www.elsevier.com/locate/neutera

Neurotoxicology and Teratol

Developmental and behavioral effects of acrylamide in Fischer 344 rats

Joan Garey*, Sherry A. Ferguson, Merle G. Paule

Division of Neurotoxicology, HFT-132, National Center for Toxicological Research, 3900 NCTR Rd., Jefferson, AR 72079-9502, United States

Received 16 December 2004; received in revised form 10 March 2005; accepted 21 March 2005

Available online 26 April 2005

Abstract

Human exposures to acrylamide (ACR), a known neurotoxicant, can occur via a variety of substances, including cigarette smoke and the

ingestion of certain carbohydrate-based foods cooked at high temperatures. In this study, Fischer 344 sperm plug-positive female rats were

treated daily with ACR (0, 0.5, 1.0, 2.5, 5.0 or 10.0 mg/kg/day) by gavage beginning on gestation day 7. Dosing of dams ended when litters

were born; pups received daily gavage at the same dose as their dam from postnatal day (PND) 1 through PND22. Pups were tested using a

battery of behavioral assessments from PNDs 4–22. Statistically significant decreases in body weight were observed in pups exposed to

ACR at doses as low as 1.0 mg/kg/day (treatment�day; repeated measures ANOVA, P <0.0001). No statistically significant differences

among treatment groups were observed in righting reflex, forelimb hang, or open field measures of activity. Statistically significant effects of

ACR were observed at the 10 mg/kg/day dose on negative geotaxis performance (P <0.01) and a linear trend in fall-time latencies on

Rotarod performance on PNDs 21–22 (P <0.05), with higher doses producing shorter latencies. These results suggest that ACR exposure

produces deficits in development and motor coordination that are observable before weaning.

D 2005 Elsevier Inc. All rights reserved.

Keywords: Acrylamide; Rats; Development; Behavior; Neurotoxicity

1. Introduction

Acrylamide monomer (ACR) is an established neuro-

toxicant found in a range of non-food products. ACR is used

in the production of dyes, adhesives, contact lenses, soil

conditioners and permanent press fabrics [13]. After

polymerization, it is used in polyacrylamide gel electro-

phoresis (PAGE) procedures and as a flocculent in water

treatment, paper production and mineral processing. Ciga-

rettes provide an additional source of ACR exposure, with

ACR levels recovered in mainstream smoke ranging from

1.1–2.34 Ag per cigarette [21].

The recent discovery that ACR is formed in certain

carbohydrate-containing foods (i.e., those containing the

amino acid asparagine) when prepared at typical high

cooking temperatures [24] has spurred a renewed effort to

determine the risk of ACR to human health [27]. Concen-

trations of ACR as high as 3500 Ag/kg (parts per billion;

0892-0362/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.ntt.2005.03.007

* Corresponding author. Tel.: +1 870 543 7905.

E-mail address: jgarey@nctr.fda.gov (J. Garey).

ppb) have been reported for french fries and potato chips

[27]. Recent exploratory measurements of acrylamide in

foods show levels (ppb) in: baby food of up to 130; breads

and bakery products up to 340; cereals up to 266; snack

foods up to 1168; nuts and nut butters up to 457; crackers up

to 504; chocolate products up to 909; coffee up to 351 and

dried foods up to 1184 [5]. Average daily exposures for the

general populace have been estimated to be in the range of

0.3–0.8 Ag/kg/day and children may have intakes two to

three times that of adults on a mg/kg basis [27]. ACR has

also been identified in breast milk and can cross the human

placenta [22].

ACR has been shown to produce a central-peripheral

neuropathy in laboratory animals, including rats and

monkeys, as well as in humans (reviewed in [18]). ACR

neurotoxicity appears to be dose- and time-dependent, with

axonal degeneration accruing over time with repeated

exposures despite no apparent accumulation of ACR at

sites of toxicity [7]. Moreover, neurotoxic effects have been

documented in rats in brain regions associated with higher

cognitive functions [17]. In this study, we examined

ogy 27 (2005) 553 – 563

J. Garey et al. / Neurotoxicology and Teratology 27 (2005) 553–563554

maternal and pre-weaning developmental and behavioral

effects of ACR with daily exposures beginning in utero on

gestation day (GD) 7 and continuing through postnatal day

(PND) 22.

2. Methods

2.1. Chemical

Acrylamide (electrophoresis grade; purity >99%) was

obtained from Sigma Chemical Co. (St. Louis, MO).

Identity was confirmed by mass spectrometry and 1H-

NMR analysis at the National Center for Toxicological

Research (NCTR). Standard reference data on ACR was

obtained using the NIST Mass Spectral Search Program for

the NIST/EPA/NIH Mass Spectral Library, Version 2.0a

(ChemSW, Fairfield, CA). Purity was confirmed at >99.6%

through the use of capillary gas chromatography with flame

ionization detection (GC/FID), GC/electron impact-mass

spectrometry and 1H-NMR analysis at NCTR.

2.2. Dosing solutions

The dosing solutions were prepared every two weeks by

mixing ACR with 0.2 micron-filtered water. Stability studies

of the high and low concentrations (i.e., 0.1 and 2.0 mg/mL

solutions) were performed using GC/FID. ACR solutions

were determined to be stable for up to 28 days at ambient

temperature when stored in amber glass bottles. Dosing

solutions were stored for no longer than oneweek prior to use.

Solutions of each concentration of ACR (i.e., 0, 0.1, 0.2,

0.5, 1.0 and 2.0 mg/mL) were analyzed using GC/FID. The

concentrations of all dosing solutions were within 10% of

the target concentrations.

