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OCR for page 11
2
Developmental Effects of
Chemical Contaminants
The discussion in this chapter is limited to embryo (fetal) death, growth
retardation, and malformations the only end points measured in the Food
and Drug Administration's (FDA) guidelines for Segment II develop-
mental toxicity studies of drugs (Collins, 1978) and, therefore, the only
end points for which there are sufficient data bases for analysis. Postnatal
functional impairment is not covered, despite its relevance, since there is
no well-established data base with which to make cross-species compar-
isons. The much broader spectrum of end points included under the head-
ing of reproductive toxicology, e.g., germ cell toxicity, infertility, and
dysfunction of the adult reproductive system, are covered in Chapter 3.
Embryo lethality is defined and reported in the literature as the ratio of
resorptions or dead fetuses in a litter at term to the number of implantation
sites. Growth retardation is measured by weighing and taking crown-to-
rump measurements of live fetuses at term. The frequency and type of
structural anomalies are determined by gross inspection of the fetuses and
by detailed skeletal and soft tissue analysis. The occurrence of embryo
death precludes measurements of growth retardation or identification of
malformations, because these two end points are noted only on live fetuses.
Developmental toxicity includes any detrimental effect produced by
exposures during embryonic stages of development. Such lesions can be
either irreversible or reversible. Embryolethal lesions result in resorption,
spontaneous abortion, or stillbirth. Persistent lesions that cause overall
growth retardation or delayed growth of specific organ systems are gen-
erally referred to as embryotoxic. For a chemical to be labeled a teratogen,
it must significantly increase the occurrence of irreversible structural or
11
OCR for page 12
|2 DRINKING WATER AND HEALTH
functional abnormalities in live offspring after it is administered to either
parent before conception, to the female during pregnancy, or directly to
the developing organism.
Many teratologists believe that any chemical administered in appropriate
dosages at certain developmental stages can cause some disturbances in
embryonic development in some laboratory species (Fabro et al., 1982;
Karnofsky, 1965; Staples, 19751. For an agent to be classified as a de-
velopmental toxicant, it must produce adverse effects on the conceptus at
exposure levels that do not induce severe toxicity in the mother (e.g.,
substantial reduction in maternal weight gain, persistent emesis, hypo- or
hyperactivity, or convulsions), so that the effects are not secondary to the
stress on the maternal system. The main reason for conducting develop-
mental toxicity studies is to ascertain whether an agent causes specific or
unique toxic effects on the conceptus. If these studies are conducted under
extreme conditions of maternal toxicity, then identification of exposures
uniquely toxic to the conceptus or pregnant animal is not possible. (This
is discussed in more detail later in this chapter.) In some cases, however,
chemical agents are deliberately administered at maternally toxic doses to
determine the threshold level for adverse effects on the offspring. As a
result, conclusions can be qualified to indicate that adverse effects on the
conceptus were obtained at maternally toxic exposure levels and may not
be indicative of selective or unique developmental toxicity.
INFLUENCE OF TIME OF EXPOSURE
Compared to adults, developing organisms undergo rapid and complex
changes within a relatively short period. Consequently, the susceptibility
of the conceptus to chemical insult varies dramatically within each of the
major developmental stages, i.e., the preimplantation, embryonic, fetal,
and neonatal stages. As shown in Table 2-1, the time between ovulation
and preimplantation development is similar among several mammalian
species, regardless of gestation length (Brinster, 1975~. Alterations in the
hormonal milieu as well as direct secretion of chemicals into uterine fluids
during this period can interfere with implantation and result in embryo
death. The preimplantation embryo appears to be more susceptible to death
than to teratogenicity following chemical insult. In studies with preim-
plantation embryo cultures, severe toxicity was manifested by rapid death
of the embryo, and the less severe effects included decreased cleavage
rates and arrested development (Brinster, 1975~. There have been few
studies on the effects of sublethal exposures to preimplantation embryos,
and the possibilities of persistent biochemical or morphological alterations
have not been adequately explored.
OCR for page 13
Developmental Effects of Chemical Contaminants 13
TABLE 2-1 Timing of Early Development in Some Mammalian Speciesa
Times of Early Development
(days from ovulation)
Length of
Blastocyst Gestation
Mammal Formation Implantation Organogenesis (days)
_ . . .
Mouse 3-4 4-5 6-15 21
Rat 3-4 5-6 6-15 22
Rabbit 3-4 7-8 6-18 30
Sheep 6-7 17-18 14-36 150
Monkey
(rhesus) 5-7 9-11 20-45 164
Human 5-8 8-13 21-56 270
aAdapted from Br~nster, 1975.
Following implantation, organogenesis takes place. During that period,
there are highly specific periods of vulnerability for different organ sys-
tems, thus making the embryo extremely susceptible to the induction of
structural birth defects. The periods when the major embryonic organ
systems of the rat are most sensitive to teratogenic insult are shown in
Figure 2-1. Administration of a teratogen on day 10 of rat gestation is
likely to result in a high level of brain and eye defects, intermediate levels
of heart and skeletal defects, and a low level of urogenital defects. If the
same agent were administered on day 11, a different spectrum of mal-
formations would be anticipated, predominantly effects on the brain and
palate. Figure 2-1 also illustrates that exposure to teratogens usually results
in a spectrum of malformations involving a number of organ systems,
reflecting the overlap of critical periods for individual organ systems. This
is most evident in species such as rodents, which have short gestation
periods, but can also be observed in humans. Most teratogens have been
found to influence the development of several organ systems in humans
and to cause clusters of malformations rather than single anomalies.
The critical period of inducing anomalies in individual organ systems
may be as short as 1 day or may extend throughout organogenesis. In the
rat, for example, urogenital effects can be produced by drug treatment
from the 9th to 18th day of gestation. This implies that development of
the urogenital system is multiphasic and that individual stages may have
different sensitivities to chemical insult. Depending on the mechanism of
action of the agent and the time of administration, it is possible that only
one or a few of these steps will be affected but that succeeding stages
will be disrupted as a result of the original alteration. The persistence of
the agent also influences the malformation pattern, as discussed later in
OCR for page 14
4 DRINKING WATER AND HEALTH
50
z
° 40
z
a:
' 30
o
o 20
z
-
~ 10
o
J
a:
o
~1
/
- I''
i//
—1,-
~ Brain
I ~ \
/Heart and \ \
/ Axial Skeleton \
Palate
Aortic \, ~_'\
Arches _ _ ~ ~ ~ <
,_
-
_
I ~~-t-
9 10 11
Urogenita I
""_
~ "~_
_`
1 - 1
8
12 13 14
DAYS OF GESTATION IN THE RAT
15 16
FIGURE 2-1 Hypothetical pattern of the susceptibility of rudimentary embryonic organs to
teratogenic insult. Adapted from Wilson, 1965.
this chapter. Processes governing embryonic differentiation are not well
understood, yet they are most likely to determine the intrinsic susceptibility
of individual organ systems to teratogenic insult.
