Preweaning behaviors, developmental landmarks, and acrylamide and glycidamide levels after pre- and postnatal acrylamide treatment in rats

Neurotoxicology and Teratology 32 (2010) 373–382

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Neurotoxicology and Teratology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / n e u t e r a

Preweaning behaviors, developmental landmarks, and acrylamide and glycidamide levels after pre- and postnatal acrylamide treatment in rats☆ Sherry A. Ferguson a,⁎, Joan Garey a, Melody E. Smith a, Nathan C. Twaddle b, Daniel R. Doerge b, Merle G. Paule a a b

Division of Neurotoxicology, National Center for Toxicological Research/FDA, United States Division of Biochemical Toxicology, National Center for Toxicological Research/FDA, United States

a r t i c l e

i n f o

Article history: Received 13 November 2009 Received in revised form 12 January 2010 Accepted 24 January 2010 Available online 29 January 2010 Keywords: Acrylamide Righting Negative geotaxis Activity Rat

a b s t r a c t At high levels of exposure, acrylamide monomer (AA) is a known neurotoxicant (LoPachin, 2004 [23]). The effects of lower levels of exposure, such as those experienced via a typical human diet, have not been widely investigated. Data at these levels are particularly relevant given the widespread human exposure through carbohydrate-containing foods cooked at high temperatures. Additionally, daily AA intake is estimated to be higher for infants and children. Earlier, we described behavioral alterations in preweaning rats resulting from developmental AA treatment (0.5–10.0 mg/kg/day) (Garey et al., 2005 [14]). In the present study, the effects of lower doses were measured as well as serum AA and glycidimide (GA) levels in dams, fetuses, and young pups. Pregnant Fischer 344 dams (n = 48–58/treatment group) were gavaged with 0.0, 0.1, 0.3, 1.0, or 5.0 mg AA/kg/day beginning on gestational day 6 and ending on the day of parturition. Beginning on postnatal day 1 (PND 1) and continuing through PND 21, all pups/litter were gavaged with the same dose as their dam. There were no AA treatment effects on offspring fur development, pinnae detachment, or eye opening. Offspring body weight was somewhat decreased by 5.0 mg/kg/day, particularly in males. However, righting reflex (PNDs 4–7), slant board (i.e., negative geotaxis) (PNDs 8–10), forelimb hang (PNDs 12–16), and rotarod behavior (PNDs 21–22) were not significantly altered by AA treatment. Male and female offspring of the 5.0 mg/kg/day group were 30–49% less active in the open field at PNDs 19–20 (p < 0.05). Serum AA levels of GD20 dams and their fetuses were comparable, indicating the ability of AA to cross the placental barrier. AA levels of pups were not affected by age (PND 1 and 22) or sex. In all rats, serum AA and GA levels exhibited a dose–response relationship. These data extend those of our previous study (Garey et al., 2005 [14]) and demonstrate that overt preweaning neurobehavioral effects are apparent in rats exposed to acrylamide preand postnatally, but only at the highest doses tested. Published by Elsevier Inc.

1. Introduction Occupational exposure to acrylamide monomer (AA) is associated with neuropathy characterized by numbness in the hands and/or feet, lower limb fatigue, and unsteady gait [2,6,17,23,31]. Similar neuropathies can be produced in laboratory animals treated with relatively high doses of AA (e.g., >10 mg/kg/day) [12,28,36]. More recently, however, the detection of measurable levels of AA in heat-treated

☆ This document has been reviewed in accordance with United States Food and Drug Administration (FDA) policy and approved for publication. Approval does not signify that the contents necessarily reflect the position or opinions of the FDA nor does mention of trade names or commercial products constitute endorsement or recommendation for use. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the FDA. ⁎ Corresponding author. HFT-132, Division of Neurotoxicology, 3900 NCTR Road, Jefferson, AR 72079, United States. Tel.: +1 870 543 7589; fax: +1 870 543 7181. E-mail address: [email protected] (S.A. Ferguson). 0892-0362/$ – see front matter. Published by Elsevier Inc. doi:10.1016/j.ntt.2010.01.010

foods containing high levels of reducing sugars and the amino acid asparagine [34,35] has spurred renewed interest in the toxicity of AA. Estimates of daily AA intake in adult humans range 0.2–0.8 µg/kg/ day (reviewed in [3,10]) and depend on lifestyle, diet, and age. For example, smokers are estimated to have intakes at the higher end of the range [3] as do those consuming higher amounts of foods known to be rich in AA [16,24]. Estimated intakes in children and adolescents are at least 50% higher than in adults (reviewed in [10] but see also [18]), in part because their smaller body size results in a higher average food intake/kg body weight than adults and because children and adolescents tend to consume more French fries, potato chips/ crisps and other snack foods with high concentrations of AA. Given that children under 10 years of age have an estimated intake of 1.0– 1.3 µg/kg/day (reviewed in [10]), the neurotoxicological alterations that may occur with developmental exposure via diet needs careful study. While the acute effects of AA treatment at high doses (≥10 mg/kg/ day) in laboratory animals mimic the neuropathies described in

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humans, the effects of long-term exposure to lower AA doses more relevant to dietary intake estimates have not been investigated. Even rarer are descriptions of effects occurring after developmental treatment. Because AA crosses the placenta [7,27,30], is found in breast milk [30], and in baby food [11,19], it is likely that human exposure begins prenatally and continues throughout life. Our laboratory has conducted a series of studies evaluating the effects of developmental and lifelong AA treatment in Fischer 344 rats. Earlier, we described minor decrements in body weight in rats that had been treated with AA beginning on gestational day 6 and continuing postnatally [14]. Mild motor coordination alterations were exhibited by the treated offspring, as demonstrated by slant board (negative geotaxis) and rotarod performance. Here, we provide data on the effects of lower AA doses as well as serum levels of AA and its epoxide metabolite, glycidamide (GA).

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 AA 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. Acrylamide treatment solutions Treatment solutions were prepared every two weeks by mixing AA with 0.2 µm-filtered water. Stability studies of the high and low concentrations (i.e., 0.02 and 1.0 mg/mL solutions) were performed using GC/FID. AA solutions were shown to be stable for up to 28 days at an ambient temperature when stored in amber glass bottles; however, treatment solutions were stored for no longer than two weeks prior to use. Solutions of each concentration of AA (i.e., 0.0, 0.02, 0.06, 0.20, and 1.0 mg/mL) were analyzed using GC/FID and all were found to be 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 followed the “Guide for the care and use of laboratory animals” [26]. Date-mated sperm plug-positive Fischer 344 (F344) female rats (n = 258) were obtained from the NCTR Breeding Colony over 15 replicates. Of these, 9 replicates (n = 130 plug-positive females) were obtained between November 2004 and January 2005 and are termed here Experiment 1; the remaining 6 replicates (n = 128 plug-positive females) were obtained February–April 2006 and are termed here Experiment 2. Funding issues necessitated the extended periods for obtaining the replicates. Plug-positive rats arrived at the vivarium on GD6 at which time they were tail tattooed and individually housed in standard polycarbonate tub cages lined with wood chip bedding. Food (see below) and water were provided ad libitum. Temperature and humidity of the housing room were maintained at 23 ± 3 °C and 45–55% relative humidity, respectively. The housing room was maintained on a 12:12 h light:dark cycle with lights on at 0700 when daylight saving time was in effect and at 0600 standard time.

