CHLORPYRIFOS EFFEC

DEVELOP

ANIM

GINGER MOSER, PHD, DA

OFFICE OF RESEARCH AND 

FIFRA SAP 
Background

 SAP in 2008 review of anima

concluded:

 “independent of exact time frame o

postnatal exposure, only doses of 1 greater demonstrate significant effebehavior”

 Two areas of concern

 Inconsistent behavioral changes Lack of dose-response and data on ChE inh

 Recommended review of critical eve

exposure and targeted endpoints

2
Focus of Current Developmen

Neurotoxicity Review

 2012 review aims to assess neuro

outcomes in terms of:

Domains of neurological functionCritical exposure periodsEvidence of brain ChE inhibition du

periods

Targeted review of publications si

New informationIntegration with existing database

3
Existing Literature

4

 26 papers* from 11 laboratories

 Include 9 since 2008, plus 2 new laborat Focus on 20 papers from 9 laboratories 25 actual studies

 Focus review on papers with:

 Dosing any time up to weaning Assessments after weaning to reflect long Lower doses, avoid toxic levels of ChE inh Rodents
Existing Literature

5

 Variability in these 25 studies include di

 Exposure paradigms

 10 gestational studies with 7 dosing periods 13 postnatal studies with 5 dosing periods 2 perinatal studies with different dosing periods

 Routes of exposure and vehicles

 4 routes of administration

 subcutaneous, oral, dermal, implant

 4 vehicles

 DMSO, peanut oil, corn oil, ethanol

 Dose selections

 1 (10 studies), 2, 3, or 5 doses

 Species

 Rat studies 

≈ mouse studies
Agency Guideline Interpre

 1998 US EPA Guidelines for Neuroto

Assessment conclude:

 Developmental neurotoxicity effects in 

indicate the potential for altered neurodevelopment in humans

 Specific types of developmental effect

experimental animal studies may not bthose seen in humans

6
Cognition Domain: Outcom

 Spatial learning and memory: radial arm 

water maze, foraging in radial arm maz

 Altered function, mostly showing deficit and f 8 of 9 studies, gestational and early postnat

 Effects at 1 mg/kg/d and above, maybe at 0.3  Lowest NOEL reported at 0.2 mg/kg/d using wa

 Alternation learning in T-maze

 No effect 5 of 5 studies, gestational and postnatal exp

 Classical conditioning using passive avoid

 No effect 2 of 2 postnatal studies

7
Cognition Domain: Consider

 Most of these tests validated and accepted 

foraging)

 Morris water maze

 Replications within one laboratory (2 studies)

 Radial arm maze

 Across laboratories, different procedures and eq Gender differences in effect and controls not co

 Activity level or motor differences can confo

 No activity differences in 5 of 6 studies (where 

cognitive changes

 Dose-response seen in 3 of 5 studies with 2  Effect sizes sometimes small  Differences sometimes transient, sometimes 

training

8
Replication of Cognitive Effe

Morris Water Maze

9

Billauer-Haimovitch et al., 2009; Turgeman et al., 2011

Multiple studies in mice within the same laboratory, 

Significantly slower time to find platform at 1 and 3 mg/kg/dNot 5 or 10 mg/kg/dNo changes in swim speed
Replication of Cognitive Effe

Radial Arm Maze

10

Significant across training for both sexesAldridge et al., 2005

Reference memory errors in rats within the same labo1 mg/kg/d, PND1-4Increased errors in males, decreased in females

Males Females

Males FemalLevin et 
Anxiety and Emotion Domain

Outcomes

 Anxiety: elevated plus maze, light-dark 

 Altered behaviors 7 of 8 studies, gestational and postnatal ex Effects at 1 mg/kg/d and above

 Emotion: novelty and chocolate prefere

 No effect with novelty exploration in 2 post Change in chocolate preference in 1 postna