Histogenesis, functional maturation, and growth are the major processes
occurring during the fetal and neonatal (i.e., perinatal) periods. Insult at
these later developmental stages leads to a broad spectrum of effects that
can generally be manifested as growth retardation, functional disorders,
or transplacental carcinogenesis. The fetus is more resistant to lethal effects
than is the embryo, but the incidence of stillbirths is measurable. The
perinatal period is a time of high susceptibility to carcinogenesis. At least
three factors contribute to this enhanced susceptibility: high cellular rep-
lication rates, ontogeny of xenobiotic-metabolizing enzymes, and low
immunocompetence. Several childhood tumors occur so early in life that
prenatal origin is considered likely. Among these are acute lymphocytic
(but not myelogenous) leukemia, Wilms' tumor, neuroblastoma, carci-
noma of the liver, and presacral teratoma (Miller, 1973~. In 1976 cancer
was the chief cause of death from disease among children under the age
of 15 in the United States, accounting for 11.3% of all childhood deaths.
Leukemias and lymphoma accounted for approximately half of these deaths,
followed by cancers of the central nervous system, soft tissues, kidney,
and bone (ACS, 19801.
Studies of direct-acting transplacental carcinogens, such as ethylnitro-
sourea (ENU), indicate that susceptibility to carcinogens in rodents begins
OCR for page 15
Developmental Effects of Chemical Contaminants ~ 5
after completion of organogenesis. In one study, tumors in offspring oc-
curred primarily when ENU was given during the fetal period, whereas
birth defects and embryo deaths predominated when exposures were ad-
ministered earlier in organogenesis (Ivankovic, 19791. This is not to imply
that teratogenesis and carcinogenesis are mutually exclusive processes,
however. Birth defects and neoplasms occur together in the same offspring
with unusually high frequency, but not necessarily at the same site. Ter-
atogenesis and carcinogenesis can be considered graded responses of the
embryo to injury, teratogenesis representing the grosser response involving
major necrosis. Bolande (1977) has postulated that certain agents cause
teratogenesis in early, relatively undifferentiated embryos; combined car-
cinogenesis and teratogenesis when older embryos are exposed; and car-
cinogenesis alone when exposure occurs during the perinatal period. Prenatal
insult may also predispose the offspring to tumor induction in later life.
PATTERNS OF DOSE RESPONSE IN LABORATORY ANIMAL
STU Dl ES
Functional deficits and perinatally induced cancers, such as those caused
by diethylstilbestrol (DES) (Herbst et al., 1977), are often not manifested
until adolescence or later. They are usually examined as end points in
themselves without correlation to outcomes observable at the time of birth.
Observations made at the time of birth indicate that the major effects from
prenatal exposure are embryo death, malformations, and growth retar-
dation. The relationship between these outcomes is complex, and varies
with the agent, the time of exposure, and the dose.
Some developmental toxicants can cause malformations in the entire
litter at exposure levels not causing embryo death. The dose-response
pattern for such agents is shown in Figure 2-2A. If the dose is increased
beyond that causing malformations of the entire litter, embryo death can
occur, but often in conjunction with maternal toxicity. Fetal malformations
are usually accompanied by growth retardation. Note that the curves for
these two end points are parallel and slightly displaced from one another
in Figure 2-2A. This pattern of response is rare, indicating that the agents
have high teratogenic potency. Both natural and synthetic glucocorticoids
cause this kind of dose-response pattern. The target-organ specificity of
glucocorticoids is related to the concentration of glucocorticoid receptor
protein, which is higher in the craniofacial region than in other parts of
the embryo (Pratt and Salomon, 19811. Thus, pharmacological doses ad-
ministered to laboratory animals at midgestation induce malformations of
the palate. Glucocorticoid induction of cleft palate in the absence of other
major malformations, embryo death, extensive necrosis, or growth retar-
OCR for page 16
6 DR! N KING WATER AND H "LTH
loo
Malformation
/
/ Growth
/ Retardation
At//
o
~ /
Lethal it`,r
A
On
LL
t 1oo
LL
7
o
CL
US o
Ct:
o
100
o
/:th,al itv
/ Growth
/ / Retardation
>/ ~ '.' Ma If ormation
B
/ Letha I ity
Growth /
Retardation ~ /
1 ~
/
C
EMBRYOTOX IC RANGE OF DOSES ~
FIGURE 2-2 Theoretical dose-response patterns for different types of
embr~rotoxic agents. Adapted from Neubert et al., 1980.
cations is an example of developmental toxicity with selective teratogenic
potency.
A more common dose-response pattern involves a combination of re-
sorptions, malformations, growth retardations, and unaffected fetuses after
exposure to a developmentally toxic range of doses of an agent (Figure
2-2B). Lower doses may cause predominantly resorptions or malforma-
tions, depending on the teratogenic potency of the agent. As the dosage
increases, however, embryo death predominates until the entire litter is
resorbed. Growth retardation can precede both outcomes or parallel the
malformation curve. This response pattern is typical of agents that are
cytotoxic to replicating cells by alterating replication, transcription, trans-
lation, or cell division. These agents include alkylating, antineoplastic,
and many mutagenic substances. The susceptibility of the embryo to these
agents derives from the high rate of cell division during organogenesis.
OCR for page 17
Developmental Effects of Chemical Contaminants 17
Low doses of cytotoxic agents administered relatively early in the critical
period may kill cells at rates low enough that the cells can be replaced
through compensatory hyperplasia, resulting in growth-retarded but mor-
phologically normal fetuses at term. Higher doses administered later during
the critical period may substantially deplete cell number, leaving insuf-
ficient time for replacement before critical morphogenetic events occur.
The resulting hypoplasia of the rudimentary organs and retarded prolif-
eration of surviving cells are the initial events leading to the induction of
malformations. High levels of exposure may damage too many cells and
organ systems to be compatible with survival, thus resulting in embryo
death (Ritter, 19771. Exposure to cytotoxic agents during organogenesis
can produce all three outcomes both within and among litters. Some litters
may be totally resorbed, others may include only growth-retarded fetuses
at term, and still others may include a mixture of malformed or growth-
retarded fetuses and resorption sites.