2.3. Animals

All procedures using animals were approved by the

NCTR Institutional Animal Care and Use Committee and

were in accordance with NIH Guidelines for the Care and

Use of Laboratory Animals.

Eighty-eight date-mated sperm plug-positive Fischer 344

(F344) female rats (Simonsen Laboratories; Gilroy, CA) were

obtained over three replicates. Of these animals, 14 were

sacrificed on gestation day (GD) 20 to obtain blood fromdams

and fetuses (data not reviewed here). Therefore, a total of 74

plug-positive rats were retained through at least GD23 to

allow for littering. Those animals that did not litter by GD23

were sacrificed on GD24 and their uteri examined for fetuses

and evidence of resorption sites. Rats that littered and

their offspring continued on study through postnatal day

(PND) 22 unless the death of the entire litter required removal.

Plug-positive rats arrived at the NCTR vivarium no later

than GD3 at which time they were tattooed (tail) and

quarantined. During quarantine, rats were individually

housed in 42.5�26.6�18.5 cm, high-temperature poly-

sulfone cages located within a SealSafe Individually

Ventilated Caging System (Model No. 2H36MAC30CACP;

Tecniplast USA, Phoenixville, PA), supplied with auto-

claved standard hardwood chip bedding. Rats were provided

food (see below for diet information) and autoclaved 0.2

micron-filtered water ad libitum. After the quarantine

period, animals were individually housed in 48.3�26.7�20.3 cm polycarbonate cages with wire lids and provided

with standard hardwood chip bedding. After parturition,

litters were housed in the cage with their dams. Food and

water were provided ad libitum. Throughout the study, the

temperature and humidity of the housing rooms were 23T3-C and 45–55% relative humidity, respectively and the

animals were maintained on a 12 h:12 h light:dark cycle

with lights on at 7 am when daylight savings time was in

effect and at 6 am standard time.

2.4. Diet

The diet was NIH-31IR (5LG-6 Irradiated Rodent Diet;

Purina Test Diet, Richmond, IN). This irradiated, powdered

diet was used because sterilization of the diet by microwave

irradiation in the absence of pelleting (which requires a

steam-extrusion process) has been observed to produce less

ACR than autoclaving [25]. Analysis by NCTR’s Division

of Chemistry using liquid chromatography/electrospray

ionization-mass spectrometry analysis (LC/EIMS) deter-

mined the ACR content of the diet to be approximately 40

ppb. The ACR content of the water used as the vehicle

control and drinking water was found by LC/EIMS to be

below the limit of detection, which was 2 ppb.

2.5. Study design

Plug-positive females were assigned to treatment groups

by body weight on the day of arrival in order to achieve

comparable average body weights for all groups. Beginning

on GD7 (GD0=day of detection of vaginal sperm plug), all

females of a treatment group received one of five doses of

ACR (0.5, 1.0, 2.5, 5.0 or 10.0 mg/kg) or vehicle (0.2

micron-filtered water) daily via orogastric gavage. Dosing

occurred at the same time each dayT1 h. All doses were

administered at volumes of 5 mL/kg. In addition, body

weights of females were determined daily from GD7 until

time of sacrifice on PND 22. Food and water consumption

of all plug-positive females and dams with litters were also

monitored daily during the same time period.

On the day of birth (PND 0), no compound was given to

any dam or pup. On PND 1, the number of delivered pups

(alive and dead) was counted and their sex determined and

recorded. From PND 1 on, pups were gavaged with ACR at

the same doses given previously to their dams [0.5, 1.0, 2.5,

5.0 or 10.0 mg/kg/day; control pups received vehicle only

(0.2 micron-filtered water)]. Thus, the last gavage to

pregnant females was given the day prior to parturition.

J. Garey et al. / Neurotoxicology and Teratology 27 (2005) 553–563 555

After pup dosing on PND 1, litters were culled to achieve

an even sex distribution among as many litters as possible

with a target litter size of eight (4 :4 or 3 :5 sex ratios) or

seven with a 3 :4 sex ratio. Cross-fostering of pups within

same treatment groups was performed where necessary to

achieve target numbers and sex distribution. Of the 74 date-

mated females, 50 gave birth. Of these, 42 litters were of

usable size. After culling and fostering, 37 litters had a

minimum of 7 pups each, with sex ratios of 4 :4, 3 :5 or 3 :4.

However, in order to maintain an appropriate number of

litters per treatment group, six additional litters were

retained which had a litter size of �5 and sex ratios as

follows: 2 :5 (1 litter; 2.5 ml/kg/day treatment group), 1 :6

(2 litters; control and 2.5 mg/kg/day treatment group), 3 :3

(1 litter; 0.5 mg/kg/day treatment group) and 1 :4 (2 litters;

0.5 and 10 mg/kg/day treatment groups). After culling, the

remaining pups were paw-tattooed for identification.

Beginning on PND 1 and continuing until sacrifice on

PND 22, all remaining pups, including fosters, were

weighed, dosed and observed daily for the occurrence of

fur development (the appearance of fur sufficient to cover

the skin), pinnae detachment (both ears completely unfolded

from the head) and eye opening (both eyes fully open).

Evidence of inconsistency in the procedure used to

determine day of pinnae detachment made it necessary to

evaluate data on this measure from the last two replicates

only. All physical and behavioral observations and measure-

ments on animals were made during the light phase of the

light:dark cycle. In addition, mortality observations were

made twice daily.

