* Springborn Laboratories, Inc., 640 North Elizabeth Street, Spencerville, Ohio 45887;
AFRL/HEST, Wright-Patterson Air Force Base, Ohio;
ManTech Environmental Technology, Dayton, Ohio; and
§ Pathco, Inc., Ijamsville, Maryland
Received February 21, 2000; accepted June 8, 2000
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ABSTRACT |
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Key Words: perchlorate; water; contamination; toxicity; rat; thyroid; hypertrophy; risk.
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INTRODUCTION |
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Large-scale production of perchlorate salts began in the United States in the mid-1940s (Fisher et al., 2000) and large volumes have been disposed of in various states since the 1950s (Urbansky, 1998
). Perchlorate is exceedingly mobile in aqueous systems and can persist for many decades under typical ground and surface water conditions (Urbansky and Schock, 1999
). Since the development of low-level (45 ppb) detection methods in the late 1990s, perchlorate contamination has been discovered in a number of water supplies across the United States (Urbansky, 1998
). There are at least 11 states with confirmed releases of perchlorate in ground or surface water (Urbansky and Schock, 1999
). Most often the sites of contamination have been associated with the manufacture or testing of solid rocket fuels (personal communication, D. Rogers, 2000). Chemical fertilizer has also been reported as a potential source of perchlorate contamination (Susarla et al., 1999
). Perchlorate is not regulated in the Unted States under the Federal Safe Drinking Water Act.
Perchlorate was once used medicinally to treat Graves disease and other hyperthyroid conditions (reviewed by Orgiassi and Mornex, 1990). However, due to cases of agranulocytosis and aplastic anemia with fatalities, perchlorate is no longer used therapeutically in the United States for this purpose (Larsen and Ingbar, 1992). The basis for perchlorate's therapeutic effect is the competitive inhibition of iodide uptake by the thyroid gland (Wyngaarden et al., 1952
). The perchlorate ion, because of its similarity in ionic size to iodide, competes with iodide for uptake into the thyroid gland by the sodium-iodide symporter. At therapeutic dosage levels, this competitive inhibition results in reduced production of the thyroid hormones, triiodothyronine (T3) and thyroxine (T4), and a consequent increase in thyroid stimulating hormone (TSH) via a negative feedback loop involving the thyroid, pituitary, and hypothalamus (reviewed by Wolff, 1998).
Perchlorate is of concern because of uncertainties in the existing toxicological database available to address the potential human health effects of low-level drinking water exposure. Although perchlorate has been used medicinally, its potential effects have not been previously investigated in a Good Laboratory Practice (GLP) compliant subchronic or chronic doseresponse study. In addition, those studies which are available have not established adequate doseresponse relationships and/or confirmed the thyroid as the critical effect organ of perchlorate toxicity.
Mannisto et al. (1979) evaluated the effects of potassium perchlorate (PP) in male Sprague-Dawley rats. The test substance was administered via the drinking water for 4 days at concentrations of 10, 50, 100, or 500 mg/l. TSH was increased while T3 and T4 were decreased in a dose-dependent manner starting at the 50 mg/l concentration. These authors did not evaluate thyroid organ weight, thyroid histopathology, or other potential systemic effects of PP. Sreebny et al. (1963) reported submaxillary gland and pancreas weight decreases in male Sprague-Dawley rats exposed for 30 or 60 days to 1% PP in the drinking water. Parotid gland amylolytic activity was also decreased in the treated rats compared to controls. The authors considered these changes to be secondary to thyroid weight increases and therefore mediated by hypothyroidism. Sreebny et al. (1963) did not evaluate thyroid hormone levels, thyroid histopathology, or other potential systemic effects. Hiasa et al. (1987) reported increases in both absolute and relative thyroid weights in male Wistar rats exposed to 1000 ppm PP in the diet for 140 days. TSH was increased and T4 was decreased in the treated rats; neither body weight nor liver weight was affected. Hiasa et al. (1987) did not evaluate thyroid histopathology or potential effects in other organ systems. Shigan (1963) investigated the effects of 0, 0.25, 2, and 40 mg/kg/day AP given in distilled water for 9 months to white rats (sex and strain not specified) on cardiac electrical activity, liver function, and conditioned reflexes. These authors found no differences between the treated and control rats for the parameters evaluated.