A third dose-response pattern consists of growth retardation and embryo
death without malformations (Figure 2-2C). The dose-response curve for
embryo death in this case is usually steep, which may imply a dose
threshold for survival of the embryo. Growth retardation of surviving
fetuses usually precedes a significant increase in lethality. Agents pro-
ducing this pattern of response would be considered embryotoxic or em-
bryolethal substances but not teratogenic. When such a pattern is observed,
it is necessary to conduct additional studies with doses within the range
causing growth retardation and embryo death. Results obtained at these
intermediate doses can indicate whether teratogenicity has been masked
by the deaths of the embryos. Agents in this class include the mitochondrial
protein synthesis inhibitors chloramphenicol and thiamphenicol (Neubert
et al., 1980~. On days 10 and 11 of treatment with thiamphenicol, the
dose-response curve for embryo death is steep, increasing from baseline
to 100% mortality at doses between 100 and 125 mg/kg body weight per
day (Bass et al., 19781. In the same study, dose-dependent inhibition of
mitochondrial respiration, adenosine triphosphate (ATP) content, and cy-
tochrome oxidase activity in embryonic tissue was correlated with growth
retardation and death of the embryos. There is no basis for target-organ
susceptibility to perturbation of such fundamental cellular processes in the
early embryo. Consequently, all tissues appear to be equally affected. An
early sign of perturbation is overall growth retardation, which progresses
to death of the entire litter once a threshold for cellular energy requirements
is exceeded. These conditions are incompatible with teratogenicity, which
can induce irreversible lesions in some tissues while sparing others, thus
permitting survival of abnormal embryos to term.
For some agents, i.e., those cytotoxic to replicating cells (Figure 2-
2B), growth retardation, embryo death, and teratogenicity are viewed as
OCR for page 18
i~ DRINKING WATER AND HEALTH
different degrees of manifestation of the same primary insult, cytotoxicity.
For others, there is a qualitative difference in response, and the primary
insult leads to embryo death alone (Figure 2-2C) or to teratogenicity alone
(Figure 2-2A). Separate evaluations of growth retardation, teratogenicity,
and embryo death for increasingly higher doses are necessary to determine
the agent's primary mode of action.
In safety studies, the usual sequence of testing begins with dose range-
finding studies in relatively small numbers of pregnant rodents. On days
6 through lS of gestation, animals are exposed to the test agent at doses
up to and including those causing limited maternal toxicity or develop-
mental toxicity (e.g., death or severe growth retardation). The purpose of
this type of study is to obtain a qualitative yes-or-no signal about the
potential developmental toxicity of the agent. At the next level of testing,
larger numbers of animals are exposed on days 6 to 15 of gestation to
obtain quantitative information on dose-response relationships. The high-
est dose should cause measurable maternal toxicity (e.g., significant
depression of weight gain) or developmental toxicity (e.g., significant
depression of fetal body weight or increased embryo death), and the low
dose should cause no observable effects. If evidence of selective devel-
opmental toxicity is obtained from this study, it may be necessary to
conduct a third study, exposing dams on single days during organogenesis
at doses that are not maternally toxic, to obtain a clear definition of the
dose-response pattern of developmental toxicity.
EXTRAPOLATION OF ANIMAL DATA TO HUMANS
Spectrum of End Points
The timing of exposure and the patterns of dose response obtained in
animal studies have important implications for extrapolating the resultant
data to humans. The major implication is that a spectrum of end points
can be produced, under the controlled conditions of timing and exposure
that can be achieved in animal studies. In some cases, the spectrum
comprises a continuum of response: depressed birth weight or functional
impairment at low doses, birth defects at intermediate doses, and death
at high doses. Less commonly, birth defects alone or deaths alone are
produced. Consequently, in estimating risks to humans, all exposure-
specific adverse outcomes must be taken into consideration not just birth
defects. Most often neglected in extrapolation of animal data to humans
is fetal growth retardation, despite the strong evidence concerning the
adverse consequences of low birth weight in human infants (Hull et al.,
19781. Fetal growth retardation in the absence of a significant reduction
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Developmental Effects of Chemical Contaminants 19
TABLE 2-2 Frequency of Selected Adverse Pregnancy Outcomes in
Humansa
Event
Frequency
per 100
Pregnancies
10-20
Spontaneous abortions, 8 to 28 weeks
Chromosome anomalies in spontaneous
abortions, 8 to 28 weeks
Chromosome anomalies detected by
amniocentesis
Stillbirths
Low birth weight (<2,500 g) among live
births
Major malformations among live births
Chromosome anomalies among live
births
Severe mental retardation among children
<15 years old
30-40
2-4
2-3
0.2
0.4
aAdapted from Edmonds et al., 1981.
in maternal weight gain is an important event to be considered in cross-
species extrapolation.
A similar spectrum of response has been observed in humans after
prenatal exposure to developmental toxicants. The specific effects in that
spectrum are determined by the time and duration of exposure, magnitude
of exposure, interindividual differences in sensitivity, interactions with
other types of exposure, and interactions among all these factors (Fraser,
1977~. Consequently, manifestations of developmental toxicity cannot be
presumed to be constant or specific across species; i.e., an animal model
cannot be expected to forecast exactly the human response to a given
exposure. For instance, an agent that induces cleft palate in the mouse
may elevate the frequency of spontaneous abortion or intrauterine growth
retardation in humans. Any manifestation of exposure-related develop-
mental toxicity in animal studies can be indicative of a variety of responses
in humans (Kimmel et al., 19841.
Table 2-2 illustrates another factor to be considered in cross-species
extrapolation. The most common adverse pregnancy outcome in humans
is spontaneous abortion or early fetal loss (before the 28th week of preg-
nancy), occurring in at least 10% to 20% of all recognized pregnancies.
Estimates from prospective studies range even higher: between 20% and
25% of all conceptions spontaneously abort (Edmonds et al., 19811. The
incidence of spontaneous abortions is high during early pregnancy, es-
pecially during the first 12 weeks, and gradually decreases to the 20th
OCR for page 20
20 DRINKING WATER AND H"LTH
week, after which fetal loss is uncommon. Approximately one-third of
the specimens obtained from spontaneous abortions occurring between 8
and 28 weeks of gestation contain chromosome aberrations. The frequency
of such aberrations is at least 60-fold higher among spontaneous abortions
than among term births. Among the spontaneous abortions without chro-
mosome aberrations, approximately half have structural malformations
(Edmonds et al., 1981~. The frequency of such malformations is not as
well documented as that of chromosome aberrations, because they are
difficult to observe in specimens that are often macerated or incomplete.
In the remaining one-third of the specimens, the incidence of placental
inflammations suggests that uterine infections can be high (Ornoy et al.,
19811.