2.6. Behavioral testing protocols

Litters were run in a random order for each behavioral

test. The days selected for testing were determined by

previous experience with these assessments [4,8–10]. These

responses represent developmental milestones; the window

of time selected for each test is the key period of time during

which a determination can be made as to whether a toxicant

is accelerating or delaying the emergence of the given

behavior. Over PNDs 4–7, 8–10 and 12–16, all pups in

each litter were assessed for righting reflex, negative

geotaxis and forelimb hang time, respectively. In addition,

one male and one female per litter were randomly selected

for testing of both open field behavior (PNDs 19 and 20)

and Rotarod performance (PNDs 21 and 22). Using only

two pups per litter for both of the latter two tests was

necessitated by the relatively long test sessions preventing

the testing of all pups in the litter. All behavior tests were

conducted using methods similar to those previously

described for our laboratory [4].

For righting reflex, negative geotaxis and forelimb

hanging, all pups of a litter scheduled for testing were

removed from their home cage and placed together in a

single shoebox-sized plastic cage prior to testing. The pups

were returned immediately to their home cage after the

entire litter completed testing. For open field and Rotarod

testing, up to six pups from three litters were placed into

their own individual shoebox-sized plastic cages prior to

testing. When testing of the group was completed,

individuals were immediately returned to their home cages,

except on the final day of testing when animals were

sacrificed. All behavioral testing was performed before

litters received their daily ACR gavage.

For all measures using a stopwatch (righting reflex, nega-

tive geotaxis and forelimb hanging), the instrument was ac-

curate to 1/100 s and measurements were made in these units.

2.6.1. Righting reflex protocol (PNDs 4–7)

Each pup was placed dorsal side down on a smooth flat

surface and the latency to right itself onto all four paws

(dorsal side up) was recorded using a stopwatch. A

maximum latency of 60 s was assigned for those subjects

that did not right within that allotted time. Each pup was

tested for a single trial on each of the four test days.

2.6.2. Negative geotaxis protocol (PNDs 8–10)

Each rat was placed on a wooden board 8.9 cm wide

covered with sandpaper for traction over a 28 cm long

region beginning at the lower end of the board. The board

was angled at 45- to the horizontal with ample padding

around the apparatus. The pup was placed on the board

between the center and the lower end of the apparatus, on its

ventral side with nose pointed toward the lower end.

Holding the pup on the sides of its body using one hand,

the pup was slightly pulled back toward the center of the

apparatus until its forepaws were on a marked line at 10.2

cm from the lower end of the board. With the body in

position and spine straight, the pup was released and a

stopwatch started. The rat was allowed 60 s to complete a

180- turn from its original starting position. Incomplete

turns and falls from the apparatus were recorded as were the

latencies to fully turn. A maximum of 60 s was assigned for

those subjects that did not make a 180- turn. The time of fall

was recorded for subjects that fell off the apparatus. Each

pup was tested for a single trial per test day.

2.6.3. Forelimb hang protocol (PNDs 12–16)

The apparatus consisted of a taut string stretched between

two blocks of wood spaced 46 cm apart. The height of the

string from the surface below was 41 cm and ample padding

was provided to prevent injury upon falling. For five

consecutive days, each pup was placed on the string by

allowing its forepaws to grasp it; the pup was thus oriented

in a chin-up fashion and the latency to fall (maximum of 60

s) was measured with a stopwatch. Each pup was tested for

a single trial per test day.

2.6.4. Open field activity protocol (PNDs 19 and 20)

On two consecutive test days, pups were removed from

their home cage and placed individually in a Plexiglas

chamber (46.5�46.5�46.5 cm) bisected by eight pairs of

6 8 10 12 14 16 18 20 225

10

15

20

25

30

35

0 mg/kg0.5 mg/kg1.0 mg/kg2.5 mg/kg5.0 mg/kg10.0 mg/kg

Am

ou

nt

Co

nsu

med

(g

)

6 8 10 12 14 16 18 20 220

20

40

60

80

100

120

140

160

0 mg/kg0.5 mg/kg1.0 mg/kg2.5 mg/kg5.0 mg/kg10.0 mg/kg

Am

ou

nt

Co

nsu

med

(g

/kg

bo

dy

wei

gh

t)

(a)

(b)

Gestation Day

Fig. 1. Effect of ACR on maternal food intake during gestation. No effect of

ACR was observed on food intake whether analyzed as absolute intake (a)

or relative to body weight (b).

6 8 10 12 14 16 18 20 2240

60

80

100

120

140

160

180

200

0 mg/kg0.5 mg/kg1.0 mg/kg2.5 mg/kg5.0 mg/kg10.0 mg/kg

6 8 10 12 14 16 18 20 2210

15

20

25

30

35

40

45

0 mg/kg0.5 mg/kg1.0 mg/kg2.5 mg/kg5.0 mg/kg10.0 mg/kg

(a)

(b)

Am

ou

nt

Co

nsu

med

(m

l)A

mo

un

t C

on

sum

ed (

ml/k

g b

od

y w

eig

ht)

Gestation Day

Fig. 2. Effect of ACR on maternal water intake during gestation. No effect

of ACR was observed on water intake whether analyzed as absolute intake

(a) or relative to body weight (b).

J. Garey et al. / Neurotoxicology and Teratology 27 (2005) 553–563556

photobeams. Photobeam breaks over each 12 min session

measured activity, duration of immobility and entries into

the center area. Six chambers were used to test a maximum

of six animals at a time, with one rat per chamber. Each

chamber was interfaced with a computer for automated data

collection and animals were assigned the same chamber on

both test days. At the end of each session, chambers were

cleaned of fecal matter and urine. Each animal was tested for

one session per test day.

