Table Of Content1
i. Origins of this project
Approximately a decade ago, I was contacted for the first time by the law offices of
Stuart Calwell to give expert advice on the law suit brought by the family of Joey Lee
Walker (henceforth the “Walker Case”). The case, to my recollection, involved the
exposure of Mr. Walker to unregulated levels of chlorpyrifos (henceforth “CPF”), which
produced both acute and delayed pathological reactions. The delayed ones including
epileptic seizures, and Mr. Calwell wanted advice as to how exposure to CPF and related
organophosphates could give rise to delayed seizures.
Numerous possible mechanisms for this connection exist, and my work, initially,
consisted of a) outlining these mechanisms;; b) giving a qualitative assessment of how
well established they are in basic and/or clinical science;; c) outlining alternative routes
that can give rise to seizure disorders;; d) assessing the likelihood that those alternative
routes occurred in the case of Mr. Walker.
In addition, there was an ancillary issue as to whether stress may have
exacerbated the neuropathology in Mr. Walker. I evaluated this possibility as well, and
concluded that it was not very likely.
In the course of doing this work, I was sent a number of published papers and
internal reports from Dow scientists concerning the potential pathological (particularly
neuropathological) consequences of CPF exposure. I found these papers to be of
remarkably poor quality and, upon reporting that informally, was asked to more formally
critique them as part of the Walker report. My overall assessment was that the quality was
so poor that I would be skeptical about the veracity of the contents, and of the contents of
any other work produced by these scientists (including both the direct bench scientists
and the supervisors that signed off on the reports). It should be noted that, from the start,
this negative assessment of the quality of the work and its intrinsic veracity does not imply
any fraudulence on the part of these scientists.
Similar work was done in the cases of Christina and Alex Ebling (henceforth, the
“Ebling Case”). To my recollection, the case involved two children exposed to unregulated
levels of CPF who had sustained extensive neurological damage. My role was the same
in explaining the science underlying such damage. Again, my report also included the
critique I had made of the handful of published and internal Dow reports regarding CPF,
with a strong emphasis on their poor quality.
Around the time of depositions in the Ebling case, the idea emerged among Mr.
Calwell, Mr. Roger Pardieck, Ms. Regan Quinn and myself to make a more formal
analysis of all of the relevant papers published by Dow scientists, with the primary goal
being to determine whether the poor quality of the handful already examined extended to
the entire body of work. The expectation at the time was that this would involve hundreds
and perhaps thousands of papers. For that reason, from the start, the idea included
recruiting a team of experts to carry out this analysis under my supervision. It was
eventually decided that this project would be carried out.
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Recruitment and selection of the team of experts to evaluate papers
A team was recruited with the following characteristics:
A. All members were neuroscientists with experimental bench work experience. In
principle, any experimental bench researcher working in the life sciences (e.g., a
hematologist) could assess the quality of the work, as the errors were of a type
recognizable to any scientist in that broader category. However, the process would be
slower and more difficult for non-neuroscientists because of the unfamiliarity with the
scientific content. The decision was made not to restrict the team to neurotoxicologists
because of the rarity of such individuals fitting the other requirements;; in fact, none were
neurotoxicologists. Despite this, all neuroscientists share a common vocabulary and core
knowledge base which allows for informed reading of papers in differing sub-disciplines of
neuroscience.
B. All members were affiliated at the time with Stanford University. This was for two
reasons. The first was because of the convenience of working with people at the same
institution. The second was because of the quality of scientists, particularly
neuroscientists, at Stanford. The team was made up of scientists affiliated with the
Neuroscience Program and/or the Biology Department. The latter was ranked as the #1
biology department in the country by U.S. News & World Report for the majority of
the years that this project was carried out;; this is considered to be the most visible and
influential ranking system that there is for American colleges and universities. The
Neuroscience Program was rated in the top 5 departments throughout this period, as well
as by the Science Citation Index (a measure of the impact of papers in their field, as
assessed by the frequency with which they are cited subsequently in the work of other
scientists).
