Acute and subacute oral toxicity of periodate salts in rats

Regulatory Toxicology and Pharmacology 83 (2017) 23e37

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Acute and subacute oral toxicity of periodate salts in rats Emily May Lent*, Lee C.B. Crouse, William S. Eck Toxicology Directorate, Army Public Health Center, Aberdeen Proving Ground, MD, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 24 May 2016 Received in revised form 7 November 2016 Accepted 8 November 2016 Available online 10 November 2016

Periodate salts are being developed as potential replacements for perchlorate due to potential health hazards associated with exposure to perchlorate. The aim of this study was to investigate acute and subacute effects of periodate salts in rats. Acute oral toxicity of potassium and sodium periodate was determined using the Sequential Stage-Wise Probit method. The LD50 for potassium periodate was 732 (95% CI ¼ 539e838, slope ¼ 13.4) and 685 mg/kg (95% CI ¼ 580e809, slope ¼ 10.6) for females and males, respectively. The LD50 for sodium periodate was 318 (95% CI ¼ 292e347, slope ¼ 24.3) and 741 mg/kg (95% CI ¼ 704e779, slope ¼ 31.2) for females and males, respectively. In the subacute study, rats were administered sodium periodate at five doses (1/16 LD50 up to LD50) or distilled water for 14days via oral gavage. Female rats in the 318 mg/kg-day group and male rats in the 185, 370, and 741 mg/kg-day groups exhibited moribundity, kidney toxicity, uremia, and a stress response. BMDL10s of 17.2 and 33.7 mg/kg-day were derived for females and males, respectively. Comparison with the NOAEL for perchlorate-induced thyroid toxicity in rats (0.009 mg/kg-day) suggests sodium periodate is less toxic than perchlorate on a subacute basis. © 2016 Published by Elsevier Inc.

Keywords: Oxidizer Thyroid Iodine Renal Perchlorate Periodate

1. Introduction Periodates, polyatomic anions with the chemical formula IO 4, are being developed as alternatives to perchlorates for use as an oxidizer in military pyrotechnic devices (Ball, 2012; Fields, 2012; Moretti et al., 2012). Alternatives to perchlorates are being pursued due to the environmental and health hazards associated with these compounds. The use of perchlorate has been widely scrutinized due to the potential for the compound to cause thyroid dysfunction and developmental abnormalities in pregnant and nursing mothers, fetuses, and newborns (Brechner et al., 2000; Buffler et al., 2006; Crump et al., 2000; Kelsh et al., 2003; USEPA, 2013; York et al., 2005). Sodium and potassium periodate have been identified as potential replacement incendiary oxidizers that fulfill the pyrotechnic requirements and are presumed, based on structure, to be less toxic than those currently in use. Periodate ions are slightly larger than perchlorate ions, leading munitions developers to speculate that the ions are too big to interact with thyroid receptors in the same manner as perchlorate (Ball, 2012; Fields, 2012; Moretti et al., 2012). However, if transported into

* Corresponding author. Army Public Health Center, 5158 Blackhawk Road, ATTN: MCHB-PH-TEV, Aberdeen Proving Ground, MD, USA. E-mail address: [email protected] (E.M. Lent). http://dx.doi.org/10.1016/j.yrtph.2016.11.014 0273-2300/© 2016 Published by Elsevier Inc.

the cell by the sodium-iodide symporter (NIS), periodate ions will bring iodide into the follicular lumen of the thyroid that could then be incorporated into thyroid hormones. Furthermore, alteration of blood iodine levels can have profound effects on thyroid status. High blood iodide levels disrupt thyroxinogenesis by blocking the release of T3 and T4 from the follicle (Capen and Martin, 1989). Therefore, the increase in available iodine with periodate dosing in the present study may result in iodine-induced thyrotoxicosis. Existing toxicity data on periodates are limited to an intraperitoneal LD50 in mice of 58 mg/kg for sodium periodate (Lewis, 1996) and an oral LD50 of 7.07 g/kg for pentacalcium orthoperiodate (PCOP) in fasted male rats (Kuhajek and Andelfinger, 1970). Metabolism studies in the rat have demonstrated prompt reduction of intravenously injected metaperiodate to iodate and subsequently to iodide (Anghileri, 1965; Taurog et al., 1966). Thus, in the present study, observed effects may be the result of rapid reduction of periodate to iodate and ultimately to iodide. The toxicity of iodates and iodides varies greatly with the route of administration and the feeding state of the animal. Potassium periodate is four to six times more toxic when administered intraperitoneally than orally to fasted mice (LD50 136 and 531e815 mg/kg, respectively). Sodium and potassium iodate are nineteen and eleven times more acutely toxic, respectively, than sodium and potassium iodide when given intraperitoneally to fasted mice (Webster et al., 1957). The cation does not appear to