A provisional reference dose (RfD) of 0.0001 mg/kg/day was proposed for perchlorate based on an acute effects study in Graves disease patients published by Stanbury and Wyngaarden in 1952 (U.S. EPA, 1992). However, the U. S. Environmental Protection Agency (U.S. EPA) did not adopt this provisional value as the regulatory level. Subsequently, the U.S. EPA (1995) proposed an RfD ranging from 0.0001 to 0.0005 mg/kg/day, with a total uncertainty factor of 300 based on Stanbury and Wyngaarden (1952) as well as rat studies by Shigan (1963) and Mannisto et al. (1979). In 1997 Toxicology Excellence for Risk Assessment (TERA, Cincinnati, OH), on behalf of an industry consortium, conducted an external peer review that concluded the database on perchlorate was insufficient to develop an RfD. The panel, which consisted of experts from government, academia, and industry, recommended a 90-day toxicity study in rats, a neurodevelopmental toxicity study in rats, and literature reviews to assess the need for additional studies (personal communication, J. Dollarhide, 1999). Another peer review meeting was held to prioritize the list of perchlorate studies needed and to develop protocols. Ultimately, testing was initiated to develop the necessary toxicological database for a quantitative human health risk assessment for perchlorate.
The purpose of this study was to evaluate the potential effects of perchlorate when administered to rats as AP in their drinking water over the course of 14 or 90 days. The study design included a nontreatment recovery period of 30 days to assess the reversibility of perchlorate-induced effects. The potential effects of AP on male sperm parameters, female estrous cycling, bone marrow micronucleus formation, and serum hormone levels (TSH, T3, and T4) were also investigated.
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MATERIALS AND METHODS |
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Drinking water solutions.
Drinking water solutions containing AP were prepared on a weekly basis during the study in reverse-osmosis (RO) deionized water. The concentration of AP in the drinking water was adjusted weekly for each sex group, based on measured body weights and water consumption, to achieve the desired dosage levels.
Animals.
The in-life study was conducted at Springborn Laboratories, Inc., Spencerville, Ohio. Male and female Sprague-Dawley Crl:CD®BR VAF/Plus® rats, approximately 5 weeks of age, were obtained from Charles River Laboratories, Inc., Kingston, NY. The rats were housed individually in suspended stainless steel cages, in an environment-controlled room (12 h light/12 h dark cycle; 22 ± 2° C; 50 ± 15% relative humidity; 1215 air changes/h). After an acclimation period of approximately 2 weeks, the rats were randomly assigned to study groups. Drinking water and Purina Certified Rodent Chow (PMI Rodent Meal #5002, Purina Mills, Inc.) were provided to the animals ad libitum. The drinking water was provided in individual water bottles to allow measurement of water and perchlorate consumption.
Experimental design.
The 90-day study was designed in general agreement with U.S. EPA Health Effects Test Guideline OPPTS 870.3100. All aspects of the study were performed in strict compliance with U.S. EPA Good Laboratory Practice regulations 40 CFR Part 160.
The experimental design is illustrated in Table 1. The study consisted of a vehicle control (water only) and five AP treatment groups that received AP dosages of 0.01, 0.05, 0.2, 1.0, and 10.0 mg/kg/day. The rats were observed daily for survival and clinical signs of toxicity. Individual body weights, food consumption, and water consumption were measured at weekly intervals. Ten animals/sex/group were euthanized and necropsied after 14 and 90 days of treatment. An additional 10 animals/sex in groups 1, 3, 5, and 6 (vehicle control, 0.05, 1.0, and 10.0 mg/kg/day groups) were designated for euthanasia after a nontreatment recovery period of 30 days. All surviving rats were euthanized by CO2 inhalation followed by exsanguination.