C. All members were post-doctoral scientists. Such scientists are individuals who
have received their PhD’s and then spend approximately 2-5 years training in a different
laboratory. This is meant to expand upon their graduate training, and to prepare them for
establishing an independent research career of their own. Most such post-docs occur in
academia, while a small percentage occur in industry (i.e., pharmaceutical and/or biotech
companies that are typically large enough to have basic science divisions). Following a
successful post-doc, individuals ideally then obtain a job in academia (i.e., a faculty
position) or industry. The decision was made to recruit post-doctoral scientists (rather
than graduate students and/or professors) for two reasons. First, the quality of scientific
training at the post-doctoral level is considerably higher than at the graduate student
level, and such a level was thought necessary for this project. Second, in contrast to
most faculty members, post-doctoral scientists are still predominately doing bench work
(i.e., carrying out experiments on their own), rather than supervising the work of others,
and this generally gives more insight into small details of scientific papers.
Individuals meeting the criteria above were excluded from the project if a) they
knew any of the scientists whose papers were to be critiqued;; b) any laboratory that they
had worked in had been supported by funds from Dow Chemical or from an affiliate of
Dow Chemical;; c) if they or family members had any stocks in Dow Chemical or its
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affiliates;; d) if they had been involved in any related legal cases, including as an expert
witness.
Team members were recruited in early 2006 by sending an announcement through
an email list of post doctoral scientists at Stanford. The project was described as involving
critiquing a body of published neuroscience research. The announcement did not make
mention of Dow, CPF, or organophosphate pesticides. The announcement did make
mention that this was related to a legal project;; no indication was made whether the work
would be done for a plaintiff or a defendant. The announcement indicated that pay would
be $50/hour.
Ten individuals applied;; one was excluded because of family investments in Dow
Chemical. In late 2007, a second team was recruited. This was because of a certain
degree of attrition of the original team, either because the individual, a) no longer had
time to work on the project, or b) had left Stanford to a position elsewhere. Six
individuals applied;; one was excluded because of a history of working in a lab receiving
funding from Dow. A few months later, an additional person was added on to pick up
the pace of the work.
Appendix I consists of the CV’s of these 15 individuals at the time of their
recruitment. Individuals had degrees from universities such as Cornell, Harvard,
Stanford, U.C. Berkeley, U. Pennsylvania and Yale. Collectively, team members had
published more than 100 papers reporting experimental research in peer-reviewed
English language scientific journals (excluding book chapters and reviews);; this
represents a high level of productivity for neuroscientists at this stage of training.
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Establishing quality control prior to data generation
The broad goal was for the team to read and critique papers by Dow scientists
concerning CPF, along with a number of types of control papers (described below).
Quality control was established before analysis began (with additional features added
once data were generated). These included:
a. Each paper was to be analyzed by two members of the team.
b. Members were instructed not to discuss papers with any other team members.
c. The data base of evaluations was not accessible to any team members (i.e.,
they could input data but could not see reports of other individuals).
An extensive section after the Results discusses additional quality controls in this
study.
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Identification of the chlorpyrifos papers by Dow scientists to be analyzed
Relevant papers were searched for on “PubMed,” which is the most commonly
used search engine for biomedical and life science papers published in professional
journals, with more than 10 million papers accessible from it. A search was carried out
with the search terms “Dow” and “chlorpyrifos.” A total of 46 papers were identified. Six
were eliminated, as they turned out not to be by Dow authors.
An additional 21 papers were eliminated as they were not relevant to the analysis.