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modify iodide and iodate acute toxicity. Oral LD50 values of 1650 and 1862 mg/kg have been reported for sodium and potassium iodide, respectively, in fasted mice; while sodium and potassium iodate are approximately 3 times more toxic with LD50 values of 505 and 531 mg/kg, respectively (Webster et al., 1957). When administered to fed mice, potassium iodate is 1.8 times less toxic than when administered to fasted mice. No difference in iodide toxicity was observed between fasted and fed mice (Webster et al., 1957). Webster et al. (1957) reported effects of sodium iodate and potassium iodate oral exposure included alternate hyperactivity and lassitude, weakness, prostration, dyspnea, and diarrhea. Transient increases in gastrointestinal pH and degeneration of parietal cells, hemolytic effects including hemoglobinuria and hemosiderin deposits in the kidneys were observed. Mortality was attributed to renal damage. Symptoms of iodide exposure were similar, except with slower onset and the absence of changes in gastrointestinal pH, degeneration of parietal cells, and hemoglobinuria (Webster et al., 1957). To determine whether sodium periodate and/or potassium periodate provide reduced health hazard alternatives to currently fielded oxidizers, acute and subacute oral toxicity tests were conducted in rats. 2. Materials and methods 2.1. Test substance Neat potassium periodate (CAS # 7790-21-8; lot 10174755; purity: 100.5%) and sodium periodate (CAS # 7790-28-5; lot B27Z025; purity: 99.07%) were purchased from Alfa Aesar, Ward Hill, MA, USA. 2.2. Experimental design This study was conducted using male and female Sprague Dawley (Crl: CD (SD) CD®) rats obtained from Charles River Laboratories, Wilmington, Massachusetts. Animal use procedures were approved by the Army Public Health Center (APHC) Institutional Animal Care and Use Committee. Animal care and use was conducted in accordance with The Guide for the Care and Use of Laboratory Animals and all applicable Federal and DOD regulations. The USAPHC Animal Care and Use Program is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. All animals were housed in temperature-, relative humidity-, and light-controlled rooms. The target conditions of the rooms were 68e72  F and 30e70 percent humidity. An automatically controlled 12:12-h light/dark cycle was maintained, with the dark period beginning at 1800 h. A certified pesticide-free rodent chow (Harlan Teklad®, 2016C Certified Rodent Diet) was available ad libitum. Filtered tap water was provided ad libitum via an automated watering system. Dosing solutions/suspensions were prepared by weighing the required amount of potassium or sodium periodate and adding a measured volume of deionized water. For the acute study, fresh dosing solutions/suspensions were prepared for each day/round of dosing. For the 14-day study, six dosing solutions, 3.1, 6.2, 12.5, 25, 50, and 100 milligrams per milliliter (mg/ml) of sodium periodate, were prepared at the start of the study in sufficient volume for use throughout the study. Dosing solution concentrations were verified via high performance liquid chromatography with ultra violet detection and were within 96e109% of nominal concentrations. As such, all results are reported using the nominal concentrations. Additionally, a stability test conducted on a representative solution indicated that the compound was stable under the test and storage