These observations suggest that the majority of human embryos bearing
chromosome aberrations or morphological abnormalities are lost through
early miscarriage. Epidemiological approaches to monitoring the fre-
quency of early fetal loss and detecting such fetal abnormalities have only
been used to a limited extent. Most studies of humans focus on the ex-
amination of adverse effects, such as major malformations, stillbirths, low
birth weight, and neonatal deaths, at the time of birth or later. Underes-
timation of adverse pregnancy outcome, and thus true risk and pattern of
response, is unavoidable in human studies whenever measurements are
made only from the time of birth onward. Moreover, it is difficult to
design studies to work within the limitations of statistical power and to
document exposure of humans, even when observations are confined to
the time of birth onward (Edmonds et al., 1981; IRLG Epidemiology
Work Group, 19811.
Developmental toxicants with dose-response patterns resembling those
in Figure 2-2A could be detected by monitoring malformations at the time
of birth, especially if the malformations were rare (such as those resulting
from thalidomide), or if the exposed populations were large (such as those
with rubella infections). The possibility of concordance in the pattern of
malformation across species would be greatest for potent teratogens op-
erating in the pattern shown in part A of Figure 2-2, because they tend
to demonstrate target-organ specificity. Agents with patterns shown in
parts B and C would\probably be missed, because early fetal loss is not
routinely monitored in human populations, even though it has been done
successfully in isolated groups (Kline et al., 19771.
Concordance of Results from Animal and Human Studies
For several well-studied developmental toxicants, there is good evidence
for dose-response correspondence between humans and animals. The cor-
respondence is nearly 100% when data on animals have been expected to
OCR for page 21
Developmental Effects of Chemical Contaminants 2 ~
provide a qualitative yes-or-no signal, i.e., when any exposure-related
adverse effect from an animal study is taken into consideration and not
just those specific outcomes that are also seen in humans. Thalidomide
is the only toxicant known to produce developmental abnormalities in
humans but not to produce such effects consistently in conventional lab-
oratory animal species (see reviews by Brent, 1972; FDA, 1980; Frankos,
1985; Fraser, 1977; Nisbet and Karch, 1983; Nishimura and Tanimura,
1976; Schardein, 1976; Shepard, 1980; Strobino et al., 1978; and Wilson,
1973~. An FDA review of the literature (FDA, 1980; Frankos, 1985)
indicates that of 38 compounds having demonstrated or suspected tera-
togenic activity in humans, all except one tested positive in at least one
animal species. Furthermore, more than 80% were positive in more than
one species. Eighty-five percent of the 38 compounds were teratogens in
mice, 80% in rats, 60% in rabbits, 45% in hamsters, and a low of 30%
in primates. Other species (i.e., the cat, ferret, and guinea pig) have been
used to test only a few of these substances.
Nisbet and Karch (1983) reported that humans appear to be as sensitive
to thalidomide as the most sensitive species tested (the cat), and 5 to 10
times more sensitive than those species with comparable target-organ
specificity for limb defects (the rabbit and various primates). These authors
also compared the minimally effective doses in animals and humans of
eight known teratogens where there was cross-species concordance in
target organs. When dose was converted to mg/kg body weight per day
for these teratogens fi.e., thalidomide, polychlorinated biphenyls (PCBs),
alcohol, aminopterin, methotrexate, methylmercury, DES, and diphenyl-
hydantoin], humans were shown to be more sensitive than laboratory
animals by factors ranging from 1.8 (for PCBs) to 50 (for methylmercury).
If exposure was expressed in units of dose per unit body surface area per
day, the ratios of human to animal sensitivity ranged from 0.3 to 8.0
(Nisbet and Karch, 19831.
These findings of qualitative (yes-or-no signals) and quantitative (dose-
response) concordance support the use of animal studies for predicting
risk to humans. However, there are qualifications that must be placed on
their direct application to risk estimation. These comparisons of response
have necessarily been limited to agents for which there have been estab-
lished effects in humans and substantial data bases from animal studies.
Only the eight agents tested could be found to meet these criteria (Nisbet
and Karch, 19831. In the Catalog of Teratogenic Agents, Shepard (1980)
has listed more than 600 agents that cause congenital anomalies at any
dose in laboratory animals. Only 20 of these are confirmed or suspected
developmental toxicants in humans. Consequently, the major concern in
risk assessment today is that far more agents have been shown to be
positive in animal studies than have been identified in human studies for
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24 DRINKING WATER AND H"LTH
TABLE 2-3 Historical Control Data on Major and Minor Fetal
Malformationsa
Laboratory No. of Fetuses Major Minor Malformations (%)
Animal Examined Malformations (%) Visceral Skeletal
-
New Zealand
white
rabbits 36,508 0.74 2.53 8.60
CD rats 51,349 0.41 2.02 2.35
CD1 mice 22,389 0.84 3.68 5.32
aAdapted from Palmer, 1978.
incidence of common variants and minor anomalies, are taken into con-
sideration, a more sensitive appraisal of developmental toxicity can be
obtained.
The influence on power attributable to the end point's historical vari-
ability is illustrated in Table 2-4, which shows the number of litters of
different strains of rats and mice that would be required to detect 5% and
10% changes in fetal weight or embryo death. From 22 to 50 litters of
mice are required to detect a 10% depression in fetal weight, whereas
only 12 to 16 rat litters would be required to detect the same magnitude
of weight depression. For embryo death, from 235 to 324 litters of mice
are necessary to detect a 10% increase in resorptions, compared to 216
to 248 rat litters. Fewer litters are needed to detect a change in fetal
weight, probably because this is a continuously distributed end point with
relatively low variability. In contrast, embryo death is a highly variable,
binomially distributed parameter, and more than 200 litters are required
to detect even a 10% change in this response (Nelson and Holson, 1978~.
.
TABLE 2-4 Number of Litters Per Group Required to Detect Designated
Changes in Fetal Weight and Embryo Lethality in Rats and Micea
Change in Change in
Fetal Weight Embryo Lethality
Test Animal 5% 10% 5% 10%
-
Mice
A/J 84 22 1,176 324
C57BL/6 198 50 992 288
CD1 84 22 805 235
Rats
CD 62 16 858 248
Osborne-Mendel 44 12 723 216
aFrom Nelson and Holson, 1978.
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Developmental Effects of Chemical Contaminants 25
Given the current testing requirements for 20 rats or mice per group, the
most sensitive end point in developmental toxicology studies is fetal body
weight, as judged by the statistical power of the study. Within the range
of normal variability for this response, a logo change in fetal body weight
would be statistically significant (p < 0.05) if that change had been ob-
served in 20 rodent litters in a group.