This was because they were a) purely methodological in nature;; b) about non-mammalian
species;; c) about agricultural issues, such as levels of chlorpyrifos residues in soil or food;;
d) merely an abstract. These 21 were:
a. A total of 11 papers were eliminated because they were purely
methodological (e.g., describing an improved or less expensive technique for measuring
chlorpyrifos breakdown products in the urine, a new statistical method for analyzing
epidemiological data, a way of assessing the accuracy of estimates of occupational
pesticide exposure, a theoretical model for analyzing exposure to a pesticide). Neither
the content nor the quality of such papers would be directly relevant to issues of the
extent of CPF neurotoxicity. These papers were:
Aggregate exposures under the Food Quality Protection Act: An approach using
chlorpyrifos. Shurdut BA, Barraj L, Francis M. Regul Toxicol Pharmacol. 1998
Oct;;28(2):165-77. Review. PMID: 9927565 [PubMed - indexed for MEDLINE]
Analysis of 3,5,6-trichloropyridinol in human urine using negative-ion chemical
ionization gas chromatography-mass spectrometry. Bartels MJ, Kastl PE. J Chromatogr.
1992 Mar 13;;575(1):69-74. PMID: 1517302 [PubMed - indexed for MEDLINE]
On-line coupling of supercritical fluid extraction with multidimensional microcolumn
liquid chromatography/gas chromatography. Cortes HJ, Green LS, Campbell RM. Anal
Chem. 1991 Dec 1;;63(23):2719-24. PMID: 1722652 [PubMed - indexed for MEDLINE]
Semiautomated preparation of 3,5,6-trichloro-2-pyridinol in human urine using a
Zymate XP laboratory robot with quantitative determination by gas chromatography-
negative-ion chemical ionization mass spectrometry. Ormand JR, McNett DA, Bartels MJ.
J Anal Toxicol. 1999 Jan-Feb;;23(1):35-40. PMID: 10022207 [PubMed - indexed for
MEDLINE]
Exposure endpoint selection in acute dietary risk assessment. Wolt JD. Regul
Toxicol Pharmacol. 1999 Jun;;29(3):279-86. PMID: 10388613 [PubMed - indexed for
MEDLINE]
Practical immunochemical method for determination of 3,5, 6-trichloro-2-pyridinol
in human urine: applications and considerations for exposure assessment. Shackelford
DD, Young DL, Mihaliak CA, Shurdut BA, Itak JA. J Agric Food Chem. 1999
Jan;;47(1):177-82. PMID: 10563869 [PubMed - indexed for MEDLINE]
Refinements in acute dietary exposure assessments for chlorpyrifos. Wright JP,
Shaw MC, Keeler LC. J Agric Food Chem. 2002 Jan 2;;50(1):235-41. PMID: 11754574
[PubMed - indexed for MEDLINE]
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Strategies to assess systemic exposure of chemicals in subchronic/chronic diet
and drinking water studies. Saghir SA, Mendrala AL, Bartels MJ, Day SJ, Hansen SC,
Sushynski JM, Bus JS. Toxicol Appl Pharmacol. 2006 Mar 15;;211(3):245-60. Epub 2005
Jul 22. PMID: 16040073 [PubMed - indexed for MEDLINE]
Human red blood cell acetylcholinesterase inhibition as the appropriate and
conservative surrogate endpoint for establishing chlorpyrifos reference dose. Chen WL,
Sheets JJ, Nolan RJ, Mattsson JL. Regul Toxicol Pharmacol. 1999 Feb;;29(1):15-22.
Review. PMID: 10051415 [PubMed - indexed for MEDLINE]
The effect of route, vehicle, and divided doses on the pharmacokinetics of
chlorpyrifos and its metabolite trichloropyridinol in neonatal Sprague-Dawley rats. Marty
MS, Domoradzki JY, Hansen SC, Timchalk C, Bartels MJ, Mattsson JL. Toxicol Sci. 2007
Dec;;100(2):360-73. Epub 2007 Oct 10. PMID: 17928393 [PubMed - indexed for
MEDLINE]
Chlorpyrifos exposure and biological monitoring among manufacturing workers.
