### Appendix 3. In vivo developm ental table, Draft. Do not cite.

Page 1 of 11 Appendix 3

. In vivo studies of chlorpyrifos administered during gestation ; effects described are only those measured after weaning. Bold indicates functional domain that showed effects

.

Study & laboratory Species & strain Dose, route, vehicle Dosing period AChE inhibition Domain Age of testing Outcome, effective dose & gender Notes a Dose

response b ? Abou

Donia et al., 2006 Duke Rat SD 1 mg/kg/d dermal in 70% ethanol GD4

- 20 0

- 30 % increase brain (regions) activity at PND90, F not M Neuromotor f unction PND90 Decreased grip time, M& F ; Decreased performance on inclined plane, F not M ; Used 2/sex/litter

, (5 litters/ treatment), did not control for litter ; C erebellar pathology NA

Icenogle et al., 2004 Duke Rat SD 1, 5 mg/kg/d

sc in DMSO GD9

- 12 Not measured Motor

a ctivity PND 28

- 42 Faster habituation in figure

- 8, 5 mg/kg/d, M& F 10/sex/dose, no more than 1 of each sex from each litter ; ACh system involvement Yes LOEL=1 mg/kg/d PND28

- 56 Lower latencies (faster) in t

-
m aze, early in testing, 1&5 mg/kg/d, M &F PND >91 Increased center crosses in elevated plus maze (more active), 5 mg/kg/d , M & F Cognition PND28

- 56 No effect on t

- maze spontaneous alternation, M&F PND 56

- 91 Increased radial arm maze working & reference memory errors, early in training, 5 mg/kg/d , M&F Neuromotor f unction PND >91 No effect on acoustic startle reflex or prepulse inhibition

, M&F Anxiety & E motion PND >91 N o effect on time in sides of elevated plus maze , M&F Billauer

Haimovitch
et al., 2009 Israel Mouse HS/lbg 1, 3, 5, 10, 20 mg/kg/d

sc in DMSO GD9

- 18 Not measured Cognition PND75 Longer latencies (slower learning) in Morris water

maze , 1&3 mg/kg/d , M& F 1/sex/litter ; N varied, 14

- 34/dose group ; All dams dosed at 20 mg/kg/d died before delivery ; Nicotine & stem cell as treatments No LOEL=1 mg/kg/d Maximal effect at 3 mg/kg/d; Hi gher doses had no effect

### Appendix 3. In vivo developm ental table, Draft. Do not cite.

Page 2 of 11 Study & laboratory Species & strain Dose, route, vehicle Dosing period AChE inhibition Domain Age of testing Outcome, effective dose & gender Notes a Dose

response b ? Turgeman et al., 2011 Italy Mouse HS/lbg 3 mg/kg/d GD9

- 18 Not measured Cognition PND80 Longer latencies (slower learning) in Morris water

maze , M&F 1/sex/litter; N=13

- 25/dose
group; Stem cells as
treatments NA

Laviola et al., 2006 Italy Mouse

, wild type on C57/Bl6 background ; Study included reeler homo

and heterozygous mice Oxon 5 mg/kg/d sc in osmostic pump with DMSO GD14

- 16 Not measured Motor

a ctivity PND > 70 No difference in activity levels during habituation, before drug challenge s Only data from

w ild type shown here ; Never mentions genders ; Oxon not parent; I ndividual, as phenotyped, used; T otal of 6 litters/treatment ; ACh & DA system involvement ; Also some preweaning testing NA

Venerosi et al., 2010 Italy Mouse CD

- 1 6 mg/kg/d
po in peanut oil GD14

- 17 Not measured Anxiety &

e motion PND90 Increased time in tunnel between sides in light

- dark box , F not M; D ecrease d latency to enter dark side (p=0.05) (increased anxiety), F not M; Litter as unit ; N= 6

- 13 /dose ; 5HT system involvement NA

No effect on forced swimming task , M&F Social behavior & maternal interaction Adult F

, after mating on postpartum day 8 Decreased attack behavior, increased social sniffing behavior to intruder male

### Appendix 3. In vivo developm ental table, Draft. Do not cite.

Page 3 of 11 Study & laboratory Species & strain Dose, route, vehicle Dosing period AChE inhibition Domain Age of testing Outcome, effective dose & gender Notes a Dose

response b ? Haviland et al., 2010 UWM Mouse Swiss Webster 1, 5 mg/kg/d sc in DMSO GD17

