Abstract
The purpose of this study was to evaluate the potential neurodevelopmental toxicity of perchlorate exposure during gestation and the first 10 days of lactation. Mated Sprague-Dawley rats (25/exposure group) were given continual access to 0, 0.1, 1.0, 3.0, or 10.0 mg/kg-day ammonium perchlorate (AP) in drinking water, starting gestation day 0 (mating) through lactation day 10 (DL 10). One pup/sex/litter/exposure group was assigned to (1) juvenile brain weights, morphometry, and neuropathology; (2) passive avoidance and watermaze testing; (3) motor activity and auditory startle habituation; and (4) adult regional brain weights, morphometry, and neuropathology. AP had no effect on body weights, feed consumption, clinical observations, or sexual maturation of pups at exposures as high as 10.0 mg/kg-day. There were no behavioral effects in the offspring exposed as high as 10.0 mg/kg-day as evaluated by passive avoidance, swimming watermaze, motor activity, and auditory startle. Increases in hypertrophy and hyperplasia of the thyroid follicular epithelium and a decrease in the thyroid follicle size were observed in culled male pups in the 10.0 mg/kg-day group on DL 5. The exposure level for effects on triiodothyroxine (T3), thyroxine (T4), and thyroid-stimulating hormone (TSH) levels for pups were 0.1, 1.0, and 3.0 mg/kg-day, respectively. There was an apparent increase in the thickness of the corpus callosum of the 10 mg/kg-day group pups on DL 12. The no-observed-adverse-effect level (NOAEL) for maternal toxicity was greater than 10.0 mg/kg-day. Based on the thyroid morphometric and histopathologic findings, the NOAEL for pup toxicity was 0.1 mg/kg-day.
Perchlorate (
Until recently, a paucity of toxicity data suitable for risk assessment purposes has been available on ammonium perchlorate. In 1997, an external peer review panel of risk assessors, perchlorate experts, and thyroid experts convened to help identify data needed to derive a reference dose (RfD) for chronic oral exposure to perchlorate (Toxicology Excellence for Risk Assessment 1997a, 1997b). This panel recommended toxicity studies evaluating perchlorate that would assist state and federal regulatory agencies in developing drinking water guidelines. The toxicity testing strategy was expedited through a unique partnership between the Department of Defense and the U.S. Environmental Protection Agency (USEPA) (1998), together with members of an Interagency Perchlorate Steering Committee, which included other government representatives from the National Institute for Environmental Health Sciences and affected state, tribal, and local governments. These studies were to use current U.S. EPA 870 guidelines (1986). Toxicity tests recommended were a rat developmental neurotoxicity (York et al. 1999 and presented here), a 90-day rat toxicity (Siglin et al. 2000), a rabbit developmental toxicity (Poirier et al. 1999; York et al. 2001b), and a rat two-generation reproductive toxicity (Dourson et al. 1999; York et al. 2001c). In the mammal, maternal thyroid hormones are critical to normal brain and physical development (Porterfield 1994). The medical literature is clear that severe maternal, fetal, or neonatal iodine deficiency or severe hypothyroidism either in laboratory animals or in humans can result in adverse neurodevelopmental outcomes in their offspring if not diagnosed and treated promptly. Production and homeostasis of thyroid hormones, in humans and rodents alike, is complex and is regulated by many factors, one being dietary iodine. Among mammals there exists considerable variability in physiological control of thyroid hormones. Unlike the rodent, the human has a high-affinity binding protein, thyroxine (T4)-binding globulin that binds T4. This protein allows T4 to be transported, preventing deiodinization of the hormone until it reaches the tissues (Robbins 2000). Rodents lack this hormone and as a result, more T4 remains bound to proteins with lower affinity and are more susceptible to removal from the blood, metabolism, and excretion. The half-life of T4 in rats is correspondingly much shorter in rats than in humans (less than 1 day versus 5 to 9 days, respectively) (USEPA 1998a). The human also has substantial stores of iodide in the thyroid gland whereas the rodent does not. The literature demonstrates antithyroid agents, such as methimazole (MMI) and propylthiouracil (PTU), given to pregnant rats at sufficient doses affect neurodevelopmental outcomes in offspring (Porterfield 2000). Neuromorphological and neurodevelopmental effects that have been reported include retarding or inhibiting the migration of the external granular layer of the cerebellum and delayed acoustic startle (Schneider and Golden 1986). Because perchlorate inhibits iodide uptake at the sodium iodide symporter of the follicular thyroid cells, there is the potential that, given sufficient dose, perchlorate could cause maternal hypothyroidism. Specifically, under normal conditions, rat fetuses rapidly increase the amount of iodide absorbed on days 19 through 21, the period before birth when the thyroid becomes highly active (Sztanyik and Turai 1988). Thus perchlorate might cause adverse effects due to maternal hypothyroidism or direct action on the thyroid gland by perchlorate passed from the dam to the fetus or neonate.
Therefore, the purpose of this study was to evaluate the potential neurodevelopmental toxicity of perchlorate, following exposure of pregnant rats to ammonium perchlorate in their drinking water during gestation and for the first 10 days of lactation. This study was designed and based on current regulatory guidelines and with the assistance of the USEPA. Neurodevelopmental outcomes were measured as morphometry and behavior. Neurobehavioral testing included passive avoidance, watermaze, motor activity, and auditory startle habituation evaluations. Also evaluated was the potential effect of perchlorate on maternal and fetal thyroid histology and maternal and fetal serum thyroid hormone levels. Data from this study are intended for use in an assessment of the potential human risk of perchlorate exposure.
MATERIALS AND METHODS
Test Material
Ammonium perchlorate, 99.8% purity, (CAS no. 7790-98-9) was obtained from Aldrich Chemical Company, Inc. (Milwaukee, Wisconsin; lot 03907LF). Formulations of ammonium perchlorate in reverse-osmosis (R.O.)/deionized water were prepared at least weekly, stored refrigerated, then allowed to reach room temperature before use. The nonchlorinated test drinking solutions were adjusted to concentrations intended to yield target doses of 0 (carrier), 0.1, 1.0, 3.0, and 10.0 mg/kg-day, based on actual weekly water consumption. The dams consumed progressively higher dosages of ammonium perchlorate during lactation due to consuming more water during nursing, a common finding in multigeneration studies using the drinking water route of exposure (Christian et al. 2002a, 2002b). During the exposure period, rats were given continual access, via drinking water, to either R.O./deionized water (carrier control group) or formulations of ammonium perchlorate in deionized water (test exposure groups). Exposures were terminated on day 10 of lactation (DL 10) by switching from formulated water bottles to control drinking water bottles so it is not likely that neonates were exposed to higher concentrations of ammonium perchlorate by drinking from their mother’s water bottle.
