Epidemiology: Case Analysis of

Pesticides (2,4-D) Commonly Used

in Agricultural Production

James S. Bus PhD, DABT, ATS

Exponent, Inc.
Argentine Toxicological Association Conference
September 26, 2014
Comodoro, Argentina
Epidemiology: Interpretation Challenges

• Study type:
• Cohort, case-control, ecologic
• Study size: limited numbers of participants
• Exposure characterization
• Survey history provides indirect evaluation
• Direct measurement limited
• Confounders
• Multiple chemical exposures
• Lifestyle factors, etc.
Toxicology: Aiding Interpretation of Epidemiology Studies

• Mode of Action
• Providing biological plausibility to epidemiology
evaluations
• Hypothesis generation
• “The dose makes the poison”
• Connecting dosimetry (dose response) in
experimental toxicity studies to human exposures
• Weight-of-Evidence
• Enhancing science-informed risk assessments
reflecting both animal and human data
2,4-D: Why an Excellent Case Example?

• Long history of use

• 1st registered crop protection chemical in North
America: 1947
• Many decades of experience
• Extensive epidemiology data
• Well characterized human exposures
• Human biomonitoring: 2,4-D in urine
• Extensive state-of-art animal toxicology database
• Tests cover health endpoints of concern
• Toxicokinetic data links 2,4-D toxicity test data to
human exposures
2,4-D Epidemiology: Association with Non-Hodgkins
Lymphoma (NHL)?
 Interpreting epidemiology for risk
assessment
• Strength of association
– Size, statistical significance and precision
– Important to consider methodological strength
• Consistency
– Across different investigators, settings
– Within a study
• Dose-response
2,4-D Epidemiology: Association with Non-Hodgkins
Lymphoma (NHL)?
 Early studies not specific to 2,4-D or NHL
Later studies larger, better design

Study OR CI Sig? CIR Comment

Smith 92 2.7 0.7-9.6 No 13.7 Least robust

Included HD,
All phenoxies
Hardell 94 5.5 2.2-11.0 Yes 5 Co-exposure with
2,4,5-T
Mills 05 3.8 1.9-7.8 Yes 4.1 Bystander exposure

Miligi 06 0.9 0.5 – 1.8 No 3.6 Largest study

DeRoos 03 0.9 0.6-1.2 No 2 Most robust

Pooled 3 studies
 Toxicology: What Health Concerns are Addressed?

• Short term (acute) effects:

– Lethality by multiple routes of exposure (oral, skin, and inhalation)
– Eye and skin irritation and corrosivity
– Sensitization – allergic response?
• Longer term (subchronic, chronic) effects: Cancer, chronic diseases
– Lifetime studies in rats and mice; 2 year studies in dogs
• Genetic toxicity – damage to genetic material, e.g., DNA, etc.?
• Reproductive toxicity – assessed over multiple generations in rats
• Developmental toxicity (birth defects?) – rats and rabbits
• Neurotoxicity, often including developmental neurotoxicity – rats
• Immunotoxicity – rats or mice – immune suppression?
• Endocrine effects: In vitro & in vivo; impact on hormone systems?
• Metabolism and pharmacokinetics – How is chemical handled in body?
• Specialized studies: Mode of action - Do animal effects predict human
effects?
 2,4-D Toxicology: Informing Epidemiology
Carcinogenicity & Subchronic Toxicity
Charles et al. Fund. Appl. Toxicol. 33: 161-165 (1996); subchronic rat
Charles et al. Fund. Appl. Toxicol. 33: 166-172 (1996) ; rat & mouse bioassays
Genotoxicity
Charles et al. Mut. Res. 444: 207-216 (1999); Ames assays & UDS
Gollapudi et al. Mut. Res. 444: 217-225 (1999); Mammalian cell genotoxicity
Charles et al. Mut. Res. 444: 227-234 (1999); in vivo Micronucleus
Developmental Toxicity
Charles et al. Toxicol. Sci. 60: 121-131 (2001); Rat and rabbit teratology
Reproductive and Endocrine Toxicity, Developmental Neuro- and Immuno-
toxicity
Coady et al., Toxicol. In Vitro 28: 1018-1025 (2014)
Coady et al., Ecotox. Env. Safety 90:143-150 (2013)
Marty et al. Toxicol. Sci. 136: 527-547 (2013); Extended one-generation study
(reproduction, developmental neuro- and immunotoxicity, endocrine)
Neurotoxicity
Mattsson et al. Fund. Appl. Toxicol. 40: 111-119 (1997); Acute & chronic, rat

