Developmental Toxicology and Diphenyl Ethers
In the history of pesticide regulation, the most frequent stated cause for restricting pesticide use has
undoubtedly been the allegation of carcinogenicity (Table 1). Environmental damage - notably because
a compound is persistent and bioaccumulative - is clearly in second place. However, reproductive and
developmental toxicity have often been the visible result of bioaccumulation, and the mechanism of the
most severe environmental damage. Thus DDT, officially banned from most U.S. uses because of its
suspect carcinogenicity, (but actually because of its bioaccumulation in food chains) exerts its ecological
damage by thinning the eggshells of birds' eggs, effectively ending successful reproduction in many
species.
In only a few cases have pesticides been restricted or withdrawn because of a feared risk to human
reproduction or development.1 The most notorious was DBCP (dibromochloropropane), a soil fumigant
that causes sterility in men. The evidence against DBCP was epidemiological: clearly dose-related
decreases in sperm numbers occurred among baggers, applicators, and others working with it.
Moreover, pre-existing animal data (published more than 10 years earlier by scientists in the employ of
the manufacturers) supported the human data. Only two pesticides have been removed from commerce
based on animal data suggesting risk to human fetuses: nitrofen and dinoseb.
Reproductive and developmental toxicity encompass the whole of life, from conception to senescence.
Even if we consider only the reproductive cycle, most aspects of life - prenatal development, postnatal
growth and maturation; behavior, hormonal homeostasis, and structural integrity of the reproductive
organs - contribute to successful production of the next generation. Chemicals and/or processes are
known that affect every aspect of the reproductive cycle adversely, at least in laboratory animals (Table
2). Therefore, determining that a chemical does not deleteriously affect reproductive capacity requires
evaluation of at least one full cycle - from fertilization to fertilization. In reality, because abnormalities
of the female reproductive tract can affect survival of her progeny, it is necessary to carry the test out for
at least 2 generations. This two generation assay is the most complete - and in most cases the only assay necessary for reproductive and developmental toxicity. If carefully done and carefully evaluated,
a 2-generation assay will answer the question: does this agent adversely affect reproduction? It will not,
however, tell us why a given agent prevents successful production of the next generation (nor even, in
many cases, whether it is the parents or the offspring which are targets of the agent). Therefore, if the
company or the investigator wishes to know how or why a given agent acts, additional tests may be
needed. Moreover, largely for historic reasons, additional assays are required by regulatory agencies.
Multi-generation Assay
A multi-generation assay is appropriate if long-term exposure to, or bioaccumulation of, an agent is
anticipated; e.g., food-additives, pesticides. It should be required for any agent used therapeutically
during pregnancy, as shown by DES2. U.S. guidelines require that one rodent and one non-rodent
species be used. Figure 1 shows one protocol for a multigeneration assay. As a general summary, the
multi-generation assay requires that the compound is administered to the parental or F0 generation,
1
TCDD (dioxin) was shown to affect primate development at extremely low levels; however, the regulatory
concern over its carcinogenesis was dominant in regulating TCDD-contaminated pesticides.
2
DES, diethylstilbestrol, is a synthetic estrogen that was used until 1973 in efforts to prevent miscarriages. It
was quite widely prescribed, often to women who were not at known risk of miscarrying, sometimes even without
identifying the drug or the rationale for administering it. In 1973 it was shown that some young women exposed
to DES in utero developed clear cell adenocarcinoma of the vagina (a very rare cancer) during adolescence. Since
then it has become clear that, although the cancer is rare (0.01% of women) the majority of women and men
exposed in utero have abnormalities of the reproductive tract that may decrease male fertility but certainly make it
more difficult for many "DES daughters" to carry pregnancies to term. The resulting premature infants have the
typical problems of low birth-weight babies, thus carrying the DES legacy to a 3rd generation.
beginning at 30-40 days of age for rats. Administration is usually in feed. Animals are mated after about
60 days on the diet. Surviving offspring of the first litters [F1a ] are necropsied3 at weaning, and the
parents are kept until 2nd litters [ F1b ] are weaned. Then the parental generation is necropsied.
Randomly selected F1b pups are kept on the test diet and mated at about 100 days of age; 1st litters [F2a
] are necropsied at weaning. The F1b are necropsied when the F2a litters are weaned. A 3-generation
study carries the procedure through one more iteration. Variations on what necropsies are done exist.