Animals and Husbandry
One hundred seventy-five female Sprague Dawley [Crl:CD (SD)IGS BR VAF/Plus] rats were supplied by Charles River Laboratories, Inc. (Kingston, NY). The female rats were 65 days of age at arrival and weighed 171 to 234 g. The rat was selected as the animal model based on USEPA 870.6300 Testing Guidelines and because it is widely used throughout the industry for nonclinical studies, not because it is a model for chemicals that affect the thyroid. The thyroid was, however, a target tissue in the rat for ammonium perchlorate exposure. After a 2-week acclimation period, female rats cohabitated with a male breeder rat, of the same source and strain, in a 1:1 ratio. Mated female rats were considered to be day 0 of gestation (DG 0), and randomly assigned to one of five exposure groups, 25 rats per exposure group. Rats were provided ad libitum Certified Rodent Chow no. 5002 (PMI Nutritional International, St. Louis, Missouri). Certified Rodent Chow no. 5002 contains seasonal amounts of soybean meal and oil but the amount was not determined. Soy-based proteins can affect thyroid function (e.g., soy isoflavones inhibit thyroid peroxidase) in rats (Chang and Doerge 2000). Calcium iodate in the feed (0.67 ppm) was the source of iodine for each rat (PMI Nutrition International). The study rooms were maintained under conditions of positive airflow (relative to the hallway) and supplied with a minimum of 10 changes per hour of 100% fresh air that had been passed through 99.97% HEPA filters (Airo-Clean rooms). Room temperature and humidity were monitored constantly throughout the study and were targeted at 64°F (18°C) to 79°F (26°C) and 30% to 70%, respectively. An automatically controlled fluorescent light cycle was maintained at 12-h light: 12-h dark. Adult rats were individually housed in stainless-steel wire-bottomed cages except during the cohabitation (5 days) and postpartum periods. Beginning no later than day 20 of gestation (DG 20), P generation female rats were individually housed in nesting boxes with Bed-O’Cobs (The Andersons Industrial Products Group, Maumee, Ohio). Each dam and delivered litter was housed in a common nesting box during the postpartum period. All cage sizes and housing conditions were in compliance with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources 1996).
Experimental Design
As shown in Figure 1, 125 pregnant rats were exposed to water or ammonium perchlorate, DG 0 through DL 10. Exposure began on DG 0 instead of DG 6, which is a longer period than recommended by EPA guidance (USEPA 1996), to ensure that the dams had achieved a state of hypothyroidism. Targeted exposures were 0 (carrier), 0.1, 1.0, 3.0, or 10.0 mg/kg-day.
For P generation dams, signs of autonomic dysfunction, abnormal postures, movements or behavior patterns, and unusual appearance were recorded blind during the exposure period. Each dam was evaluated for duration of gestation, litter sizes, and pup viability at birth, in addition to maternal behavior. Feed and water consumption values were recorded daily during the exposure and postexposure periods. At birth, both male and female F1 generation rats were evaluated for viability. On postpartum day 5 (PPD 5), each litter was randomly reduced to eight pups (four males and four females, when possible) such that litter size, maternal attention and care, and availability of milk were not factors in pup development.
The idea of collection of blood and thyroid tissues from the 1200-plus culled pups was to obtain as much information as possible and came after the study protocol was finalized and so was added by amendment. Culled pups were sacrificed over a 5-h time period and bled via the inferior vena cava. Blood samples were pooled by litter for thyroid-stimulating hormone (TSH), triiodothyroxine (T3), and T4 for possible determinations. The trachea and larynx (with the thyroids attached) of the F1 generation pups were removed and retained in 10% neutral-buffered formalin for possible evaluation but not weighed. Subsequently, it was decided that the thyroid tissues would be prepared for histological evaluation by standard hemotoxyline and eosin (H&E) (6-μm slides; three sections/thyroid) and scored blind for follicular hypertrophy and severity using subjective microscopic assessment (i.e., none, minimal, mild, and moderate). Five representative follicles, from 6 thyroids/sex/group (later increased to 10 thyroids/sex/group; Baker 1999) were then quantitatively measured for lumen diameter and cell height. Quantitative assessments were conducted using a Leica Quantiment 570C Image Analysis System and a standard microscope fitted with a Sony (DXC-760MD) CCD camera and ocular scale (0.02-mm calibration scale). Large, colloid-distended, inactive follicles at the periphery of the lobes were not evaluated.
On DL 10, the group size was reduced from 25 mated females to 20 dams with litters by culling first nonpregnant rats and then, culling dams with the smallest litters. The purpose was to standardize each group size to 20, with sufficient litter size for the selection of 4 male and 4 female pups for the four subsets (refer to Figure 1). The five rats per group selected in this manner were sacrificed and had their thyroids removed and weighed. This culling process resulted in only three, three, one, three, and five pregnant rats in the 0 (carrier), 0.1, 1.0, 3.0, and 10.0 mg/kg-day exposure groups, respectively, for evaluation at this time-point (data not shown).
On PPD 12, 4 pups/sex/litter were selected for continued observation (see Figure 1). One pup/sex/litter per exposure group was randomly assigned to each subset as follows: brain weights and juvenile neurohistopathology examinations on PPD 12 (subset 1); passive avoidance on PPDs 23 to 25 and 30 to 32 and watermaze testing on PPDs 59 to 63 and 66 to 70 (subset 2); motor activity on PPDs 14, 18, 22, and 59 and auditory startle habituation on PPDs 23 and 60 (subset 3); regional brain weight and adult neurohistopathology examinations on PPDs 81 to 86 (subset 4).
During the postweaning period, body weights and feed consumption were recorded weekly and again at sacrifice. Female pups were evaluated for the age of vaginal patency, beginning on PPD 28, and male pups were evaluated for the age of preputial separation, beginning on PPD 39.
Adult Thyroid Histopathology
Microscopic evaluation was conducted on the thyroid gland and gross lesions from 25 mated female rats per exposure group (125 rats total). Twenty females in each exposure group had been exposed DG 0 through DL 10 and were sacrificed on DL 22 (i.e., 12 days of recovery). The remaining five females in each exposure group were sacrificed on DL 10 (i.e., with no recovery). The thyroid glands were processed, examined, and scored as previously described.
Subset 1 (Brain Weights and Juvenile Neurohistopathology on PPD 12)
One hundred and ninety-seven (97 male and 100 female) pups representing 20 litters per exposure group were randomly assigned to subset 1 on PPD 12 for fixation and brain weight assessments. These rats had been exposure to ammonium perchlorate in utero and during the neonatal period by lactational exposure. Of these pups, 6 pups/sex/exposure group were subsequently selected for neurohistopathological examination. The heads were removed from each pup, and immersed in neutral buffered 10% formalin. The remaining pups were sacrificed and necropsied for evaluation of gross lesions.
Dissection, Trimming, and Histotechnical Procedures
Neuropathological procedures and analyses were performed in accordance with Office of Prevention, Pesticides and Toxic Substances (OPPTS) 870.0062 guidelines (USEPA 1998b). All brains were removed from their cranial vaults. The 36 fixed brains in groups I, IV, and V had two linear measurements (in millimeters) taken with a Vernier caliper without knowing the exposure group. These measurements were (1) the cerebrum from the anterior to posterior pole, exclusive of the olfactory bulbs and (2) the cerebellum extending from the anterior edge of the cortex to the posterior pole. Each brain was weighed and then divided by six coronal slices: (1) half-way between the ventral base of the olfactory bulbs and the optic chiasm, (2) through the optic chiasm, (3) through the infundibulum, (4) through the midbrain just posterior to the mammillary body, (5) through the cerebellum just anterior to its midpoint, and (6) through the anterior portion of the medulla. Cuts 1 through 4 were made from the ventral aspect of the brain and cuts 5 and 6 from the dorsal aspect of the brain.