Burns CJ, Garabrant D, Albers JW, Berent S, Giordani B, Haidar S, Garrison R,
Richardson RJ. Occup Environ Med. 2006 Mar;;63(3):218-20. PMID: 16497866 [PubMed -
indexed for MEDLINE]
b. A total of five papers were eliminated because they were concerned with
chlorpyrifos effects on non-mammalian species (fish, insects or shellfish). While such
studies are of relevance to issues of agriculture and aquaculture, they are only distantly
related to the core issues of CPF effects on mammalian physiology. These papers were:
Evidence for negative cross resistance to insecticides in field collected Spodoptera
littoralis (Boisd.) from Lebanon in laboratory bioassays. Miles M, Lysandrou M. Meded
Rijksuniv Gent Fak Landbouwkd Toegep Biol Wet. 2002;;67(3):665-9. PMID: 12696435
[PubMed - indexed for MEDLINE]
The bioconcentration and metabolism of chlorpyrifos by the eastern oyster,
Crassostrea virginica. Woodburn KB, Hansen SC, Roth GA, Strauss K. Environ Toxicol
Chem. 2003 Feb;;22(2):276-84. PMID: 12558158 [PubMed - indexed for MEDLINE]
An ecological risk assessment for chlorpyrifos in an agriculturally dominated
tributary of the San Joaquin River. Poletika NN, Woodburn KB, Henry KS. Risk Anal.
2002 Apr;;22(2):291-308. PMID: 12022677 [PubMed - indexed for MEDLINE]
Hansen SC, Roth GA, Strauss K. The bioconcentration and metabolism of
chlorpyrifos by the eastern oyster, Crassostrea virginica. Environ Toxicol Chem. 2003
Feb;;22(2):276-84. PMID: 12558158 [PubMed
Barron MG, Plakas SM, Wilga PC. Chlorpyrifos pharmacokinetics and metabolism
following intravascular and dietary administration in channel catfish. Toxicol Appl
Pharmacol. 1991 May;;108(3):474-82. PMID: 1708533 [PubMed – indexedkljlkj\
c. One paper was eliminated because it was merely an abstract (i.e., not a full-
length, peer-reviewed paper). The paper was:
Chlorpyrifos (Dursban) and Dow employees. Sherman JD. Environ Health
Perspect. 1999 Mar;;107(3):A132-4. No abstract available. PMID: 10391732 [PubMed -
indexed for MEDLINE]
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d. Four papers were eliminated because they focused on quantities of CPF residue
in soil or food, or the effects of food processing techniques on quantities of residues.
Such studies would be pertinent to determining the amount of CPF people might be
exposed to from particular food sources, but are not relevant to issues of the physiological
and pathophysiological effects of CPF in the body. These papers were:
Stability of chlorpyrifos for termiticidal control in six Australian soils. Murray RT,
von Stein C, Kennedy IR, Sanchez-Bayo F. J Agric Food Chem. 2001 Jun;;49(6):2844-7.
PMID: 11409976 [PubMed - indexed for MEDLINE]
Determining kinetic and nonequilibrium sorption behavior for chlopyrifos using a
hybrid batch/column experiment. Cryer SA. J Agric Food Chem. 2005 May
18;;53(10):4103-9. PMID: 15884846 [PubMed - indexed for MEDLINE]
The effect of cooking on chlorpyrifos and 3,5,6-trichloro-2-pyridinol levels in
chlorpyrifos-fortified produce for use in refining dietary exposure. Byrne SL, Pinkerton SL.
J Agric Food Chem. 2004 Dec 15;;52(25):7567-73. PMID: 15675805 [PubMed - indexed
for MEDLINE]
U.S. market basket study to determine residues of the i nsecticide chlorpyrifos.
Bolles HG, Dixon-White HE, Peterson RK, Tomerlin JR, Day EW Jr, Oliver GR. J Agric
Food Chem. 1999 May;;47(5):1817-22. PMID: 10552457 [PubMed - indexed for
MEDLINE]
Of the remaining papers, nine were identified as the “core” literature. They had
the following characteristics: a) study subjects were mammals (rats, mice, rabbits);; b) the
studies were experimental in nature (i.e., where subjects were exposed experimentally to
CPF, and endpoints were then measured;; c) endpoints were physiological or behavioral.
d) the structures of the experiments were such that the paper gave insight as to the safety
of CPF.