conditions. The acute toxicity of sodium periodate and potassium periodate was assessed using the Sequential Stage-Wise Probit (SSWP) method (ASTM, 2010; Feder et al., 1991a, 1991b). In the absence of historical data or literature values, doses for the first stage of dosing were set at the default starting value of 175 mg/kg with half-log dose intervals (3.2 dose progression factor) (USEPA, 2002a). The Approximate Lethal Dose (ALD) method was also used to determine the acute toxicity of sodium periodate in fed female rats (Deichman and LeBlanc, 1943). This test was conducted to determine if differences exist between the lethal dose in fasted (overnight) (i.e., SSWP LD50) and fed rats and to assist in determining dose levels for the 14-day study. The ALD consisted of six doses and a control, with one female rat receiving each dose. Dose selection for the sodium periodate ALD was based on the results of the SSWP but included an adjustment factor based on the 2-fold increase in LD50 between fasted and fed animals that has been demonstrated for iodate (Webster et al., 1959). Dose intervals were set at approximately 1.5 the previous dose to a maximum of 2000 mg/ kg. Fifty-eight female and forty-seven male Sprague Dawley (Crl: CD (SD) CD®) rats were used for the acute portion (SSWP and ALD) of this study. Females were approximately 10 weeks old and weighed 221.1 ± 15.3 grams (g), while males were approximately 9 weeks old and weighed 307.5 ± 19.0 g. All sodium periodate and potassium periodate doses were administered according to body weight measured on the day of dosing using a stainless steel 16gauge gavage needle. Constant concentration dosing was used within each round of dosing. However, due to difficulties encountered in delivering the very concentrated suspensions without injuring the animals, concentrations were reduced after the first round of dosing; dosing volume limitations (i.e., 10 ml/kg) were not exceeded. All animals were observed for a period of 14 days. A 14-day oral toxicity study was performed with the most acutely toxic of the two periodate salts (sodium periodate) to determine the effects of repeated daily dosing. Sixty female (215.4 ± 11.1 g) and sixty male (269.8 ± 11.7 g) Sprague Dawley (Crl:CD(SD) CD®) rats approximately 10 weeks old were assigned to dose groups using a stratified random procedure, with animals stratified within sex according to body weight and dose groups assigned randomly. Body weight did not differ among dose groups prior to initiation of dosing. Dose selection for the 14-day study was based on the results of the acute study (e.g., 0.25, 0.13, 0.063, 0.031, 0.016 the LD50) with an adjustment factor for differences between fed and fasted rats calculated based on the results of the ALD. The solutions/suspensions were administered in volumes of 6.36 and 7.41 milliliter per kilogram (ml/kg) based on daily body weight for females and males, respectively, using a stainless steel 16-gauge gavage needle. The vehicle control group received a volume of deionized water equivalent to the highest exposure group. Females and males were each divided into three time-separated groups to facilitate necropsy. Animals from each dose group were approximately evenly distributed across necropsy groups. 2.3. Observations, body weight, food consumption During the dosing period, observations for mortality and signs of toxic effects were made at least twice daily. Acute study animals were weighed daily while 14-day animals were weighed on study days 0, 1, 3, 7, and 13. Terminal (fasted) body weight was obtained the morning of necropsy following overnight fasting. Food consumption was determined on study days 0, 1, 3, 7, and 13 for each pair of animals. Because rats were pair housed and individual food consumption could not be determined, food consumption was then calculated per gram of rat by dividing the food consumed by the

