United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
v-xEPA
Research and Development
EPA-600/S1-81-034 June 1981
Project Summary
Skeletal Variants as an
Indicator of Biological
Effects of Environmental
Contaminants
Sidney L Beck
The project consisted of an attempt
at developing a Skeletal Variant Assay
System (SVAS) for mice whereby it
might be possible to detect subtle bio-
logical effects of in utero exposure to
noxious substances even in the
absence of gross malformations, by an
examination of a series of variations in
development in adult skeletons.
The compounds tested included
several pesticides and a number of
known teratogens. Animals were CD-1
mice. Doses were chosen to include
potentially teratogenic and no effect
levels; they were administered by the
most appropriate route and at appro-
priate times; choices were culled from
published protocols wherever these
were available. Toxicity tests were
performed when necessary. Vehicle
controls and untreated controls were
included in each series.
Eighty-eight variants were selected
for study. They were all qualitative in
nature to increase ease of typing,
objectivity, and reliability. Most were
polymorphic in CD-1 mice and thus
what was measured was change in
frequency of occurrence in response
to treatment.
In many cases a prenatal study was
also performed in order to provide
comparability with traditional tera-
togen studies. A series of fetal skeletal
parameters was included in the
prenatal studies.
A series of adult skeletons of four-
way outcross mice which had been
selected for susceptibility and resist-
ance to the teratogen trypan blue were
available and were also examined for
the SVAS variants as a separate con-
firming study.
This Project Summary was develop-
ed by EPA's Health Effects Research
Laboratory. Research Triangle Park.
NC. to announce key findings of the
research project that is fully docu-
mented in a separate report of the
same title (see Project Report ordering
information at back).
Introduction
The experimental design was very
similar for all compounds. Mice were
purchased as needed and maintained
for, usually, three to six weeks before
the start of the experiments, in shoebox-
type cages, five animals of one sex per
cage, at 72 ± 2°F, relative humidity at
50 ± 5%, on light:dark regime of
18hr:6hr, with Purina Mouse Chowand
tap water available ad libitum Groups of
five females were mated with a single
male at 4:00 p.m. and checked for
vaginal plugs the following morning
Mated females were isolated to experi-
mental pens, with five females per pen,
each from a different mating, receiving
the same treatment. Each male contri-
buted to different experimental groups
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The order of the experimental pens was
randomized on the shelves.
Females were isolated after their last
treatment and bore and raised their
litters in isolation. Litters were counted
and weighed within one day of birth and
examined for gross abnormalities. They
were again examined, sexed, and
weighed -at weaning and allowed to
remain together until sacrifice at 62 ±2
days of age. Litters were killed, identi-
fied with unique numbers, processed
for alizarin staining of articulated
skeletons, examined for a battery of 44
variants, reprocessed for study of an
additional battery of 44 variants in
papain-digested, disarticulated
skeletons, and stored.
Frequencies of each variant m each
experimental group were calculated
and each experimental was compared
with Untreated and Vehicle Control
values for each variant. Of interest were
the number of variants which differed in
the experimental groups, the magnitude
and direction of the differences, and the
specific variants which were affected.
Statistical analysis initially involved
both Mann-Whitney U tests on mean
litter percent derived frequencies with
significance being defined as P-C05 and
t-tests on arcsin square-root trans-
formed frequencies with significance
being defined at P<.01 to compensate
forType I errors It was subsequently felt
that the Mann-Whitney test discarded
potentially useful data and the t-test
became the standard test of signifi-
cance for the preliminary analyses.
In the prenatal studies fetuses were
dissected on the 1 8th day of gestation,
examined for gross malformations,
weighed, and preserved in alcohol or
formalin for alizarin staining Fetal
skeletons were studied for ossification
of cervical centra, caudal vertebrae,
number of metatarsals and metacar-
pals, number of phalangeal rows, and
number of ribs and sternebrae.
All of the variants which occurred in
these experiments were photographed
as were additional representative
specimens of interest, thus providing a
permanent record. Some specimens
were photographed through a dissect-
ing microscope. A photographic atlas of
variants is presented in the project
report.
Conclusions
Skeletons of CD-1 mice were exam-
ined in a systematic way at 62 + 2 days
postnatal for a series of 88 normally
occurring variations. Subjects had been
exposed in utero, by treatment of their
dams, to one of a group of pesticides—
Trifluralin (TFL), 2,4,5-T (245-T),
Captan, Maneb, Dicamethrin (DMT); or
known teratogens—Thahdomide,
trypan blue (TB), acetazolamide(ACZM),
bromodeoxyundine (BUDR), diphenyl-
hydantoin (DPH), cortisone (CORT), at
both high and no-effect levels. Frequen-
cies of occurrence of each variant in
each treatment group were contrasted
with vehicle (VEH) control and
untreated (UNTD) values. Sixty-eight of
the skeletal variants were useful in
these studies; the others occurred
rarely or not at all.
