TERATOLOGY 59:323–324 (1999)
Letter to the Editor
Comments on an Article by Roy et al.
(Teratology 58:62–68, 1998)
To the Editor:
A recent article by Roy et al. (Teratology 58:62–68,
1998) reported cytotoxicity, apoptosis, and mitotic ef-
fects in rat embryos exposed in vitro to the organophos-
phate insecticide chlorpyrifos dissolved in dimethylsulf-
oxide (DMSO). They conclude that the effects in cultured
embryos, along with effects on DNA synthesis and
adenylylcyclase after subcutaneous injection of chlor-
pyrifos in DMSO (Song et al.,’97; Whitney et al., ’95;
Dam et al., ’98) signal the potential for adverse effects
that may have gone undetected in conventional develop-
mental and reproductive toxicity studies with chlorpy-
rifos.
Effects on such basic processes as DNA synthesis,
apoptosis, mitotic activity, and adenylyl cyclase re-
ported by these authors, if they were relevant to actual
in vivo exposures, should manifest themselves in the
form of dysmorphogenic effects. Yet developmental and
reproductive toxicity testing has been conducted with
chlorpyrifos according to accepted test guidelines in
rats and mice, with no indication of developmental
effects unless accompanied by significant maternal
toxicity (Breslin et al., ’96; Deacon et al., ’80). Although
fetotoxicity occurred at maternally toxic doses in the
developmental studies, a teratogenic effect did not
occur at any dosage level.
Kimmel (’90, ’98) has pointed out the limitations of in
vitro studies that include compromise of the maternal–
placental–embryo/fetal relationship, and interruption
both of normal embryo/fetal nutrition and of the influ-
ences of maternal metabolism. While in vitro studies
may serve as adjuncts to conventional testing, ‘‘(i)t is
not envisioned that in vitro testing will replace stan-
dard in vivo testing’’ (Kimmel, ’90). While acknowledg-
ing the inherent problems of extrapolation of in vitro
conditions to in vivo exposure, Roy and colleagues
stated that ‘‘assessment of ... developmental neurotoxic-
ity’’ needs to be conducted. Dow AgroSciences, as part of
our product stewardship program, has in fact con-
ducted a guideline developmental neurotoxicity study
in rats using gavage dose levels of ⱕ5 mg/kg/day during
gestation and lactation (Maurissen et al., ’99). At a dose
level of 5 mg/kg/day, which produced clinical cholinergic
r 1999 WILEY-LISS, INC.
signs as well as substantial AChE inhibition in the
brains of the dams, there were no indications of selec-
tive developmental neurotoxic effects, including a lack
of cognitive effects, in the offspring.
The high CPF concentrations used in the in vitro
studies by Roy et al., and in the pulse dosage test
strategies in in vivo studies (e.g., gavage studies, SQ
dosages in DMSO) lead to unrealistic exposure levels.
Roy et al. used concentrations of 0.5–50 µg chlorpyri-
fos/ml for 48 hr for in vitro rat embryo incubations. On
the other hand, measurement of chlorpyrifos blood
levels in pregnant and lactating rats following repeated
oral gavage at doses of 5 mg/kg/day revealed blood
levels on gestation day 20 of only 0.1 µg/ml in the dams,
and approximately 0.05 µg/ml in fetal blood (Mattsson
et al., ’99). Higher dose levels would have resulted in
excessive maternal toxicity which would have pre-
cluded further study. Thus, the concentrations of chlor-
pyrifos used by Roy et al. for whole embryo culture at
steady state were 1–3 orders of magnitude higher than
the fetal blood levels seen from the dams treated at the
maximally tolerated dose. The highest concentration of
chlorpyrifos in human volunteers given a single oral
dose of 0.5 mg/kg (i.e., 50-fold higher than the EPA
short-term exposure guideline for chlorpyrifos) in corn
oil never exceeded 0.03 µg/ml (Nolan et al., ’84).
The relevance of the incubation concentrations and
dose levels used in animal studies should be contrasted
with actual human exposure levels. Fortunately, data
are available that enable human exposure to be esti-
mated from the amount of the chlorpyrifos metabolite,
3,5,6-trichloro-2-pyridinol (TCP), excreted in the urine.
Based on the amount of TCP excreted in the urine (Hill
et al., ’95), the highest chlorpyrifos exposure in a
sample of 1,000 randomly selected adults from through-
out the United States was only 0.0017 µg/g body
weight. Similar population-based estimates derived
*Correspondence to: Thomas R. Hanley, Jr., Global Toxicology, Dow
AgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN 46268–
1054. E-mail: trhanley@dowagro.com
Received 11 November 1998; Accepted 13 November 1998
324 T.R. HANLEY ET AL.
from urinary TCP measurements from children suggest
exposures to chlorpyrifos are comparable to those of
adults (Shurdut, ’98). Thus, even the most highly
exposed individual would absorb only low levels of
chlorpyrifos, which would likely result in miniscule
blood CPF levels (based on animal studies). Hence, the
relevance of results derived from studies in which
exaggerated doses were administered by routes incon-
sistent with actual human exposure and uptake pat-
terns is highly questionable. A recent communiqué in
the Society of Toxicology newsletter (Conolly et al., ’98)
cautions that ‘‘doses that are many multiples of conceiv-
able human exposures and unrealistic routes of expo-
sure [including gavage] raise serious questions of rel-
evance.’’ This communiqué further states that ‘‘(i)t is
time for more widespread acknowledgment of the fact
that dose influences mechanism and it is expected that
mechanism will change with changing dose. Thus,
effects observed at high doses do not necessarily have to
occur following exposure to low doses.’’
THOMAS R. HANLEY, JR.*
BRADLEY A. SHURDUT
RICHARD J. NOLAN
Dow AgroSciences, LLC
Indianapolis, Indiana
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