2.4. Diet The diet was NIH-311R (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 AA than autoclaving [37]. Analysis by the Division of Chemistry at NCTR using liquid chromatography/electrospray ionization-mass spectrometry analysis (LC/EIMS) determined the AA content of the diet to be approximately 40 ppb. The AA content of the water used as the vehicle control and drinking water was found by LC/EIMS to be below the detection limit of 2 ppb. 2.5. Study design Plug-positive females were assigned to treatment groups on the basis of GD4 or GD5 body weight such that all treatment groups had approximately equal average body weights. Beginning on GD6 (GD0 = day of detection of vaginal sperm plug), each rat received one of four doses of AA (0.1, 0.5, 1.0, or 5.0 mg/kg) or vehicle (0.2 µmfiltered water) daily via gavage. Treatment occurred at the same time each day ±1 h. All doses (including vehicle) were administered at volumes of 5 mL/kg. Body weight, food consumption and water intake were measured daily beginning on GD6. On the day of birth (postnatal day (PND) 0), the number of pups/ litter were counted; no dosing was performed that day. Pup sex was recorded on PND 1. From PND 1 through 22, body weights were recorded and all pups were gavaged with AA at the same volume and dose given previously to their dam. Thus, the last gavage to dams was given the day prior to parturition and each pup was gavaged beginning on the day after parturition. After gavage of the pups on PND 1, litters were culled to achieve a litter size of eight (4:4 or 3:5 sex ratios) or seven (3:4 sex ratio). Where necessary, cross-fostering of pups within treatment groups was performed to achieve targeted litter size and sex distribution; only rarely was cross-fostering done to different treatment groups. Some culled pups were used for assay of AA and glycidamide (GA) levels (see below). After culling, the pups to be retained on study were paw tattooed for identification. Beginning on PND 1, all retained pups, including foster pups, were weighed, gavaged, and observed daily for the occurrence of developmental landmarks. Specifically, these were 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). All physical and behavioral measurements were conducted during the light phase of the light:dark cycle. In addition, twice daily mortality checks were conducted. Data from fostered pups were not included in any analyses. Some plug-positive dams were euthanized on GD20, some had normal size litters but were unable to be maintained due to housing space restrictions, some had litters that were too small to be maintained on study, and some dams did not deliver any pups by what would have been GD26. Some of each of these groups were used for assay of AA and GA levels (see below). Uteri of those dams that did not deliver by what would have been GD26 were examined at necropsy and a number of grossly visible resorption sites (uterine scars) were counted. Subsequently, uteri were placed in 10% ammonium sulfide for at least 15 min, removed, rinsed, and placed on a light table for trans-illumination to determine the number of embryo implants that died before resorption sites were formed. The numbers for each of these outcomes are tabulated in the results. 2.6. Acrylamide and glycidamide (GA) levels AA and GA levels in serum were measured in 17 GD20 females (n = 8 from Expt. 1; n = 9 from Expt. 2) and their fetuses, 79 females

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that did not deliver by what would have been GD26 (n = 39 from Expt. 1; n = 40 from Expt. 2), 222 PND 1 male and female pups at culling (n = 102 from Expt. 1; n = 120 from Expt. 2), and 108 PND 21 male and female pups at weaning (n = 37 from Expt. 1; n = 71 from Expt. 2). For GD20 measures, pregnant dams were randomly selected after inspection for visual confirmation of pregnancy. All were gavaged as normal and approximately 60 min later were anesthetized with CO2 and blood collected via cardiac puncture. This timepoint for blood collection was chosen as AA levels peak at 60 min post-gavage [9]. The blood was transferred to a serum separator tube, allowed to clot for a minimum of 30 min, and then centrifuged at 3000 rpm (1000 g) for 10 min, after which the serum was removed, placed in a polypropylene tube, and stored at −80 °C until assay. After blood collection from the dam, the fetuses were removed from the uterus and euthanized by a single cut which severed the spinal cord and the carotid artery. Fetal blood was collected into microcapillary tubes from the severed carotid artery. The microcapillary tubes were spun in an IE Micro Hematocrit Centrifuge for 5 min, after which the tubes were cut at the base of the serum, the serum expelled into a polypropylene tube, and stored at −80 °C until assay. Nonpregnant dams and PND 21 pups were anesthetized with CO2 and blood collected via cardiac puncture. The blood was transferred to a serum separator tube, allowed to clot for a minimum of 30 min, and then centrifuged at 3000 rpm (1000 g) for 10 min, after which the serum was removed, placed into a polypropylene tube, and stored at −80 °C until assay. PND 1 pups were anesthetized using CO2 and euthanized by a single cut which severed the spinal cord and the carotid artery. Blood was collected into microcapillary tubes from the severed carotid artery and spun in an IE Micro Hematocrit Centrifuge for 5 min, after which the tubes were cut at the base of the serum, the serum expelled into a polypropylene tube and stored at −80 °C until assay. Analyses of AA and GA in serum were performed using a validated high throughput LC-ES/MS/MS method previously described [38]. Briefly, labeled internal standards were added to each thawed serum sample (10–100 µL), samples were then purified using solid phase extraction in 96-well plates, and analyzed using liquid chromatography with electrospray tandem mass spectrometry in the selected reaction monitoring mode by monitoring specific transitions for labeled and unlabeled AA and GA. The limits of quantification (LOQ) were 0.01 µM for AA and 0.1 µM for GA. Quality control measures were performed during every sample set and included the analysis of blank and AA- and GA-spiked serum samples, blank injections, and injections of authentic standards.

behavior is not geotaxic in nature [1,25]. Thus, the suggested term “slant board” is used here. All pups/litter were removed from the home cage and placed in a small holding cage prior to testing. Each pup was then placed, ventral side down, with its head pointed toward the lower end on a sandpaper-covered board angled at 45° to the horizontal. Latency to turn 180° from the original starting position was measured with a maximum of 60 s allowed. Time of fall was recorded if the pup fell from the apparatus. Each pup was tested for a single trial on each of the three test days.

2.6. Behavioral testing protocols

“Experiment” was a factor in most analyses and was defined as either Experiment 1 (those plug-positive dams received November 2004–January 2005) or Experiment 2 (those plug-positive dams received February 2006–April 2006). Litter was the fundamental randomization unit in all analyses. All analyses were conducted using SAS (version 9.1, SAS Institute Inc., Cary, NC) and interactions were further analyzed using appropriate paired t-tests to compare each treated group to the control. For serum levels of AA and GA, developmental landmarks, open field activity and rotarod performance, pairwise comparisons of means of dose group were made using Dunnett adjusted contrasts. For datasets in which a repeated measures, mixed model ANOVA was used for analysis (body weights, food/water intake, righting reflex, open field activity, and rotarod performance), within-group correlations were modeled using the heterogeneous AR1 correlation structure [22]. Statistical significance was defined as p < 0.05.