For an accurate biological interpretation of depressed fetal weight and
embryo death, however, the occurrence of maternal toxicity must be taken
into consideration. Most experimental studies of developmental toxicity
have been designed to provide information on the basic mechanisms that
result in birth defects. Agents are administered under conditions that cause
a high incidence of abnormal fetuses, without concern for whether these
were secondary to toxic effects in the maternal system. In safety studies,
however, failure to recognize that developmental toxicity will inevitably
occur at exposure levels causing severe maternal toxicity can lead to false-
positive identification of many agents.
Relationship between Maternal and Developmental Toxicity
Regulatory guidelines for determining the developmental toxicity of
chemicals call for dose-response studies in pregnant animals, the highest
dose being of sufficient magnitude to induce maternal toxicity (slight but
statistically significant weight loss and not more than 10% maternal deaths).
The rationale for using a maternally toxic dose is to maximize the potential
to detect lesions in the fetus (Palmer, 1981~. Effects observed in offspring
at maternally toxic doses are used as a landmark to focus attention on
outcomes at lower doses. If a statistically significant incidence of a par-
ticular lesion is found in the high-dose group, the biological significance
of a lower and perhaps nonsignificant incidence at lower dose levels is
magnified. It can be difficult, however, to interpret some effects observed
only at maternally toxic dose levels. Are they indicative of unique and
selective developmental toxicity, or are they a function of nonspecific
alterations in maternal homeostasis?
It is generally accepted that developmental toxicity in the form of in-
creased resorptions and decreased fetal body weight can occur at mater-
nally toxic dose levels. The role of maternal toxicity in the induction of
congenital malformations is not clear, however. Recently, Khera (1984)
reviewed more than 85 published studies in mice to examine the rela-
tionship of birth defects to maternal toxicity and embryo toxicity. He noted
that doses of test agents that caused maternal toxicity, as indicated by
reduced maternal body weight, clinical signs of toxicity, or deaths, com-
monly caused reduction in fetal body weight, increased resorptions, and,
rarely, fetal deaths. He identified three patterns of association between
OCR for page 26
26 DRINKING WATER AND H"LTH
maternal toxicity and malformations: (1) for some compounds, maternal
toxicity was not associated with malformations; (2) for others, maternal
toxicity was associated with a diverse pattern of malformations, which
often included cleft palate; and (3) the maternal toxicity of still others was
associated with a characteristic and unique pattern of malformations.
Compounds in the second category are the most difficult to classify in
terms of teratogenic potential. Cleft palate has been reported as the prin-
ciple malformation resulting from food and water deprivation during preg-
nancy in mice (Szabo and Brent, 19751; however, cleft palate is also a
malformation specifically induced in mice by a number of teratogens,
most notably the glucocorticoids, without any apparent maternal toxicity.
Complete ascertainment of food and water consumption, maternal body
weights, and, occasionally, alterations in maternal homeostasis (i.e., organ
histopathology, kidney or liver dysfunction, hematological alterations,
pharmacologic reactions, and other possible toxic effects) are necessary
to distinguish between cleft palate caused by a teratogenic effect of a
chemical on the embryo and a nonspecific toxic effect on the dam that
secondarily influences embryonic development.
Compounds in the third category were structurally unrelated to test
agents administered at maternally toxic doses that caused increased re-
sorptions and decreased fetal body weight. The characteristic pattern of
defects induced by these agents was exencephaly; open eyes; fused, miss-
ing, or supernumerary ribs; and fused or scrambled sternebrae. The se-
verity and incidence of these defects could be directly related to the degree
of maternal toxicity. They were absent or rare at doses that were nontoxic
to the dam. Khera (1984) concluded that these defects resulted from
maternal toxicity and did not reflect the teratogenic potential of the com-
pounds.
Kavlock et al. (1985) also examined the association between maternal
toxicity and malformations by administering 10 chemicals to mice at doses
causing low (Low) to moderate (LD40) maternal mortality rates. Three
compounds caused a dose-related increase in the incidence of resorptions,
decreased fetal body weight, and malformations that appeared to be in-
dicative of developmental toxicity and not the result of indirect maternal
action. For seven compounds, an increased incidence of supernumerary
ribs was observed. There was a significant (p < 0.001) inverse linear
relationship between maternal weight gain during pregnancy and the in-
cidence of extra ribs in groups treated with these chemicals, compared to
the respective controls. Under the conditions of this study, there appeared
to be a quantitative relationship between supernumerary ribs and nonspe-
cific maternal toxicity. This quantitative relationship needs to be estab-
lished for the defects attributed to maternal toxicity in Khera's study
OCR for page 27
Developmental Effects of Chemical Contaminants 27
(1984), especially for defects classified as major malformations (e.g.,
exencephaly and open eyes).
As implied from this discussion, not all effects observed in animal
studies may be appropriate for use in risk assessment. Although there is
little doubt that major effects, such as irreversible and life-threatening
malformations and severe embryotoxicity, are deleterious to animals and
humans, other effects are of considerable less importance. Common skel-
etal variants, such as retarded ossification of the sternum or vertebrae, are
considered to be reversible and indicative of slight developmental delay
and not of teratogenesis. In humans, the incidence of congenital anomalies
of the ribs and vertebrae is low (between 0.02% and 0.03%), and these
are considered minor variants with little functional consequence (Heinonen
et al., 19771. A low level of concern should be attached to common
variations observed in animals, especially if they are the only effects
observed and if they only occur in conjunction with maternal toxicity.
Greater importance should be given to variations that are dose related or
that occur at doses not maternally toxic.
Selection of the NOEL/LOEL
If the data are of sufficient quality and quantity, it should be possible
to identify the NOEL or the LOEL, the maternally toxic dose levels, and
the specific types and incidences of adverse effects in the fetus. When
the data are insufficient to identify these parameters, the agent should not
be subjected to quantitative risk assessment. In the absence of sufficient
data, a qualitative assessment should be conducted to rank agents on the
basis of high, moderate, or low potential for developmental toxicity in
humans. Agents that selectively induce irreversible developmental toxicity
in animals, at low doses that are not maternally toxic, have the highest
potential for causing developmental toxicity in humans. If maternally toxic
exposure causes irreversible developmental toxicity, or if nontoxic ma-
ternal exposure causes reversible variants or minor malformations in an-
imals, the agent should be considered a moderate hazard to humans. A
low hazard to humans can be projected if prolonged exposure of animals
to high levels of the compound does not result in developmental toxicity.