Brain slices, 7-μm cuts by a rotary microtome, were embedded in paraffin and stained with H&E and/or with luxol fast blue/cresyl violet (LFB/CV). The first four slides from control and high-dose group brains received both stains. Section of brains from rats in the three intermediate exposure groups evaluated morphometrically were stained only with LFB/CV; other brains sections were stained with only H&E. Slides were randomized and blinded with opaque tape for pathological analyses.
Nine linear morphometric measurements were taken on each of the 24 PPD 12 brains (6 brains/sex from each of the maternal high-dose and control groups). As indicated above, two of the measurements were taken from the intact brains prior to trimming and processing. The other seven measurements were taken from the histologic sections using a calibrated ocular micrometer as follows:
Thickness of the frontal cortex. This measurement was of the dorsal portion of the cerebral cortex within the coronal section passing through the optic chiasm and was taken at a magnification of 40×.
Thickness of the parietal cortex. This measurement was of the dorsolateral portion of the cerebral cortex within the coronal section taken through the optic chiasm. This measurement was taken at a magnification of 40×.
Diagonal width (maximum cross sectional width) of the caudate putamen and underlying globus pallidus. This measurement was also performed of the coronal section taken at the level of the optic chiasm but was taken at a magnification of 20×.
Thickness of the corpus callosum at its midpoint within the section taken at the level of the optic chiasm. This measurement was taken at a magnification of 100×.
Thickness of the hippocampal gyrus. This measurement was performed on the dorsolateral portion of the dentate gyrus within the section taken just posterior to the mammillary body. Measurements were taken from the hippocampus from both sides of the brain slice and the mean value recorded. This measurement was taken at a magnification of 40×.
Height of the cerebellum at the level of the deep cerebellar nuclei, including lobes 1 through 6 and extending from the roof of the fourth ventricle to the dorsal surface. This is the maximum height of the cerebellum and was taken at a magnification of 20×.
Thickness of the external germinal layer of the cerebellum. Because the thickness of this layer varied considerably from region to region, multiple areas were measured over the dorsum of the cerebellum and the median value recorded as one measurement. This measurement was taken at a magnification of 400×.
Histopathological evaluations were performed on 38 specific neuroanatomic regions of the same brain sections.
Subset 2 (Passive Avoidance on PPDs 23 to 25 and 30 to 32 and Watermaze Testing on PPDs 59 to 63 and 66 to 70)
Two hundred (100 male and 100 female) pups were assigned to subset 2 and represents 1 male and 1 female offspring per litter per maternal exposure group. On PPDs 23 to 25, learning, short-term retention, long-term retention, and hyperactivity were evaluated using an automated passive avoidance test system located in a separate behavior area of the Testing Facility. The testing apparatus consists of a two-compartment stainless-steel chamber (Coulbourn Instruments, Allentown, PA) with transparent Plexiglas lids in which one compartment is outfitted with a bright light and transparent Plexiglas floor. The remaining chamber is outfitted with a grid floor to which a brief (1-s) pulse of a mild electric current (1 mA) can be delivered. The two compartments are separated by a sliding door. White noise attenuated the impact of any extraneous sound and the overhead room lights were turned off during the testing session; a 5-V light provided the only illumination for the technician. A rat’s natural tendency, when placed in a lighted open area, is to explore the area and move to a darker place. The rat must ‘learn’ not to seek the dark area in order to avoid an adverse consequence and then retain this memory over a short-term (30-s) and long-term (1-week) period. Each rat was required to reach a “learning” criterion by remaining in the “bright” compartment for two consecutive 60-s trials without crossing over to the “dark” compartment. The maximum number of trials in any test session was 15. Each rat was tested twice separated by a 1-week interval, the criterion being the same for both days of testing, and testing was counterbalanced across exposure groups.
Exposure groups were compared using the mean values of the following dependent measures: (1) the number of trials to meet the learning criterion in the first session (overall learning performance); (2) the latency (in seconds) to enter the “dark” compartment from the “bright” compartment on trial 1 in the first test session (activity levels and exploratory tendencies in a novel environment); (3) the latency (in seconds) to enter the “dark” compartment from the “bright” compartment on trial 2 in the first test session (short-term retention); (4) the number of trials to the criterion in the second test session (long-term retention); and (5) the latency (in seconds) to enter the “dark” compartment from the “bright” compartment on trial 1 in the second session (also an indication of long-term retention).
On PPDs 59 to 63, all rats tested in the passive avoidance paradigm in subset 2 were also evaluated in a water-filled M-maze for overt coordination, swimming ability, learning, and memory. Testing was conducted in the study room under standard room conditions, and each rat was removed from its home cage immediately prior to testing. The modified M-maze was constructed of stainless-steel and filled with water to a depth of approximately 9 inches and was sanitized in the cagewasher prior to the start of testing. At the end of each day of testing, the M-maze was drained, rinsed, and disinfected. The water temperature was maintained at 21°C ±1°C during testing.
The rat was required to swim from the base of the M-maze to the end of one of the arms in order to be removed from the water. An error was counted when the rat made a wrong turn. Each rat was required to reach a criterion of five consecutive errorless trials to terminate the test session. Each rat was given a maximum number of 15 trials per session to meet the learning criterion. Latency (measured in seconds) to choose the correct arm of the maze was measured. Each rat was tested twice (separated by a 1-week interval), the correct goal and the criterion were the same for both test sessions and testing was counterbalanced across exposure groups.
Exposure groups were compared using the mean values of the following dependent measures: (1) the number of trials to meet the learning criterion on the first day of testing (overall learning performance); (2) the number of errors (incorrect turns in the maze) for each trial on the first day of testing (also an indication of overall learning performance); (3) the latency (in seconds) to reach the correct goal on trial 2 of the first day of testing (short-term retention); (4) the number of trials to criterion on the second day of testing (long-term retention); (5) the number of errors for each trial on the second day of testing (long-term retention); and (6) the latency (in seconds) to reach the correct goal on trial 1 of day 2 of testing (another indicator of long-term retention).
Rats assigned to subset 2 were sacrificed and necropsied on PPDs 90 to 92; all thyroids were weighed and all gross lesions evaluated. One randomly selected F1 generation rat/sex/litter per exposure group was bled via the vena cava for TSH, T3, and T4 determinations.
Subset 3 (Motor Activity on PPDs 14, 18, 22, and 59 and Auditory Startle Habituation on PPDs 23 and 60)
Two hundred (100 male and 100 female) pups were assigned to subset 3. Behavioral testing consisted of evaluations of motor activity testing and acoustic startle habituation. This represents 20 litters/maternal exposure group. Motor activity was evaluated on PPDs 14, 18, 22, and 59 using an automated apparatus (Foss and Lochry 1991) consisting of a passive infrared sensor mounted on the outside of a stainless-steel wire-bottomed cage. This apparatus measures locomotion and other forms of movement as a means of examining the animal’s exploratory behavior in the new environment. All rats in subset 3 were individually tested during the four testing sessions. The sensor measured the total activity of the rat, and could detect both increases and decreases in movement. Each test session was 1 hour in duration with the number of movements and time spent in movement tabulated at each 5-min interval. Because this apparatus could not measure distance or type of movement of offspring, this test distinguished movement from rest. Groups were counterbalanced (sex, exposure group) across test sessions and cages.