- 20 Not measured Cognition PND60

- 81 Slower learning of food recognition & position, 1& 5 mg/kg/d, F not M; Faster food recognition learning, 1 not 5 mg/kg/d, M not F; Litter as unit; N=8/sex/dose; Thyroid involvement Yes (F only) LOEL=1 mg/kg/d

In males, 5 mg/kg/d had no effect Spurious changes in radial arm

maze (says no effect, only 9 trials) Motor

a ctivity PND60

- 81 No effect on foraging rate, M&F Levin et al., 2002 Duke Rat SD 1, 5 mg/kg/d sc in DMSO GD17

- 20 Not measured Motor

a ctivity PND28

- 42 Lower latencies (faster) in t

maz e, early in testing, 1& 5 mg/kg/d, M& F 1/sex/dose from 10 litters Yes LOEL=1 mg/kg/d Sl ower habituation in figure

- 8, 1 &5 mg/kg/d, F only

PND56

- 91 Lower latencies (faster) in radial arm maze during certain blocks only, 1& 5 mg/kg/d, M&F Cognition PND28

- 42 No effect on t

- maze spontaneous alternation, M&F PND56

- 91 Increased radial arm maze working & reference memory errors, 1 mg/kg/d only, F not M No Radial arm

maze effect at 1 mg/kg/d only Ricceri et al., 2006 Italy Mouse CD

- 1 3, 6 mg/kg/d po in peanut oil Plus vehicle on PND11

- 14 GD15

- 18 Dam on GD19 (pilot study): 40% brain ChE inhibition at 6 mg/kg/d, serum inhibition at both doses Pup on GD19: no brain Motor

a ctivity PND70 Increased crossing in open field, 6 mg/kg/d, only M tested Litter as unit ; 10 litters/ gestational dose Yes A ctivity NOEL= 3 mg/kg/d PND120 No effect on elevated plus

maze activity, M&F Anxiety&

e motion PND120 Decreased head dips in elevated plus maze (decreased anxiety), 3 mg/kg/d only, M not F No Elevated plus maze effect only at 3 mg/kg/d

### Appendix 3. In vivo developm ental table, Draft. Do not cite.

Page 4 of 11 Study & laboratory Species & strain Dose, route, vehicle Dosing period AChE inhibition Domain Age of testing Outcome, effective dose & gender Notes a Dose

response b ? inhibition,

serum inhibition at both doses Pup on PND15: no brain or

serum inhibition Social behavior & maternal interaction PND75

- 80 Increased offensive posture behaviors, 6 mg/kg/d, only M tested; No effect on non

- social behaviors Yes Aggression NOEL=3 mg/kg/d

PND90 No effect on induced maternal behaviors

Venerosi et al., 2006 Italy Mouse CD

- 1 3, 6 mg/kg/d sc in peanut oil Plus vehicle on PND11

- 14 GD15

- 18 Cites Ricceri 2006 findings Social behavior & maternal interaction PND120 Increased ultrasonic vocalizations, 6 mg/kg/d; only F tested Increased social investigation, 6 mg/kg/d Litter as unit; 10 litters/ gestational dose Yes NOEL=3 mg/kg/d

a Sample sizes and analyses refer only to behavioral measures
b Graded response across doses or else effect at higher but not lower dose(s) M, F, Male, Female

### Appendix 3. In vivo developm ental table, Draft. Do not cite.

Page 5 of 11 Appendix 3 .2 In vivo studies of chlorpyrifos administered postnatally . E ffects described are only those measured after weaning

. Bold indicates functional domain showed effects

.

Study & laboratory Species & strain Dose

, route, vehicle Dosing period AChE inhibition Domain Age of testing Outcome, effective dose & gender Notes a Dose

response b ? Dam et al., 2000 Duke Rat SD 1 mg/kg/d sc in DMSO PND1

- 4 ~60% brain (regions) inhibition in M, ~20% in F; more at 2 hr than 4 hr after single dose on PND1 Motor

a ctivity PND21, 30 Decreased open field activity & rearing, M not F 5

- 7/sex/dose ; Maybe split

- litter dosing but not explicit ; Also some preweaning testing NA

Levin et al., 2001 Duke Rat SD 1 mg/kg/d sc in DMSO PND1

- 4 Not measured Motor

a ctivity PND28

- 42 Longer latencies (slower) in
t

- maze, M not F 10/sex/dose ; Split

- litter dosing ; No ACh system involvement NA

No effect on habituation in figure

- 8, M&F Cognition PND28

- 42 No effect on t

- maze spontaneous alternation, M&F PND56

- 91 Increased radial arm maze working & reference errors, early in training, M not F ; Decreased radial arm maze working & reference errors, F not M Adridge et al., 2005 Duke Rat SD 1 mg/kg/d sc in DMSO PND1