2.6.5. Rotarod performance protocol (PNDs 21 and 22)

Motor coordination was assessed using an automated

Rotarod system (Smart Rod; AccuScan Instruments, Inc.,

Columbus, OH). The apparatus consisted of a rubber rod 2.5

cm in diameter and 11.5 cm in length housed in a Plexiglas

chamber, with the rod placed 36.0 cm from the floor of the

apparatus. A computer interfaced with the apparatus

controlled the rotation of the rod. Each rat was placed on

the rod which then began to slowly rotate. The rat had to

continuously maintain its position on the top surface of the

rod to avoid falling off. The apparatus was programmed to

accelerate over six 20 s increments of 2–4 rpm each to

reach a maximum speed of 20 rpm at the end of 2 min. The

rod continued to rotate at 20 rpm for another 3 min and then

slowed to a stop over 30 s. Rats were tested over three

successive trials on each of two consecutive days. A trial

ended when either the rat fell or the time reached 5 min with

the rat remaining on the rod. The computer recorded latency

to fall (in seconds) and rpm at fall time. If a rat remained on

the rod for the entire 5 min, a latency of 300 s and a speed of

20 rpm were recorded.

2.7. Statistics

For body weight change in dams, a repeated measures

two-way ANOVAwas performed with day and weight (g) as

factors. For food and water intake in dams, repeated

measures two-way ANOVAs were performed with day

and intake (g and ml, respectively) as factors. For fur

development, pinnae detachment and eye opening day

analyses, as well as the analysis of pup body weight on

PND1, two-way ANOVAs with treatment and sex as factors

were performed. For body weight change in pups, as well as

all behavioral measures, data were analyzed using repeated

measures ANOVAs with sex, treatment and day as factors.

All two-way ANOVAs were performed using SigmaStat 3.0

(Systat Software, Inc., Point Richmond, CA). Repeated-

measures analyses were performed using either SAS or JMP

5.0 (both from SAS Institute, Cary, NC). Post-hoc tests were

J. Garey et al. / Neurotoxicology and Teratology 27 (2005) 553–563 557

performed when results of initial analyses were significant.

Additional statistical tests were used as indicated in the text.

For analyses of data on multiple pups per litter, the male and

female average for each litter was obtained, providing two

data points per litter.

2.8. Quality assurance methods

These range-finding studies were conducted in compli-

ance with the Food and Drug Administration Good

Laboratory Practice Regulations (Code of Federal Regu-

lations, Title 21, Part 58).

Fig. 3. Effect of ACR on pregnancy in plug-positive females. No effect of

ACR was observable on pregnancy, although the three highest treatment

groups combined had a lower average percent pregnancy rate than the three

lowest treatment groups combined.

3. Results

3.1. Maternal measures

3.1.1. Maternal food and water intake

Maternal food and water intake during gestation among

dams with litters surviving to PND22 are shown in Figs. 1

and 2. No significant main effects of treatment were

observed in food intake by repeated-measures ANOVA,

measured as either absolute intake [F(5,43)=1.7; P=0.17]

or intake relative to body weight [F(5,43)=1.7; P=0.15].

No significant main effects of treatment were observed in

either absolute [F(5,40) = 2.3; P = 0.06] or relative

[F(5,40)=1.7; P <0.15] water intakes. No significant

interactive effects of treatment�day were observed in any

of these measures, although significant effects of day were

observed for all measures (P <0.0001 in all cases).

3.1.2. Maternal body weight

An analysis of body weight gain between GD7 and

GD21 of dams with litters surviving until PND22 demon-

strated no evidence of an effect of ACR (one-way ANOVA;

P=0.67; data not shown).

3.2. Gestation and birth measures

3.2.1. Number of pregnancies, gestation length

Of the 74 date-mated females retained for littering, 51

were pregnant. Litter parameters are shown in Table 1.

Table 1

Litter parameters

Treatment group (mg/kg/day)

0 0.5

No. of litters 8 10

Mean litter size at birth

(meanTS.E.M.)

9.5T0.6 9.3T1.0

No. of males at PND1 35 45

No. of females at PND1 40 32

No. dead pups on PND1 1 12

While no effect of ACR on pregnancy was observed, the

three highest treatment groups combined had a lower

average percent pregnancy rate than the three lowest

treatment groups combined (Fig. 3).

With one exception, all pregnant dams littered on

GD22 or GD23. The exception was one dam that was

sacrificed on GD24 and found to have a single fetus in

the uterus. While a graph of percent of births vs.

treatment indicates that a higher percentage of GD23

births occurred among the three highest treatment groups

(see Fig. 4), a Fisher’s Exact Test (SAS) of the data

demonstrated that this result was not statistically signifi-

cant (P=0.31).

3.2.2. Litter size and sex ratio

No significant effects of acrylamide were found on litter

size on the day of birth (see Fig. 5a) or sex ratio of litters

(one-way ANOVA; F(5,43)=2.1; P=0.09). However, an

analysis of litter size using PND1 data (Fig. 5b) indicates a

statistically significant smaller litter size in the 2.5 mg/kg/

day dose group when compared to the vehicle control (t-

test; t=2.2; P <0.05).

1.0 2.5 5.0 10.0

9 8 10 6

9.3T0.9 7.0T1.2 8.4T0.9 9.3T1.0

44 21 36 23

49 29 33 28

1 6 11 5

Fig. 5. Effect of ACR on litter size. a) At birth, there was no apparent effect of

significant effect was observed, with the 2.5 mg/kg/day treatment group having a

Fig. 4. Effect of ACR on gestation length. The majority of GD23 births

occurred in high treatment group litters; however, this result was not

statistically significant.

J. Garey et al. / Neurotoxicology and Teratology 27 (2005) 553–563558

3.2.3. Litter condition

In the 10.0 mg/kg/day group, there was one litter in

which there was no milk in the pups’ stomachs

(observable to the naked eye by viewing the ventral side

of the pups) and all pups in this litter died within five

days of birth. In a separate 10.0 mg/kg/day litter, all pups

were observed to have a mottled appearance to their skin.