Auditory startle habituation test evaluates a basic form of behavioral plasticity: the decrease in the intensity of a reflex response to a repeated stimulus of an abrupt, loud sound. The response to each stimulus is monitored by recording the force exerted on a platform when the animal reflexes. The rats were evaluated on PPDs 23 and 60. All pups in subset 3 were tested on both days for their reactivity to auditory stimuli and habituation of responses with repeated presentation of that stimulus. The rats were tested in sets of four rats within a sound-attenuated chamber using an automated acoustic startle apparatus (Coulbourn Instruments, Allentown, PA) located in a separate behavior area of the Testing Facility. The rats were presented with a 20-ms, 120-dBA burst of noise at 10-s intervals for 50 trials. Exposure groups were compared for (1) average peak amplitude of responses, and (2) the rate of decline in the amplitude of the responses over time. The average response magnitude and the pattern of responses over ten trial blocks were compared across the exposure groups.
Rats assigned to subset 3 were sacrificed and necropsied on PPDs 67 to 69 and all gross lesions were evaluated. Thyroids were weighed for 20 randomly selected rats/sex/exposure group after 48 h fixation.
Subset 4 (Regional Brain Weight and Adult Neurohistopathology Examinations on PPDs 81 to 86)
Two hundred (100 male and 100 female) pups were assigned to subset 4. Of these pups, 6 rats/sex/exposure group were randomly selected for regional brain weights on PPDs 81 to 86. Of the remaining rats, 6 rats/sex/exposure group were randomly selected for adult neurohistopathological examination on PPDs 82 to 85. Up to five remaining rats from five litters per exposure group were randomly selected for assessment of thyroid parameters, including TSH, T3, and T4 serum levels, thyroid weights, and histopathology. Only rats selected for regional brain weights were sacrificed by whole body vascular perfusion of heparin and pentobarbital followed by neutral-buffered 10% formalin. The calvaria were removed, and the brain blotted, chilled, and weighed. A transverse section was made to separate the rhombencephalon and the cerebellum isolated from the medulla oblongata/pons. A transverse section was made at the level of the optic chiasm, passing through the anterior commissure. The cortex and hippocampus were peeled from the posterior section (diencephalon/mesencephalon) and added to the anterior section (telencephalon). The four sections (telencephalon, diencephalon/mesencephalon, medulla oblongata/pons, and cerebellum) were then weighed. All control and high-exposure group specimens of the brain were selected for neurohistological evaluation.
Dissection, Trimming, and Histotechnical Procedures
Each brain was weighed and then divided by nine coronal slices similar to those used on the juvenile rat brains. After removal of the brain, the gasserian ganglia and associated trigeminal nerve tissue were removed from the floor of the cranial vault, using a rongeur and a no. 11 scalpel blade. The cord was lifted out of the spinal canal in the vertebral column with the spinal nerve roots attached. As the cord was lifted out (starting in the lumbar region and progressing to the cervical region), the spinal nerves were severed from their more distal attachments using microdis-section scissors. After the spinal cord was removed, the dorsal and ventral nerve roots (including the dorsal root ganglia) were removed. Cross sections of the spinal cord were taken from each of the cervical and lumbar swellings and from the mid-thoracic cord. One horizontal and one paramedian sagittal section were also taken of the cervical cord.
The sciatic nerve and its branches were removed from one of the hind legs. Cross and longitudinal sections of the sciatic nerve and tibial nerve were taken, as well as longitudinal sections of both the common peroneal (fibular) and the sural nerves.
Brain slices, 2 to 3 mm thick, spinal cord, gasserian ganglia, nerve roots, and dorsal root ganglia were all embedded in paraffin. Sections of the peripheral nerves were embedded in glycol methacrylate. Five-micrometer sections were prepared from paraffin tissue blocks by a rotary microtome, and stained with H&E and/or with LFB/CV and the Bielschowsky’s technique. The first four slides from control and high-dose group brains received both stains. Section of brains from rats in the three intermediate-exposure groups that were evaluated morphometrically were stained only with LFB/CV; other brains sections were stained with only H&E. The glycol methacrylate blocks were sectioned at 2 μm and stained with H&E, toluidine blue, and the Bielschowsky’s technique.
Morphometric Procedures
After the brain slices had been processed, sectioned, stained, coverslipped, and appropriately labeled (animal ID and dose group), the slide labels were blinded with opaque tape and the slides randomized. Eight linear morphometric measurements were taken on each of the 24 brains (6 brains/sex from each of the maternal high-dose and control groups). As indicated above, two of the eight measurements were taken from the intact brains prior to trimming and processing. The other six measurements were taken from the histologic sections using a calibrated ocular micrometer. Theses were the same measurements as conducted on the PPD 12 pups except the thickness of the external germinal layer of the cerebellum was not measured.
Rats assigned to subset 4 were sacrificed and necropsied on PPDs 81 to 86; all gross lesions were evaluated. Thyroids were weighed after 48 h fixation from 20 randomly selected rats/sex/exposure group.
Hormone Analyses
Blood was collected from the inferior vena cava. Serum samples were aliquoted into three vials for TSH, T3, and T4 determinations and shipped (frozen on dry ice) for determination of hormone levels. Radioimmunoassay (RIA) T3 assay kits were purchased from Diagnostic Product Corp. (Los Angeles, CA), and canine T3 antibody–coated tubes were used. RIA T4 assay kits and T4 antibody–coated tubes were purchased from Diagnostic Product Corp. RIA TSH assay kits were purchased from Amersham Corp. (Arlington Heights, IL), and lyophilized rabbit anti-rat TSH serum and Amerlex-M second antibody (donkey anti-rabbit serum-coated onto magnetized polymer particles containing sodium azide) were both used. RIA kits used for all of the standard and unknown hormone measurements were of the same batch number and had the same expiration date. All assays were performed in triplicate and according to the manufacturer’s recommended procedures.
Statistical Analysis
Adult data were evaluated with the individual rat as the unit of measurement. Litter values were used in evaluation of pup data, as appropriate. Variables with interval or ratio scales of measurement (body weights, feed consumption values, latency, and errors per trial scores in behavioral tests and percent mortality per litter) were analyzed as parametric data. Bartlett’s test of homogeneity of variances (Sokal and Rohlf 1969a) was used to estimate the probability that the exposure groups had different variances. A nonsignificant result (p >.05) indicated that an assumption of homogeneity of variance was appropriate, and the data were compared using the analysis of variance test (Snedecor and Cochran 1967a). If that test was significant (p ≤ .05), the groups given the test substance were compared with the control group using Dunnett’s test (Dunnett 1955). If the Bartlett’s test was significant (p ≤ .05), the analysis of variance test was not appropriate, and the data were then analyzed as nonparametric data. When 75% or fewer of the scores in all the groups were tied, the Kruskal-Wallis test (Sokal and Rohlf 1969b) was used to analyze the data. In the event of a significant result (p ≤ .05) using the Kruskal-Wallis test, Dunn’s test (Dunn 1964) was used to compare the groups given the test substance with the control group. When more than 75% of the scores in any exposure group were tied, Fisher’s exact test (Siegel 1956) was used to compare the proportion of ties in the exposure groups.