The endpoints of such a multi-generation assay are
>>> fertility index, or [pregnancies/mating] x 100;
>>> gestation index, or liveborn/litter [or, liveborn/total born];
>>> sex ratio, at age 1, 4, 7, 14, 21 days of age
>>> weaning index, or [number alive day 21] / number at 4 days4
>>> growth index, or mean weights of two generations of male and female offspring at 1, 4, 7, 14, 21
days of age.
The strengths of a multigeneration assay are that it identifies transplacental carcinogens [e.g.,
DES] and 2nd generation reproductive agents [e.g., the aromatase inhibitor fenarimol 5], bioaccumulative
compounds [e.g., mirex]; and postnatally lethal agents [e.g., nitrofen (q.v.)]. The major weakness of the
multi-generation assay is that it is expensive, costing over $500,000 for each species. It is also tedious
and labor-intensive. Because two generations of animals must be maintained and bred, it also takes a
long time to obtain results.
One Generation 3 Segment Assay :
The one generation assay attempts to overcome the long elapsed time required by the multi-generation
assay by evaluating reproduction, prenatal toxicity and postnatal toxicity separately. In theory, it is
possible to conduct the three segments in parallel, shortening the time elapsed before a result is
available.
SEGMENT 1: General fertility and reproductive performance :.
Ten males are dosed for 60 days, and 20 females for 14 days, before mating, and dosing
continues throughout the study. Two females are mated with each male. This allows one female to be
killed in midgestation, while the other is allowed to carry her litter through term to weaning. The
endpoints include information on pregnancy incidence, implantation rate, pre- and postnatal survival,
and pre- and postnatal growth rates.
The major strength of this segment is that it measures gonadal function of both sexes, as well as
estrus cycle, mating behavior, conception rates, early stages of development. It can also be used to
identify postnatal effects on lactation and behavior, postnatally apparent malformations, and growth
3
"Necropsy" includes all necessary postmortem evaluations, from gross dissection to histopathology.
4
In many studies, litters are culled to specific size, often 8, on day 4, to avoid effects of different litter sizes on weight
gain. In behavioral teratology, it is considered necessary, since litter size affects development of pups. Many
teratologists think it is a serious error to cull litters when physical development is being examined.
5
Fenarimol is a fungicide (marketed by Eli Lilly). In the 2-generation assay, no F2 generation was produced at
the highest dose, even though F1 males and females were physically normal and healthy. The company was able
to show that fenarimol inhibits aromatase, the steroid-metabolizing enzyme needed to produce testosterone in
neonatal male rats so that their brains become properly male-imprinted. Because this imprinting occurs
prenatally in humans, and because fenarimol does not cross the placenta (although it is transmitted through milk),
and because the extremely high doses needed for aromatase inhibition are unlikely to occur even in accidents,
fenarimol was registered.
retardation. The major weakness of the assay is that, if a problem occurs, localizing it will take another
round of experiments. This is also true of the multi-generation assays, however.
SEGMENT II: Teratology Segment
U.S. guidelines require one rodent and one non-rodent species. Rats are the most common
rodent species, while the non-rodent species is usually the rabbit. Females are mated, and treated during
organogenesis [if the day the semen plug is seen is day 0 of gestation, treatment is on days 6-15
inclusive in rats and mice, days 6-18 in rabbits]. At least 2 doses, one of which causes maternal toxicity
and one which does not, must be used. Concurrent controls are required, and should be treated with the
vehicle in order to equalize stress effects between treated and control animals. All females are killed
before parturition, and net maternal weight gain [excluding the uterus and its contents] determined. Half
the pups are examined externally, for soft-tissue malformations, and for skeletal abnormalities.
The major weakness of the teratology assay is that there is no certainty that the results can be
extrapolated to humans, and overwhelming evidence that interspecies differences are significant.
Therefore, both positive and negative results can be attacked - and are - either in regulatory proceedings
or in torts. On the other hand, there has not been another thalidomide disaster since these requirements
were instituted.7
SEGMENT III: Perinatal and Postnatal Segment
For the investigation of perinatal and postnatal toxicity, 20 females per dose are treated from the
last quarter of gestation through lactation to weaning. In the U.S., 2 treated groups plus concurrent
controls are required. In the U.K., 3 treated groups plus concurrent controls are required.
The major strengths of the perinatal/postnatal segment are that it identifies postnatal toxicity, including
effects on lactation. It also identifies malformations that are not prenatally apparent, delayed maternal
toxicity, and behavioral toxicity in weanlings. This third assay can actually be combined effectively
with either the 2-generation or the Segment I assays. It is uniquely useful in identifying "other effects"
of chemicals that prevent implantation or alter mating behavior. In theory, it could also identify effects
that would be masked by mortality if exposure occurs before implantation or during organogenesis. The
major weaknesses of the perinatal/postnatal assay is that it is quite limited, both in the effects identified
and in the dosing regimen. Alone, it would not identify either structural teratogens or agents affecting
male fertility, implantation, or mating behavior [to name a few]. In combination, it can be replaced by
modifications of other assays. Thus it becomes a burdensome addition to other reproductive and
developmental toxicity assays.