 Emotion and despair: forced swim test

 No effect in 1 gestational study

11
Anxiety and Emotion Domain

Considerations

 Most of these tests validated and accep Specific endpoints not consistently measu

across studies

 Different procedures across studies and with Mouse: of 6 studies, 3 showed increased and 

anxiety

 Rat: of 2 studies, 1 showed decreased anxie

 Activity level differences can confound mea

 No differences in 5 of 6 studies, when reported, 

 Dose-response in only 1 of 4 studies with 2  Effect sizes sometimes small

12
Replication of Anxiety Effects:

Elevated Plus Maze & Light-Da

13

Increased anxiety in mice using two different tests; Female offspring, exposed GD15-LD14

Braquenier et al., 2010

Light-Dark Box

Elevated Plus Maze

Decreased anxiety maze; Offspring ex

Ricceri et al., 2
Social/Maternal Behavior Dom

Outcomes and Considerations

 Same-sex social interactions

 Altered behaviors 6 of 10 studies, gestational and postnatal exposur Increased social interactions or agonistic responses 

 Maternal behaviors

 4 of 5 studies, gestational and postnatal exposure Decreased reactions to intruder, increased or decrea

towards pups

 All studies from same laboratory, in mice Endpoints not standardized, even within same l Sometimes just one or few significant changes o

measured behaviors

 Tests of social behavior have not been widely 

developmental neurotoxicity studies

14
Motor Activity Domain: 

Outcomes and Considerations

 Spontaneous, exploratory activity, including 

rearing: figure-8 chambers, open fields

 Altered activity levels 8 of 14 studies, gestational and postnatal Rat: increased (1), decreased (2), or no effect (2

exposures; no effect with gestational or perinata

 Mouse: increased (3), gestational and postnatal 

 Habituation over test session, different from 

 Altered in 3 of 6 studies where analyzed Different statistical approaches across laborato

 Motor activity is accepted and widely used 

of general behavioral changes

15
Neuromotor Domain: 

Outcomes and Consideration Auditory startle response

 No effects in 2 studies in rats, gestation

exposures

 Neuromuscular performance

 Altered in 1 gestational study in rats

 Very limited literature

16
Measurements of ChE Inhib

17

Exposure period

Species Dosing Time of measuremen

Gestational Rat

Rat

GD6-20 po1GD17-20 sc

2

No inhibition 4 hr afte(also at 0.3 mg/kg/dNo inhibition 24 hr aft

Earlypostnatal

RatMouse 

Single dose sc

3

PND1-4 sc

4

Inhibition 2 and 4 hr aInhibition 1 hr, not 4 h

Late postnatal

RatMouse

Single dose sc

3

PND11-14 sc4,5

Inhibition 2 and 4 hr aNo inhibition 1, 4, or 2

ChE inhibition in pup or fetal brain at 1 mg/kg, given once oThis dose produced effects in almost all domains across stud

 No information about: 

Fetal ChE inhibition at shorter times after dosing

or on other days

No-effect dose for PND1 to 4
Sources of Variability in Out

18

 Experimental design

 Procedural and equipment differences, within and  Insufficient evaluation of dose-response Species, strains, routes of exposure, vehicles Ages of exposure and subsequent testing

 Outcomes

 Control values and treatment effects not always e Brain ChE inhibition in fetus/pup

 Gestational exposures maybe not, postnatal yes

 Interpretations

 Statistical analyses and acceptance criteria not app Subtle effects vs false positives Potential for false negatives by conservative accept

underpowered studies

 Publication bias against studies of negative data
Summary

19

 Multiple studies and laboratories repor

aspects of neurobehavioral function in following developmental exposure

 Effects seen long after chemical exposure and 

if present, had recovered

 Behavioral alterations show some consi

of broad domains of neurological funct

 Most consistent effects on cognition, anxiety, so Differences in direction and magnitude of cha

specificity

 Differences in procedures, exposures, etc. imp

across studies
Summary

20

 No clear evidence of critical period of 

species differences

 New publications support previous ana

strengthen conclusion of 2008 SAP

 Neurobehavioral effects at doses ≥1 mg/kg/ Insufficient dose-response data for effects and 

 Not enough data to definitely conclude 

sensitivity between developmental outcinhibition

 Need robust dose-response and time-course d

systematic replications of exposure periods
21 Questions?