If the data are sufficient for a quantitative risk assessment, the next
decision to be made is whether to establish a NOEL or a LOEL. This
decision depends largely on which dose level can most accurately be
identified from the data base. Greater experimental confidence can be
placed on the LOEL, insofar as this value is empirically derived, whereas
the NOEL can be orders of magnitude below the exposure level that would
induce developmental toxicity.
OCR for page 28
28 DRINKING WATER AND HEALTH
Selection of the LOEL need not be restricted to responses that are
statistically significant. Trends in the data indicating biologically relevant
elevations in the incidence of adverse effects at low doses can be used if
there are statistically significant increases in the occurrence of these effects
at higher doses. LOELs are most accurately selected when the response
is minimal (i.e., it is slightly elevated above background and involves
reversible developmental toxicity), indicating that the NOEL is being
approached. When statistical significance cannot be used as a guide to
select the LOEL, which will often be the case, minimal responses can be
regarded as those causing a doubling of the background rate (from con-
current or historical controls) for the particular response. To protect against
the possibility that humans may have double the background rate of the
response (which for major malformations would represent an unacceptable
increase from approximately 60,000 malformed infants per year to 120,000),
a large safety factor can be used for LOELs selected under these conditions.
RISK ASSESSMENT
Quantitative Assessment of Developmental Toxicity
Investigators concerned with the regulatory aspects of risk assessment
have focused on the development of a quantitative index for comparing
developmental toxicity across species, taking into account concurrent ma-
ternal toxicity. Underlying this approach is the perceived need to distin-
guish between compounds that are uniquely toxic to the embryo and those
that induce developmental toxicity at exposure levels that are also toxic
to the mother. Agents in the latter category should be regulated on the
basis of their maternal toxicity, whereas those in the former would be
regulated on the basis of their unique toxicity to the embryo.
Johnson (1980) has developed a testing system for addressing this issue
quantitatively. He defined teratogenic hazard potential as the ratio of adult
to developmental toxicity, or the A:D ratio, i.e.,
lowest adult toxic (lethal) dose
g lowest developmental toxic dose
He has calculated this ratio for more than 70 compounds using data from
an in vitro system of Hydra attenuata adult and embryonic tissues. The
A:D ratio from the hydra assay has been 0.1 to 10 times greater than the
mammalian A:D ratio. Most compounds had ratios near 1, several had
ratios larger than 5, and very few had ratios larger than 10 (Johnson and
Gabel, 19831. This system has been proposed for setting priorities for
further testing of agents in mammalian developmental toxicity studies.
OCR for page 29
Developmental Effects of Chemical Contaminants 29
Fabro et al. (1982) have explored the quantitative characteristics of a
similar type of index in mammalian studies. Dose-response data for adult
mortality and fetal malformations were fitted (probit of response against
log of dose) for eight compounds. The observed log-probit dose-response
lines for lethality and teratogenicity were not parallel, and there was not
a constant ratio between the slopes for the two lines. Consequently, a
simple ratio between the median lethal dose and the median effective dose
(i.e., LD50:EDso) could not be used. To calculate a relative teratogenic
index (RTI), Fabro and colleagues established a ratio between one point
on each dose-response line. The Loo value was chosen to represent adult
mortality on the basis that a low LD value is necessary to guard against
compounds with a shallow dose-response curve for adult mortality. The
teratogenic dose tDos was chosen for teratogenicity that is, the dose
causing a 5% elevation of the malformation rate above background. The
investigators believed that the tDos could be estimated with confidence
for most teratogens, because induced malformations often occur at a fre-
quency between 1% and 20% in animal studies. The committee concluded
that this approach appears to be satisfactory for ranking the candidate
compounds according to teratogenic potency, provided the relationship of
dose to teratogenic response is not complicated by significant adult mor-
tality.
This ranking system was developed to evaluate structure-teratogenicity
relationships between structurally related compounds. For this purpose,
the RTI seems adequate. The potential usefulness of this index for inter-
species comparisons and risk estimation, however, has not been estab-
lished. In their evaluation of the RTI, Hogan and Hoel (1982) argued that
because of the lack of parallelism between the probit lines for lethality
and teratogenicity, the index will not be invariant in the selection of other
LD and tD values; e.g., if a ratio of LD~o:tDos were chosen instead of
LDo~:tDos, a different ranking order for the RTI would be obtained. In
addition, the index would be subject to the established deficiencies of the
probit model, which tends to be insensitive in the low dose region near
the origin of the dose-response curve.
Therefore, until the RTI has been more extensively applied and eval-
uated, it should not be used for risk assessment. It is apparent, however,
that a uniform method for ranking agents according to their selective
toxicity to the conceptus needs to be established. Such a method would
provide a yardstick against which all agents could be compared and would
standardize the selection of the NOEL or LOEL for the risk assessment
equation. Selection of the safety factor could then be based on the severity
of the end point.
Existing models for quantitative risk assessment do not appear to be
adequate for developmental toxicity data. Multistage models used for
OCR for page 30
30 DRI N KI NG WATER AND H "LTH
mutagenicity and carcinogenicity data are based on a no-threshold as-
sumption (Anderson and CAG, 1983), whereas it is generally accepted
that thresholds do exist for developmental toxicity (Wilson, 19731. A more
thorough discussion of models can be found in Chapter 8. The Environ-
mental Protection Agency (EPA, 1983) used a number of models to eval-
uate developmental toxicity data on PCBs and found that the safe dose
varied by a factor of 7,000 for one set of data, depending on the model
used. The EPA and Oak Ridge National Laboratory concluded that existing
mathematical models are inappropriate for assessing developmental tox-
icity data and that the safety factor approach is appropriate for establishing
exposure levels expected to yield acceptable levels of risk (EPA-ORNL,
1982~.
The FDA has also indicated that it will use the safety factor approach
in developmental toxicity risk assessment but has not given specific details
on how the safety factors will be chosen. It is likely that safety factors
between 100 and 1,000 will be applied to NOELs identified in develop-
mental toxicity studies of drug residues in human food. Smaller factors
may be used when the prenatal effect can be ascribed to nonspecific
maternal toxicity (Norcross and Settepani, 19831. Thus, due to the absence
of other widely accepted approaches, the use of safety factors seems to
be the only suitable approach to the quantitative assessment of develop-
mental toxicity data (see Chapter 8~.
Selection of the Safety Factor
The preceding discussion forms the basis of the committee's proposal
that the following criteria be considered when selecting a safety factor:
· Minimum quality and quantity of data are required to perform a
quantitative risk assessment. Compounds without a sufficient data base
should be qualitatively assessed for high, moderate, and low potential to
cause developmental toxicity in humans.