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Ophthalmology.
Ophthalmological examinations were performed on all animals prior to study initiation, near conclusion of treatment, and near completion of the 30-day recovery period. Eyes were dilated using 0.5% Mydriacyl® ophthalmic solution prior to bimicroscopic slit-lamp and indirect ophthalmoscopic examinations by a Board-certified veterinary ophthalmologist.
Clinical pathology and TSH, T3, and T4 analyses.
Blood samples were collected from all animals at scheduled euthanasia after 14, 90, or 120 days for evaluation of routine hematology and clinical chemistry parameters, as well as TSH, T3, and T4. The blood samples were obtained via the vena cava immediately prior to necropsy. The rats were euthanized in a randomized block design across groups to minimize any potential bias related to blood and tissue sampling times. The time of euthanasia and blood sample collection was recorded for each animal. Blood samples for hematology were analyzed using a Coulter S Plus IV hematology analyzer. Differential leukocyte counts were performed manually under oil immersion (100x) from Wright-Giemsastained slides. Serum samples for clinical chemistry were analyzed using a Beckman Synchron CX-5 chemistry analyzer. Appropriate controls were used to monitor the accuracy and precision of the hematology and clinical chemistry instruments.
For each animal, serum for TSH, T3, and T4 determinations was divided into three vials (500 µl/vial), which were immediately frozen and stored at 70°C until analysis. The hormone assays were conducted using radio-immunoassay (RIA) kits, following the manufacturers' instructions. For each hormone evaluated, RIA kits with the same lot number and expiration date were used. Tracer (125I) radioactivity was measured using a gamma counter (Packard Instrument Co., Meriden, CT). T3 RIA kits (T3 standards lot numbers C30-3 to C30-8; 125I-T3 lot #TC320166) were purchased from Diagnostic Product Corp. (Los Angeles, CA) and canine T3 antibody tubes (lot #TC31034) were used. T4 RIA kits (T4 standards lot numbers T4C3 to T4C8; 125I-T4 lot #TT42-0241) were purchased from Diagnostic Product Corp. (Los Angeles, CA), and rabbit T4 antibody tubes (lot #TT41-1200) were used. TSH RIA kits (assay code #RPA554) 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 containing sodium azide) were both used. All samples and standards were analyzed in triplicate.
Estrous cycling.
Vaginal smears were examined daily to assess estrous cyclicity for 3 weeks prior to scheduled euthanasia at 90 or 120 days. The smears were examined under low power magnification (10x) and classified into four stages (i.e., proestrus, estrus, metestrus, and diestrus), according to the methodology of Yuan and Carlson (1987).
Sperm analysis.
Semen samples were obtained from all male rats euthanized after 90 or 120 days for evaluation of sperm count (106/g cauda), concentration (106/ml), motility (%), and morphology. Sperm count and concentration were determined from samples obtained from the left cauda epididymis. Sperm motility and morphology were evaluated from samples collected from the vas deferens. A Hamilton Thorne IVOS 10 semen analyzer (Hamilton Thorne Research, Beverly, MA) was used for the sperm count, concentration, and motility assessments. Sperm morphology was assessed by microscopic examination of a minimum of 200 sperm/animal at 300500x magnification. Morphological endpoints were based primarily on head and tail abnormalities, based on the classification systems of Linder et al. (1992) and Seed et al. (1996). The mean percentage of morphologically normal sperm was calculated for each group.
Gross necropsy and histopathology.
All animals were subjected to a complete gross necropsy examination at the time of death or scheduled euthanasia. For each rat, a complete set of tissues and organs was preserved by immersion in 10% neutral buffered formalin (NBF), including adrenals, aorta, brain, cecum, colon, duodenum, epididymides, esophagus, exorbital lachrymal glands, eyes, larynx, femur, gross lesions, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes, mammary gland, nose, ovaries, pancreas, pharynx, pituitary, prostate, rectum, salivary gland, sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord, spleen, sternum, stomach, testes, thymus, thyroid/parathyroid, tongue, trachea, urinary bladder, uterus and vagina. Fresh organ weights were obtained for the liver, kidneys, testes, ovaries, brain, spleen, lungs, epididymides, uterus, pituitary, and heart. Following complete fixation in 10% NBF, all thyroid/parathyroid glands were carefully trimmed and weighed by a single technician.
