Human testing: Sass and Needleman respond to industry.

In the March 2004 issue of EHP, we reported on two examples of industry-sponsored studies that intentionally dosed human subjects with toxic pesticides (Sass and Needleman 2004). The four industry-sponsored respondents (Charnley and Patterson 2004; Chart et al. 2004; McAllister 2004; Tobia et al. 2004) focused primarily on their claims that the studies were conducted in accordance with formal ethical guidelines. Although that contention is debatable, it misses fundamental scientific criticisms we raised: a) sample sizes and statistical power were too small to find an effect, if one were present; b) study populations were inappropriate to establish no observed effect levels (NOELs) for children, the most sensitive population; c) outcome measures were unsuitable to establish NOELs for children; and d) study interpretations were biased to ignore or dismiss evidence of adverse health effects. The pesticides under consideration were organophosphate neurotoxins that are chemically related to military nerve gas.

We would like to respond to Charnley and Patterson (2004), Chart et al. (2004), McAllister (2004), and Tobia et al. (2004) in detail.

First, if there is no chance of obtaining valid conclusions, one cannot ethically expose humans to risks. A recent National Research Council (NRC) report (NRC 2004) stated that underpowered studies "cannot be ethically acceptable if [they are] scientifically invalid." The industry human experimentation studies conducted to date employed samples of < 60 subjects (AMVAC 1997; Haines 1971; Wyld et al. 1992). The calculated statistical power to find an effect was in the range of 0.2. This means that they had a one-in-five chance of detecting an effect if it were present, practically guaranteeing a finding of no effect.

Second, the outcome measures in the industry studies were peripheral red blood cell acetyl cholinesterase (AChE) levels, a symptom checklist, and blood pressure (AMVAC 1997; Haines 1971; Wyld et al. 1992). The insensitivity of these measures stands in contrast with recent literature on low-dose noncholinergic toxic effects of the organophosphate pesticides (OPs), including effects on neurogenesis and behavior (Aldridge et al. 2004; Icenogle et al. 2004; Meyer et al. 2004a, 2004b). Although the primary outcome of interest in protecting children is the effect of OPs on the developing brain, neurobehavioral toxicity was not evaluated in any of the industry reports (AMVAC 1997; Haines 1971; Wyld et al. 1992). In addition, whereas evidence of carcinogenic properties has been published on a number of OP pesticides, no attempt has been made to evaluate this or any other chronic end point.

Third, there was no urgent or compelling need for human data, given the existing evidence of toxicity from laboratory experiments. Industry sought information from human studies primarily to avoid the 10-fold safety factor when extrapolating from animal studies to human risk. Eliminating the safety factor would result in setting higher levels of allowable exposure and permit greater sales of the pesticides.

Fourth, the AMVAC Chemical Corporation, sponsor of the dichlorvos (DDVP) study we discussed (AMVAC 1997), reported that although there were statistically significant differences in cholinesterase inhibition between treated and placebo groups, "none of these differences were considered to be of biological significance," and the dose was considered a NOEL. However, the U.S. Environmental Protection Agency (EPA) rejected AMVAC's interpretation of the results, instead concluding that "the reduction in RBC cholinesterase activity was considered by the Hazard ID [identification] Committee to be biologically significant," and the dose tested was considered to be a lowest observed effect level (LOEL) (U.S. EPA 1998b). This illustrates our concern that, "when studies are sponsored by chemical manufacturers with a financial interest in the study outcome, the studies may be biased in design and in interpretation" (Sass and Needleman, 2004).