- 4 Not measured Cognition PND64+ Increased radial arm maze working & reference

me mory errors , M not F ; Decreased working memory errors in radial arm maze , F not M 9/sex/dose; Probably split

- litter dosing but not explicit NA

Anxiety &

e motion PND52

- 53 Increased open arm time in elevated plus maze (decreased anxiety), M not F PND54 Decreased chocolate milk preference (anhedonia), M&F Motor

a ctivity P ND52

- 5 3 Increased center crossings in elevated plus maze , M not F

### Appendix 3. In vivo developm ental table, Draft. Do not cite.

Page 6 of 11 PND64+ No differences in radial arm

maze latencies, M&F Ricceri et al., 2003 Italy Mouse CD1 1, 3 mg/kg/d sc in DMSO PND1

- 4 20, 23% brain inhibition at low & hi gh dose 1 hr after dose on PND4; no inhibition at 4, 24 hr Motor activity PND25 No effect on open field

ac tivity Split

- litter dosing ; n=8

- 18/sex/dose;
says all mice got all
tests but there are unexplained d ifferences i n sample size across tests; Bonferroni

corrected post

- hocs followed ANOVA main effects with p>0.05 ; Also some preweaning testing No, ChE & agonistic behaviors similar at both doses

, LOEL=1 mg/kg/d PND35

- 38 Increased activity at door opening in 2

- chamber box, only 3 mg/kg/d tested, M&F Anxiety & emotion PND38 No effect on novelty preference in 2

- chamber box, only 3 mg/kg/d tested, M&F Social behavior & maternal interaction PND45 No effect investigative, affiliative, or soliciting behaviors, M&F ; Decreased self

- grooming, 1&3 mg/kg/d, M&F Increased frequency & duration agonistic behavior sum, differs across time, 1&3 mg/kg/d, M not F Cognition PND60 No effect on passive avoidance, only M tested Dam et al., 2000 Duke Rat SD 5 mg/kg/d sc in DMSO PND11

- 14 ~20

- 30% brain (regions) inhibition, M&F, more at 4 hr than 2 hr after single dose on PND11 Motor

a ctivity PND21, 30 Increased open field rearing & grooming, M&F 5

- 7/sex/dose ; Maybe split

- litter dosing but not explicit ; Figure 3 s ays male only but gender differences not significant NA

Levin et al., 2001 Duke Rat SD 5 mg/kg/d sc in DMSO PND11

- 14 Not measured Motor

a ctivity PND28

- 42 Longer latencies (slower) in
t

- maze, M not F 10/sex/dose ; Split

- litter dosing ; ACh system involvement NA

Decreased habituation (slower) in figure

- 8, M&F Cognition PND28

- 42 No effect on t

- maze spontaneous alternation, M&F PND56

- 91 No effect on radial arm maze working or reference
memory , M&F

### Appendix 3. In vivo developm ental table, Draft. Do not cite.

Page 7 of 11 Ricceri et al., 2003 Italy Mouse CD1 1, 3 mg/kg/d sc in DMSO PND11

- 14 No brain inhibition at 1, 4, 24 hr after dose Motor

a ctivity PND25 Increased distance traveled in open field, 1 & 3 mg/kg/d

, M&F Split

- litter dosing ; n=8

- 18/sex/dose ; says all mice got all
tests but there are unexplained differences i n sample size across tests; Bonferroni

corrected post

- hocs followed ANOVA main effects with p>0.05 Yes & No Dose

response on activity, 1 has more effect on agonistic behaviors LOEL=1 mg/kg/d PND35

- 38 Increased activity following door opening in 2

- chamber box, only 3 mg/kg/d tested, M&F Anxiety & emotion PND3 8 No effect on novelty preference in 2

- chamber box, only 3 mg/kg/d tested, M&F Social behavior & maternal interaction PND45 No effect on investigative or affiliative behaviors, M&F ; Increased soliciting behavior, 3 mg/kg/d , M&F Increased frequency & duration agonistic behaviors throughout, 1 &3 mg/kg/d , M not F Cognition PND60 No effect on passive avoidance, only M tested Ricceri et al., 2006 Italy Mouse CD

- 1 1, 3 mg/kg/d sc in peanut oil Plus vehicle to dam GD15

- 18 PND11

- 14 24 hr after last dose (PND15): no brain inhibition,

serum inhibition both doses Motor

a ctivity PND70 Increased crossing in open field, only 1 st 5 min, 3 mg/kg/d , only M tested Litter as unit ; 10 litters/gestational dose, postnatal split