All tissues from control and high-dose animals euthanized after 14 or 90 days were examined microscopically. Additionally, the liver, kidneys, lungs, thyroids, and gross lesions from all intermediate dose groups euthanized after 14 and 90 days and from all recovery animals were examined for histopathological changes. The tissues were trimmed, embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin. Microscopic examinations were performed by a Board-certified veterinary pathologist experienced in rodent pathology.
Micronucleus formation.
Bone marrow samples were collected from all animals euthanized after 90 and 120 days for possible evaluation of bone marrow micronucleus formation. Six additional (satellite) rats were administered a single intraperitoneal injection of cyclophosphamide (20 mg/kg body weight) to serve as positive controls for the micronucleus assay. Bone marrow slides from the vehicle control, positive control, and high-dose (10 mg/kg/day) groups were stained and evaluated as described previously (Schmid, 1976). The frequency of micronucleated cells was determined by random observation of 1000 polychromatic erythrocytes (PCEs) per slide. The ratio of PCEs/NCEs (normochromatic erythrocytes) was also determined to assess potential cytotoxicity due to perchlorate.
Analytical chemistry.
A sensitive and selective ion chromatography (IC) method for the analysis of perchlorate and nitrate, a possible interference ion, was developed by Tsui et al. (1998) to support this study. The IC method was shown to be capable of detecting both perchlorate and nitrate at 5 ppb in reagent grade water with excellent accuracy and precision. The IC method was used to verify the stability of aqueous AP solutions and to periodically confirm target concentrations of AP in the drinking water solutions prepared for the study.
The IC was performed using a dionex DX-300 High Performance Liquid Chromatograph with a Dionex CDM-3 conductivity detector. An ASRS-II anion suppresser operating in auto suppression-external mode was used. The system included a Dionex AI 350 autosampler. Anion analyses were performed using a Dionex IonPak AS-11 ion chromatography column (4.0 x 250 mm), Dionex ATC-1 anion trap column, and Dionex AG-11 guard column (4.0 x 50 mm). The mobile phase, consisting of 45 mM NaOH in 55:45 water:methanol, was set at 1 ml/min flow rate. The injection loop volume was 50 µl, and the regenerant flow rate was 10 ml/min. All IC analyses were performed at 30°C.
Statistical analysis.
Body weight, body weight gain, food consumption, water consumption, clinical pathology parameters, organ weights, estrous cycle lengths, and semen parameters were analyzed by one-way analysis of variance (ANOVA) (Snedecor and Cochran, 1967) followed by the Tukey-Kramer test (Dunnett, 1980
) when appropriate. The Chi-square test (Siegal, 1956
) was used to analyze the incidence of females in each group exhibiting estrous cyclicity. Serum hormone levels (TSH, T3, and T4) were statistically analyzed by ANOVA, followed by the Bonferroni multiple comparisons test (Rosner, 1990
). Bone marrow micronuclei counts and PCE/NCE ratios were analyzed using ANOVA and Chi-square, respectively. All statistical comparisons utilized a minimum significance level of p < 0.05.
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RESULTS |
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Body Weights and Weight Gain
Animal growth curves for the 90-day study are illustrated in Figure 1. There were no statistically significant differences in mean body weights among the groups. Mean body weight gain was slightly but significantly (p < 0.05) decreased in 10 mg/kg/day males during the first week. Specifically, the 10 mg/kg/day males gained an average of 57 g, whereas control males gained an average of 64 g on study week 1. Although statistically significant, this difference was not considered toxicologically meaningful. No other statistically significant differences in body weight gain were noted.