Behavioral testing was conducted identically to that described earlier [14]. Behavioral testing of the offspring was always conducted prior to the daily gavage. Briefly, litters were assessed in a random order for each test described below. For righting reflex, slant board behavior (formerly termed negative geotaxis) and forelimb hang, all pups/litter were assessed by testers blind to treatment condition. For open field activity and rotarod coordination, one male and one female/litter were tested with the same male/female pair tested for both tests and on each day. 2.6.1. Righting reflex (PNDs 4–7) All pups/litter were removed from the home cage and placed in a small holding cage prior to testing. Each pup was placed dorsal side down on a smooth flat surface and the latency to right onto all four paws was recorded with a maximum of 60 s allowed. One trial/day occurred for each of the four test days. 2.6.2. Slant board behavior (PNDs 8–10) This test has formerly been termed negative geotaxis in previous publications [5,14]; however, recent reports suggest that such

2.6.3. Forelimb hang (PNDs 12–16) All pups/litter were removed from the home cage and placed in a small holding cage just prior to testing. Each pup was held close to a taut string (41 cm above a padded surface) stretched between two wooden blocks and then permitted to grasp the string with its forepaws, at which point it was released, such that it was suspended from the string by its forepaws. The latency to fall from the string (maximum of 60 s) was measured and each pup was tested for a single trial on each of the five test days. 2.6.4. Open field activity (PNDs 19–20) The male and female pups to be tested were removed from their home cage and placed individually in small holding cages for transport to the testing room. The test apparatus was a Plexiglas chamber bisected by photobeams. Duration of immobility (s), total number of photobeam breaks/12-min session and number of photobeam breaks/3-min period (4 periods/test session) were measured. Each of the two pups/litter was tested on two consecutive days. 2.6.5. Rotarod coordination (PNDs 21–22) The male and female pups to be tested were removed from their home cage and placed individually in small holding cages for transport to the testing room. Each pup was placed on a 2.5 cm diameter rotating rod (Smart Rod; AccuScan Instruments, Inc., Columbus, OH) which gradually accelerated 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 at 20 rpm for another 3 min and then slowed to a stop over the last 30 s. Each of the two pups/litter was tested for 3 consecutive trials/day. Latency to fall was automatically recorded via the computer interface. 2.7. Statistics

2.7.1. Serum levels of AA and GA Several measurements of serum AA and GA were below the LOQ. For the purpose of analyses, the LOQ was taken as a conservative value for those measurements below the LOQ (0.01 µM for AA and 0.1 µM for GA). For nonpregnant dams, a two-way analysis of variance

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(ANOVA) was performed for AA and GA levels with Experiment and AA dose as factors. There were insufficient data for pregnant dams and fetuses for meaningful statistical inference; however, descriptive statistics were calculated for AA and GA levels. Pup AA and GA levels were analyzed using a four-way ANOVA with factors of Experiment, dose group, sex and age (PND 1 or 22) and all interactions. 2.7.2. Dam body weight, food intake and water consumption Daily gestational body weights were analyzed separately from daily lactational body weights as not all litters were retained on study through weaning; however, the datasets were analyzed similarly. Gestational (GD6–21) or lactational (postparturitional days (PPDs) 1–21) body weights were analyzed via a repeated measures analysis of covariance (ANCOVA) with Experiment, AA dose, day, and all interactions. For gestational body weights, the GD6 body weight (prior to AA treatment) was used as the covariate. For postparturitional body weights, the PPD 1 body weight was used as the covariate. Gestational and lactational food intake and water consumption were analyzed separately. Initially, gestational food and water intake (g or mL/day) were averaged into three periods: GDs 7–11, 12–16, and 17–21. Lactational food and water intake (g or mL/day) were averaged into six periods: PPD 0–3, 4–7, 8–11, 12–15, 16–19, and 20–22. These were then analyzed via separate repeated measures, mixed model ANOVAs with Experiment, AA dose, GD or PPD period, and all interactions. 2.7.3. Offspring body weight Because litters were culled, birth weights (PND 1) which included all pups/litter were analyzed separately from PND 2 to 22 body weights. Birth weights were averaged by sex for each litter and each sex was separately subjected to a two-way ANOVA with factors of Experiment, AA dose, and the interaction. PND 2–22 body weights were averaged by sex for each litter (excluding fostered offspring) and each sex was then separately subjected to a repeated measures, mixed model ANOVA with Experiment, AA dose, PND, and all interactions. 2.7.4. Developmental landmarks For each endpoint (day of eye opening, pinna detachment, and fur development), the data were first averaged within litter by sex (excluding fostered offspring) such that each litter contributed two datapoints (one/sex) for each endpoint. A mixed model ANOVA was performed with Experiment, AA dose, sex, and all interactions as fixed effects, and litter as a random effect. Contrasts were generated to check for linear trends in dose and log (dose + 1). Because the doses were not evenly spaced, the contrast coefficients were generated using orthogonal polynomials. 2.7.5. Righting reflex The latency to right data were not normally distributed; thus, a log transformation was performed to reduce skewness. Initially, a repeated measures, mixed model ANOVA with factors of Experiment, AA dose, PND, and sex was used to analyze the log latency time. Interactions not reaching statistical significance were excluded from the final model; thus, the final repeated measures, mixed model ANOVA was performed by sex with Experiment, AA dose, PND, and treatment × PND as fixed effects and litter as a random effect. The heterogeneous AR1 structure was used to fit a separate covariance for each PND accounting for the variability over time. Contrasts were generated to check for linear trends in PND, AA dose, and log (dose + 1). Because the doses were not evenly spaced, the contrast coefficients were generated using orthogonal polynomials. 2.7.6. Slant board behavior To analyze the odds of failure (falling from the apparatus or failure to turn within the alloted 60 s), a repeated measures logistic