Prenatal exposure to 2,4,5-T, TFL,
DMT, TB, ACZM, and BUDR could be de-
tected by the existence of substantial
numbers of skeletal variants the fre-
quencies of which were significantly
different from VEH and UNTD controls,
at the higher dose levels. Prenatal
exposure to Captan, Thahdomide, corti-
sone, diphenylhydantoin, and probably
Maneb, could not be detected by this
criterion. This suggests that the SVAS is
a sensitive, yet not indiscriminate,
method for detecting prenatal exposure
to a variety of noxious substances, even
in the absence of gross malformations.
Considering the particular variants
which were affected, it would appear
that a smaller subgroup could serve as a
reasonable screen for detecting subtle
biological effects, by virtue of their
having responded in a large number of
the experimental series. These include
the presence of an interfrontal bone, the
existence of parted f rentals and preoptic
sutures in the skull, imperfect trans-
verse foramina (F.T.I.) of the atlas,
reduction of articular processes of the
thoracic vertebrae, occurrence of 27
rather than the usual 26 presacral
vertebrae, sacral fusions in the
vertebral column, possible carpal
fusions in the appendicular skeleton,
and the occurrence of 14 ribs.
Some variants were only affected by
teratogenic or high dose levels. These
included malformations of the ribs, and
fusions in the lumbar vertebrae and in
the transverse processes of the sacral
vertebrae. Some of these might be con-
sidered cryptic malformations. In most
of the series there were variants which
were enormously affected only by one
or two compounds. These included
Interfrontal and Carpal Fusions in TB
and BUDR experiments, F.T.I, of the
atlas by TB, Accessory Parietal and 27
presacral vertebrae (on day 8 only) by
ACZM, fusions of dorsal processes of
sacral vertebrae by 2,4,5-T and ACZM,
fusions of the vertebral body in sacral
vertebrae by ACZM (especially in the
days 9-11 treatment) and by TB, the
occurrence of fewer than 30 caudal
vertebrae in the 245-T, TFL and perhaps
ACZM treated groups, and caudal
fusions by ACZM and TB. Most of the
treatment effects on the important vari-
ants in all studies resulted in increases
in frequencies of occurrence.
In many of the experiments there
were also variants which were uniquely
affected. These included: Fused
Frontals, Frontal Foramen Double and a
really major increase in absence of
prominent dorsal spine of the second
thoracic vertebra in 245-T, doubling of
the posterior foramen minus in the
palatal bone by TFL, F.T.I of C3-C6 by
DMT, dyssymphysis of the atlas, malfor-
mations and dyssymphyses of the
cervical vertebrae by TB (also in Captan
treated animals). Accessory Parietal
and Abnormal Metoptic Roots by ACZM
on day 8, lumbar fusions and malforma-
tions in both ACZM regimes and in
BUDR treatments on day 9.
Replication of experiments with TB,
ACZM, and BUDR resulted in good to
excellent agreement between the two
series. Considerations of spectrum and
magnitude of response as well as idio-
syncratic or specific responses add
strength to the ability of the SVAS to
detect prenatal exposure to 245-T, TFL,
DMT, TB, ACZM, and BUDR.
Prenatal studies with most of the
compounds were in essential agree-
ment with published results; DPH was
neither teratogenic nor fetotoxic in
these studies and was also negative in
the SVAS; cortisone, although it had no
effect on the skeletons of survivors, was
a potent cleft palate producer. From the
prenatal studies there was also the sug-
gestion that ossification of the centra of
the cervical vertebrae and of caudal
vertebrae might be indicative of expo-
sure to high doses of all or nearly all of
the compounds.
Finally, examination of available
skeletons from adult mice in several
generations of a four-way outcross
mouse population selected for suscep-
tibility or resistance to trypan blue tera-
togenesis as well as an unselected line
revealed better agreement across gen-
erations for each line that was seen in
different batches of CD-1 mice. The
variability of different "batches" of CD-1
mice require an examination of un-
treated animals in each group of
animals purchased.
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Sidney L Beck is with the Biology Department, Wheaton College, Norton, MA
02766.
Neil Chernoff is the EPA Project Officer (see below).
The complete report, entitled "Skeletal Variants as an Indicator of Biological
Effects on Environmental Contaminants." (Order No. PB 81-186 025; Cost:
$17.00, subject to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
1 US GOVERNMENT PRINTING OFFICE 1981 -757-012/7126
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