· The committee has concluded that humans should be considered at
least 50 times more sensitive than animals to agents causing well-defined
developmental toxicity in animal studies. This is in keeping with the fact
that safety factors of 100 and 1,000 are most commonly applied to NOELs
for developmental end points.
· Compounds causing developmental toxicity at levels well below those
causing maternal toxicity constitute a greater level of risk than compounds
causing developmental toxicity only at maternally toxic doses. This greater
degree of risk should be reflected by application of a larger safety factor.
· The degree of risk associated with a compound is determined by the
severity of response in animal tests and the conditions of time and route
OCR for page 31
Developmental Effects of Chemical Contaminants 3 ~
of exposure under which the response occurs. The greatest degree of hazard
is presented by compounds causing serious effects under conditions of
exposure that are relevant to humans.
Ultimately, criteria for determining an acceptable risk will have to be
developed (Bass and Neubert, 19801. This will involve defining the terms
acceptable and risk. What magnitude of a risk is acceptable for a person
or for a given population? Is, for example, the doubling of a background
rate for an adverse response of 1 in 1,000 acceptable? To what extent
should reversible effects and common variants be taken into consideration
in risk assessment? These questions cannot be answered from a scientific
point of view alone but will require public policy decisions that take into
account the benefit of the chemicals under consideration and priorities for
protecting public health. Decisions at this level will greatly influence the
requirements for safety testing and risk assessment.
REFERENCES
ACS (American Cancer Society). 1980. Cancer Facts & Figures. National Headquarters,
American Cancer Society, Inc., New York. 31 pp.
Anderson, E. L., and CAG (Carcinogen Assessment Group of the U.S. Environmental
Protection Agency). 1983. Quantitative approaches in use to assess cancer risk. Risk
Anal. 3:277-295.
Bass, R., and D. Neubert. 1980. Testing for embryotoxicity. Arch. Toxicol. (Suppl. 4):256-
266.
Bass, R., D. Oerter, R. Krowke, and H. Spielmann. 1978. Embryonic development and
mitochondrial function. III. Inhibition of respiration and ATP generation in rat embryos
by thiamphenicol. Teratology 18: 93- 102.
Bolande, R. P. 1977. Teratogenesis and oncogenesis. Pp. 293-325 in J. G. Wilson and F.
C. Fraser, eds. Handbook of Teratology. Vol. 2. Mechanisms and Pathogenesis. Plenum,
New York.
Brent, R. L. 1972. Drug testing for teratogenicity: Its implications, limitations and appli-
cation to man. Pp. 31-43 in M. A. Klingberg, A. Abramovici, and J. Chemke, eds.
Drugs and Fetal Development. Plenum, New York.
Brinster, R. L. 1975. Teratogen testing using preimplantation mammalian embryos. Pp.
113-124 in T. H. Shepard, J. R. Miller, and M. Marois, eds. Methods for Detection of
Environmental Agents That Produce Congenital Defects. Proceedings of the Guadeloupe
Conference Sponsored by l'Institut de la Vie. American Elsevier, New York.
Collins, T. F. X. 1978. Reproduction and teratology guidelines: Review of deliberations
by the National Toxicology Advisory Committee's Reproduction Panel. J. Environ.
Pathol. Toxicol. 2:141-147.
Edmonds, L., M. Hatch, L. Holmes, J. Kline, G. Letz, B. Levin, R. Miller, P. Shrout,
Z. Stein, D. Warburton, M. Weinstock, R. D. Whorton, and A. Wyrobek. 1981. Report
of Panel II: Guidelines for reproductive studies in exposed human populations. Pp. 37-
110 in A. D. Bloom, ed. Guidelines for Studies of Human Populations Exposed to
Mutagenic and Reproductive Hazards. Proceedings of conference held January 26-27,
OCR for page 32
32 DRINKING WATER AND HEALTH
1981, in Washington, D.C. March of Dimes Birth Defects Foundation, White Plains,
N.Y.
EPA (U.S. Environmental Protection Agency). 1983. Quantitative Risk Assessment of
Reproductive Risks Associated with Polychlorinated Biphenyl (PCB) Exposure. Office
of Pesticides and Toxic Substances, U.S. Environmental Protection Agency, Washington,
D.C. [52 pp.]
EPA-ORNL (U.S. Environmental Protection Agency-Oak Ridge National Laboratory).
1982. Pp. 99, 111 in Assessment of Risks to Human Reproduction and to Development
of the Human Conceptus from Exposure to Environmental Substances. Report No. EPA-
600/9-82-001. U.S. Environmental Protection Agency, Washington, D.C.
Fabro, S., G. Shull, and N. A. Brown. 1982. The relative teratogenic index and teratogenic
potency: Proposed components of developmental toxicity risk assessment. Teratogen.
Carcinogen. Mutagen. 2:61-76.
FDA (U.S. Food and Drug Administration). 1970. Food and Drug Administration Advisory
Committee on Protocols for Safety Evaluations: Panel on Reproduction report on repro-
duction studies in the safety evaluation of food additives and pesticide residues. Toxicol.
Appl. Pharmacol. 16:264-296.
FDA (U.S. Food and Drug Administration). 1980. Caffeine; deletion of GRAS status,
proposed declaration that no prior sanction exists, and use on an interim basis pending
additional study. Fed. Regist. 45:69817-69838. (21 CFR Parts 180 and 182)
Frankos, V. H. 1985. FDA perspectives on the use of teratology data for human risk
assessment. Fund. Appl. Toxicol. 5:615-625.
Fraser, F. C. 1977. Relation of animal studies to the problem in man. Pp. 75-96 in J. G.
Wilson and F. C. Fraser, eds. Handbook of Teratology. Vol. 1. General Principles and
Etiology. Plenum, New York.
Heinonen, O. P., D. Stone, and S. Shapiro. 1977. Pp. 127, 450 in Birth Defects and
Drugs in Pregnancy. Publishing Sciences Group, Inc., Littleton, Mass.
Herbst, A. L., R. E. Scully, S. J. Robboy, W. R. Welch, and P. Cole. 1977. Abnormal
development of the human genital tract following prenatal exposure to diethylstilbestrol.
Pp. 399-412 in H. H. Hiatt, J. D. Watson, and J. A. Winsten, eds. Origins of Human
Cancer. Book A. Incidence of Cancer in Humans. Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y.
Hogan, M. D., and D. G. Hoel. 1982. Extrapolation to man. Pp. 724-727 in A. W. Hayes,
ed. Principles and Methods of Toxicology. Raven Press, New York.