This mottling was apparent even as fur developed, but

pup appearance became normal once fur development was

complete.

3.3. Pup developmental measures

3.3.1. Body weight

Body weights for pups after PND1 are shown in Fig. 6.

Data were analyzed by an AR-1 mixed model repeated

measures ANOVAwith heterogeneous variance components

ACR on litter size. b) On PND1, after which time some pups had died, a

smaller litter size on average than the control.

0 5 10 15 200

5

10

15

20

25

30

35

40

0 mg/kg (n=7)0.5 mg/kg (n=9)1.0 mg/kg (n=9)2.5 mg/kg (n=7)5.0 mg/kg (n=8)10.0 mg/kg (n=5)

0 5 10 15 200

5

10

15

20

25

30

35

40

0 mg/kg (n=8)0.5 mg/kg (n=9)1.0 mg/kg (n=9)2.5 mg/kg (n=7)5.0 mg/kg (n=8)10.0 mg/kg (n=5)

Postnatal Day

Bod

y W

t (g)

Bod

y W

t (g)

Males

Females

Fig. 6. Effect of ACR on pup body weights from PNDs 1–22. ACR

treatment had a small but significant effect on pup body weight on PND22

when data were considered with both sexes combined; no significant effect

of sex was observed. See text for details.

J. Garey et al. / Neurotoxicology and Teratology 27 (2005) 553–563 559

for males and females. A statistically significant treat-

ment�day effect was observed [F(105,1594) = 1.42;

P <0.01]. Post-hoc analyses revealed significantly lower

weights in the 1.0, 2.5, 5.0 and 10.0 mg/kg/day treatment

groups compared to the control group on PND22 (P <0.05

overall), with differences ranging between 2–8% for

females and 5–10% for males. There was, however, no

Table 2

Developmental measures in acrylamide-treated rat pups

Event Dose of acrylamide (mg/kg body weight)

0 0.5 1

Males

Day of eye opening 17.4T0.2 17.3T0.3 1

Day of fur development 9.7T0.2 10.0T0.0 1

Females

Day of eye opening 17.3T0.3 17.2T0.3 1

Day of fur development 9.7T0.2 10.0T0.0 1

Values represent postnatal day of observationTS.E.M. Animals were inspected for

until the relevant day was established for all animals.

significant effect of sex� treatment�day [F(105,1591)=

0.62; P=1.00].

3.3.2. Day of fur development, pinnae detachment and eye

opening

No statistically significant differences were observed in

day of fur development or eye opening (see Table 2).

However, a statistically significant treatment effect was

observed for day of pinnae detachment [F(5,63)=7.8;

P <0.001], with post-hoc analyses demonstrating that the

10.0 mg/kg/day treatment group had a later day of pinnae

detachment than that observed in all other treatment groups

(all pairwise multiple comparison procedures, Holm–Sidak

method; P <0.001; see Fig. 7).

3.4. Behavioral measures

3.4.1. Righting reflex, negative geotaxis, forelimb hanging

and open field activity

No significant treatment effects were observed on

performance of righting reflex or duration of forelimb hang

time (Table 3). ACR also had no significant effects on any

open field measure (total activity or level of inactivity; see

Table 3). However, a statistically significant treatment effect

was observed on negative geotaxis performance (F[5,

79]=3.9; P <0.01; see Fig. 8). Pups in the 10 mg/kg/day

treatment group had a significantly shorter latency to turn

180- than pups in all other groups except for those treated

with 5.0 mg/kg/day (Tukey HSD; P <0.05).

3.4.2. Rotarod performance

No statistically significant differences in Rotarod

latency to fall time were observed. However, analysis of

the data for both sexes combined over the two days of

testing revealed a statistically significant linear trend

toward decreased fall latency with increased ACR dose

(repeated measures ANOVA with orthogonal linear con-

trasts, P <0.05). A linear trend was in evidence whether

the data were analyzed as two days of averaged trial data

(see Fig. 9) or as six trials (three trials over two days)

considered separately. A significant effect of trial was also

.0 2.5 5.0 10.0

7.3T0.3 17.7T0.2 17.0T0.3 17.3T0.4

0.0T0.1 10.1T0.3 9.8T0.3 10.3T1.0

7.2T0.2 17.3T0.3 16.8T0.4 17.5T2.5

0.0T0.2 10.1T0.1 9.6T0.3 10.4T1.5

these developmental milestones beginning on PND1; inspections continued

0

2

4

6

8

10

12

*

0(8)

0.5 (10)

1.0 (10)

2.5 (7)

5.0 (8)

10.0 (5)

Treatment group (mg/kg/day)

Day

of

pin

nae

det

ach

men

t

( ) = number of litters

female

male

Fig. 7. Effect of ACR on day of pinnae detachment. Pups in the 10 mg/kg/day treatment group had a significantly later day of pinnae detachment than that seen

in all other treatment groups ( P <0.05).

J. Garey et al. / Neurotoxicology and Teratology 27 (2005) 553–563560

found, indicating that the pups in all treatment groups

tended to improve their performance with repeated trials

(P <0.001).