regression analysis was used with a binomial distribution and logit link function. This analysis determines if AA treatment affects falling or failing to turn. To analyze the latency to turn time, a Cox Proportional Hazards model [20] was run with Experiment, AA dose, sex, and PND as factors. This analysis determines if AA treatment affects the latency to turn. 2.7.7. Forelimb hang behavior To analyze the latency to fall, a Cox Proportional Hazards model [20] was run with Experiment, AA dose, sex, and PND as factors. 2.7.8. Open field activity For each endpoint (activity and duration of immobility), a repeated measures, mixed model ANOVA was performed with Experiment, AA dose, sex, session, and all interactions. Period (3-min periods within each 12-min session) was nested within session in the model. A contrast was generated to check for a linear dose trend. Because the doses were not evenly spaced, the contrast coefficients were generated using orthogonal polynomials. 2.7.9. Rotarod performance For latency to fall, a repeated measures, mixed model ANOVA was performed with Experiment, AA dose, sex, PND, and all interactions. Interactions of more than two levels were not included in the analysis because they would not impact the interpretation of results. The heterogeneous AR1 structure was used to fit a separate covariance for each PND accounting for the variability over time. Contrasts were generated to check for linear trends in dose and log (dose + 1). Because the doses were not evenly spaced, the contrast coefficients were generated using orthogonal polynomials. 2.8. Quality assurance methods These studies were conducted in compliance with the Food and Drug Administration Good Laboratory Practice Regulations (Code of Federal Regulations, Title 21, Part 58). 3. Results 3.1. AA and GA serum measures AA and GA levels are shown in Figs. 1–3. Higher doses of AA produced higher serum levels of AA and GA in all groups. Analysis of AA levels in nonpregnant dams (Fig. 1) indicated significant effects of Experiment (F(1, 69) = 5.08, p < 0.03) and AA dose (F(4, 69) =239.98, p < 0.0004). Post-hoc tests indicated lower AA levels in Experiment 2 (p < 0.05) and significant differences between the control and the 1.0 and 5.0 mg/kg AA groups (p < 0.05). Analysis of GA levels in nonpregnant dams indicated a significant effect of AA dose (F(4, 69) = 220.37, p < 0.0004). Post-hoc tests indicated significant differences between the control and the 1.0 and 5.0 mg/kg AA groups (p < 0.05). Because no serum was collected from control GD20 dams in Experiment 1 and there were no more than 3 datapoints in any other combination of Experiment and dose, there were insufficient data for meaningful statistical inference. However, the data for AA levels indicated higher mean levels for Experiment 1 than Experiment 2 and a dose–response relationship. Similarly, no serum was collected from control fetuses in Experiment 1 and no more than 3 datapoints in any other combination of Experiment and dose; thus, there were insufficient data for meaningful statistical inference. Similarly, however, the AA levels indicated higher mean levels for Experiment 1 than Experiment 2 and a dose–response relationship. Analysis of AA levels in pups indicated a significant interaction of Experiment × dose group × PND (F(4, 202) = 2.94, p < 0.03). Post-hoc tests indicated that AA levels in PND 1 pups of Experiment 1 treated

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Fig. 1. Serum levels of AA and GA from female rats which did not deliver by what would have been GD26. Blood was collected approximately 60 min after gavage. Group numbers range 6–10/Experiment/AA group.

with 5.0 mg/kg were significantly more than control PND 1 pups of Experiment 1 (p < 0.05), AA levels in PND 22 pups treated with 0.3 or 5.0 mg/kg of Experiment 1 were significantly more than control PND 22 pups of Experiment 1 (p < 0.05), and AA levels in PND 1 and 22 pups treated with 5.0 mg/kg of Experiment 1 were significantly more than control same-age pups of Experiment 2. Analysis of GA levels in

pups indicated a significant interaction of Experiment × dose group × PND (F(4, 204) = 5.02, p < 0.001). Post-hoc tests indicated no significant differences among GA levels in PND 1 pups within Experiment. Further, GA levels of PND 22 pups treated with 1.0 or 5.0 mg/kg in Experiment 2 were significantly more than control PND 22 pups in Experiment 2 (p < 0.05).

Fig. 2. Serum levels of AA and GA from GD20 dams and their fetuses. Blood was collected approximately 60 min after gavage. Group numbers range 1–3/Experiment/AA group; however, no GD20 dams of the 0.0 mg/kg group were assayed in Experiment 1. All data for fetuses were first averaged within litter. A and B: Serum levels of AA and GA in GD20 dams. C and D: Serum levels of AA and GA in fetuses.

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Fig. 3. Serum levels of AA and GA from PND 1 and PND 22 pups. Blood was collected approximately 60 min after gavage. Group numbers range 3–9/Experiment/AA group. All data were first averaged within litter. A and B: Serum levels of AA and GA in PND 1 pups. Note: One Experiment 1 PND 1 female of the 5.0 mg/kg group had a GA level of 7.324 µM while all others were below 1.0 µM. C and D: Serum levels of AA and GA in PND 22 pups.

3.2. Dam measures Few resorptions or implantations were observed in those females that did not deliver by what would have been GD26. Three females had one observable implantation each (one control, one in the 0.3 mg/kg AA group, and one in the 5.0 mg/kg AA group). Resorptions were more common. Two females exhibited one resorption each (one in the 0.1 mg/kg AA group and one in the 0.3 mg/kg AA group). Two females had two resorptions each (both in the 0.1 mg/kg AA group), two females had five resorptions each (one in the 1.0 mg/kg AA group and one in the 5.0 mg/kg group), and one female in the 5.0 mg/kg AA group had 8 observable resorptions. Fig. 4 shows gestational and lactational body weights of dams by Experiment and AA dose. Analysis of gestational body weights indicated a significant main effect of AA dose (F(4, 14) = 4.92, p < 0.02) and a significant interaction of Experiment × gestational day (F(14, 266) = 2.26, p < 0.007). Post-hoc tests indicated that gestational body weights of the 0.1, 0.3, and 1.0 mg/kg AA groups were significantly more than the control group (p < 0.05 for all contrasts). Post-hoc tests of the Experiment × gestational day interaction did not indicate any significant effects on any day. Analysis of lactational body weights indicated a significant interaction of Experiment × AA dose × PPD (postparturitional day) (F(76, 168) = 3.47, p < 0.001). Post-hoc tests indicated that in Experiment 1, dams of the 0.3 mg/kg group weighed significantly less than control dams on PPD 10 (p < 0.05) and dams of the 1.0 mg/kg group weighed significantly more than control dams on PPD 18 (p < 0.05). There were several significant post-hoc tests within Experiment 2: dams of the 0.1 mg/kg group weighed significantly more than control dams on PPDs 5, 8–12, 14, 16, 18, and 20 (p < 0.05

for all contrasts), dams of the 0.3 mg/kg group weighed significantly less than control dams on PPD 16 (p < 0.05), dams of the 1.0 mg/kg group weighed significantly more than control dams on PPD 20 (p < 0.05), and dams of the 5.0 mg/kg group weighed significantly more than control dams on PPD 20 (p < 0.05). Analysis of gestational water intake indicated a significant interaction of AA dose × gestational period (i.e., GDs 7–11, 12–16, 17–21) (F(8, 28) = 2.58, p < 0.03); however, post-hoc tests indicated no significant differences between any AA group and the control group at any of the three time periods. Analysis of lactational water intake indicated a significant interaction of Experiment × lactational period (i.e., PPDs 0–3, 4–7, 8–11, 12–15, 16–19, 20–22) (F(5, 45) = 6.58, p < 0.0009). On all PPD periods except PPDs 16–19, dams in Experiment 1 consumed more water than dams in Experiment 2 (p < 0.05). Analyses of gestational food consumption indicated a significant interaction of Experiment × gestational period (F(2, 38) = 13.49, p < 0.0009). Post-hoc tests indicated that on GDs 7–11 and 17–21, dams of Experiment 1 consumed more food than dams of Experiment 2 (p < 0.05). Analysis of lactational food consumption indicated a significant interaction of Experiment × lactational period (F(5, 45) = 18.02, p < 0.0009). Post-hoc tests indicated that dams in Experiment 1 consumed more food during PPDs 0–3 and 12–15 than did dams in Experiment 2; however, during PPDs 16–19, dams in Experiment 1 consumed less food than dams in Experiment 2. 3.3. Litter results and offspring body weight Descriptive statistics for litters, including pregnancies, total number of pups delivered, sex ratios, PND 1 pup weights, and numbers