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Ophthalmology
Ophthalmological examinations did not reveal any test articlerelated ocular effects in the AP-treated rats. Occasional findings of slight corneal crystals and moderate conjunctival exudate were noted at study termination; however, these findings were of low incidence and randomly distributed among the groups.
Routine Clinical Pathology
Hematology and clinical chemistry results at conclusion of the 90-day study are summarized in Tables 25. A few statistically significant (p < 0.05) differences in hematology and clinical chemistry parameters were observed; however, these did not follow any pattern that would indicate a relationship to AP treatment.
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Following the 30-day recovery period, TSH levels were significantly increased (p < 0.05) in all three female recovery groups (0.05, 1.0, and 10 mg/kg/day), whereas no significant differences in TSH levels were observed in the male recovery groups. The increases in TSH levels of females ranged from 16% higher in 0.05 mg/kg/day females to 22% higher in the 10 mg/kg/day females. In contrast to the TSH results, mean T4 levels were significantly lower (p < 0.05) than controls in all three male recovery groups (0.05, 1.0, and 10 mg/kg/day), whereas no significant differences in T4 levels were observed in the female groups. The decreases in T4 levels of males ranged from 23% lower than control males in the 0.05 and 1.0 mg/kg/day groups to 39% lower than control males in the 10 mg/kg/day group. Statistically significant differences in T3 were limited to a lower mean T3 value in 10 mg/kg/day females (12% lower than control females). No statistically significant differences in T3 levels were observed in the male recovery groups.
Sperm Analysis and Estrous Cyclicity
Male sperm parameters and female estrous cyclicity data are summarized for the 90-day interval in Table 6. There were no statistically significant differences in sperm count, concentration, motility, or morphology at the end of 90 days or following the 30-day recovery period (data not shown). Similarly, no significant differences in estrous cyclicity data were noted after 90 days of treatment or following the recovery period (data not shown). The number of females cycling in each group and the mean cycle lengths remained comparable between the control and AP-treatment groups.
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Analytical Chemistry
A detailed description of analytical chemistry results can be found in the technical report by Tsui et al. (1998). Briefly, analysis of stability samples showed that AP was stable in reagent-grade water for at least 109 days at concentrations encompassing those used in the 90-day study. Periodic analysis of animal drinking water solutions demonstrated that they were accurately prepared, with all solutions being within ± 10% of the targeted concentrations. No perchlorate was detected in the vehicle control drinking water solutions, and no nitrate was found in any of the solutions analyzed, including the RO deionized water used to prepare the AP drinking water solutions.
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DISCUSSION |
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Effects of perchlorate at the 10 mg/kg/day level were not surprising given its mode of action as an inhibitor of iodide uptake by the thyroid. These effects consisted of increased absolute and relative thyroid weights, decreased T3 and T4 levels, increased TSH levels, and thyroid histopathological changes. In a previous pilot study in which AP was administered in the drinking water for 14 days to male and female Sprague-Dawley rats at approximate dosage levels of 0, 0.1, 0.5, 1.2, 2.7, 4.6, 11.5, and 23.5 mg/kg/day, T3 and T4 were decreased in a dose-dependent manner in both sexes (Caldwell et al., 1996; personal communication, D. Caldwell, 2000). In the present study, statistically significant (p < 0.05) differences in TSH, T3, and T4 were observed in one or both sexes following 14 and 90 days of AP treatment (Figs. 2 and 3
). In general, these differences tended to follow a dose-related pattern consisting of significantly increased mean TSH levels and significantly decreased mean T3 and T4 levels compared to respective control values. After only 14 days of treatment, statistically significant reductions in thyroid hormones were observed at dosage levels as low as 0.01 mg/kg/day. Thus, the present results appear to support the previous thyroid hormone and TSH findings by Caldwell et al. (1996). Caldwell and coworkers also reported a dose-related decrease in follicular lumen size and dose-related increase in cell hypertrophy (graded as slight to mild) in their 14-day pilot study starting at the 0.5 mg/kg/day AP level (personal communication, D. Caldwell, 2000).