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total weight of the rats in each cage. Food efficiency ratios were calculated as a ratio of body weight gained to food consumed. 2.4. Urinalysis During the night prior to scheduled necropsy for the 14-day study, each surviving animal was placed in a metabolism cage capable of separating urine and feces for approximately 12 h during which free-catch urine was collected. Animals were fasted during this period to coincide with fasting prior to necropsy. Water was available ad libitum via a water bottle containing a measured volume of water. Urine samples were transferred to clear, graduated conical centrifuge tubes and volume, color, and appearance of each sample were recorded. Specific gravity was tested using a refractometer. Chek-Stix™ Test Strips were used to conduct chemical analyses including pH, protein, glucose, ketones, bilirubin, blood, urobilinogen, nitrites, and leukocytes. Chek-Stix™ is a registered trademark of Siemens Healthcare Diagnostics (Tarrytown, NY). 2.5. Necropsy After 14-days of treatment, or when determined to be moribund, all surviving rats were anesthetized with CO2, blood was collected by intracardiac puncture, and rats were euthanized using CO2. Animals from the acute study and animals that died during the study, if the animals were determined to have died recently, were submitted for gross necropsy. Tissues that were not grossly autolytic were submitted for histopathologic evaluation. At necropsy, the adrenals, brain, heart, kidneys, epididymides, liver, ovaries (without oviducts), spleen, testes, thymus, thyroid (with attached portion of trachea), and uterus were removed, trimmed, and weighed (except the thyroid/trachea, which was preserved prior to weighing). In addition to the organs listed above, samples of peripheral nerve, muscle, spinal cord, eye plus optic nerve, gastrointestinal tract, urinary bladder, lung, bone marrow, pituitary, and vagina were collected. Tissues, except testes and epididymides which were preserved in Davidson's fixative, were preserved in 10% buffered formalin for histological examination. 2.6. Histopathology Tissues from the 14-day study were selectively trimmed and slides prepared and submitted for histologic examination by a board certified veterinary pathologist. Sections of adrenal glands, brain, epididymides, heart, intestine (large and small), kidneys, liver, ovaries, spleen, stomach, testes, thymus, thyroid, and uterus (as appropriate) were trimmed, slides prepared, and these were submitted for histologic examination. In addition, selected tissues with gross observations saved at necropsy were also trimmed and processed. All processed and embedded tissues were microtomed at 5 micrometers (mm) thick and stained with hematoxylin and eosin. The pathologist examined slides for compound-induced histopathologic changes via light microscopy. A variety of non-neoplastic lesions were noted in various tissues, and were semi-quantitatively graded across a 4-point scale based on severity of effect, where Grade 1 (minimal) referred to a minor change of negligible biologic significance or which affected less than 10 percent of the presented tissue area, and Grade 2 (mild) referred to a greater change which affected 10 to 19 percent of the tissue area. Grade 3 (moderate) was scaled to refer to a change of clear biologic relevance and which affected at least 20 percent of the tissue area, and Grade 4 (marked) was scaled for lesions considered to be of maximal morphologic change. For the thyroid gland, an additional microscopic notation was made for all rats. Thyroid follicular epithelium was evaluated for increased size

25

(hypertrophy and height) and graded on a 1 to 5 scale. Follicular epithelium that was predominately flattened was graded as 1; whereas follicular epithelium that was all tall columnar in morphology was graded as 5. Mostly cuboidal-shaped epithelium constituted a grade of 3. Grades 2 or 4 were used for variations between these appearances. 2.7. Clinical chemistry and hematology Blood for clinical chemistry analyses was transferred to collection tubes free of additives and was allowed to clot at room temperature for 30e40 min, and was centrifuged for approximately 2.5 min at 12,000g. Serum was removed and immediately analyzed for clinical chemistry parameters. Aliquots (100 microliters (ml)) were also placed in siliconized tubes and stored at approximately 35  C for subsequent thyroid hormone assays (triiodothyronine (T3), total thyroxine (T4), (TSH)). The following clinical chemistry parameters were evaluated using the Idexx VetTest® 8008 Chemistry Analyzer and the VetLyte® Electrolyte Analyzer: albumin (ALB), alkaline phosphatase (ALKP), alanine aminotransferase (ALT), amylase (AMYL), blood urea nitrogen (BUN), calcium (CA), cholesterol (CHOL), creatinine (CREA), globulin (GLOB), glucose (GLU), total bilirubin (TBIL), total protein (TP), sodium (Na), phosphate (PHOS), potassium (K), and chloride (Cl). (VetTest® and VetLyte® are registered trademarks of IDEXX Laboratories, Inc.). Blood for hematological analyses was transferred to tripotassium ethylenediamine-tetraacetic acid (K3EDTA) containing tubes. The following hematology parameters were evaluated using the Cell-Dyn 3700 Hematology Analyzer (Abbott Laboratories, Abbott Park, IL 60064): erythrocyte count (RBC), hematocrit (HCT), hemoglobin concentration (HGB), mean cell volume (MCV), mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), red blood cell distribution width (RDW), total white blood cell count (WBC), and differential leukocyte count (neutrophils: NEU, lymphocytes: LYM, monocytes: MONO, eosinophils: EOS, basophils: BASO), platelet count (PLT), and mean platelet volume (MPV). To determine average activated prothrombin time, blood was transferred to a tube containing sodium citrate, the blood mixed, then centrifuged for approximately 2.5 min at 12,000g. The plasma was analyzed using the MCA 210 Microsample Coagulation Analyzer (BioData Corporation, Horsham, PA 19044). 2.8. Thyroid hormone assays Triiodothyronine and total thyroxine were determined using the TOSOH® Bioscience AIA®-360 Automated Enzyme Immunoassay System. Analysis of TSH was conducted using a commercially available rat TSH Enzyme linked Immunosorbent Assay kit purchased from ALPCO™ Immunoassays. Due to detection of interfering substances during assay validation, samples were pre-treated by precipitation with 25% polyethylene glycol (PEG-6000) prior to use in the assay (Dimeski, 2008; Sakai et al., 2009; Tate and Ward, 2004). PEG-6000 (100 ml) was added to each sample, the sample was vortexed and then centrifuged for approximately 5 min at 4000g. Assays were then conducted using the supernatant according to the manufacturer's instructions. The optical density of each well was determined at 450 nanometers (nm) and 630 nm using a BioTek® Synergy HT Multi-Mode microplate reader with Gen5™ data analysis software. Mean absorbance for each sample was calculated after adjustment for the absorbance at 630 nm. The external quality control standards (Rat Control 1 and 2 Lot 001) were within the target reference ranges. The intra-assay coefficients of variation were 2.6% and 1.8%, respectively, and the inter-assay coefficients of variation were 6.7% and 8.8%, respectively.

