
OF SKELETAL
ANOMALIES IN
MICE
Vinylidene Chloride (ppm)
Number of
Litters inspected
Fetuses inspected
Skeletal anomalies
Skull collapsed: slight
marked
Nasal bones: incompletely ossified
curved, medially
Occipital fontanel enlarged
Supraoccipital: unossified
incompletely ossified
Incus unossified
Hyoid bone unossified
Sternebrae: ossified normally
unossified
incompletely ossified
split
malaligned
extra
Centri: ossified normally
unossified
Pelvis: incompletely ossified
Paws: unossified
incompletely ossified
phalanges unossified
Oa/
10
50
4. 0+2. 7^
0+0
0+0
7.7+6.0
4.0+2.7
2.0+2.0
9.3+5.0
28.0+9.3
0+0
44.3+8.2
10.0+8.0
26.3+5.1
9.3+4.0
25.3+5.9
2.0+2.0
100+0
0+0
0+0
4.0+2.7
0+0
13.3+6.4
56
7
29
12.4+9.5
0+0
14.3+9.2
29.3+11.3
7.1+7.1
39.3+17.1-
56.0+17.7
90.0+7.2
7 5. 7+14. &
91. 9+5. 8^/
38.1+14.4
30.7+13.2
5.7+5.7^
0+0
100+0
0+0
0+0
14.3+9.2
0+0
92.9+7.1
81
7
32
40. 0+12. 6i/
21.9+10.1^
31.0+18.0
0+0
25.2+13.6
52.9+14.6^'
24.8+9.4
64.3+18.0
40.5+19.2
O+O^/
100+0^
16.7+14.1
4.8+4.8
O+O^/
0+0
97.1+2
2.9+2.9
2.9+2.9
50.0+11.7-'
11.9+7.9
45.2+13.6
0-2-
6
22
0+0
0+0
25.0+17.1
9.7+6.2
29.2+16.4
33.3+21.1
30.6+10.0
80.6+16.3
6.9+4.5
o+ol/
93. 1+4. 3d-/
47.2+12.7
58.3+14.0^
6. 9+4. Si/
0+0
100+0
0+0
0+0
0+0
25.0+17.1
45.8+15.0
a/ Control group.
Jb/ Feed restricted group.
£/ Mean + SE of the percent of fetuses with the indicated anomaly calculated on a per
litter basis.
d/ Significantly different from control (two sample rank test).
44
-------
TABLE 29
EFFECT OF VINYLIDENE CHLORIDE EXPOSURE ON GESTATIONAL
DAYS 8 TO 20 ON MATERNAL WELFARE AND REPRODUCTION IN RATS
Vinylidene Chloride (ppm)
Number Exposed
Pregnant
Alive
Non-pregnant
Alive
o-'
24
17
17
7
7
56
20
18
18
2
2
283
19
16
16
3
3
0£/
17
17
17
0
0
Body Weight Change3-7
Days 8 to 13
Days 13 to 20
Days 8 to 20
Pregnant Survivors
Implants/dam
Dams with complete resorptions
8+8
68+9 53+9
76+12 -7+8^
-54+2^
54+2
-45+10^ -0.6+4^
17 18 16 17
13.3+0.4 12.4±0.8 11.8+1.0 12.8+0.7
00 30
Live Litters
Pups/dam
Actual
Normalized
Pup weight (gm)
Pups/implants x 100
17
18.
13
17
12.4+0.5 11.7+0.8 9.5+1.1 11.6+0.6
8 8 88
7.0+0.2 5.7+0.21/ 5.5+0.2-' 6.3+0.2^
93+2 94+2 78+6^-' 92+2
aj Cm/animal/interval for pregnant rats.
b_/ Control group.
c_/ Feed restricted group.
d_/ Significantly different from control (Tukey's omega procedure).
£/ Significantly different from control (two sample rank test).
45
-------
a. Preweanlng tests:
(1) Surface righting: All of the groups were signifi-
cantly different from the control group with respect to the age at which
the pups were able to right themselves in 1 sec or less (Figure 2). The
feed restricted group consistently mastered the task sooner than the con-
trols did, and the two treatment groups were slower. However, the low and
high dose groups could not be distinguished from each other.
(2) Pivoting: Although the feed restricted group spent
the least time pivoting and the high dose group and low dose group, respect-
ively, spent the most time pivoting, these differences did not reach sta-
tistical significance (Figure 3).
(3) Auditory startle: There were no significant effects
due to treatment. The females did show the startle response sooner than
males, but this was true across all treatments (Figure 4).