Data from the motor activity and auditory startle habituation tests, with measurements recorded at intervals (blocks) throughout each test session, were analyzed using an analysis of variance with repeated measures (SAS Institute, Inc. 1988). A significant result (p ≤ .05) in that test could have appeared as an effect of exposure (differences among exposure groups in the totals of all measurements in a session) or as an interaction between exposure and block (differences in the patterns of exposure group values across the measurement periods). If the exposure effect was significant, the totals for the control group and each of the exposure groups were compared using Dunnett’s test. If the Exposure × Block interaction was significant, an analysis of variance test (Snedecor and Cochran 1967a) was used to evaluate the data at each measurement period, and a significant result (p ≤ .05) was followed by a comparison of the exposure groups using Dunnett’s test.
Variables with graded or count scores (litter size, the number of trials to reach criterion in a behavioral test, or the day a developmental landmark appeared) were analyzed as nonparametric data. Clinical observation incidence data, as well as other proportion data, were analyzed as contingency tables using the variance test for homogeneity of the binomial distribution (Snedecor and Cochran 1967b).
Kruskal-Wallis one-way analysis of variance on ranks, with a Bonferroni procedure to correct for multiple comparisons (Simes 1986), was used to analyze the histopathology and morphometric portions of the PPD 5 culled pup thyroid data. Sexual maturation parameters were analyzed by subset and then were combined across subsets for presentation.
RESULTS
Consumed Dosages
The average actual consumed doses of ammonium perchlorate for the P generation maternal rats were 0.0, 0.1, 1.1, 3.5, and 11.6 mg/kg-day (100%, 100%, 110%, 117%, and 116% of target, respectively) during the 3-week gestation period and 0.0, 0.2, 1.6, 4.7, and 15.4 mg/kg-day (100%, 200%, 160%, 157%, and 154% of target, respectively) during the 10-day lactation exposure period (Table 1).
General Toxicity
No dams died as a result of exposure to ammonium perchlorate. One 0 (carrier) mg/kg-day exposure group dam and one dam in the 1.0 mg/kg-day exposure group died during delivery, and one 0 (carrier) mg/kg-day exposure group dam was sacrificed on DL 1 because of a prolapsed uterus.
No clinical observations, signs of overt toxicity, observations of autonomic dysfunction, or necropsy observations that occurred during the gestation, or lactation periods were considered related to the test substance. Average body weights (Figure 2) and body weight changes for the dams were comparable among the five exposure groups for all days of gestation and lactation. Absolute (g/day) and relative (g/kg/day) water and feed consumption values were comparable among the five exposure groups during each tabulated interval of the gestation and lactation periods (data not shown).
Delivery, Morbidity, and Litter Data (PPD 1 to PPD 22)
There were 22 to 25 pregnant dams in each exposure group. Ammonium perchlorate exposures as high as 10.0 mg/kg-day did not adversely affect any parameter evaluated at natural delivery or during the 22-day lactation period (Table 2). Gestation length, litter size, number of stillborn, gestation index, and pup viability index were comparable across exposure groups and did not differ significantly. Average weights of offspring on PPDs 1, 8, 14, and 22 were not different by maternal perchlorate exposure. All clinical and necropsy observations in the F1 generation pups were unrelated to ammonium perchlorate exposure.
Culled F1 Generation Pup Thyroid Hormone and Histopathlogy Data (PPD 5)
On the day of culling litters, culled pups were subsequently evaluated for serum T3, T4, and TSH levels and thyroid histopathology. A statistically significant increase in serum TSH (22% increase) was observed for offspring born to dams treated with 10.0 mg/kg-day (p ≤ .01; Table 3). The levels for the 0.1 mg/kg-day exposure group were comparable to carrier group values. Serum T3 values were significantly reduced (p ≤ .05 or p ≤ .001) when compared to the carrier group levels for the 1.0, 3.0, and 10.0 mg/kg-day exposure groups. The percent decreases from the control group values were 10.2%, 55.7%, and 56.8%, respectively. Serum T4 values were significantly reduced (p ≤ .001) from the carrier group level in the 3.0 and 10.0 mg/kg-day exposure groups (21.4% and 25.8%, respectively).
The incidences of follicular cell hypertrophy were elevated in offspring of dams in all exposure groups for both sexes (Table 3). Thyroid weights were not taken. There was a significant increase (p ≤ .05) in the severity of thyroid hypertrophy in male pups in the 10.0 mg/kg-day exposure group and in the female pups in the 3.0 and 10.0 mg/kg-day exposure groups. Morphometrically, there was an exposure-related decrease in thyroid follicular lumen diameter and area and increase in follicular epithelium height for both sexes. The decrease in lumen diameter reached significance (p ≤ 0.05) for the culled male pups from the 1.0 mg/kg-day and higher exposure groups. The decrease in lumen area was significant for the 0.1, 3.0, and 10.0 mg/kg-day exposure groups but not the 1.0 mg/kg-day exposure group. The decrease in lumen area for the male pups in the 0.1 mg/kg-day exposure group was not considered treatment related because there was no response at the next higher exposure. The thyroid follicular cell lumen diameter was significantly decreased for female pups in the 3.0 and 10.0 mg/kg-day groups and the lumen area was significantly decreased in the 10 mg/kg-day exposure group.
Maternal Thyroid Histopathlogy and Hormone Data (DL 22)
Terminal body weights, absolute thyroid weights, and the ratios of thyroid weight to terminal body weight of the P generation dams were comparable among the five exposure groups on DL 22 (Table 4). There were observations of decreased colloid in the thyroid glands of some dams. However, these observations were nonlinear to increasing dose and nonsignificant. No other treatment-related microscopic changes were observed for follicular hypertrophy or follicular hyperplasia in the thyroid glands of the female P generation rats exposed to ammonium perchlorate.
F1 Generation Male and Female Rats
Sexual Maturation
Ammonium perchlorate exposure to the dams had no effect on the sexual maturation of the offspring. Target exposures as high as 10.0 mg/kg-day ammonium perchlorate administered to the P generation dams did not affect the day of vaginal patency or the day of preputial separation in the F1 generation rats from all three subsets combined (Table 5).
General Toxicity
One F1 generation male rat in the 10.0 mg/kg-day maternal exposure group and four F1 generation female rats in the 0.1 mg/kg-day maternal exposure group died before study termination. A gross necropsy revealed no lesions that were considered exposure related. These deaths were not considered treatment related because no other rats in the 10.0 mg/kg-day exposure group died and the incidence of deaths in the 0.1 mg/kg-day exposure group was not exposure dependent.
All clinical observations in the F1 generation male and female rats were considered unrelated to the test substance. Average body weights and body weight changes for F1 generation male and female rats were generally comparable among the five exposure groups throughout the postpartum period. Body weight gains for the tabulated intervals PPDs 5 to 12 (subsets 1 through 4), PPDs 5 to 65 (subsets 2, 3, and 4), PPDs 5 to 79 (subsets 2 and 4), and PPDs 5 to 86 (subset 2 only) were comparable among the five exposure groups. Absolute and relative feed consumption for F1 generation male and female rats were comparable among the five exposure groups throughout the postpartum period.