4. Discussion

In this study on the effects of ACR on developmental

and behavioral measures in rats, ACR exposure signifi-

cantly decreased pup body weight gain over the last

several days of treatment. A delay in the day of pinnae

detachment and reduced negative geotaxis latencies were

observed in the 10 mg/kg/day group only. ACR reduced

pup body weight at doses as low as 1.0 mg/kg/day. While

previous studies have documented body weight reductions

in Fischer 344 rats exposed to ACR in utero, the lowest

dose reported to cause significant pup body weight

decreases was 5.0 mg/kg/day and only in males [26]; the

next lowest dose tested was 0.5 mg/kg/day. Wise et al. [28]

used Sprague–Dawley rats in a study in which dams were

dosed from GD6 through lactational day 10; they also

found 5 mg/kg/day to be the lowest dose at which pup

Table 3

No significant effects of ACR were observed on righting reflex, forelimb hangin

Righting reflex (s) Forelimb hanging (s)

PND4 PND5 PND6 PND7 PND12 PND13 PND1

0 mg/kg 5.2T1.5a 2.7T0.8 1.8T0.2 3.1T1.1 9.3T1.0 13.2T2.0 23.7T

0.5 mg/kg 4.2T0.4 2.9T0.6 1.7T0.2 2.1T0.4 8.1T0.7 11.4T1.1 17.7T

1.0 mg/kg 5.3T1.4 2.3T0.3 1.9T0.2 2.9T0.9 9.9T1.4 10.0T0.7 19.5T

2.5 mg/kg 8.1T2.2 3.0T0.8 2.7T0.9 2.2T0.3 10.5T1.5 16.1T2.9 20.7T5.0 mg/kg 3.7T1.1 3.2T0.6 2.1T0.3 2.1T0.2 9.0T1.3 15.1T1.7 22.8T

10.0 mg/kg 8.3T1.7 4.0T1.1 2.4T0.5 2.6T0.4 12.4T2.0 12.8T2.1 23.9Ta meanTS.E.M.

body weight was reduced by ACR exposure, although it

was seen only transiently and only in females. Wise et al.

[28] indicated that of all the measures in their study, pup

body weight was the most sensitive indicator of devel-

opmental toxicity.

The ACR-induced delay in pinnae detachment by

approximately two days has not been reported previously

for ACR; however, a review of previous developmental

studies of ACR suggests that pinnae detachment was not

assessed (e.g., [11,28]). While ACR appeared to have no

significant main effect of treatment in a repeated-measures

ANOVA of Rotarod latency to fall time, the linear trend

analysis for Rotarod performance indicated the existence of

a dose-response relationship, with the highest dose of ACR

producing the shortest fall-time latency. Previous studies of

ACR using Rotarod testing have reported significant ACR

effects on Rotarod performance in a variety of rodent

models (e.g. [14,16,20,23]). All of these studies indicate that

ACR produces detectable effects on Rotarod performance,

prior to the occurrence of other observable effects. The lack

of ACR effects on forelimb hang time or open field activity

observed here suggests that motor deficits may be just

g, or open field measures

Open field measures

Inactivity (s) Total activity

(# beam breaks)

4 PND15 PND16 PND18 PND19 PND18 PND19

1.4 27.4T2.0 29.9T2.0 699.4T7.5 657.3T30.4 6.0T1.1 17.3T7.9

1.5 24.2T2.2 28.0T2.0 703.0T1.2 626.4T30.5 5.2T0.2 32.0T9.7

1.7 21.5T1.8 28.2T1.7 675.4T18.7 690.7T14.7 11.6T4.4 10.8T5.3

2.4 25.5T2.9 27.2T2.5 676.1T16.5 616.9T38.9 9.6T3.0 25.9T8.61.8 24.0T2.1 28.5T2.4 678.4T11.8 626.9T26.7 12.2T3.8 30.9T8.5

3.6 25.9T4.0 23.4T3.1 683.8T20.0 674.8T21.7 13.7T6.8 16.3T7.4

0

10

20

30

40

50

60

70 0 mg/kg0.5 mg/kg1.0 mg/kg2.5 mg/kg5.0 mg/kg10.0 mg/kg

*

PND8 PND9 PND10

Lat

ency

to

tu

rn (

sec)

Fig. 8. Effect of ACR on negative geotaxis. A statistically significant main effect of treatment was observed.

J. Garey et al. / Neurotoxicology and Teratology 27 (2005) 553–563 561

starting to appear in rats at the time they were tested in the

Rotarod; (i.e., PNDs 21 and 22).

Performance on the multi-modal negative geotaxis and

Rotarod tasks requires the involvement of numerous CNS

and PNS components. Thus, ACR effects on these two tasks

may impact a variety of systems and/or regions, such as

muscle strength, response to fatigue, and cerebellar func-

tioning. High dose ACR exposure has been shown to affect

the dopaminergic system at high doses [1]), but whether

similar effects might occur at the lower doses used here is

unknown. However, appropriate functioning of the vestib-

ular system is essential for negative geotaxis and Rotarod

Fig. 9. Effect of ACR on Rotarod performance. Data presented are averaged

over the two days of testing. A statistically significant trend was observed,

with higher doses corresponding to lower fall time latencies (repeated

measures ANOVA with orthogonal linear contrasts; P <0.05).

performance [2,15]. Further, the ACR-induced delay in

pinnae detachment is intriguing given that the inner ear

develops in part from the same embryonic germ cell layer

(i.e., ecotoderm) as the outer ear [3,19]. It may be that one

effect of developmental ACR exposure is to alter the

trajectory of normal ear development. That the righting

reflex (also dependent upon vestibular function [2] was not

significantly altered by ACR here may be a function of the

earlier time of assessment (PNDs 4–7) when total ACR

exposure may have been below a threshold level. Tasks that

were less dependent upon inner ear function (open field

exploratory locomotor activity and the forelimb hang test for

neuromuscular strength [6]) were also unaffected by the

ACR levels in this study. Ultimately, the mechanisms

underlying changes in negative geotaxis and Rotarod

performance may also play a role in the gait abnormalities

described in rodents exposed to higher ACR doses.