(4) Bar holding: Although the high dose group did reach
the criterion of hanging on the bar for 15 sec sooner than the other groups
the difference did not reach statistical significance (Figure 5). However,
when the data were analyzed for each group as the total time spent hanging
on the bar, then the high dose group did significantly better (p < 0.001).
In other words, the high dose group did hang on the bar longer than the
other three groups, but they did not reach the 15 sec criterion signifi-
cantly sooner.
(5) Righting in air; In this test, the feed restricted
and low dose groups took significantly longer to show the response, while
the high dose group was not distinguishable from the control group (Figure 6).
(6) Visual placing; This test proved to be a difficult
one to perform. The rat had to approach the ledge very close to elicit a
response and then it was difficult to be sure that vibrissal contact had not
occurred. It was also nearly impossible to standardize this test. Because
of these problems the data were not analyzed.
(7) Swimming ability; There were no significant effects
due to sex or treatment in the swimming test (Figure 7). The feed restricted
and high dose groups did reach the score of 2 a little sooner than the other
two groups, but the difference was only marginally significant (p < 0.06).
(8) Physical maturation: The high dose group showed de-
tachment of the external ear earlier and eruption of the teeth significantly
later than the controls (p < 0.05 and 0.01, respectively) (Figure 8). For
the teeth, the feed restricted and low dose groups also showed later matura-
tion relative to the controls. There was a significant sex effect for time
of eye opening, with the females maturing earlier. However, this was true
across treatment groups.
46
-------
• Control (N = 34)
O Feed Restricted (N =34)
* Low Dose (N = 34)
DHigh Dose (N =26)
100
Z 80
O
O
Z
x
u
LLJ
u
60
40
20
0
SURFACE RIGHTING
I
I
I
I
I
j
0
45678
AGE IN DAYS
10 11 12
Figure 2 - Cumulative Percent of Rats, Sexes Combined, Reaching
a Criterion of 1 Sec or Less for Righting on a Flat
Surface. All groups were significantly different
from controls at the 0.01 level.
47
-------
30
27
24
,/> 21
Q
Z Iff
o 18
u
LU
00 15
Z
^ 12
P
9
6
3
0
PIVOTING
D--
—•Control (N =34)
—o Feed Restricted (N = 34)
—A Low Dose (N =34)
—Q High Dose (N =26)
6 8
AGE IN DAYS
10
12
14
Figure 3 - Amount of Time Spent Pivoting During a 3-Min Test
Period, Sexes Combined.
48
-------
10
to
X
a 8
0)
a
0
O-
$• 6
"c
in
« 4
O)
2
0
—
—
.
*^B
-
Control Feed Low High
Restricted Dose Dose
N = 34 N=34 N=34 N = 26
Figure 4 - First a Complete Startle Response Was Observed for
Various Groups of Rats, Sexes Combined.
49
-------
100 r-
BAR HOLDING
•Control (N =34)
OFeed Restricted (N =34)
A Low Dose (N =34)
^ High Dose (N =26)
13 14 15 16 17
AGE IN DAYS
18 19 20 21
Figure 5 - Cumulative percent of Rats, Sexes Combined, Reaching
a Criterion of 15 Sec on the Bar-Holding Apparatus.
These differences are not significant, but the high
dose group did spend significantly (p < 0.001) more
time hanging on the bar (see text).
50
-------
14
12
10
X
0
Q
-o 8
V
.SJ
_E
O
U-
0)
ro
4
2
0
Righting
i^
—
—
In Air
*
*
Control Feed Low High
Restricted Dose Dose
N = 34 N=34 N = 34 N = 26
Figure 6 - First Day of Appearance for Righting in Air for Groups
of Rats, Sexes Combined. * = statistically signifi-
cant from the control group at the 0.05 level.
51
-------
14
Swimming Ability
12
10
C = Control (N = 34)
FR= Feed Restricted (N = 34)
L=Low Dose (N = 34)
H=High Dose (N = 26)
0)
>
0>
IE
u
o
0>
o
u
1/1
0)
CD
C
FR L
Score 2
H
FR L H
Score 3
FR L H
Score 4
Figure 7 - First Day for Score of 2, 3, or 4 ia the Swimming Test,
Sexes Combined. The difference for pair-fed and
high dose in reaching the score 2 was marginally
significant (p < 0.06).
52
-------
14
12
10
Physical Maturation
C-Control (N = 34)
FR=Feed Restricted (N = 34)
L=Low Dose (N = 34)
H=High Dose (N = 26)
X
o
-o
I
0)
to
_Q
O
C
0)
0)
O)
C FR L H
Pinnae Unfolded
C FR L H
Incisor Eruption
C FR L H
Eyes Opened
Figure 8 - First Day of Appearance of Physical Parameters for
Groups of Rats, Sexes Combined. * = statistically
significant from the control group at the 0.05
(pinnae) and 0.01 (incisor) levels.