Pup Brain Weights and Neurohistological Evaluation (Subset 1)
All F1 generation male and female pups in subset 1 appeared normal at PPD 12 gross necropsy. The terminal body weights, brain weights, and ratios of the brain weight to the terminal body weight for F1 generation male and female pups in subset 1 were comparable among the carrier and two highest exposure groups and did not differ significantly (Table 6).
There were no treatment-related neuropathologic changes observed histologically in the 36 brains (6 per sex in each of the 3.0 and 10.0 mg/kg-day and carrier control exposure groups) of the male and female F1 generation pups on PPD 12. Morphometric evaluation of brain and nine specific regional areas indicated the mean value for the thickness of the corpus callosum of the 10.0 mg/kg-day exposure group female pups was significantly greater (p ≤ 0.01) than that for the carrier control group female pups (Table 6). Albeit not statistically significant, the size of the corpus callosum in the male pups at the 10.0 mg/kg-day exposure level was also increased by 30.8%. The difference between the means of the control and high-dose group was less than 64 μm. Although the reason for the greater mean thickness of the corpus callosum in the high-dose female rats was not apparent (this white matter tract was microscopically normal in appearance), these increases in the size of the corpus callosum of the 10.0 mg/kg-day exposure group pups prompted an evaluation of the next lower exposure group (3.0 mg/kg-day). The additional analysis on the 3.0 mg/kg-day exposure group revealed a significant decrease (p ≤ .01) in the hippocampal gyrus size in the F1 generation male pups, a significant increase (p ≤ .01) in the anterior to posterior cerebellum size, and a significant decrease (p ≤ .05) in the caudate putamen of the F1 generation female pups, but no significant difference in the corpus callosum (the size of the corpus callosum in the 3.0 mg/kg-day exposure group was actually below control values for more male and female pups).
Behavioral Evaluations (Subsets 2 and 3)
There were no exposure-dependent differences for learning, short-term retention, long-term retention, or response inhibition in the F1 generation male or female rats in subset 2, as evaluated by performance in the passive avoidance and watermaze paradigms. Beginning on PPD 22 and retested 1 week later, no statistically significant differences for passive avoidance occurred in the F1 generation male or female pups in the number of trials to criterion, trial latencies, or numbers of rats that failed to learn for the five exposure groups (Table 7).
Beginning on PPD 59 and retested 1 week later, no exposure-dependent differences occurred for watermaze performance in the F1 generation male or female rats regarding learning, short-term retention, long-term retention, or response inhibition. No statistically significant differences occurred in the number of trials to criterion, trial latencies, or numbers of rats that failed to learn for the five exposure groups (Table 8). There was a significant increase (p ≤ .05) in errors per trial for session 2 in the 1.0 mg/kg-day maternal exposure group male rats; however, this was a single occurrence and was not exposure dependent.
No necropsy observations in the F1 generation male and female rats in subset 2 were considered related to ammonium perchlorate exposure. The terminal body weights, thyroid weights, and the ratio of the thyroid weight to terminal body weight for F1 generation male and female rats in subset 2 were comparable among the five exposure groups (Table 9).
Administration of ammonium perchlorate at target exposures up to 10.0 mg/kg-day to the P generation dams did not affect the overall average number of movements or time spent in movement (Tables 10 A to D)1 for the F1 generation male or female rats in subset 3 as evaluated by motor activity in a 1.5 h session (18 5-min blocks) at four different time points (PPDs 14, 18, 22, and 59). The data for time in movement were comparable to the total number of movements and are not presented. The time spent in movement on PPD 14 (Table 10A) was significantly increased (p ≤ .05) for the male rats in block 5 for the 3.0 mg/kg-day maternal exposure group and significantly decreased (p ≤ .05) for the female rats in block 1 for the 1.0 mg/kg-day maternal exposure group. The time spent in movement on PPD 18 (Table 10B) was also significantly increased (p ≤ .05) for the male rats in block 1 for the 0.1 mg/kg-day maternal exposure group. The time spent in movement on PPD 22 and 59 (Tables 10C and 10D) were comparable across all exposure groups. Overall, there were no significant differences in data for total number of movements for any of the exposure groups, sex of animal, or time point tested.
Target exposures as high as 10.0 mg/kg-day to P generation rats did not affect the auditory startle (Table 11) response magnitude in the F1 generation male or female rats following a series of 20-ms, 120-dBA bursts of noise at 10-s intervals for 50 trials at either time period tested (PPDs 23 and 60).
No necropsy observations in the F1 generation male and female rats in subset 3 were considered related to the test substance. Terminal body weights, absolute thyroid weights, and the ratios of thyroid weight to terminal body weight for the F1 generation male and female rats in subset 3 were comparable among the five exposure groups (Table 12).
Adult Thyroid Histopathology, Brain Neuropathology, and Morphology (Subset 4)
No necropsy observations that occurred in the F1 generation male and female rats in subset 4 on PPD 89 were considered related to the test substance. Terminal body weights, absolute thyroid weights, and the ratio of thyroid weight to terminal body weight for the male and female rats were comparable among the five exposure groups (Tables 13A and 13B). The incidence of decreased colloid and follicular hypertrophy were slightly increased at the three highest exposure levels.
The terminal body weights, average absolute, and relative to terminal body weights of the brain regions (telencephalon, diencephalon/mesencephalon, medulla oblongata/pons, and cerebellum) for the F1 generation male and female rats were comparable among the five exposure groups (Table 14). There were no treatment-related neuropathologic changes observed histologically in the 24 brains (6 per sex in each of the high-and control exposure groups) of the male and female F1 generation rats on PPD 89. Whole brain weights of the F1 generation males sacrificed on PPD 89 for morphometric measurements were significantly increased (p ≤ .05) in the 10.0 mg/kg-day exposure group (Table 15). Morphometry evaluation of eight specific regional brain areas indicated the mean value for the thickness of the frontal cortex, caudate putamen, and corpus callosum of the 10.0 mg/kg-day exposure group male pups were significantly increased (p ≤ .05 or p ≤ .01). There were no treatment-related morphometric changes observed in the F1 generation female rats on PPD 89.
DISCUSSION
The medical literature is clear that severe or moderate maternal hypothyroidism, whether caused by iodine deficiency or not, can result in adverse neurodevelopmental outcomes in children (Man and Jones 1969; Rovet, Ehrlich, and Sorbara 1987; Glorieux et al. 1988; Fenzi et al. 1990; Vermiglio et al. 1990; Tillotson et al. 1994; Pop et al. 1995, 1999; Haddow et al. 1999). However, these adverse neurodevelopmental effects in children have only been associated with moderate to severe iodine deficiency or moderate to severe hypothyroidism, whatever the cause. Further, studies in rats have shown that hypothyroidism, induced by known antithyroid agents such as MMI or PTU or by severe iodine deficiency, can result in reduction in the size of the cerebellum, cerebral cortex, and hippocampus due to altered cell proliferation and cell death and reduced myelination, and an increase in the volume of the external granular layer of the cerebellum (Koibuchi and Chin, 2000; Mwangi, 1998, 2001; Rodriguez-Pena et al. 1993; Schalock, Brown, and Smith 1979). Maternal rat exposure to MMI and PTU has also produced a range of neurobehavioral and developmental outcomes in offspring, including effects on motor activity and auditory startle, and delays in eye opening (Albee et al. 1989; Comer and Norton 1982, 1985; Goldey et al. 1995; Knipper et al. 2000; Schaefer et al. 2001; Schneider and Golden 1986; Tamasy et al. 1984, 1986; Weller et al. 1996). Again, these effects are associated with severe maternal hypothyroidism, and the threshold for these effects is not known.