Although a previous ACR study [28] has been conducted

in rats exposed in utero and during the pre-weaning period

(GDs 6-PND 10), Sprague–Dawley rats were used and

the behavioral endpoints assessed in the present study were

not examined. Wise et al. [28] examined horizontal motor

activity in the open field and auditory startle response; the 15

mg/kg/day dose produced significant decreases in open field

activity at PND 21 in females only; the same dose produced

significant decreases in auditory startle response in PND 22

males and females, as well as PND 59 female weanlings.

Lower doses tested (10 mg/kg was the next lowest dose)

produced no significant results in these paradigms.

The absence of milk in the stomachs of one litter of pre-

weaning pups observed in the present study has also been

reported in previous ACR rat studies [12,28]. In their

studies, lack of milk in the pups was observed as early as

J. Garey et al. / Neurotoxicology and Teratology 27 (2005) 553–563562

PND4 for pups in a 25.0 mg/kg/day treatment group [12]

whereas Wise et al. [28] observed the same effect at 15.0

and 20.0 mg/kg/day (rats were also tested at 10.0 mg/kg/day

but the effect was not observed in them). Friedman et al.

[12] suggested that the lack of milk in their pups seemed to

be due to an inadequate milk supply from dams compro-

mised by their own high-dose ACR treatment prior to giving

birth and was not a direct effect of ACR on the pups.

While much is known about relatively high-dose ACR

exposure and its direct effects on the CNS and PNS, little

is known about the effects of developmental exposures to

ACR on behavior. Therefore, the main focus of this study

was to determine what, if any, effects ACR may have on

early developmental behaviors. The results obtained here

must be considered in the context of the exposure regime

utilized. Since exposures began early after implantation

and continued until weaning, it is not possible to determine

whether the noted effects of ACR were due to pre- or

postnatal exposures, or a combination of both. Determining

the relative influence on behavior of ACR exposure during

specific periods of development will require additional

studies which were beyond the scope of the present report.

An additional focus of the current study was to assess the

effects of ACR on the developing nervous system in the

absence of concurrent maternal toxicity. Thus, pups were

exposed directly to ACR via gavage rather than via milk

from treated dams which, according to previous work

[11,12] would have exhibited overt toxicity with continued

treatment at the higher doses. Direct dosing of pups also

allowed precise control of ACR exposure levels, at least

during the postnatal period. Clearly, direct prenatal

exposure of fetuses is not possible so maternal dosing

was necessary to attain fetal exposure during pregnancy.

Interestingly, however, analyses of a small number of

blood samples collected on GD20 indicate that the

acrylamide levels are approximately equal in both maternal

and fetal blood, at least for the lower doses (unpublished

observations).

Another key element of the current study was the use of a

rodent diet containing very low intrinsic levels of ACR.

Previous ACR neurotoxicity studies in rodents were

conducted prior to the knowledge of an ACR presence in

the diet; thus standard rodent chows were used. Standard

NIH-31 autoclaved diet pellets contained 14 times the

amount of ACR found in the irradiated meal used in this

study [25]. Thus, in earlier ACR rodent studies, animals

were most likely exposed to higher cumulative background

levels of ACR which may have compromised assay

sensitivity, especially where relatively low levels of ACR

(i.e., <5.0 mg/kg/day) were provided as treatment.

The results presented here suggest that exposure to ACR

monomer during the earliest stages of development has the

potential to produce developmental and motoric disturban-

ces which are detectable at a relatively early age. Coupled

with the findings of previous studies suggesting that ACR

produces cumulative neurotoxic damage with continuous

exposure, it is clear that additional work needs to be done

using a wider range of behavioral measures and daily

exposure levels extending over much longer periods of time.

This is particularly true now that we know that human

exposures occur throughout the entire lifespan and that ACR

is found nearly ubiquitously in typical human diets.

Acknowledgments

This study was supported in part by Interagency Agree-

ment #224-93-0001 between NCTR/FDA and the National

Institute for Environmental Health Sciences/National Tox-

icology Program. J. G. gratefully acknowledges support of a

fellowship from the Oak Ridge Institute for Science and

Education through an interagency agreement between the

US Department of Energy and the US Food and Drug

Administration. The authors gratefully acknowledge the

animal care staff of NCTR and Dr. Daniel Doerge and

Nathan Twaddle for their evaluation of acrylamide levels in

rat chow.

References

[1] A.K. Agrawal, R.E. Squibb, Effects of acrylamide given during

gestation on dopamine receptor binding in rat pups, Toxicol. Lett. 7

(1981) 233–238.

[2] V. Bouet, R.J. Wubbels, H.A.A. deJong, A. Gramsbergen, Behavioral

consequences of hypergravity in developing rats, Dev. Brain Res. 153

(2004) 69–78.

[3] M. Brown, R. Keynes, A. Lumsden, The Developing Brain, Oxford

University Press, NY, 2001.

[4] A.M. Cada, E.P. Gray, S.A. Ferguson, Minimal behavioral effects

from developmental cerebellar stunting in young rats induced by

postnatal treatment with a-difluoromethylornithine, Neurotoxicol.

Teratol. 22 (2000) 415–420.

[5] CFSAN, Exploratory Data on Acrylamide in Foods, US Department

of Health and Human Services, US Food and Drug Administration,

2002.

[6] J.N. Crawley, Behavioral phenotyping of transgenic and knockout

mice: experimental design and evaluation of general health, sensory

functions, motor abilities, and specific behavioral tests, Brain Res. 835

(1999) 18–26.