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less than same-sex control pups (p < 0.05). PND 1 body weight of female pups in Experiment 1 was approximately 3% higher than those of Experiment 2. Fig. 5 shows preweaning body weights for males (top) and females (bottom). Analysis of preweaning body weights of male offspring indicated a main effect of AA dose (F(4, 7) = 11.57, p < 0.003) and a significant interaction of Experiment × PND (F(20, 162) = 2.15, p < 0.005). Post-hoc tests indicated that males of the 5.0 mg/kg group had significantly lower body weights than control males (p < 0.05) and male offspring of Experiment 1 weighed more than male offspring of Experiment 2 on all PNDs (p < 0.05). In the analysis of preweaning body weights of female offspring, all main effects and interactions were significant including the 3-way interaction of Experiment × AA dose × PND (F(80, 146) = 8.67, p < 0.0001). Posthoc tests indicated that control females of Experiment 1 weighed less than the 0.1, 1.0, and 5.0 mg/kg female offspring on PNDs 2–13 and PND 15 (p < 0.05). On PND 14, the control females of Experiment 1 weighed less than the 0.1 and 5.0 mg/kg female offspring and on PNDs 16–22, the control females of Experiment 1 weighed less than all AA dose groups (p < 0.05 for all contrasts). In Experiment 2, the control female offspring weighed more than all AA dose groups on all PNDs (p < 0.05 for all contrasts). 3.4. Developmental landmarks Table 2 shows age at eye opening, fur development, and pinna detachment. There was a main effect of Experiment on age at eye opening (F(1, 87) = 36.07, p < 0.0001) indicating that eye opening occurred slightly later in Experiment 2. There were no significant effects of sex or AA dose, nor any significant interactions of these. There was a main effect of Experiment on age at fur development (F(1, 86) = 4.44, p < 0.04) indicating that fur development occurred slightly later in Experiment 2. There were no significant effects of AA dose or sex, nor any significant interactions of these. There was a significant interaction of Experiment × AA dose × sex (F(4, 86) = 2.96, p < 0.03) on age at pinna detachment. However, there were no interpretable significant post-hoc tests. Fig. 4. Mean (± SEM) dam body weight by treatment group. A. Gestational body weights. As percent of GD 6 body weight, dams of the 0.1, 0.3, and 1.0 mg/kg AA groups weighed significantly more than the control group (p < 0.05). B. Postparturitional body weights. A significant interaction of Experiment × AA dose × postparturitional day indicated several significant post-hoc tests (see text for details).

that died preweaning are shown in Table 1. Seven dams (two in Experiment 1 and five in Experiment 2) produced viable litters that were unable to be maintained on study due to housing space restrictions. Analysis of PND 1 body weight of male pups indicated a main effect of AA dose (F(4, 125) = 4.20, p < 0.003). Analysis of PND 1 body weight of female pups indicated a main effect of Experiment (F(1, 127) = 7.27, p < 0.008) and AA dose (F(4, 127) = 4.47, p < 0.002). For both sexes, those pups treated with 5.0 mg/kg weighed significantly

3.5. Righting reflex Average latency to right by AA dose and PND is shown in Table 3. Analysis of latencies in the full factorial repeated measures mixed model ANOVA indicated somewhat shorter latencies (i.e., faster righting) in males. Thus, results from the more appropriate analyses separately by sex are presented. In the analysis of female average latency time, neither Experiment, AA dose, nor any interaction of these were significant. Further, the linear trend dose analysis was not significant. The main effect of PND was significant (F(3, 276) = 30.93, p < 0.001) and indicated improving performance (i.e., shorter latencies) with age in all groups (data not shown). In the analysis of male average latencies, neither Experiment, AA dose, nor any interaction of

Table 1 Litter results (mean ± SEM). AA mg/kg

# dams Expt 1, Expt 2a

# pregnant Expt 1, Expt 2

Pups/litter (incl. dead)b

# litters with too few pupsc

Males/litterd

Females/litterd

PND 1 male body weight (g)

PND 1 female body weight (g)

0.0 0.1 0.3 1.0 5.0

27, 25, 25, 20, 25,

14, 15 12, 17 13, 16 13, 14 16, 17

7.0 ± 0.6 8.1 ± 0.6 8.2 ± 0.6 7.7 ± 0.6 8.2 ± 0.5

11 11 5 6 10

3.76 ± 0.43 4.34 ± 0.39 3.58 ± 0.36 3.89 ± 0.44 3.63 ± 0.31

3.13 ± 0.41 3.59 ± 0.38 4.34 ± 0.51 3.85 ± 0.35 4.50 ± 0.37

5.76 ± 0.08 5.75 ± 0.08 5.69 ± 0.08 5.87 ± 0.08 5.47 ± 0.05e

5.56 ± 0.08 5.46 ± 0.09 5.46 ± 0.07 5.55 ± 0.06 5.20 ± 0.05e

a b c d e

24 27 23 23 23

Totals do not include dams removed on GD20 (Expt. 1 n = 8; Expt. 2 n = 9) for measurement of AA and GA levels (see text). Number of pups/litter was obtained on the day of birth (PND 0). Too few pups to be maintained on study was typically defined as 5 or fewer total pups with no cross-fostering options. Measured on PND 1. Significantly less than same-sex control.

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S.A. Ferguson et al. / Neurotoxicology and Teratology 32 (2010) 373–382 Table 3 Mean ( ± SEM) righting reflex, slant board, forelimb hang, open field, and rotarod coordination endpoints.a AA mg/kg Righting reflex 0.0 0.1 0.3 1.0 5.0 PND 4 2.99 ± 0.30 2.66 ± 0.22 3.45 ± 0.38 3.19 ± 0.49 2.95 ± 0.35 PND 5 2.35 ± 0.27 2.71 ± 0.35 1.99 ± 0.17 2.16 ± 0.16 2.57 ± 0.34 PND 6 1.61 ± 0.12 1.85 ± 0.16 1.66 ± 0.09 1.78 ± 0.12 1.83 ± 0.15 PND 7 1.57 ± 0.09 1.45 ± 0.09 1.35 ± 0.07 1.57 ± 0.14 1.62 ± 0.12 Slant board PND 8 51.8 ± 0.7 57.9 ± 0.6 52.8 ± 0.5 54.7 ± 0.9 55.8 ± 0.5 PND 9 54.3 ± 0.9 54.0 ± 0.9 55.9 ± 0.9 55.0 ± 1.0 54.7 ± 1.0 PND 10 45.9 ± 1.3 49.8 ± 1.3 52.6 ± 1.3 48.1 ± 1.5 48.8 ± 1.5 Forelimb hang PND 12 11.8 ± 0.7 12.5 ± 0.8 11.4 ± 0.7 11.7 ± 0.8 13.8 ± 0.8 PND 13 20.4 ± 1.2 18.8 ± 1.0 17.0 ± 0.9 18.9 ± 1.0 20.3 ± 1.1 PND 14 22.3 ± 1.2 22.0 ± 1.0 22.2 ± 1.1 22.5 ± 1.0 20.4 ± 0.9 PND 15 23.1 ± 1.2 23.5 ± 1.1 21.9 ± 1.1 24.1 ± 1.2 22.3 ± 1.1 PND 16 21.3 ± 1.2 19.3 ± 1.0 20.8 ± 1.1 21.7 ± 1.2 20.9 ± 1.1 Open field PND 19 594.3 ± 35.1 595.8 ± 25.2 599.6 ± 21.4 588.5 ± 22.5 644.1 ± 18.7b PND 20 518.4 ± 33.3 494.8 ± 22.4 556.8 ± 23.4 523.3 ± 18.5 569.5 ± 28.8b Rotarod PND 21 43.4 ± 3.1 43.7 ± 2.2 42.1 ± 1.7 43.5 ± 2.0 48.6 ± 2.7 PND 22 57.6 ± 3.0 56.6 ± 3.2 54.3 ± 2.5 62.3 ± 3.5 66.5 ± 3.0 a Righting reflex and slant board data are latency (s) to right or turn; open field data are duration (s) of freezing behavior; forelimb hang and rotarod data are latency (s) to fall. b Significantly more than 0.0 mg/kg group (p < 0.05).