Summary of Testing
There is no single assay that can adequately predict developmental toxicity, much less the full spectrum
of reproductive and developmental toxicity. On the other hand, if the several assays required by
regulatory agencies are adhered to, and if a modicum of intelligence is applied to the analysis of all of
the data obtained, adequate warning of reproductive toxicity can be obtained. Even thalidomide, if it
had been evaluated either in the 3 segment reproductive assay or the 2-generation assay, would have
prevented reproduction in rats because it prevents implantation. Thus a warning would have sounded
6
Teratology, the "study of monsters" is the study of structural malformations, and the terms teratogen and
teratogenesis are usually restricted to agents (respectively, processes) that cause structural malformations. The
more general term developmental toxicant is used for agents that cause prenatal death, growth retardation, or
functional deficits (in any combination, with or without also causing malformations).
7
The one major human teratogen that has been introduced to commerce since teratology testing became obligatory is
the vitamin A derivative AcutaneTM, whose developmental toxicity was expected on the basis of numerous animal
studies demonstrating that both hyper- and hypo-vitaminosis A results in developmental toxicity. Acutane was
also identified as a teratogen in testing.
even though thalidomide does not cause structural malformations in rodents. If, on the other hand, the
rules are followed blindly, and interpreted without thought, atypical compounds can slip through any
regulatory net.
Additional Comments
One consideration in developmental toxicology is the significance of transplacental carcinogenesis.
This phenomenon has been well-documented in animal studies, and there is no reason to consider that
human fetuses are immune. The first human example was the 1973 identification of the transplacental
carcinogenicity of diethylstilbestrol [DES]. The risk of transplacental carcinogenesis provides another
incentive for using the 2-generation assay, since the F1 generation is kept long enough (to weaning of its
2nd litters) that early onset cancers can be detected at necropsy.
In contrast to mutagenesis and carcinogenesis, developmental toxicity is a threshold phenomenon. It is
generally accepted that nonmutagenic teratogens must be present in significant concentrations to exert a
deleterious effect.8
One of the difficulties in determining the risk a chemical poses to human development is that we have
no good means of extrapolating risks of developmental toxicity between species. Although there is
roughly a 60% concordance between laboratory species in chemically induced malformations, there is a
40% discordance, and - in contrast to the situation in genetic toxicology - no theoretical basis for
predicting what chemicals are likely to be developmental toxicants. In practice, all teratogens but two
have been identified by "alert clinicians": doctors in practice who recognize a common cause for a cluster of malformations. From Gregg's identification of rubella-induced cataracts to the 1983 identification
of Acutane (an anti-acne medication derived from Vitamin A), the "alert clinician" has been the most
effective warning system we have. Only smoking (1970s) and the anti-convulsive valproic acid (1970s)
were identified by surveillance. In the case of smoking, the evidence was purely epidemiological; in the
case of valproic acid, a case-control monitoring of pregnancies at risk identified the teratogen.
8
This statement includes the exception: mutagens can, in theory, alter DNA if even one molecule of the mutagen is
present; they could similarly act as teratogens via their effect on DNA.
TABLE 1: RESTRICTIONS OF PESTICIDE USE BY FEDERAL OR STATE AGENCIES
____________________________________________________________________________________
PESTICIDE
CLASS
RESTRICTED CAUSE
Aramite
acaricide, insecticide 1955
carcinogeni
ii
Aminotriazole
herbicide
1959
carcinogen
Alkyl mercury compounds
fungicides
1970
chronic toxicity
DDT, DDD
insecticides
1971
bioaccumulationiii
Strychnineiv, sodium fluoroacetatev rodenticides
1972
acute toxicity
Aldrinvi, dieldrinvii
insecticides
1974
carcinogensviii
ix
Heptachlor, chlordane
insecticides
1976
carcinogens8
Strobane
insecticide
1976
carcinogen8
Mirex
insecticide
1976
bioaccumulation
Leptophos
insecticide
1976
delayed neurotoxicity
DBCP
fumigant
1977
male sterility
Chloranil
fungicide
1977
carcinogen
Chlordimeform
insecticide, acaricide 1977x
Carcinogen
Chlordecone
insecticide
1978
neurotoxicity
BHCxi
insecticide
1978
off flavors
xii
2,4,5-T and Silvex
herbicides
1979
dioxin contamination
Endrin
insecticide
1979
toxicity to fishxiii
xiv
Chlorobenzilate
acaricide
1979
carcinogen, male sterility
Pyriminil
rodenticide
1980
Pancreatic poisonxv
Nitrofen
herbicide
1980
developmental toxicant
Aldicarb
insecticide
1981
ground-water pollutant
Ethylene dibromide
fumigant
1983
carcinogen
Toxaphene
insecticide
1983
fish toxicity; carcinogen
Dicofol
miticide
1986
bioaccumulation
Dinoseb
herbicide
1986
dev. toxicity, male sterility
Captafol
fungicide
1986
carcinogen
xvi
Alachlor
herbicide
1987
carcinogen; water pollutant
Chlordane
insecticide
1987
indoor air pollutantxvii
xviii
Captan
fungicide
1989
carcinogen
Diazinon
insecticide
environ, contamination
xix
Amitraz
acaricide
carcinogen
xx
Benomyl
fungicide
1982?