The responders from Bayer CropScience (Tobia et al. 2004) stated that in our letter we omitted the critical fact that the Bayer-sponsored aldicarb study on human subjects had been reviewed and found "acceptable and appropriate" by a U.S. EPA Scientific Advisory Panel (SAP) in 1992 and reaffirmed in 1998. In 1992 the SAP had no ethical guidelines governing acceptance of human studies. As to the 1998 review, the statement by Tobia et al. (2004) is misleading. In fact, the U.S. EPA statement issued in 1998 to the SAP/Science Advisory Board (SAB) (U.S. EPA 1998a) reads in its entirety:

   [The U.S.] EPA is deeply concerned that some
   pesticide manufacturers seem to be engaging in
   health-effects studies on human subjects as a way
   to avoid more protective results from animal tests
   under the new Food Quality Protection Act. The
   government has in place very stringent standards
   that apply to federally funded research to ensure
   the protection of human subjects. [The U.S.]
   EPA will be asking its independent Science
   Advisor, Board to apply these same standards to
   pesticide data submitted to [The U.S.] EPA by
   companies for review. No human test data [have]
   been used by [The U.S.] EPA for any final decisions
   about acceptable levels of pesticide under
   the new food safety law. The protection of public
   health from adverse effects of pesticides can be
   achieved through reliance on animal testing and
   use of the highest ethical standards.

The U.S. EPA SAP/SAB members were in strong agreement that neurotoxic agents should be given to humans only if there was an urgent and compelling need for the information, and if there was no other way to obtain it (U.S. EPA 2000).

There is growing literature on noncholinergic effects of organophosphates. Meyer et al. (2004a) recently reported developmental alterations in adenylyl cyclase signaling in rat pups exposed to chlorpyrifos, confirming neurodevelopmental effects through noncholinergic mechanisms. Icenogle et al. (2004) reported behavioral changes in adolescent and adult rats after low-dose chlorpyrifos administration. In humans, Berkowitz et al. (2004) reported a small but significant reduction in head circumference in infants born to mothers with detectable levels of chlorpyrifos in their blood, coupled with low maternal paraoxonase (PON1) activity. Perera et al. (2003) reported that chorpyrifos and diazinon found in mother's blood and umbilical cord blood was associated with lower infant birth weight and length, suggesting poorer predicted health outcomes. In an update, Whyatt et al. (2004) reported that regulatory restrictions on the two pesticides measurably lowered exposure and resulted in increased infant head size, providing encouragement for strict regulations to prevent or limit exposure.

The regulation of pesticides must protect against neurodevelopmental and neurobehavioral effects from even low-dose exposures during critical stages in fetal and neonatal development. Measures of cholinesterase in adult peripheral blood are a poor surrogate for these critical effects. The examples of industry-sponsored human tests of pesticides are indefensible on both the scientific and ethical grounds, and U.S. EPA acceptance of such data for setting standards opens the door to serious harm to the general public as well as the subjects of such tests.

The authors declare they have no competing financial interests.

REFERENCES

Aldridge JE, Soldier FJ, Slotkin TA. 2004. Developmental exposure to chlorpyrifos elicits sex-selective alterations of serotonergic synaptic function in adulthood: critical periods and regional selectivity for effects on the serotonin transporter, receptor subtypes, and cell signaling. Environ Health Perspect 112:148-155.

AMVAC. 1997. Dichlorvos: A Single Blind, Placebo Controlled, Randomized Study to Investigate the Effects of Multiple Oral Dosing on Erythrocyte Cholinesterase Inhibition in Healthy Male Volunteers. Report No CTL/P/5392. Study No. XH6063 MRID No. 442488-01, Newport Beach, CA:AMVAC Chemical Corporation.

Berkowitz GS, Wetmur JG, Birman-Deych E, Obel J, Lapinski RH, Godbold JH, et al. 2004. In utero pesticide exposure, maternal paraoxonase activity, and head circumference. Environ Health Perspect 112:388-391.

Charnley G, Patterson J. 2004 Ethical standards of studies involving human subjects [Letter]. Environ Health Perspect 112:A152-A153.

Chart IS, Manley A, Youngren SH. 2004. Study criticisms unjustified [Letter]. Environ Health Perspect 112:A151-A152.

Haines RG 1971. Ingestion of Aldicarb by Human Volunteers: A Controlled Study of the Effect of Aldicarb on Man. Union Carbide Corporation Study No. ALD-03-77-2215 MRID No. 00101911. HED Doc Nos. 007601, 010450. Washington, DC: U.S. Environmental Protection Agency.