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2.9. Data collection and statistical analyses Experimental data generated during the course of this study were recorded by hand and tabulated, summarized, and/or statistically analyzed using Microsoft® Excel, SAS®, and SPSS® 21.0. Environmental data were automatically recorded using MetaSys® Building Management System. Data from the SSWP were analyzed according to previously published methods (Feder et al., 1991a, 1991b) to determine the LD50, 95% confidence interval and slope of the dose response curve. The ALD was determined as the lowest dose which was lethal where two successively higher doses were lethal and three lower doses were not lethal. Data from the subacute study were analyzed based on the type of data collected and the frequency of collection. Variables measured only at the end of the study were analyzed using a onefactor Analysis of Variance (ANOVA) with dose group as the main effect. Organ-to-brain and organ-to-body weight ratios were calculated and analyzed similarly to the other parameters measured at the end of the study. Absolute organ weight was analyzed by Analysis of Covariance (ANCOVA), with dose group as the main effect and body weight at the end of the study as the covariate (Bailey et al., 2004). Interpretation of changes in absolute organ weight, organ-to-body weight ratio, and organ-to-brain weight ratio in the evaluation of compound related effects was based on published analysis of control animal data (i.e., organ-tobody weight ratio: liver and thyroid; organ-to-brain weight ratio: adrenals and ovaries; ANCOVA: brain, heart, kidney, testes) (Bailey et al., 2004). Parameters measured multiple times during the study (i.e., body weight, food consumption) were analyzed using repeated measures ANOVA with sample day as the within subject factor and dose group as the between subject factor. If the interaction between within and between subject factors was significant, the effect of dose group on the parameter was determined within each sampling day using a one-factor ANOVA. Ordinal urinalysis data were coded for analysis by ANOVA. When significant main effects were observed (p < 0.05), appropriate post hoc tests were used to compare dose groups to the control group [e.g., Tukey's multiple comparison test, Dunnett's T3 (if variances are unequal), or Sidak (for ANCOVA)]. Data were evaluated for homogeneity of variance by Levene's test. The following hematology, clinical chemistry, and hormone parameters violated the assumptions of normality and were analyzed using nonparametric tests (I Mann-Whitney U): T3, TSH, NEU, %NEU, %LYM, MONO, %MONO, EOS, HGB, HCT, MCHC, RDW, ALT, AMYL, BUN, CREA, GLOB, TBIL, Na, Cl, PLT, Ca. 3. Results 3.1. Acute study Clinical signs (lethargy, rough coat, labored breathing, prostration, squinting, dark eyes, hunched posture, chromodacryorrhea, diarrhea, bloody urine, and red discharge from nose) of acute toxicity occurred in rats given single oral doses of potassium periodate of 560 mg/kg and greater and rats given doses of sodium periodate of 175 mg/kg and greater, appearing approximately fifteen to 30 min after dosing and persisted throughout the first day. Gross pathology observations included mottled kidneys with dark red medulla, enlarged kidneys (potassium periodate), pale medulla of kidneys, fluid filled intestines, bright red mucosa of the glandular stomach, vascular congestion in stomach and cecum (sodium periodate), focal grey and focal red areas in stomach (sodium periodate), thickened stomach lining (sodium periodate), viscous material in non-glandular stomach (sodium periodate), contents of intestines orange/red (sodium periodate), mottled liver (potassium periodate),