[7] K.M. Crofton, S. Padilla, H.A. Tilson, D.C. Anthony, J.H. Raymer,

R.C. MacPhail, The impact of dose rate on the neurotoxicity of

acrylamide: the interaction of administered dose, target tissue concen-

tration, tissue damage and functional effects, Toxicol. Appl. Pharma-

col. 139 (1996) 163–176.

[8] S.A. Ferguson, A.M. Cada, Developmental treatment with difluoro-

methylornithine has few effects on behavior or body weight in

Sprague–Dawley rats, Neurotoxicol. Teratol. 26 (2004) 83–93.

[9] S.A. Ferguson, E.P. Gray, A.M. Cada, Early behavioral development

in the spontaneously hypertensive rat: a comparison with the Wistar–

Kyoto and Sprague–Dawley strains, Behav. Neurosci. 117 (2003)

263–270.

[10] S.A. Ferguson, M.G. Paule, R.R. Holson, Neonatal dexamethasone on

day 7 in rats causes behavioral alterations reflective of hippocampal,

but not cerebellar, deficits, Neurotoxicol. Teratol. 23 (2001) 57–69.

[11] E.A. Field, C.J. Price, R.B. Sleet, M.C. Marr, B.A. Schwetz, R.E.

Morrissey, Developmental toxicity evaluation of acrylamide in rats

and mice, Fundam. Appl. Toxicol. 14 (1990) 502–512.

J. Garey et al. / Neurotoxicology and Teratology 27 (2005) 553–563 563

[12] M.A. Friedman, R.W. Tyl, M.C. Marr, C.B. Myers, F.S. Gerling, W.P.

Ross, Effects of lactational administration of acrylamide on rat dams

and offspring, Reprod. Toxicol. 13 (1999) 511–520.

[13] B.G. Gold, H.H. Schaumberg, Acrylamide, in: P.S. Spencer, H.H.

Schaumberg, A. Ludolph (Eds.), Experimental and Clinical Neu-

rotoxicology, 2nd edR, Oxford University Press, New York, 2000,

pp. 124–132.

[14] M.L. Kaplan, S.D. Murphy, Effect of acrylamide on rotarod perfor-

mance and sciatic nerve b-glucuronidase activity in rats, Toxicol.

Appl. Pharmacol. 22 (1972) 259–268.

[15] W.J. Kinnard Jr., N. Watzman, Techniques used in the evaluation

of psychotropic drugs on animal activity, J. Pharm. Sci. 55 (1966)

995–1012.

[16] M.H. Ko, W.P. Chen, S.Y. Lin-Shiau, S.T. Hsieh, Age-dependent

acrylamide neurotoxicity in mice: morphology, physiology and

function, Exp. Neurol. 158 (1999) 37–46.

[17] E.J. Lehning, C.D. Balaban, J.F. Ross, R.M. LoPachin, Acrylamide

neuropathy: III. Spatiotemporal charcteristics of nerve cell damage in

forebrain, Neurotoxicology 24 (2003) 125–136.

[18] R.M. LoPachin, The changing view of acrylamide neurotoxicity,

Neurotoxicology 25 (2004) 617–630.

[19] K.L. Moore, The Developing Human, W.B. Saunders Co, Philadel-

phia, PA, 1988.

[20] L. Shell, M. Rozum, B.S. Jortner, M.F. Ehrich, Neurotoxicity of

acrylamide and 2,5-hexanedione in rats evaluated using a functional

observational battery and pathological examination, Neurotoxicol.

Teratol. 14 (1992) 273–283.

[21] C.J. Smith, T.A. Perfetti, M.A. Rumple, A. Rodgman, D.J. Doolittle,

IARC group 2A carcinogens’ reported in cigarette mainstream smoke,

Food Chem. Toxicol. 39 (2001) 175–180.

[22] F. Sorgel, R. Weissenbacher, M. Kinzig-Schippers, A. Hofmann, M.

Illauer, A. Skott, C. Landersdorfer, Acrylamide: increased concen-

trations in homemade food and first evidence of its variable absorption

from food, variable metabolism and placental and breast milk transfer

in humans, Chemotherapy 48 (2002) 267–274.

[23] H. Tanii, K. Hashimoto, Neurotoxicity of acrylamide and related

compounds in rats. Effects on rotarod performance, morphology of

nerves and neurotubulin, Arch. Toxicol. 54 (1983) 203–213.

[24] E. Tareke, P. Rydberg, P. Karlsson, Analysis of acrylamide, a

carcinogen formed in heated foodstuffs, J. Agric. Food Chem. 50

(2002) 4998–5006.

[25] N.C. Twaddle, M.I. Churchwell, L.P. McDaniel, D.R. Doerge,

Autoclave sterilization produces acrylamide in rodent diets: im-

plications for toxicity testing, J. Agric. Food Chem. 52 (2004)

4344–4349.

[26] R.W. Tyl, M.A. Friedman, P.E. Losco, L.C. Fischer, K.A. Johnson,

D.E. Strother, C.H. Wolf, Rat two-generation reproduction and

dominant lethal study of acrylamide in drinking water, Reprod.

Toxicol. 14 (2000) 385–401.

[27] WHO, Health Implications of Acrylamide in Food: Report of a Joint

Food and Agriculture Organization (FAO)/WHO Consultation, WHO

Headquarters: Pub Food Safety Programme, WHO, Geneva, Switzer-

land, 2002.

[28] L.D. Wise, L.R. Gordon, K.A. Soper, D.M. Duchai, R.E. Morrissey,

Developmental neurotoxicity evaluation of acrylamide in Sprague–

Dawley rats, Neurotoxicol. Teratol. 17 (1995) 189–198.