p < 0.001), indicating shorter latencies with age, and sex (χ2(1) = 4.09, p < 0.05), indicating slightly faster turning in males, but the main effect of AA dose was not significant nor was any interaction with AA dose (Table 3). 3.7. Forelimb hang Fig. 5. Mean (± SEM) preweaning body weights of AA-treated offspring averaged over Experiment (males, top; females, bottom). Males of the 5.0 mg/kg/day group weighed less than control males (p < 0.05) throughout the preweaning period.

these were significant. Again, the linear trend dose analysis was not significant. The main effect of PND was significant (F(3, 276) = 26.64, p < 0.001) and indicated improving performance with age in all groups.

3.6. Slant board behavior In the analysis of odds of failure, the full factorial model with Experiment, AA dose, sex, and PND resulted in none of the interaction effects being statistically significant; thus, they were dropped from the model, except for the AA dose × PND interaction which was retained such that mean failure rate profiles could be produced. Thus, the final model consisted of the main effects and the AA dose × PND interaction. Of these, only the main effect of PND was significant (χ2(2) = 34.9, p < 0.001) which indicated decreasing odds of failure with increasing age (data not shown). In the analysis of latency to turn, there were main effects of PND (χ2(2) = 51.73, Table 2 Age (PND) at developmental landmarks (mean ± SEM). AA mg/kg

Eye opening

Fur development

Pinna detachment

0.0 0.1 0.3 1.0 5.0

18.5 ± 0.2 18.5 ± 0.2 18.8 ± 0.2 18.5 ± 0.2 18.9 ± 0.2

10.7 ± 0.1 10.4 ± 0.1 10.6 ± 0.1 10.6 ± 0.1 10.7 ± 0.1

19.77 ± 0.26 19.37 ± 0.26 19.85 ± 0.24 19.79 ± 0.24 19.68 ± 0.24

There was a main effect of PND in the analysis of latency to fall (χ2(4) = 252.70, p < 0.001) which indicated increasing latencies with age (data not shown). The main effects of AA dose and sex were not significant nor were any interactions (Table 3). 3.8. Open field activity In the full model, no interactions were significant; thus, a reduced model excluding interactions of more than two levels was used. For total activity, there were two significant main effects: AA dose (F(4, 365) = 2.71, p < 0.03) and session (F(1, 365) = 34.27, p < 0.0001). Post-hoc tests indicated that the 5.0 mg/kg group exhibited significantly less activity than the control group (p < 0.05) and activity was higher on the second session (i.e., PND 20) than on the previous day's session (p < 0.05) (Fig. 6). Similarly, the analysis of duration of immobility indicated main effects of AA dose (F(4, 365) = 2.90, p < 0.03) and session (F(1, 365) = 28.65, p < 0.0001). Again, post-hoc tests indicated the 5.0 mg/kg group had a higher duration of immobility than did the control group (p < 0.05) and immobility duration was lower on the second session relative to the first (p < 0.05) (Table 3). 3.9. Rotarod motor coordination Analysis of latency to fall indicated significant main effects of AA dose (F(4, 178) = 3.02, p < 0.02), sex (F(1, 178) = 8.31, p < 0.004), and PND (F(1, 187) = 127.53, p < 0.00009) as well as a significant interaction of Experiment × PND (F(1, 187) = 4.96, p < 0.03). Post-hoc tests indicated that no AA group was significantly different from the control group (Table 3), females remained on the rotating rod longer than males (p < 0.05), and performance was improved on the second day of testing (p < 0.05) (data not shown). There was an overall

S.A. Ferguson et al. / Neurotoxicology and Teratology 32 (2010) 373–382

Fig. 6. Mean (± SEM) open field activity of AA-treated offspring averaged over Experiment. Offspring treated with 5.0 mg/kg/day were less active than control offspring (p < 0.05) on both test days.

significant dose linear trend (p < 0.002); however, no AA group was significantly different from the control group.

4. Discussion Pregnant Fischer 344 rats were treated orally with 0.0–5.0 mg/kg/ day of AA and their offspring were treated directly with the same dose as their dam beginning on postnatal day (PND) 1 and continuing through weaning. This treatment regimen did not substantially alter gestational body weight, food or water intake of the dams. Male and female offspring of dams treated with 5.0 mg/kg weighed 5–6% less than control offspring on postnatal day (PND) 1. However, developmental landmarks such as age at eye opening, fur development, and pinna detachment were not affected by AA treatment at any dose. Neonatal righting reflex, slant board, forelimb hang, and motor coordination behaviors were unaltered by AA treatment. However, male and female offspring of the 5.0 mg/kg AA group were significantly less active in an open field assessment. AA and GA levels reflected a dose response in serum from gestational day 20 dams and fetuses, adult nonpregnant females, and PND 1 and 22 male and female pups. The oral AA doses in this study (0.1–5.0 mg/kg) are higher than the estimated daily AA intake for humans [3,10]; however, most evidence to date indicates that AA-induced neurotoxic effects follow a linear dose–response relationship [15,21,33]. This would indicate that at the typical estimated human daily intakes, there are likely to be few neurotoxic effects as only the highest dose of AA (i.e., 5.0 mg/kg) had any demonstrable effect. Further support for this derives from physiologically based pharmacokinetic/pharmacodynamic modeling specifically targeted for neurotoxicity risk estimation from which only “minimal” risks from dietary AA exposure were determined [8]. Still, birthweights were lower in both sexes and preweaning body weights were lower in males. Similar AA treatment with 5.0 mg/kg in an earlier study was also shown to decrease preweaning body weight in Fischer 344 rats [14]. However, after chronic exposure beginning postweaning, decreased gestational weight gain in rats has been reported by as little as 2.0 mg/kg/day [39]. The serum levels of AA and GA reported here are comparable to those described by Doerge et al. in rats gavaged with 0.1 mg AA/kg [9]. In that study, kinetic parameters derived from gavage dosing were significantly different between males and females. For example, area under the curve (AUC) for AA was several folds higher in adult female rats. Here, we described AA serum levels in male and female fetuses and PND 1 and 22 pups of both sexes. At these young ages, there were no similar indications of sex differences in AA or GA levels. The