The nine papers were:
#1. Deacon MM, Murray JS, Pilny MK, Rao KS, Dittenber DA, Hanley TR Jr, John
JA. Embryotoxicity and fetotoxicity of orally administered chlorpyrifos in mice.
Toxicol Appl Pharmacol. 1980 Jun 15;;54(1):31-40. No abstract available. PMID: 6156524
[PubMed - indexed for MEDLINE]
#2. Maurissen JP, Hoberman AM, Garman RH, Hanley TR Jr. Lack of selective
developmental neurotoxicity in rat pups from dams treated by gavage with
chlorpyrifos. Toxicol Sci. 2000 Oct;;57(2):250-63. PMID: 11006355 [PubMed - indexed
for MEDLINE]
#3. Maurissen JP, Shankar MR, Mattsson JL. Chlorpyrifos: lack of cognitive
effects in adult Long-Evans rats. Neurotoxicol Teratol. 2000 Mar-Apr;;22(2):237-46.
PMID: 10758353 [PubMed - indexed for MEDLINE]
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#4. Yano BL, Young JT, Mattsson JL. Lack of carcinogenicity of chlorpyrifos
insecticide in a high-dose, 2-year dietary toxicity study in Fischer 344 rats. Toxicol Sci.
2000 Jan;;53(1):135-44. PMID: 10653531 [PubMed - indexed for MEDLINE]
#5. Hanley TR Jr, Carney EW, Johnson EM. Developmental toxicity studies in rats
and rabbits with 3,5,6-trichloro-2-pyridinol, the major metabolite of chlorpyrifos. Toxicol
Sci. 2000 Jan;;53(1):100-8. PMID: 10653527 [PubMed - indexed for MEDLINE]
#6. Mattsson JL, Wilmer JW, Shankar MR, Berdasco NM, Crissman JW,
Maurissen JP, Bond DM. Single-dose and 13-week repeated-dose neurotoxicity
screening studies of chlorpyrifos insecticide. Food Chem Toxicol. 1996 Apr;;34(4):393-
405. PMID: 8641666 [PubMed - indexed for MEDLINE]
#7. Breslin WJ, Liberacki AB, Dittenber DA, Quast JF. Evaluation of the
developmental and reproductive toxicity of chlorpyrifos in the rat. Fundam Appl Toxicol.
1996 Jan;;29(1):119-30. PMID: 8838647 [PubMed - indexed for MEDLINE]
#8. Gollapudi BB, Mendrala AL, Linscombe VA. Evaluation of the genetic toxicity of
the organophosphate insecticide chlorpyrifos. Mutat Res. 1995 Mar;;342(1-2):25-36.
PMID: 7533892 [PubMed - indexed for MEDLINE]
#9. Corley RA, Calhoun LL, Dittenber DA, Lomax LG, Landry TD. Chlorpyrifos: a
13-week nose-only vapor inhalation study in Fischer 344 rats. Fundam Appl Toxicol. 1989
Oct;;13(3):616-8. PMID: 2482212 [PubMed - indexed for MEDLINE]
Henceforth, throughout this report, these will be referred to as the “core”
Dow CPF papers.
The remaining 10 papers were identified as the “secondary” set of papers. All of
were capable of producing results pertinent to the safety of CPF. They were either:
A. Epidemiological (i.e., the relationship between spontaneous patterns of disease
and of CPF exposure in populations of humans). These were not included in the core set
of papers because of the non-experimental nature of the studies (i.e., in contrast to the
studies with animals in which there was intentional exposure to CPF). The nature of the
errors detected in the initial set of Dow papers was mostly intrinsic to experimental
studies.
B. Concerned with pharmacodynamics of CPF (i.e., the dynamics of how it
distributes in various compartments in the body). These were not included in the core
set of papers because they did not concern whether CPF had an adverse effect on the
body. Instead, they concerned the time course of CPF presence in the body.