In the present study routine histopathological examination revealed a test articlerelated effect in the thyroids of 10 mg/kg/day males and females after 14 or 90 days of AP treatment. The effect was characterized primarily by follicular cell hypertrophy with microfollicle formation and colloid depletion. There also appeared to be an increased number of microfollicles in the central portion of the affected thyroids; however, there was no evidence of focal hyperplasia as would be indicated by epithelial stratification or increased mitotic figures. Interestingly, while most of the thyroid lesions were graded as minimal to mild, lesions of moderate severity were noted only in 10 mg/kg/day males at the 14-day interval. Morphological changes similar to those seen in the present study have been reported in male Fischer 344 rats fed an iodine-deficient diet (Kanno et al., 1992) and in female Wistar rats following chemical inhibition of iodine organification (Gerber et al., 1994
). Kanno et al. (1992) reported that dietary iodine deficiency in male Fischer 344 rats produced diffuse thyroid hyperplasia, characterized by small follicles with tall epithelium and reduced colloid, together with a decrease in T4 and an increase in TSH. Gerber et al. (1994) reported similar morphological changes in female Wistar rats, which they referred to as microfollicular goiter, following inhibition of iodine organification by methimazole or inhibition of iodide uptake by sodium perchlorate. These results indicate that the thyroid changes observed in the present study are not unique, but rather consistent with histomorphological effects reported previously in rats following dietary iodine deficiency or impaired iodine organification. In an effort to characterize in a consistent manner the microscopic changes associated with perchlorate treatment, thyroid slides from this and other recent perchlorate studies are being reexamined by a pathology working group initiated by the U.S. EPA.
Despite efforts to minimize variability in the serum hormone analyses (e.g., animals were sampled in a randomized block design across groups; all samples were analyzed in triplicate around the same approximate time; and only RIA kits with the same lot number and expiration date were used), some variability was evident in the mean control levels of the various hormones during the course of the study. For example, in control males, mean TSH levels (± SD) ranged from 14.8 ± 1.5 ng/ml at 14 days to 20.9 ± 1.6 ng/ml at 120 days. In control females, mean TSH levels (± SD) ranged from 10.5 ± 0.8 ng/ml at 14 days to 16.5 ± 1.5 ng/ml at 90 days. Mean T3 levels of control males and mean T4 levels of control males and females tended to be less variable over time. In contrast, mean T3 levels of control females exhibited marked variability over the course of the study, ranging from 133 ± 15 ng/dl at 14 days to 224 ± 21 ng/dl at 120 days. It was not clear if the observed variability in mean control hormone levels was reflective of normal age-related variations or due to other factors such as the relatively small sample sizes (n = 9 or 10). Sex, strain and age-related differences in thyroid hormone levels of rats have been described by several investigators (Azizi, 1979; Donda et al., 1987
; Tang, 1985
). Consequently, it is difficult to define normal levels of T3 and T4 in rats and to compare values between laboratories, as these are highly dependent on diet and other factors. Additionally, there is a marked diurnal rhythm in TSH secretion, as with other pituitary hormones (Thomas and Williams, 1994
). In any case, the present results in control animals underscore the importance of balanced sample collection across groups, consistency in animal husbandry and sample handling/analysis, and the critical need for concurrent control data when assaying TSH and thyroid hormones in rats. Because these procedures were employed in the present study, results of the serum hormone analyses are regarded as accurate and reflective of the hormone levels in the animals from which samples were obtained.