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comparable levels of AA and GA in the GD20 fetuses relative to the dams clearly indicate the ability of AA to cross the placental barrier and are suggestive of the same for GA, although the possibility of fetal conversion of AA to GA cannot be eliminated. However, fetal or neonatal conversion of AA to GA seems unlikely given the much lower levels of GA measured in PND 1 pups gavaged with AA compared with fetal serum levels and those of PND 22 pups. While this is only indirect evidence of the inability of young pups to convert AA to GA, it is suggestive of such. Additional evidence are data indicating substantially higher whole body adduct DNA levels in PND 3 mice after injection with GA relative to AA which was interpreted as consistent with a deficiency of cytochrome P450 activity in neonatal mice, limiting GA formation and formation of adducts [13]. Earlier, we also reported that similar treatment with 10 mg/kg AA significantly delayed day of pinna detachment and slant board performance [14]; lower doses (i.e., 0.5–5.0 mg/kg) had more minimal effects. AA treatment in our previous study had no effects on open field activity when measured at PND 18 and 19 [14]. Further, Wise et al. reported that developmental AA treatment (GD6 through lactational day 10) at 5 mg/kg/day had no effects on the open field activity of either male or female Sprague–Dawley rats, although higher doses had sporadic effects [40]. Here, however, males and females in the 5.0 mg/kg AA group were substantially less active in the open field, particularly on PND 19. This hypoactivity did not appear to be a result of alterations in habituation as this treatment group was less active than the control group throughout the 12-min session and not during any specific 3-min period within the session. It is difficult to reconcile the discrepant findings of the current study and our previous study [14]. Supplier of the rats differed, but testing experience was identical. In our previously published experiment, the date-mated rats were obtained from an outside vendor and shipped to the NCTR. In the current studies, the datemated rats were obtained through our own on-site breeding colony. A comparison between the previous and current study indicates that all groups in our previous study were much less active than those of the current study (e.g., the control group of the previous study averaged 6–17 total beam breaks/session vs. 45–65 total beam breaks/session in the current study). In fact, the behavior of subjects of the current study seemed more developmentally advanced; that is, the current subjects exhibited shorter latencies to right, longer forelimb hang durations, and longer latencies to fall from the rotarod. Yet, day of eye opening and fur development was somewhat later in the subjects reported here. It is possible that inbred or environmental differences relevant to the differences in animal sources are responsible, at least in part, for the differences in developmental results. It should be noted that, although few developmental effects were observed in the preweaning ages, the rats described here as Experiment 2 subjects were tested until 24 months of age in various behavioral assessments. One of those assessments indicated an early post-weaning age decreased motivation in subjects at the highest dose as measured by operant behavior [15]. That suggests that, despite a seeming lack of early developmental changes (except for open field activity), other effects of AA treatment were apparent at a later age. Continued exposure and/or difference in endpoint measured could be responsible for the observed changes in motivation with AA treatment. The lack of AA effects on motor coordination as measured with rotarod performance is consistent with previous reports of rats tested at 5.0 mg/kg/day, including our own [14]. Further, chronic AA doses of 15 mg/kg/day or greater seem necessary to induce hindlimb splay or gait abnormalities in adult male or pregnant/lactating rats [4,32,40]. The subjects of the current study were re-assessed for rotarod performance at PNDs 341–343 and 523–525 after lifetime AA exposure. At the later age (PNDs 523–525) females exposed to ≥1.0 mg/kg/day displayed poorer motor coordination than same-sex controls, although males did not seem to be affected by AA exposure [29].

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In the current study, developmental AA treatment (0.1–5.0 mg/kg/ day) had few effects on preweaning behaviors or developmental landmarks, although a significant effect was observed on activity in the open field at an age just prior to weaning. Serum levels of AA and GA from adult female rats, GD20 female rats and their fetuses, and PND 1 and 22 pups exhibited clear dose–response relationships. Behavioral data from these subjects will be assessed through two years of age with animals having received daily AA exposure throughout. The results, which will include additional learning/ memory assessments, as well as grip strength and foot splay tests, will provide an increased database in order to enable a more accurate risk assessment of AA for humans. Conflict of interest statement Nothing declared. Acknowledgements The authors gratefully appreciate the technical expertise provided by Mr. Delbert Law of the Bionetics Corporation and his incredibly talented staff of animal care technicians. This study was supported by Interagency Agreement #224-07-007 between the NCTR/FDA and the National Institute for Environmental Health Sciences/National Toxicology Program. J. Garey and M. Smith were supported by a postdoctoral fellowship and a Science Internship, respectively, from the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the U.S. Food and Drug Administration. References [1] J.R. Alberts, B.A. Motz, J.C. Schank, Positive geotaxis in infant rats (Rattus norvegicus): a natural behavior and a historical correction, J. Comp. Psychol. 118 (2004) 123–132. [2] M. Bachmann, J.E. Myers, B.N. Bezuidenhout, Acrylamide monomer and peripheral neuropathy in chemical workers, Am. J. Ind. Med. 21 (1992) 217–222. [3] T. Bjellaas, P.T. Olesen, H. Frandsen, M. Haugen, L.H. Stolen, J.E. Paulsen, J. Alexander, E. Lundanes, G. Becher, Comparison of estimated dietary intake of acrylamide with hemoglobin adducts of acrylamide and glycidamide, Toxicol. Sci. 98 (2007) 110–117. [4] J.F. Bowyer, J.R. Latendresse, R.R. Delongchamp, L. Muskhelishvili, A.R. Warbritton, M. Thomas, E. Tareke, L.P. McDaniel, D.R. Doerge, The effects of subchronic acrylamide exposure on gene expression, neurochemistry, hormones, and histopathology in the hypothalamus–pituitary–thyroid axis of male Fischer 344 rats, Toxicol. Appl. Pharmacol. 230 (2008) 208–215. [5] A.M. Cada, E.P. Gray, S.A. Ferguson, Minimal behavioral effects from developmental cerebellar stunting in young rats induced by postnatal treatment with alpha-difluoromethylornithine, Neurotoxicol. Teratol. 22 (2000) 415–420. [6] C.J. Calleman, Y. Wu, F. He, G. Tian, E. Bergmark, S. Zhang, H. Deng, Y. Wang, K.M. Crofton, T. Fennell, et al., Relationships between biomarkers of exposure and neurological effects in a group of workers exposed to acrylamide, Toxicol. Appl. Pharmacol. 126 (1994) 361–371. [7] K.L. Dearfield, C.O. Abernathy, M.S. Ottley, J.H. Brantner, P.F. Hayes, Acrylamide: its metabolism, developmental and reproductive effects, genotoxicity, and carcinogenicity, Mutat. Res. 195 (1988) 45–77. [8] D.R. Doerge, J.F. Young, J.J. Chen, M.J. Dinovi, S.H. Henry, Using dietary exposure and physiologically based pharmacokinetic/pharmacodynamic modeling in human risk extrapolations for acrylamide toxicity, J. Agric. Food Chem. 56 (2008) 6031–6038. [9] D.R. Doerge, J.F. Young, L.P. McDaniel, N.C. Twaddle, M.I. Churchwell, Toxicokinetics of acrylamide and glycidamide in Fischer 344 rats, Toxicol. Appl. Pharmacol. 208 (2005) 199–209. [10] E. Dybing, P.B. Farmer, M. Andersen, T.R. Fennell, S.P. Lalljie, D.J. Muller, S. Olin, B.J. Petersen, J. Schlatter, G. Scholz, et al., Human exposure and internal dose assessments of acrylamide in food, Food Chem. Toxicol. 43 (2005) 365–410. [11] P. Fohgelberg, J. Rosen, K.E. Hellenas, L. Abramsson-Zetterberg, The acrylamide intake via some common baby food for children in Sweden during their first year of life—an improved method for analysis of acrylamide, Food Chem. Toxicol. 43 (2005) 951–959. [12] P.M. Fullerton, J.M. Barnes, Peripheral neuropathy in rats produced by acrylamide, Br. J. Ind. Med. 23 (1966) 210–221.