- litter dosing ; Says anxiety effect in both sexes but not significant in males Yes NOEL=1 mg/kg/d (most endpoints) PND120 No effect on elevated plus

maze activity , M&F
Social behavior & maternal interaction PND75

- 80 Increased attack behaviors, 3 mg/kg/d, only M tested; N o effect on M non

- social behaviors Yes maternal LOEL=1 mg/kg/d PND90 Increased induced maternal behaviors , 1&3 mg/kg/d Anxiety PND120 Increased time in open arms in elevated plus maze (decreased anxiety), 3 mg/kg/d , F only Yes NOEL=1 mg/kg/d

Venerosi et al., 2006 Italy Mouse CD

- 1 1, 3 mg/kg/d sc in peanut oil Plus vehicle to dam GD15

- 18 PND11

- 14 Not measured, cites Ricceri 2006 findings Social behavior & maternal interaction PND120 No effect on ultrasonic vocalizations or social investigation, only F tested Litter as unit 10 litters/gestational dose, postnatal split

- litter dosing Yes NOEL=3 mg/kg/d

### Appendix 3. In vivo developm ental table, Draft. Do not cite.

Page 8 of 11 Venerosi et al., 2008 Italy Mouse CD

- 1 3 mg/kg/d sc in peanut oil PND11

- 14 Not measured Social behavior & maternal interaction PND40

- 45 No effect on social interaction with same

- sex conspecific, M&F Litter as unit ; Split

- litter dosing, 2/sex/dose/litter ; N=15 litters ; N=9

- 14/dose for some maternal tests ; Female offspring mated on PND60, maternal tests after giving birth NA

LD1

- 7 In lactating dams: Longer latency to build nest ; S horter latency to lick pups ; Increased time digging (non

maternal behavior) LD7 In lactating dams: Decreased defensive, increased social investigative, & increased digging behaviors when exposed to intruder male Anxiety & emotion LD2 In lactating dams: Faster entry & more mice entering light side (decreased anxiety) Motor

a ctivity PND40

- 45 Decreased distance moved during acclimation before social test, 1 st time block, M&F Johnson et al., 2009 MSU Rat SD Incrementing doses: low, 1 mg/kg/d; mid, 1 up to 4 mg/kg/d; hi, 1 .5 up to 6 mg/kg/d Po in corn oil PND1

- 21 PND20 (time after dose not given)

– 14

- 53 % brain (hippocampal) inhibition, all doses significant ; PND30

- 40, mid & high doses still show inhibition (17

- 23%) ; No inhibition PND50; M&F same Cognition PND36

- 60 Increased radial arm maze working memory errors, hi dose throughout training , all doses during last week, M only; Decreased radial arm maze reference memory errors, middle to end of training

, mid & hi doses, F only; I ncreased radial arm maze reference e rrors, middle week on training , mid & hi doses, M only Split

- litter dosing; n=10

- 14/sex/dose ; Table 1 says PND20, not 21 Yes LOEL= low dose ( 1 mg/kg/d PND1

- 21)

Motor

a ctivity PND36

- 60 No effect on response time in radial arm maze

Carr et al., 2001 MSU Rat SD Incrementing doses: low, 3 mg/kg/d; PND1

- 21, every other day PND25, 30

– 14

- 26% brain (regions) Motor

a ctivity PND25, 30 Decreased open field activity, mid & hi, M&F Between

- litter dosing ; N=5 litters/dose, Yes A ctivity NOEL= low

### Appendix 3. In vivo developm ental table, Draft. Do not cite.

Page 9 of 11 mid, 3 up to 6 mg/kg/d; hi, 3
up to 12 mg/kg/d Po in corn oil inhibition at low dose; all doses significant, M&F same 2/sex from each litter ; U sed within

- litter factor in analysis ; Also some preweaning testing dose ( 3 mg/kg/d PND1

- 21 )

Jett et al., 2001 JHU Rat LE 0.3, 7 mg/kg/d sc in peanut oil PND7, 11, 15 “preweaning study” No brain inhibition on PND7 (3 hr after dose), 8, 16, or 28 Cognition PND24

- 28 Longer latencies to find platform in Morris water maze, 7 mg/kg/d throughout, 0.3 mg/kg/d on day 1 (p=0.05), M& F; Less time spent in training quadrant, 7 mg/kg/d, M&F Between

- litter dosing after random culling; N=8

- 10/sex/dose, “2 or more litters used” ; “Postweaning” study not presented Yes NOEL or LOEL=0.3 mg/kg/d (p=0.05) Authors say both ways

a Sample sizes and analyses refer only to behavioral measures
b Graded response across doses or else effect at higher but not lower dose(s) M, F, Male, Female

### Appendix 3. In vivo developm ental table, Draft. Do not cite.

Page 10 of 11 Appendix 3

. 3 In vivo studies of chlorpyrifos administered during gestation and postnatally , con tinuous or discontinuous exposures. E ffects described are only those measured after weaning

. Bold indicates functional domain showed effects

.