Hull, D., J. Dobbing, R. W. Miller, F. Naftolin, M. Ounsted, H. Rehder, J. S. Robinson,
C. Tudge, and R. H. Usher. 1978. Definition, epidemiology, identification of abnormal
fetal growth: Group Report. Pp. 69-83 in F. Naftolin, ed. Abnormal Fetal Growth:
Biological Bases and Consequences. Dahlem Konferenzen, Berlin.
IRLG (Interagency Regulatory Liaison Group) Epidemiology Work Group. 1981. Guide-
lines for documentation of epidemiologic studies. Am. J. Epidemiol. 114:609-613.
IRLG (Interagency Regulatory Liaison Group) Testing Standards and Guidelines Work
Group. 1981. Recommended Guidelines for Teratogenicity Studies in the Rat, Mouse,
Hamster or Rabbit. Washington, D.C. [13 pp.] (Available from National Technical
Information Service, Springfield, Va., as Publication No. PB-82-119488.)
Ivankovic, S. 1979. Teratogenic and carcinogenic effects of some chemicals during prenatal
life in rats, Syrian golden hamsters, and minipigs. Pp. 103-115 in Perinatal Carcino-
genesis. National Cancer Institute Monograph 51. NIH Publication No. 80- 1633. National
Cancer Institute, U.S. Department of Health, Education, and Welfare, Bethesda, Md.
Johnson, E. M. 1980. A subvertebrate system for rapid determination of potential terato-
genic hazards. J. Environ. Pathol. Toxicol. 4(5):153-156.
OCR for page 33
Developmental Effects of Chemical Contam
inants 33
Johnson, E. M., and B. E. G. Gabel. 1983. An artificial 'embryo' for detection of abnormal
developmental biology. Fund. Appl. Toxicol. 3:243-249.
Karnofsky, D. A. 1965. Mechanism of action of certain growth-inhibiting drugs. Pp. 185-
194 in J. G. Wilson and J. Warkany, eds. Teratology: Principles and Techniques.
University of Chicago Press, Chicago.
Kavlock, R. J., N. Chernoff, and E. H. Rogers. 1985. The effect of acute maternal toxicity
on fetal development in the mouse. Teratogen. Carcinogen. Mutagen. 5:3-13.
Khera, K. S. 1984. Maternal toxicity A possible factor in fetal malformations in mice.
Teratology 29:411-416.
Kimmel, C. A., J. F. Holson, C. J. Hogue, and G. L. Carlo. 1984. Reliability of Exper-
imental Studies for Predicting Hazards to Human Development. Final Report. NCTR
Technical Report for Experiment No. 6015. National Center for Toxicological Research,
Jefferson, Ark. 56 pp.
Kline, J., Z. Stein, B. Strobino, M. Susser, and D. Warburton. 1977. Surveillance of
spontaneous abortions: Power in environmental monitoring. Am. J. Epidemiol. 106:345-
350.
Miller, R. W. 1973. Prenatal origins of cancer in man: Epidemiological evidence. P. 175
in L. Tomatis and U. Mohr, eds. Transplacental Carcinogenesis. IARC Scientific Pub-
lications No. 4. International Agency for Research on Cancer, Lyon, France.
Nelson, C. J., and J. F. Holson. 1978. Statistical analysis of teratologic data: Problems
and advancements. J. Environ. Pathol. Toxicol. 2:187-199.
Neubert, D., H. J. Barrach, and H.-J. Merker. 1980. Drug-induced damage to the embryo
or fetus: Molecular and multilateral approach to prenatal toxicology. Curr. Top. Pathol.
69:241-331.
Nisbet, I. C. T., and N. J. Karch, eds. 1983. Chemical Hazards to Human Reproduction.
Noyes Data Corporation, Park Ridge, N.J. 245 pp.
Nishimura, H., and T. Tanimura. 1976. Clinical Aspects of the Teratogenicity of Drugs.
American Elsevier, New York. 453 pp.
Norcross, M. A., and J. A. Settepani. 1983. Current guides to establishing safe levels of
animal drug residues. Paper presented at the International Symposium on the Safety
Evaluation of Animal Drug Residues, Berlin, Federal Republic of Germany, 27-29
September 1983.
Ornoy, A., J. Salamon-Arnon, Z. Ben-Zur, and G. Kohn. 1981. Placental findings in
spontaneous abortions and stillbirths. Teratology 24:243-252.
Palmer, A. K. 1978. The design of subprimate animal studies. Pp. 215-253 in J. G. Wilson
and F. C. Fraser, eds. Handbook of Teratology. Vol. 4. Research Procedures and Data
Analysis. Plenum, New York.
Palmer, A. K. 1981. Regulatory requirements for reproductive toxicology: Theory and
practice. Pp. 259-287 in C. A. Kimmel and J. Buelke-Sam, eds. Developmental Tox-
icology. Raven Press, New York.
Pratt, R. M., and D. S. Salomon. 1981. Biochemical basis for the teratogenic effects of
glucocorticoids. Pp. 179-199 in M. R. Juchau, ed. The Biochemical Basis of Chemical
Teratogenesis. Elsevier/North-Holland, New York.
Ritter, E. J. 1977. Altered biosynthesis. Pp. 99-116 in J. G. Wilson and F. C. Fraser, eds.
Handbook of Teratology. Vol. 2. Mechanisms and Pathogenesis. Plenum, New York.
Schardein, J. L. 1976. Drugs as Teratogens. CRC Press, Inc., Cleveland. 291 pp.
Shepard, T. H. 1980. Catalog of Teratogenic Agents, 3rd ed. The Johns Hopkins University
Press, Baltimore, Md. 410 pp.
OCR for page 34
34 DRINKING WATER AND H"LTH
Staples, R. E. 1975. Definition of teratogenesis and teratogens. Pp. 25-26 in T. H. Shepard,
J. R. Miller, and M. Marois, eds. Methods for Detection of Environmental Agents That
Produce Congenital Defects. American Elsevier, New York.
Strobino, B. R., J. Kline, and F. Stein. 1978. Chemical and physical exposure of parents:
Effects on human reproduction and offspring. J. Early Hum. Dev. 1:371-399.
Szabo, K. T., and R. L. Brent. 1975. Reduction of drug-induced cleft palate in mice.
Lancet 1: 1296-1297.
Wilson, J. G. 1965. Embryological considerations in teratology. Pp. 251-261 in J. G.
Wilson and J. Warkany, eds. Teratology: Principles and Techniques. University of
Chicago Press, Chicago.
Wilson, J. G. 1973. Environment and Birth Defects. Academic Press, New York. 305 pp.
Representative terms from entire chapter:
maternal toxicity