Icenogle LM, Christopher NC, Blackwelder WP, Caldwell DP, Qiao D, Soldier FJ, et al. 2004. Behavioral alterations in adolescent and adult rats caused by a brief subtoxic exposure to chlorpyrifos fluring neurulation. Neurotoxicol Teratol 26(1):95-101.

Meyer A, Seidler FJ, Aldridge JE, Tate CA, Cousins MM, Slotkin TA. 2004a. Critical periods for chlorpyrifos-induced developmental neuretoxicity: alterations in adenylyl cyclase signaling in adult rat brain regions after gestational or neonatal exposure. Environ Health Perspect 112:295-301

Meyer A, Seidler FJ, Slotkin TA. 2004b Developmental effects of chlorpyrifos extend beyond neurotoxicity: critical periods for immediate and delayed onset effects on cardiac and hepatic cell signaling. Environ Health Perspect 112:170-178.

McAllister RS. 2004. Statement of CropLife America on pesticide testing involving human subjects [Letter]. Environ Health Perspect 112:A154-A155.

NRC (National Research Council). 2004. Intentional Human Dosing Studies for EPA Regulatory Purposes: Scientific and Ethical Issues, Washington, DC: National Academies Press. Available: http://www.nap.edu/books/0309091721/ html/[accessed 31 March 2004].

Perera FP, Rauh V, Tsai WY, Kinney P, Camann D, Barr D, et al. 2003. Effects of transplacental exposure to environmental pollutants on birth outcomes in a multiethnic population. Environ Health Perspect 111:201-205.

Sass JB, Needleman HL. 2004, Industry testing of toxic pesticides on human subjects concluded "no effect," despite the evidence [Letter]. Environ Health Perspect 112:A150-A191.

Tobia A, Ayers A, Blacker A, Hodges L, Carmichael N. 2004. Aldicarb study misrepresented in human testing debate [Letter]. Environ Health Perspect 112:A155-A156.

U.S. EPA. 1998a. EPA Statement on Human Testing Washington, DC: U.S. Environmental Protection Agency, Available: http://www.epa.gov/oscpmont/sap/1998/ december/epastmt.htm [accessed 31 March 2004].

U.S. EPA. 1998b. Memorandum from JE Stewart, Registration Action Branch II, to C Scheltema, Risk Characterization and Analysis Branch. Review of Toxicity Studies on DDVP Using Human Volunteers (Data Evaluation Reports for MRID Nos. 44317901, 442488-01, and 442488-02). Washington, DC: U.S. Environmental Protection Agency.

U.S. EPA. 2000. Comments on the Use of Data from the Testing of Human Subjects A Report by the Scientific Advisory Board and the FIFRA Scientific Advisory Panel. EPA-SAB-EC-00-017. Washington, DC:U.S. Environmental Protection Agency. Available: http://www.epa.gov/sab/ pdf/ec0017.pdf [accessed 31 March 2004].

Whyatt RM, Barr DB, Camann DE, Kinney PL, Barr JR, Andrews HF, et al. 2003. Contemporary-use pesticides in personal air samples during pregnancy and blood samples at delivery among urban minority mothers and newborns. Environ Health Perspect 111:749-756.

Wyld PJ, Watson CE, Nimmo WS, Watson N. 1992. A Safety and Tolerability Study of Aldicarb at Various Dose Levels in Healthy Male and Female Volunteers, Rhone-Poulenc, Lyon, France, Inveresk Clinical Research Report No. 7786. MRID No. 42373-01. HED Doc No 010459. Washington, DC: U.S. Environmental Protection Agency.

Jennifer B. Sass

Natural Resources Defense Council

Washington, DC

E-mail: [e-mail]

Herbert L. Needleman

University of Pittsburgh School of Medicine

Pittsburgh, Pennsylvania

E-mail: [e-mail]