Study & laboratory Species & strain Dose

, route, vehicle Dosing period AChE inhibition Domain Age of testing Ou tcome, effective dose & gender Notes a Dose

response b ? Maurissen et al., 2000 Dow Rat SD 0.3, 1, 5 mg/kg/d po in corn oil GD6

- LD10 Dam on GD20, 4

- 5 hr after dose, 45% blood but no brain inhibition at 0.3; greater blood and brain inhibition at higher doses Neuromotor function PND22 Increased auditory startle latency did not meet lower p

value, 5 mg/kg/d, M&F; “effects may have been r eal”

– p

- value =0.03 Litter as unit ; 1/sex/litter ; N=8/sex/dose for delayed alternation (same rats at both ages) ; N=20/sex/dose for startle and motor activity (same rats at both ages) ; alpha set at 0.02 ; Also some preweaning tests ; Maternal toxicity at 5 m g/kg/d No doses showed effects using higher statistical threshold PND61 No effect on auditory startle measures, M&F Motor activity PND21 and PND60 No effect on total counts or habituation at either age, M&F ; High dose “may have had ..slight increase in activity on PNDs 17, 21, and 60” Cognition PND22

- 24 and PND61

- 91 No effect on spatial delayed alternation learning at either age, M&F Braquenier et al., 2010 Belgium Mouse CD

1 0.2, 1, 5 mg/kg/d po in corn oil GD15

- LD14 Pup on PND1, 3 hr after dose to dam, 14% brain inhibition, only 5 mg/kg/d group tested Motor activity “Adult” No effect on locomotor activity 1/litter for each test ; only F tested ; N=8

- 10/dose ; Dunnetts post hoc after main effect p<0.10 No Effect only at 1 mg /kg/d NOEL=0.2 mg/kg/d PND80 No effect on elevated plus

maze total arm entries Anxiety & emotion “Adult” Decreased time in chamber center in light

- dark box and decreased box switches (increased anxiety), 1mg/kg/d only ; PND80 Decreased time & entries in elevated plus maze open arm (increased anxiety), 1 mg/kg/d only Ricceri et al., 2006 Italy Mouse CD

1 3, 6 mg/kg/d po in peanut oil Plus 1, 3 mg/kg/d sc in peanut oil on GD15

- 18 “pre” plus PND11

- 14 “post” Dam on GD19 (pilot study): 40% brain ChE inhibition at 6 mg/kg/d, serum inhibition at Motor activity PND70 Increased crossing in open fi eld, 6 mg/kg/d prenatal plus 1, not 3 mg/kg/d, postnatal NA

PND120 No pre

- post interaction on elevated plus maze activity, M&F

### Appendix 3. In vivo developm ental table, Draft. Do not cite.

Page 11 of 11 Study & laboratory Species & strain Dose

, route, vehicle Dosing period AChE inhibition Domain Age of testing Ou tcome, effective dose & gender Notes a Dose

response b ? PND11

- 14 both doses Pup on GD19: no brain inhibition,

serum inhibition at both doses Pup on PND15: no brain or

serum inhibition Anxiety & emotion PND120 No pre

- post interaction on elevated plus maze, M&F Social behavior & maternal interaction PND75

- 80 No pre

- post interaction on social behaviors, M&F

PND90 No pre

- post interaction on induced maternal behaviors

Venerosi et al., 2006 Italy Mouse CD

1 3, 6 mg/kg/d sc in peanut oil Plus 1, 3 mg/kg/d sc in peanut oil on PND11

- 14 GD15

- 18 “pre” p lus PND11

- 14 “post” Cites Ricceri 2006 findings Social behavior & maternal interaction PND120 Increased ultrasonic vocalizations & social investigation at 6 mg/kg/d prenatally reversed by 1&3 mg/kg/d postnatally NA

a Sample sizes and analyses refer only to behavioral measures
b Graded response across doses or else effect at higher but not lower dose(s) M, F, Male, Female