53
-------
There were highly significant differences in the body
weights (Figure 9). On the average, the feed restricted group was lower
than the control group, with the low dose and high dose groups lower still.
These differences decreased with time and disappeared by 3 weeks of age
(average weight: control, 52 g; feed restricted, 53 g; low dose, 52 g;
high dose, 52 g).
b. Activity test; The only significance found in the
activity data was an overall higher level of activity for the female rats
(Figure 10). There was no interaction of sex with treatment, however. As
expected for a nocturnal animal, the rats were more active during the dark.
Even though the rats were placed into the mazes during the
light phase of their cycle, the first few hours produced the greatest counts
of all. This is generally called exploratory behavior. We looked at ex-
ploratory behavior separately in the analysis by considering the first 2 hr
apart from the general analysis. Here again, however, there were no signifi-
cant differences attributable to the treatments.
IV. DISCUSSION
Pregnant mice and rats were exposed for approximately 23 hr a day
to various concentrations of vinylidene chloride. The exposure periods in-
cluded both organogenesis and portions of organogenesis. The purpose of
this study was to determine if VDC altered normal development. Development
was monitored in terms of the morphological and behavioral parameters of
offspring from both control and VDC exposed animals. In addition, aspects
of maternal welfare were also monitored in order to evaluate the toxicity
of VDC in the dam.
A. Morphological Study
1. Maternal welfare: VDC was more toxic to adult mice than
adult rats. No pregnant mice survived in atmospheres that contained either
144 or 300 ppm of VDC (Table 2). In contrast, more than half of the preg-
nant rats survived exposure to 300 and 449 ppm of VDC (Table 3). Maternal
welfare, as measured in terms of weight change and feed consumption, was
affected by exposure to VDC. The weight gain of rats was reduced during a
10 day exposure to 15 ppm of VDC (Table 3). At the end of exposure, these
dams gained more weight than controls. Similar effects on weight gain were
seen in rats at 57 and 300 ppm of VDC. However, at 449 ppm of VDC, this
effect on weight gain was not reversible. Feed consumption was reduced in
rats at VDC concentrations of 57 ppm and above. Although feed consumption
and weight gain were normal in mice exposed to 15 ppm of VDC, these parameters
were reduced at higher concentrations.
54
-------
60 i-
50
< 40
O
Z
x 30
O
8 20
CO
10
• Control (N = 136)
o Feed Restricted (N = 136)
A Low Dose (N = 136)
D High Dose (N = 104)
1
1
1
1
1
1
0
8 10 12 14
AGE IN DAYS
16 18 20 22
Figure 9 - Average Body Weight of All Rat Pups. All groups were
significantly different from the control group (p <
0.001).
55
-------
Ui
2200 r
2000 -
ACTIVITY
UJ
O
O
O
u
1000 -
800 -
600
400 -
200 -
Control (N = 13 groups of 3)
o O Feed Restricted (N = 13 groups of 3)
a A Low Dose (N = 13 groups of 3)
D o High Dose (N = 13 groups of 3)
32 36
TIME IN HOURS
Figure 10 - Continuous Activity of Groups of Three Rats (same sex) in a Residental Maze, Both Sexes
Comb ined.
-------
Reduced feed consumption may be due either to toxicity of VDC in
the adult or palatability problems associated with VDC contamination of the
feed. Since the feed was changed daily in order to minimize possible con-
tamination the former explanation is more likely. In contrast to these
observations, virgin male and female mice survived a 6 hr/day, 5 day/week
exposure to 55 ppm VDC without adverse effects on survival or weight gain
(manuscript in preparation). These animals were given feed in the evening
after the exposure period. These observations indicate that (1) the con-
tinuous exposure to low concentrations of VDC (15-41 ppm) is associated with
adverse effects on maternal welfare which are generally reversible, (2) high
concentrations of VDC produce both irreversible effects on maternal welfare
and death, (3) mice are more sensitive than rats to the acute lethal effects
of VDC, and (4) VDC is more toxic in mice during a continuous, rather than
intermittent, exposure.
2. Reproduction and anomalies; Mice and rats were exposed to
various concentrations of VDC from gestational days 6 to 16 in Part 1 of
this study. This exposure was associated with a high incidence of early
resorptions and an increased number of dams with complete resorptions in
both mice and rats. These effects occurred in mice at 30 and 57 ppm of VDC
(Table 2) and in rats at 57 and 449 ppm of VDC (Table 3). The surviving rat
fetuses had a reduced body weight at VDC concentrations of 57 ppm and above.