carcinogen
xxi
Bromoxynil
herbicide
developmental toxicity
xxii
Daminozide
growth regulator
1989
carcinogenic metabolite
____________________________________________________________________________________
TABLE 2: REPRODUCTION AND DEVELOPMENT
DEVELOPMENTAL
STAGE
PREREQUISITES or
PROCESSES
AGENTS/PROCESSES
THAT INTERFERE (IN HUMANS)
DNA replication; meiosis
mutagens,
chromosome aberrations,
aged/defective sperm, ova
DBCP
Cell division
cell migration
ovum transport,
uterine receptivity
Hormonal imbalance, uterine
anomalies, chromosome imbalance,
nutrition?
Cell division
cell migration
uterine receptivity
teratogens (e.g.: retinoids, maternal
disease, metabolic toxins,
antimetabolites ...)
cell division
tissue differentiation
functional maturation
alcohol, DES, smoking, hormones,
antimetabolites, malnutrition?
hormonal changes
maternal malformations, maternal-fetal
size disparity
growth, cell division
tissue differentiation
functional maturation
hormonal changes
chromosomal abnormalities
hormonal abnormalities, nutritional
deficiencies (Zn), mutations, structural
abnormalities
hormonal balance,
acquired behaviors,
pre/postnatal brain
development
hormones
physical deficits,
behavioral anomalies
FERTILIZATION
IMPLANTATION
ORGANOGENESIS
FETAL MATURATION
BIRTH
PUBERTY
MATING
DIPHENYL ETHER HERBICIDES
The diphenyl ether herbicides comprise a large and versatile class of herbicides. As herbicides, the
diphenyl ethers inhibit the enzyme protoporphyrinogen oxidase, in the pathway of chlorophyll synthesis.
In plants, this enzyme leads to accumulation of high levels of protoporphyrinogen, which leaves the
chloroplast and is rapidly oxidized (by enzymes outside the normal porphyrin pathway).9 In the
presence of molecular oxygen, the product(s) of the abnormal oxidation generate singlet oxygen, which
in turn causes cellular damage. Protoporphyrinogen oxidase is also an enzyme in the synthesis of heme,
and similar chains of oxidative damage have been documented in humans with hereditary defects of this
enzyme. (However, there are no data suggesting that mammalian diphenyl ether toxicity acts by this
pathway).
Among the diphenyl ether herbicides that have been registered for use on crops in the U.S. are bifenox,
nitrofen, acifluorfen, and oxyfluorfen (Figure 1).
Nitrofen: used on rice in far East; on root and other vegetables in U.S, Europe. LD50, po in rats is
variously given as 2,630 mg/kg (Rohm and Haas) or 1,470 mg/kg (UIUC) or 740 mg/kg (EPA).
Ecological Magnification (EM) in fish, 1,546; in snails, 2769. Biodegradation Index (BI) in fish, 0.29;
in snails, 0.35. (Withdrawn from the U.S. market because of its developmental toxicity.)
Bifenox: also used on rice; in U.S., pre-emergent herbicide for corn and soybeans. LD50 in rats, 1,630
mg/kg (UIUC); 6,400 (EPA). EM in fish, 49; in snails, 651. (Residues in snails were 13 times as high
as in fish, suggesting that metabolism proceeds primarily by cytochrome P450s, which are more
important in fish than in snails.) BI in fish was 1.08; in snails, 0.44.