The present study showed that ammonium perchlorate given to pregnant rats during gestation and lactation (up to 10 mg/kg-day) selectively altered thyroid hormone profiles and thyroid histology in their offspring, but did not affect offspring behaviour. Behavior is usually considered a sensitive end point of neurodevelopment but in this study, thyroid indicators were the most sensitive measure of offspring effects.
The results of administration of up to 10 mg/kg-day ammonium perchlorate from the start of pregnancy through lactation demonstrate few effects to the dam, none of which were adverse. Serum thyroid and pituitary hormone (T3, T4, and TSH) values decreased, but not to a degree that rendered the dams ill or offspring adversely affected. Histopathological analyses of thyroid glands of dams exposed from DG 0 to DL 10 demonstrated several changes. Yet, even with those changes, effects on off-pring are also limited to the thyroid hormones and histopathological thyroidal changes.
Brain Neuropathology and Morphometry Evaluations
Neurohistological examination of the brains of the F1 generation pups sacrificed on PPD 12 (subset 1) revealed a significant increase in the size (23.3%) of the corpus callosum in the female pups at the 10.0 mg/kg-day exposure level, as the only dose-dependent statistical finding. The size of the corpus callosum in the male pups at the 10.0 mg/kg-day exposure level was also increased 30.8%, although this was not statistically significant.
One would expect that any process having an adverse effect on brain development would result in a thinning of structures from neuronal death, decreased brain weights, and changes in cell density (McIntosh et al. 1983) or delayed neuron migration. An increase in size of a brain region, which is what appeared to happen in this study, should result in an increase in the number of cells (hyperplasia) or in cell size (hypertrophy), both of which should have been evident histologically. The corpora callosi of the PPD 12 pups were reexamined and remeasured and no evidence of heightened cellularity (gliosis), fibrosis, or any other pathologic change were observed. However, it was noted that the thickest corpora callosi were slightly paler than those that were thinner. This could indicate that there was a decrease in myelin and an increase in water content in these tracts. A silver stain may have allowed for an evaluation of the axons (number and diameter); however, a myelin stain may not reveal a change because a PPD 12 rat brain is poorly myelinated at this age. The apparent increase size of the corpora callosum following ammonium perchlorate exposure should be further investigated.
Brain morphometric procedures applied in this work were in adherence to EPA guidance (USEPA 1998b), which suggests that measures should be taken, at a minimum, of the thickness of major layers at representative locations within the neocortex, hippocampus, and cerebellum. However, for several brain structures (e.g., the corpus callosum, hippocampus, cerebellum), collection of thickness measures in coronal sectioning has a high probability of introducing between animal variance due to effects of minor differences in the plane of sectioning and is inconsistent with the bulk of the scientific literature in this area. The thickness of the corpus callosum showed great variability in measurements, and is likely associated with variations in the anterior to posterior shape of this structure. The optic commissure was the landmark used for slicing these brains, but because of the small size of this structure, it was often difficult to visualize in PPD 12 rat pups. Though an effect of treatment on the thickness of the corpus callosum cannot be ruled out, there were no pathological alterations of this structure.
Statistical reanalysis of these data by the USEPA (1998c, 2002) of the control, 3, and 10 mg/kg-day exposure groups was originally restricted to the corpus callosum because this was the area of the brain with the largest effect. The analysis revealed no interaction of gender and treatment; however, there was a significant effect of treatment in the 10 mg/kg-day group reported. Group means were 288, 278, and 366 μm for the control, 3, and 10 mg/kg-day exposure groups, respectively. Incorporation of historical control data from the Testing Facility (264 μm for PPD 9 and 255 μm for PPD 11) supports the conclusion that the control values for the corpus callosum size were within “normal” range for this study (Parker et al. 2000; York et al. 2001a).
Thyroid Weights and Histopathology Evaluations
There was no effect on absolute or relative thyroid weights or thyroid histopathology for the dams sacrificed on DL 12 following exposure for at least 32 days at levels up to 10.0 mg/kg-day. There was also no effect on absolute or relative thyroid weights of the F1 generation male and female rats sacrificed on PPDs 92, 69, or 85 (subsets 2, 3, or 4, respectively). However, the histological evaluation for the culled PPD 5 male pups revealed a nonsignificant increase in the incidence of hypertrophy and severity at 0.1 mg/kg-day and higher. Morphometric evaluation revealed a reduction in follicular lumen diameter and area at this same exposure. These same findings were evident in the PPD 5 female pups, but starting at the 1.0 mg/kg-day exposure level. The exposure of these pups to ammonium perchlorate was presumed be in utero or via the milk during nursing because the pups were too young to reach the ammonium perchlorate solutions in the water bottles. The scientific validity of the PPD 5 pup findings is equivocal because the tissues were collected from culled pups using a nonroutine necropsy (not randomized across groups, by day nor time of day). Additional variables that may have affected the data from these pups include limited sample sizes, litter bias (litter mates were selected and not all litters were represented), statistical analyses based on individual pups rather than the litter (the standard unit of measure), and the difficultly in measuring and differentiating hypertrophy from hyperplasia in a rapidly growing neonatal thyroid. In addition, these effects (thyroid weight and histopathology changes) were not observed in the remaining pups from the same litters that were sacrificed on PPD 59. Therefore, the effects observed in the PPD 5 pups were considered reversible or possible an artifact.
All thyroid slides from this study were reevaluated, without knowledge of sex or exposure group, using criteria that were developed jointly by pathologists from the EPA, the National Toxicology Program and Experimental Pathology Laboratories (EPL), Inc. (Wolf 2000; Wolf 2001). Following the reevaluation, a peer review Pathology Working Group (PWG) was conducted (EPL 2000) in 2000, on all sections of the thyroids. A consensus was reached on all thyroids where there was an initial difference of opinion as to the presence or severity of a particular finding. The P generation dams sacrificed on DL 12 exhibited decreased colloid and increases in both hypertrophy and hyperplasia, but no clear dose response was noted with the exception of colloid depletion at exposures above 0.1 mg/kg-day. Thyroid histopathology findings for the PPD 5 culled pups was more pronounced, with colloid depletion and increases in hypertrophy at 0.1 and 3.0 mg/kg-day exposures. Hyperplasia appeared to be an effect at 3.0 mg/kg-day. Reevaluation of the thyroid slides from PPD 59 rats found only slight and non-significant increases in colloid depletion and hypertrophy in the male rats from litters maternally exposed to 1.0 mg/kg-day and higher.