[13] G. Gamboa da Costa, M.I. Churchwell, L.P. Hamilton, L.S. Von Tungeln, F.A. Beland, M.M. Marques, D.R. Doerge, DNA adduct formation from acrylamide via conversion to glycidamide in adult and neonatal mice, Chem. Res. Toxicol. 16 (2003) 1328–1337. [14] J. Garey, S.A. Ferguson, M.G. Paule, Developmental and behavioral effects of acrylamide in Fischer 344 rats, Neurotoxicol. Teratol. 27 (2005) 553–563. [15] J. Garey, M.G. Paule, Effects of chronic low-dose acrylamide exposure on progressive ratio performance in adolescent rats, Neurotoxicology 28 (2007) 998–1002. [16] L. Hagmar, E. Wirfalt, B. Paulsson, M. Tornqvist, Differences in hemoglobin adduct levels of acrylamide in the general population with respect to dietary intake, smoking habits and gender, Mutat. Res. 580 (2005) 157–165. [17] F.S. He, S.L. Zhang, H.L. Wang, G. Li, Z.M. Zhang, F.L. Li, X.M. Dong, F.R. Hu, Neurological and electroneuromyographic assessment of the adverse effects of acrylamide on occupationally exposed workers, Scand. J. Work Environ. Health 15 (1989) 125–129. [18] A. Hilbig, N. Freidank, M. Kersting, M. Wilhelm, J. Wittsiepe, Estimation of the dietary intake of acrylamide by German infants, children and adolescents as calculated from dietary records and available data on acrylamide levels in food groups, Int. J. Hyg. Environ. Health 207 (2004) 463–471. [19] J. Jiao, Y. Zhang, Y. Ren, X. Wu, Development of a quantitative method for determination of acrylamide in infant powdered milk and baby foods in jars using isotope dilution liquid chromatography/electrospray ionization tandem mass spectrometry, J. Chromatogr. A 1099 (2005) 198–202. [20] J.D. Kalbfleisch, R.L. Prentice, The Statistical Analysis of Failure Time Data, WileyInterscience, Hoboken, NJ, 2002 Editoin Edition. [21] B. Ling, N. Authier, D. Balayssac, A. Eschalier, F. Coudore, Assessment of nociception in acrylamide-induced neuropathy in rats, Pain 119 (2005) 104–112. [22] R.C. Littell, G.A. Milliken, W.W. Stroup, R. Wolfinger, SAS System for Mixed Models, SAS Publishing, Cary, NC, 1996 Editoin Edition. [23] R.M. LoPachin, The changing view of acrylamide neurotoxicity, Neurotoxicology 25 (2004) 617–630. [24] C. Matthys, M. Bilau, Y. Govaert, E. Moons, S. De Henauw, J.L. Willems, Risk assessment of dietary acrylamide intake in Flemish adolescents, Food Chem. Toxicol. 43 (2005) 271–278. [25] B.A. Motz, J.R. Alberts, The validity and utility of geotaxis in young rodents, Neurotoxicol. Teratol. 27 (2005) 529–533. [26] National Research Council, Guide for the care and use of laboratory animals, Editoin Edition, National Academy Press, Washington DC, 1996. [27] T. Schettgen, B. Kutting, M. Hornig, M.W. Beckmann, T. Weiss, H. Drexler, J. Angerer, Trans-placental exposure of neonates to acrylamide—a pilot study, Int. Arch. Occup. Environ. Health 77 (2004) 213–216. [28] G.E. Schulze, B.G. Boysen, A neurotoxicity screening battery for use in safety evaluation: effects of acrylamide and 3′, 3′-iminodipropionitrile, Fundam. Appl. Toxicol. 16 (1991) 602–615. [29] M.E. Smith, M.G. Paule, J.D. Garey, S.A. Ferguson, Effects of lifelong acrylamide (AA) treatment on motor coordination and grip strength in adult rats. Program No 83928 2008 Neuroscience Meeting Planner Washington, DC: Society for Neuroscience (2008). [30] F. Sorgel, R. Weissenbacher, M. Kinzig-Schippers, A. Hofmann, M. Illauer, A. Skott, C. Landersdorfer, Acrylamide: increased concentrations 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. [31] M. Takahashi, T. Ohara, K. Hashimoto, Electrophysiological study of nerve injuries in workers handling acrylamide, Int. Arch. Arbeitsmed. 28 (1971) 1–11. [32] M. Takahashi, M. Shibutani, K. Inoue, H. Fujimoto, M. Hirose, A. Nishikawa, Pathological assessment of the nervous and male reproductive systems of rat offspring exposed maternally to acrylamide during the gestation and lactation periods—a preliminary study, J. Toxicol. Sci. 33 (2008) 11–24. [33] E. Tareke, B.D. Lyn-Cook, H. Duhart, G. Newport, S. Ali, Acrylamide decreased dopamine levels and increased 3-nitrotyrosine (3-NT) levels in PC 12 cells, Neurosci. Lett. 458 (2009) 89–92. [34] E. Tareke, P. Rydberg, P. Karlsson, S. Eriksson, M. Tornqvist, Acrylamide: a cooking carcinogen? Chem. Res. Toxicol. 13 (2000) 517–522. [35] E. Tareke, P. Rydberg, P. Karlsson, S. Eriksson, M. Tornqvist, Analysis of acrylamide, a carcinogen formed in heated foodstuffs, J. Agric. Food Chem. 50 (2002) 4998–5006. [36] H.A. Tilson, P.A. Cabe, The effects of acrylamide given acutely or in repeated doses on fore- and hindlimb function of rats, Toxicol. Appl. Pharmacol. 47 (1979) 253–260. [37] N.C. Twaddle, M.I. Churchwell, L.P. McDaniel, D.R. Doerge, Autoclave sterilization produces acrylamide in rodent diets: implications for toxicity testing, J. Agric. Food Chem. 52 (2004) 4344–4349. [38] N.C. Twaddle, L.P. McDaniel, G. Gamboa da Costa, M.I. Churchwell, F.A. Beland, D.R. Doerge, Determination of acrylamide and glycidamide serum toxicokinetics in B6C3F1 mice using LC-ES/MS/MS, Cancer Lett. 207 (2004) 9–17. [39] R.W. Tyla, M.A. Friedman, P.E. Losco, L.C. Fisher, 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. [40] 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.