C. Concerned with the effects of CPF on cholinesterases. This differs from the
“core” set of papers. In that group, it is not known if CPF causes toxicity, and the studies
are meant to determine that. In this category, inhibition of cholinesterases is the defining
action of CPF and is not in question;; these papers look at the effects of CPF on different
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cholinesterases in the body, determining, for example, whether they are all inhibited by
CPF to the same extent.
a. Papers (total of 5) that were epidemiological in nature:
Albers JW, Garabrant DH, Mattsson JL, Burns CJ, Cohen SS, Sima C, Garrison
RP, Richardson RJ, Berent S. Dose-effect analyses of occupational chlorpyrifos
exposure and peripheral nerve electrophysiology. Toxicol Sci. 2007 May;;97(1):196-204.
Epub 2007 Feb PMID: 17324952 [PubMed - indexed for MEDLINE]
Brenner FE, Bond GG, McLaren EA, Green S, Cook RR. Morbidity among
employees engaged in the manufacture or formulation of chlorpyrifos. Br J Ind Med. 1989
Feb;;46(2):133-7. PMID: 2466478 [PubMed - indexed for MEDLINE]
Byrne SL, Shurdut BA, Saunders DG. Potential chlorpyrifos exposure to residents
following standard crack and crevice treatment. Environ Health Perspect. 1998
Nov;;106(11):725-31. PMID: 9799188 [PubMed - indexed for MEDLINE]
Burns CJ, Cartmill JB, Powers BS, Lee MK. Update of the morbidity experience of
employees potentially exposed to chlorpyrifos. Occup Environ Med. 1998 Jan;;55(1):65-
70. PMID: 9536166 [PubMed - indexed for MEDLINE]
Gibson JE, Peterson RK, Shurdut BA. Human exposure and risk from indoor use
of chlorpyrifos. Environ Health Perspect. 1998 Jun;;106(6):303-6. Review. PMID: 9618344
[PubMed - indexed for MEDLINE]
b. Pharmacodynamics papers (total of 2):
Brzak KA, Harms DW, Bartels MJ, Nolan RJ. Determination of chlorpyrifos,
chlorpyrifos oxon, and 3,5,6-trichloro-2-pyridinol in rat and human blood. J Anal Toxicol.
1998 May-Jun;;22(3):203-10. PMID: 9602936 [PubMed –
Nolan RJ, Rick DL, Freshour NL, Saunders JH. Chlorpyrifos: pharmacokinetics in
human volunteers. Toxicol Appl Pharmacol. 1984 Mar 30;;73(1):8-15. PMID: 6200956
[PubMed - indexed for MEDLINE]
c. Papers (total of 3) concerned with cholinesterase as an endpoint:
Marable BR, Maurissen JP, Mattsson JL, Billington R. Differential sensitivity of
blood, peripheral, and central cholinesterases in beagle dogs following dietary exposure
to chlorpyrifos. Regul Toxicol Pharmacol. 2007 Apr;;47(3):240-8. Epub 2006 Dec PMID:
17141929 [PubMed - indexed for MEDLINE]
Mattsson JL, Maurissen JP, Nolan RJ, Brzak KA. Lack of differential sensitivity to
cholinesterase inhibition in fetuses and neonates compared to dams treated perinatally
with chlorpyrifos. Toxicol Sci. 2000 Feb;;53(2):438-46. PMID: 10696792
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Mattsson JL, Holden L, Eisenbrandt DL, Gibson JE. Reanalysis with optimized
power of red blood cell acetylcholinesterase activity from a 1-year dietary treatment of
dogs to chlorpyrifos. Toxicology. 2001 Mar 7;;160(1-3):155-64. PMID: 11246135
The initial wave of analyses was done on a) the “core” 9 papers, and b) the
core plus the 10 “secondary” papers.
Description:Stability of chlorpyrifos for termiticidal control in six Australian soils. Murray RT, effects in adult Long-Evans rats power of red blood cell acetylcholinesterase activity from a 1-year dietary treatment of dogs to .. in levels of acetylcholine-esterase (“ChE,” a key enzyme in the brain) in