Thyroid and Pituitary Hormone Evaluations
The thyroid hormone data show sensitivity to very low exposures to ammonium perchlorate. This observation is consistent with its known mechanism-of-action (inhibition of iodine uptake into the thyroid that disrupts thyroid hormone homeostasis, as well as the activation of the pituitary-thyroid feedback mechanism). Hormone measurements from PPD 5 culled pups pooled samples indicated there were measurable differences in T3 levels at 1.0 mg/kg-day and higher, in T4 levels at 3.0 mg/kg-day and higher, and TSH levels at 10.0 mg/kg-day. The scientific validity of these hormone findings was equivocal because of the confounding factor of collecting blood from culled pups over a 5-h time frame, each day (morning to early afternoon) for 10 days, and pooling the samples by litter. There are known time-dependent and sex-related differences in serum thyroid and pituitary hormone levels in maternal, fetal, and neonatal rodents (Lewis et al. 2003). The adult rat control values for T3, T4, and TSH observed in this study were lower than the control values for the 14-day study conducted by Caldwell et al.(1996) and 90-day study conducted by Siglin et al.(2000). However, the dams in this study were lactating (DL 10) and pregnancy tends to affect all aspects of thyroid hormone economy (Larsen and Ingber 1992). The PPD 5 pup control values for T3 and T4 observed in this study were greater than levels determined in term rat fetuses (DG 21) and lower than levels determined in PPD 10 pups, demonstrating a time-dependent increase with age for these hormones (Lewis et al. 2003). The control values for TSH were slightly lower, but consistent with DG 21 and PPD 10 rat fetuses and pups levels, respectively (4.86 to 7.22 ng/ml).
Behavioral Evaluations
There were no exposure-dependent differences for learning, short-term retention, long-term retention or response inhibition detected in the F1 generation rats in subset 2 or 3, as evaluated by performance in a passive avoidance, watermaze, motor activity, or auditory startle paradigms. The exception was a non-significant increase in motor activity in male rats in the 10.0 mg/kg-day exposure group on PPD 14 for block 6, suggesting they were not habituating or had a slower rate of habituation and maintained a higher level of activity when compared with the untreated pups. This increase was not observed in the PPD 14 female pups and was not evident in either sex at later time points (PPD 18, 22, or 59). The total number of movements and time-spent-in movement was originally collected in 18 5-min intervals (90 min) and this exposure group had a 50%, albeit not significant, increase. The data has been reanalyzed and presented in this manuscript by the first six 10-min blocks (60 min), which is now the industry standard and comparable to the four developmental neurotoxicity studies conducted at the Testing Facility after this current study (Barnett et al. 2001, 2002). The effect observed may be random variation or a type I error but the increase in motor activity in males in the 10 mg/kg-day exposure group on PPD 14 was a concern (USEPA 1998c). A separate study was conducted using the same exposure levels and similar study design (Bekkedal et al. 2000). The motor activity equipment was the Opto-Varimex and recorded nine different measures of activity. No statistical significant differences were observed for main effect nor any interactions related to dose on PPD 14, 18, or 22 (the PPD 59 evaluation was not conducted). A statistical evaluation that integrated various measurements to compare the two studies was performed (Dunson 2001). A Bayesian hierarchical model (Gelfand et al. 1990) was chosen to assess the weight-of-evidence of a dose-response trend in motor activity. A linear mixed-effect regression model (Laird and Ware 1982) related dose, sex, age, habituation time, and various interactions to expected motor activity outcome. The analyses indicated that the results of the two studies were remarkably similar. The authors concluded that there was not a significant change in general locomotor activity due to pre- and neonatal exposure to ammonium perchlorate and demonstrated at three postweaning ages, there were no differences found between any of the exposure groups for a variety of motor variables. The study conducted at Argus displayed, however, displayed a greater variability in the PPD 14 data.
CONCLUSION
The objective of this study was to provide information for use in evaluating the potential for neurodevelopmental effects in offspring after exposure to ammonium perchlorate in utero and during the neonatal period. Results for the dams exposed to 0.1, 1.0, 3.0, and 10 mg/kg ammonium perchlorate per day in the drinking water revealed no exposure-related effects on clinical observations, parturition, body weights, feed, or water consumption values. The no-observed-adverse-effect level (NOAEL) for maternal toxicity in this study was greater than 10.0 mg/kg-day. The reproductive NOAEL for this study was also greater than 10.0 mg/kg-day, based on no effects on natural delivery or litter observations.
Maternal exposure to perchlorate resulted in changes in serum thyroid hormone levels in the F1 generation offspring that were exposed in utero and as nursing neonates. Pooled serum TSH, T3, and T4 levels from the culled PPD 5 pups in the 1.0, 3.0, and/or 10.0 mg/kg-day exposure groups were significantly different from control group values. The incidences of follicular cell hypertrophy in the PPD 5 pups were elevated in all exposure groups for both sexes. Morphometrically, there was an exposure-related decrease in thyroid follicular lumen diameter and area and increase in follicular epithelium height for both sexes for the 3.0 and 1.0 exposure groups. Based on the histologic and morphometric findings in the PPD 5 pup thyroids, the NOAEL for pup thyroid toxicity was 0.1 mg/kg-day.
Ammonium perchlorate did not cause dose-related neurobehavioral or sexual maturational effects in the offspring when it was administered to dams during gestation and lactation at target exposures up to 10.0 mg/kg-day. Cognitive functions (learning, memory, and habituation) were not impaired in the pups at any time at any exposure. An effect of treatment on the thickness of the corpus callosum was present in the PPD 12 female pups in the 10.0 mg/kg-day exposure group, but there were no pathological alterations of this structure. No other effects in the F1 generation rats were observed. Based on the morphometric findings in the PPD 12 brain, the NOAEL for pup neurotoxicity was 3.0 mg/kg-day.
To provide added scientific support and a rationale for these findings for hazard identification and dose-response assessment in determining an RfD, additional studies were recommended by the EPA (USEPA 2002), and will be presented in companion papers. One publication has been recently presented, a standard rat developmental toxicity study using similar exposures as this publication (York et al. 2003), and additional publications will refine the effects seen in this study by collection and analyses of maternal and fetal blood, brains, and thyroid samples on DG 21, PPD 10, and PPD 22, and repeat the motor activity analysis on PPDs 14, 18, and 22 (Dollarhide et al. 2001).
Footnotes
Figures and Tables
Acknowledgements
The authors gratefully acknowledge the services provided by William H. Baker, DVM, Staff Pathologist at AFRL/HEST, for day 5 postpartum pup thyroid histopathology; Captain David T. Tsui, AFRL/HEST, for analytical analysis of formulations; Latha Narayanan, GEO-CENTERS, Inc., at AFRL/HEST, for adult and pup T3, T4, and TSH hormone analyses; Richard C. Pleus, Intertox, Seattle, Washington, for his expert review; and Jennifer Hawkins for her skillful technical assistance in preparing this manuscript. Funding for this study was by the United States Air Force through the Air Force Research Laboratory (AFRL/HEST) and contract number F33615-00-C-6060, Man Tech Geo-Centers Joint Venture.
1
The data was subsequently truncated to 1 h (six 10-min blocks) to match industry standards.