Despite the fact that mean TSH levels were significantly increased in males at levels of 0.2 mg/kg/day and above and in females at levels of 0.05 mg/kg/day and above, there were no indications of a thyroid growth response at perchlorate levels 1 mg/kg/day. At the 14-day interval, mean TSH levels in the 0.2, 1.0, and 10 mg/kg/day males were approximately 21, 27, and 62% higher than control males, respectively. At the 90-day interval, mean TSH levels were similar between the 0.2, 1.0, and 10 mg/kg/day male groups, being approximately 1718% higher than control males. In females, mean TSH levels at 14 days in the 0.05, 0.2, 1.0, and 10 mg/kg/day groups were approximately 17, 18, 20, and 58% higher than control females, respectively. At 90 days, only the mean TSH level of the 10 mg/kg/day females was significantly different from control females, being approximately 21% higher than the control value. Together, these findings indicate that the increases in TSH at levels
1.0 mg/kg/day were not sufficient to induce an observable growth response in the rat thyroid after 90 days. On the other hand, the marked increases in TSH observed at the 10 mg/kg/day level were sufficient to induce a growth response in the thyroid of the male and female rats after only 14 days of AP exposure. Thus, in this study, perchlorate-induced increases in TSH at levels of
1.0 mg/kg/day appeared to be tolerated without consequent alteration of follicular cell morphology, as revealed by standard histopathological techniques.
The question of what AP dosage level constitutes the No-Observed-Adverse-Effect Level (NOAEL) for this study is open to debate. Whereas the high-dose level of 10 mg/kg/day was clearly an effect level, remarkable changes at AP dosage levels of 1.0 mg/kg/day and lower were limited to statistically significant differences in TSH and thyroid hormones. Because these hormonal changes are mechanistically linked to perchlorate's effect on the thyroid, it is reasonable to conclude that the 90-day study failed to establish a definitive NOEL. However, based on the absence of traditional indicators of toxicity such as statistically significant body weight changes, adverse clinical signs, clinical pathology changes, and histopathological effects, it is also reasonable to conclude that 1.0 mg/kg/day represents the NOAEL for this study. Selection of 1.0 mg/kg/day as the NOAEL requires that the hormonal changes at AP dosage levels of 1.0 mg/kg/day and lower not be regarded as adverse effects per se. Again, this approach seems reasonable since 1) the noted hormonal changes had no effect on the general health of the rats, as measured by their normal growth and the other experimental parameters examined in the study, 2) despite the noted changes in TSH, there were no indications of compensatory thyroid growth at AP levels 1.0 mg/kg/day, and 3) even at the 10 mg/kg/day level, the thyroid growth response was reversible after the 30-day recovery period.
As compared to humans, the rat is known to be more sensitive to perturbations of the pituitary-thyroid axis. This greater sensitivity is related to shorter plasma half-lives of T3 and T4 in the rat and to differences in T3 transport proteins between rats and humans (Capen, 1997). Both rodents and humans have nonspecific low-affinity protein carriers of thyroid hormones (e.g., albumin). However, humans also possess a high-affinity binding protein, thyroxine-binding globulin, that is virtually undetectable in adult rats (Vranckx et al., 1994
). Because this protein is missing in adult rats, more T4 is susceptible to removal from the blood and hence to metabolism and excretion. Consequently, the serum half-life of T4 in rats (< 1 day) is substantially shorter than in humans (approximately 5 to 9 days). It has been estimated that this difference results in a 10-fold greater requirement for exogenous T4 in the rat with a nonfunctioning thyroid compared to the adult human (Döhler et al., 1979
). Serum T3 levels also show a species difference with the half-life in rats being around 6 h, whereas that in humans is about 24 h (Larsen, 1982
; Oppenheimer, 1979
).
In summary, oral administration of AP to rats via the drinking water produced thyroid organ weight increases and corresponding histopathological changes in the thyroid after 14 and 90 days at a dosage level of 10 mg/kg/day. These changes were reversible after a nontreatment recovery period of 30 days. Statistically significant changes in TSH and thyroid hormones were observed at AP dosage levels as low as 0.01 mg/kg/day; however, no thyroid organ weight or histopathological effects were observed at AP dosage levels of 1.0 mg/kg/day and lower. Based on these findings, 1.0 mg/kg/day is regarded as the NOAEL for this study.
In the absence of thyroid organ weight and histopathological effects, the toxicological significance of TSH and thyroid hormone changes at AP dosage levels 1.0 mg/kg/day remains to be determined.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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