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2,4-D Toxicokinetics (TK): Informing Interpretation of
Toxicology Studies
• 2,4-D subject to non-linear TK
– Renal OAT-1 Transporter
• Subject to saturation
• Males have higher levels of OAT-1 expression than females
• Rats and humans express OAT-1, dogs do not

5 mg/kg 50 mg/kg
TK Parameter
Male Female Male Female
Rat
Cmax (µg eq./g) 10 14 190 267
AUC(0-t) (µg eq.h/g) 20 57 1222 2358
Dog
Cmax (µg eq./g) 32 34 233 224
AUC(0-t) (µg eq.h/g) 2579 2853 18,310 19,956

vanRavenzwaay et al., Xenobiotica 33: 805-821, 2003

Implications of Dietary Toxicokinetic Evaluations for Toxicity
Test Design and Interpretation: 2,4-D

Modified from Saghir et al. TAAP

211: 245-260, 2006

Oral Gavage:
5 mg/kg: AUC = 21; Cmax = 9.8
50 mg/kg: AUC = 1228; Cmax = 190
 Toxicokinetics: Facilitating Toxicity Test Dose Selection
and Study Interpretation
• ILSI-HESI ACSA publications
– Carmichael et al., Agricultural Chemical Safety Assessment: A
Multisector Approach to the Modernization of Human Safety
Requirements. Crit.Rev.Toxicol. 36: 1-7, 2006
– Barton et al., The acquisition and application of absorption,
distribution, metabolism, and excretion (ADME) data in agricultural
chemical safety assessments. Crit. Rev. Toxicol. 36:9–35, 2006

• ACSA recommends setting top dose levels for toxicity studies which are at
or slightly above the inflection point of saturated metabolic and/or
clearance mechanisms, as long as the inflection point is well separated
from human exposures
– “Kinetically-Derived Maximum Dose” (KMD) – Saghir etal,
Regul.Toxicol.Pharmacol. 63: 321-332, 2012.
– KMD concept incorporated into OECD Test Guideline 443 Extended
One-Generation Reproductive Toxicity Study (2010)

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 2,4-D Toxicokinetics: Implications or Toxicity Study Design
(Kinetically Derived Maximum Dose (KMD) Determination)
6000

5000
( ug/ml/h )

4000
MTD: 52mg/kg/day
24 h

(decreased body wts)

3000 KMD: < 26 mg/kg/day
Plasma AUC

(saturation of renal

Expected dose clearance)

2000
proportional increase
based on test material
1000
intake

Human Exposure
(>10,000X below 0
low dose) 0 10 20 30 40 50 60 70 80 90

-
2,4 D Dose (mg/kg/day)

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Low High Traditional
Dose Dose High Dose
 Exposure: Plausibility Context to Epidemiology

“Biomonitoring Equivalents” (BE) addresses the questions:

If humans are exposed to 2,4-D at doses equal to the regulatory RfD, what
would be the expected urine and/or blood concentrations, i.e., BE of an
RfD exposure?

How do the modeled urine/blood concentrations compare to what is

actually detected in human biomonitoring studies?

BE values for 2,4-D substantially higher than actual real-world

urine/blood concentrations detected in biomonitoring studies indicates a
low concern for adverse human health risks

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 2,4-D Biomonitoring Equivalents: Improving Context of
Animal Toxicity Studies to Human Exposures

BE Worker
20,000 µg/L

BE Population
2,000 µg/L
Farmer
biomonitoring

Rural
biomonitoring

Adapted from Aylward, 2010, Environ Health Perspect 118,177-181

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Relating in vitro Concentrations to Human Exposures
(“Reverse Dosimetry” or “Dosimetric Anchoring”)

Rotroff etal., Tox.Sci. 117: 348-358, 2010

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2,4-D: The Epidemiology/Toxicology/Exposure Case

“Epidemiologic studies provide scant evidence that exposure to 2,4-D is

associated with soft tissue sarcoma, non-Hodgkin’s lymphoma, Hodgkin’s
disease, or any other cancer. Overall, the available evidence from epidemiologic
studies is not adequate to conclude that any form of cancer is causally associated
with 2,4-D exposure.”

Garabrant and Philbert, Crit. Rev. Toxicol. 32: 233-257 (2002)

“The 2,4-D epidemiology literature after 2001 is broad and includes studies of
cancer, reproductive toxicity, genotoxicity, and neurotoxicity. In general, a few
publications have reported statistically significant associations. However, most
lack precision and the results are not replicated in other independent studies. In
the context of biomonitoring, the epidemiology data give no convincing or
consistent evidence for any chronic adverse effect of 2,4-D in humans.”

Burns and Swaen, Crit. Rev. Toxicol. 42: 768-786 (2012)

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