In addition, fetuses from dams exposed to 15 ppm of VDC and above had an in-
creased incidence of hydrocephalus of the lateral ventricles (Table 5) and
sternabrae anomalies (Table 7). No fetal mice were available for examina-
tion at VDC concentrations of 30 ppm and above, as a result of both maternal
death and the high rate of resorptions. In this phase of the study, develop-
ment in both rats and mice was altered at VDC concentrations that affected
maternal welfare.
Since it is not possible to adequately evaluate the teratogenic
potential of a compound with doses that produce a high incidence of resorp-
tions, additional studies were designed. In these studies, VDC was admini-
stered for various phases of development. Such an exposure is valuable both
in determining the sensitivity of the embryo to the compound at various
stages of development and reducing the toxic effects of the compound. Mice
were selected for these additional teratology studies because adult mice
were more sensitive to the toxic effects of VDC than adult rats.
In Part 2, pregnant mice were exposed to 0, 41, 54 and 74 ppm of
VDC for intervals that started at various times during gestation and ended
on gestational day 15. This treatment was associated with a reduced weight
gain during exposure at VDC concentrations of 41 ppm and above (Table 8).
As a result of starting exposure on gestational days 8, 10 or 12 rather than
6, it was possible to reduce the incidence of resorptions and increase the
number of viable fetuses (Table 9). However, exposures that started on ges-
tational days 10 and 12 were still associated with a high incidence of re-
sorptions. Consequently, exposures that started on gestational day 8 were
57
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associated with fewer effects on development than exposures that started
later even though all exposures ended at the same time. This observation
suggests that (1) mice may adapt to a VDC environment after several days
and (2) adverse effects on development, which were associated with VDC
exposure, occurred later in development. According to this hypothesis,
mice exposed to VDC from gestational days 8 to 15 adapted to the VDC en-
vironment during the first few days of exposure and, consequently, the
embryos survived the critical period without any adverse effects. This
hypothesis does not imply that VDC acts directly on the embryo to affect
development. For example, the adaptation to VDC could be in terms of mater-
nal physiology and the critical period could be that phase of development
that is affected by such a change in maternal homostasis. VDC exposure was
also associated with an increased incidence of both soft tissue and skeletal
anomalies (Tables 12-15). However, this effect occurred at concentrations
that adversely affected maternal welfare.
In Part 3, pregnant mice were exposed to 0, 56, 81 and 112 ppm of
VDC for 2- to 3-day intervals during gestation. The feed restricted group,
in this part, was starved during the exposure period in an effort to dis-
sociate VDC related effects from those produced by malnourishment. All VDC
concentrations were associated with a reduced weight gain of dams during at
least one of the exposure periods (Tables 16-19). Starvation from gestational
days 6 to 9 reduced the ratio of pregnant to plug-positive mice and increased
the incidence of early resorptions (Table 16). Similar effects were seen in
all the VDC exposed groups during this interval; however, the litters in the
group exposed to 56 ppm of VDC appeared to be normal. Adverse effects on
development were generally greater with VDC concentrations of 81 and 112 ppm
than 56 ppm. The most severe pattern of anomalies seemed to occur when dams
were exposed on gestational days 15 to 17. Hydrocephalus of the lateral
ventricle and small kidneys occurred following VDC exposure (Table 24).
However, since hydrocephalus occurred at a similar, although not statisti-
cally significant, rate in the feed restricted group, it is difficult to
attribute this anomaly directly to VDC. There was, in addition, a diverse
pattern of skeletal anomalies which occurred in both VDC exposed groups as
well as the feed restricted group (Table 28). Ossification problems with
the incus occurred more frequently in the VDC exposed groups; however,
there was no dose related increase in this anomaly.
3. Summary: The morphological observations indicate that develop-
ment in rats and mice is altered by exposure to VDC. However, these effects
on development occur at VDC concentrations that adversely affect maternal
welfare, as measured by weight gain, feed consumption, and survival. Since
VDC interfered with development at doses that produced toxicity in the adult
VDC was judged to have little primary effect on development.
58
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B. Behavioral Study
1. Discussion: In Part 4, the behavioral development of rats,
which were exposed in utero to VDC, was monitored. The results of this study
tend to support the conclusion that the VDC treatment during pregnancy had
no drastic effects on the 'subsequent behavior of the offspring. This con-
clusion then leads to the conclusion that VDC exposure of the dam during
the period of gestation when brain development occurs, does not lead to
neural impairment in the offspring.
