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 culturedembryos, along with effects on DNA synthesis andadenylylcyclase 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 effectsthat 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 actualin vivo exposures, should manifest themselves in theform of dysmorphogenic effects. Yet developmental andreproductive toxicity testing has been conducted withchlorpyrifos according to accepted test guidelines inrats and mice, with no indication of developmentaleffects unless accompanied by significant maternaltoxicity (Breslin et al., ’96; Deacon et al., ’80). Althoughfetotoxicity occurred at maternally toxic doses in thedevelopmental studies, a teratogenic effect did notoccur at any dosage level.

Kimmel (’90, ’98) has pointed out the limitations of invitro studies that include compromise of the maternal–placental–embryo/fetal relationship, and interruptionboth of normal embryo/fetal nutrition and of the influ-ences of maternal metabolism. While in vitro studiesmay serve as adjuncts to conventional testing, ‘‘(i)t isnot envisioned that in vitro testing will replace stan-dard in vivo testing’’ (Kimmel, ’90). While acknowledg-ing the inherent problems of extrapolation of in vitroconditions to in vivo exposure, Roy and colleaguesstated that ‘‘assessment of ... developmental neurotoxic-ity’’ needs to be conducted. Dow AgroSciences, as part ofour product stewardship program, has in fact con-ducted a guideline developmental neurotoxicity studyin rats using gavage dose levels of #5 mg/kg/day duringgestation and lactation (Maurissen et al., ’99). At a doselevel of 5 mg/kg/day, which produced clinical cholinergic

signs as well as substantial AChE inhibition in thebrains of the dams, there were no indications of selec-tive developmental neurotoxic effects, including a lackof cognitive effects, in the offspring.

The high CPF concentrations used in the in vitrostudies by Roy et al., and in the pulse dosage teststrategies in in vivo studies (e.g., gavage studies, SQdosages 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. Onthe other hand, measurement of chlorpyrifos bloodlevels in pregnant and lactating rats following repeatedoral gavage at doses of 5 mg/kg/day revealed bloodlevels on gestation day 20 of only 0.1 µg/ml in the dams,and approximately 0.05 µg/ml in fetal blood (Mattssonet al., ’99). Higher dose levels would have resulted inexcessive maternal toxicity which would have pre-cluded further study. Thus, the concentrations of chlor-pyrifos used by Roy et al. for whole embryo culture atsteady state were 1–3 orders of magnitude higher thanthe fetal blood levels seen from the dams treated at themaximally tolerated dose. The highest concentration ofchlorpyrifos in human volunteers given a single oraldose of 0.5 mg/kg (i.e., 50-fold higher than the EPAshort-term exposure guideline for chlorpyrifos) in cornoil never exceeded 0.03 µg/ml (Nolan et al., ’84).

The relevance of the incubation concentrations anddose levels used in animal studies should be contrastedwith actual human exposure levels. Fortunately, dataare 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 (Hillet al., ’95), the highest chlorpyrifos exposure in asample of 1,000 randomly selected adults from through-out the United States was only 0.0017 µg/g bodyweight. Similar population-based estimates derived

*Correspondence to: Thomas R. Hanley, Jr., Global Toxicology, DowAgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN 46268–1054. E-mail: trhanley@dowagro.com

Received 11 November 1998; Accepted 13 November 1998

TERATOLOGY 59:323–324 (1999)

r 1999 WILEY-LISS, INC.

from urinary TCP measurements from children suggestexposures to chlorpyrifos are comparable to those ofadults (Shurdut, ’98). Thus, even the most highlyexposed individual would absorb only low levels ofchlorpyrifos, which would likely result in minisculeblood CPF levels (based on animal studies). Hence, therelevance of results derived from studies in whichexaggerated doses were administered by routes incon-sistent with actual human exposure and uptake pat-terns is highly questionable. A recent communique inthe 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 communique further states that ‘‘(i)t istime for more widespread acknowledgment of the factthat dose influences mechanism and it is expected thatmechanism will change with changing dose. Thus,effects observed at high doses do not necessarily have tooccur following exposure to low doses.’’

THOMAS R. HANLEY, JR.*BRADLEY A. SHURDUTRICHARD J. NOLANDow AgroSciences, LLCIndianapolis, Indiana

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Song X, Seidler FJ, Saleh JL, Zhang J, Padilla S, Slotkin TA. 1997.Cellular mechanisms for developmental toxicity of chlorpyrifos:Targeting the adenylyl cyclase signaling cascade. Toxicol ApplPharmacol 145:158–174.

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324 T.R. HANLEY ET AL.