Acifluorfen is a relatively nonpersistent herbicide registered for post-emergent use on rice, soybenas,
and peanuts. EPA lists the LD50 in rats as 1,300 mg/kg, po; the UIUC Extension weed specialists, as
3,300 mg/kg, po, rats. Neither gives a reference; a typo is more probable in the UIUC publication (which
is put out annually).
Oxyfluorfen, with a water solubility, 0.1 ppm, was registered as a pre- and post-emergence herbicide in
nonbearing fruit trees (prune family) and in conifers; registration was sought for soybeans and field
corn. Tolerances were sought by the manufacturer to accommodate expected residues in corn, soybeans,
eggs, milk, meat (0.05 ppm); for soybean oil, tolerance for residues of 0.25 ppm was sought. LD50,
5,000 mg/kg, po in rats. Review of oxyfluorfen registrations was carried out by EPA in 1980. primarily
because the herbicide is contaminated with perchloroethylene, a probable human carcinogen. During
this review, EPA concluded that neither the carcinogenicity data nor the developmental toxicity data
supplied by the manufacturer were adequate to assess the risk posed. Oxyfluorfen was also considered a
probable mutagen, and a hepato- and thyro-toxicant. EPA also concluded that bioaccumulation of
oxyfluorfen under normal conditions of use was quite probable. Persistence in a model aquatic
environment (not the Metcalf microcosm) was estimated at 127 days, and concern was raised about the
20 endangered species of fresh water molluscs in the Mississippi ecosystem if oxyfluorfen were widely
9
Nandihalli, UB; MV Duke, SO Duke, J Agric Food Chem 40:1993-2000, 1992.
used on soybeans. In normal, medium-textured Illinois soil, herbicidal persistence was estimated to be
2-4 months10 Most U.S. uses were cancelled in 1982.
Notes for Table 1
i
Registration for use in USA voluntarily cancelled by manufacturer.
ii
Now called amitrole
iii
Officially DDT was banned because it is a carcinogen: that was the only legal cause for banning pesticides at that time.
However, most of the evidence at the hearings dealt with DDT's effects on avian reproduction.
iv
Strychnine was also used as an avicide.
v
Sodium fluoroacetate is more commonly known as Compound 1080; most US uses were suspended in 1972; Most US
uses of strychnine and compound 1080, as well as of sodium cyanide, were cancelled in 1985.
vi
Aldrin continued to be used as a termiticide until 1987
vii
Dieldrin use was first restricted in 1971; since it is a major transformation product of aldrin, the restrictions were not very
meaningful until aldrin use was also ended.
viii
These compounds are also highly persistent and quite bioaccumulative
ix
Heptachlor and chlordane may still be used to control fire ants in power plants.
x
Cancelled, USA, 1989. Chlordimeform is a potent bladder carcinogen.
xi
BHC stands for "benzene hexachloride", but the chemical is actually 1,2,3,4,5,6-hexachlorocyclohexane. Its removal may
have been voluntary, due to off flavors, rather than because of its bioaccumulation, carcinogenicity, fetotoxicity,
reproductive and bone marrow toxicity.
xii
Cancellation of all uses, 1985.
xiii
Endrin is a cyclodiene insecticide related to aldrin and dieldrin; it is also quite persistent.
xiv
Use restricted; not fully banned.
xv
Pyriminil causes irreversible diabetes, and also peripheral neuropathy and severe postural hypotension. Nicotinamide
may act as an antidote against some of its chronic effects as well as against its acute lethality. Pyriminil is also interesting
because it its LD50 in primates is >2000 mg/kg (oral, Rhesus monkeys); in humans, as little as 5.6 mg/kg has led to diabetes
and neuropathy.
xvi
Banned in Canada, Sweden; restricted use, USA
10
UIUC Crop Science Dept, 1979
xvii
When chlordane was used as a termiticide, it often volatilized into indoor air. Use as a termiticide was cancelled because
it is both neurotoxic and a suspect carcinogen. Chlordane may still be used to control fire ants in power plants.
xviii
xix
Most uses on food crops were cancelled in 1988
Suspect carcinogen; cancelled (date not known).
xx
Use restricted, USA, 1982, for suspected carcinogenicity. Benomyl is also blamed for problems in orchid growing;
mechanisms not known, but incorrect application was not thought to be the cause.
xxi
Use restricted for teratogenicity; date not given. This is problematic, since teratogenicity bromoxynil's is not considered
to be a serious cause for restriction by teratologists.
xxii
Cancelled of food crop uses, at request of manufacturer, following publicity of residues in apple juice of the
carcinogenic transformation product, unsymmetrical 1,1-dimethylhydrazine