On most of the behavioral tests there was no effect detected due
to treatment. The high dose animals appeared to mature faster with respect
to their ability to hang onto the horizontal bar, and the time at which the
pinnae became detached. Also, along with the feed restricted group, the
high dose group reached the first maturational level (Score 2) somewhat
sooner in the swimming test. Since there were no differences at the other
maturation levels, this effect on swimming is probably spurious.
Other significant differences were a slower maturation of the
low and high dose groups for surface righting, the feed restricted and low
dose groups for righting in air, and all groups showed later eruption of
the incisors relative to the controls. One of the strongest effects was
that on body weight. The feed restricted group was lighter than the con-
trols, and both the low and high dose groups were lighter than the feed
restricted group. These differences were gone by day 21.
There were no differences detected which were due to treatments
in pivoting, auditory startle, eye opening, or adult activity levels. Of
the significant differences discussed above, the slower maturation of the
feed restricted and low dose groups in the righting in air test, and the
marginal earlier maturation of the first level of swimming ability of the
feed restricted and high dose group do not appear consistent with other
data. They probably bear no practical significance.
In summary, the results indicate that the high dose rats matured
at a faster rate with respect to bar holding ability and detachment of
the pinnae. Both the feed restricted and treatment groups had depressed
body weights, which may be related to the delayed eruption of the incisors
observed in these groups. However, with respect to righting ability on a
surface, the feed restricted group was successful the earliest and the low
and high dose groups were significantly later.
2. Summary: No drastic problems with neural development were
indicated. The VDC treatment may have altered the maturational rate of
normal behavioral reflexes and patterns, with some maturing earlier, some
later, and some not affected at all.
59
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C. General Summary
The results of this study indicate that (1) VDC is more toxic in
adult mice than adult rats, (2) adverse effects on maternal welfare, as
measured 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 VDC these effects were observed at concentra-
tions that also affected maternal welfare, (4) no problems with neural
development, as measured by behavioral parameters, were observed in rats
exposed to VDC, and (5) VDC was judged to be only a weak teratogen with
little primary effect on development.
60
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"Results of a Vinyl Chloride Teratology Study in Mice, Rats, and Rabbits,"
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TECHNICAL REPORT DATA
(I'lcasc read Instructions on tlif reverse before completing)
1. REPOi'tV NO. 2.
EPA-560/6-76-018
3. RECIF
4. TITLE AND SUBTITLE 5. REPO
The Developmental Toxicity of Vinylidene Chloride Febi
Inhaled by Rats and Mice During Gestation
6. PERF
7. AUTHOR(S) 8. PERF
R.D. Short, J.L. Minor, P. Peters, J.M. Winston,
B. Ferguson, T. Unger, M. Sawyer, and C.C. Lee
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Midwest Research Institute
425 Volker Boulevard
Kansas City, Missouri 64110
12. SPONSORING AGENCY NAME AND ADDRESS
Office of Toxic Substances
U.S. Environmental Protection Agency
Washington, D.C. 20460
15. SUPPLEMENTARY NOTES
This task was initiated to investigate the
birth defects by the inhalation route.
10. PRO
11. CON
68-C
13. TYP
Fin*
14. SPO
MENT'S ACCESSION-NO.
RT DATE
ruary 1976
ORMING ORGANIZATION CODE
ORMING ORGANIZATION REPORT NO.
GRAM ELEMENT NO.
TRACT/GRANT NO.
)l-3242, Task No. 2
E OF REPORT AND PERIOD COVERED
il
NSORING AGENCY CODE
ability of Vinylidene Chloride to produce
16. ABSTRACT
The purpose of this study was to determine the teratogenic potential of vinyli-
dene chloride inhaled by rats and mice during organogenesis . This report describes
the effects of vinylidene chloride (inhaled at various concentrations for different
intervals during gestation) on development. Morphological and behavioral parameters
were used to monitor development. Vinylidene chloride was judged to be a weak
teratogen with little primary effect on reproduction.
17. KEY WORDS AND DOCUMENT ANALYSIS
.1. DESCRIPTORS
Vinylidene Chloride
iy. DISTRIBUTION STATEMENT
Document available to public through
National Technical Information Service
Springfield, Virginia 22151
b.lDENTIFIERS/OPEN ENDE
19. SECURITY CLASS (This i
Unclassified
20. SECURITY CLASS (This ;
Unclassified
ID TERMS C. COSATI l-'icld/Croup
Report) 21. NO. OF PAGES
69
•>age) 22. PRICE
EPA Form 2220-1 (9-73)
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