dark liver (sodium periodate), liver pale and firm (sodium periodate), focal pale and red areas with irregular edges on liver (sodium periodate), dark spleen, yellow fluid in throat, foamy white fluid in esophagus, red urine in bladder (potassium periodate), lungs grey and spongy (sodium periodate), and dilated vasculature of the heart and cecum (sodium periodate). The resulting LD50s were 732 mg/kg (95% CI: 539e838, slope: 13.4) and 685 mg/kg (95% CI: 580e809, slope: 10.6) for females and males, respectively, for potassium periodate. For sodium periodate, the LD50s were 318 mg/kg (95% CI: 292e347, slope: 24.3) and 741 mg/kg (95% CI: 704e779, slope: 31.2) for females and males, respectively. In the ALD, clinical signs of toxicity were noted in females given single oral doses of sodium periodate of 283 mg/kg and greater. The only mortality occurred at 1431 mg/kg. As such these data do not conform to the prescribed framework for defining an ALD. For the purposes of deriving a fasted versus fed adjustment factor, the ALD was defined as 1431 mg/kg in fed female rats. The resulting adjustment factor was four. 3.2. 14-Day study 3.2.1. Clinical observations and mortality Lethargy, squinting, congested breathing, prostration, hunched posture, rough coat, bloody bedding in cage, bloody urination, dried red material on front paws, brown perianal staining, diarrhea, red discharge from nose, and barbering were noted in female rats in the 318 mg/kg-day group and male rats in the 185, 370, and 741 mg/kgday groups. Five females in the 318 mg/kg-day sodium periodate group were terminated after 6e11 days of dosing due to moribund condition. All males in the 370 and 741 mg/kg-day sodium periodate groups died or were terminated due to moribund condition after 4e13 and 4e6 days, respectively. One male in the 185 mg/kg group was terminated due to moribund condition at day 13 (Table 1). 3.2.1.1. Body weight and food consumption. In female rats treated with sodium periodate, body weight generally increased throughout the study for all dose groups; however, body weight of females in the 318 mg/kg-day group was reduced (11.7%) compared to the control group at day 13 (p ¼ 0.028). In male rats treated with sodium periodate, body weight increased over time in the 46, 93, and 185 mg/kg-day groups, but decreased in the 370 and 741 mg/ kg-day groups (Table 2). Body weight was reduced, compared to the control, by 8.8% and 15.6% at day 3 in the 370 and 741 mg/kg-day groups (p < 0.001 and p < 0.001, respectively), and by 19.3% at day 7 Table 1 Summary of study design and mortality/morbidity for female and male rats orally dosed with sodium periodate for 14-days. Group

Females Control 20 mg/kg 40 mg/kg 80 mg/kg 159 mg/kg 318 mg/kg Males Control 46 mg/kg 93 mg/kg 185 mg/kg 370 mg/kg 741 mg/kg

N

Mortality

Early Sacrifice

N

Study Day (N)

N

Study Day (N)

10 10 10 10 10 10

0 0 0 0 0 0

na na na na na na

0 0 0 0 0 5

na na na na na 6, 9, 10, 11 (2)

10 10 10 10 10 10

0 0 0 0 2 4

na na na na 13 (2) 3, 5 (3)

0 0 0 1 8 6

13 4, 5, 8 (3), 10, 12, 13 4, 5(3), 6(2)

na ¼ not applicable; N ¼ number of animals.

E.M. Lent et al. / Regulatory Toxicology and Pharmacology 83 (2017) 23e37

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Table 2 Summary of body weight, food consumption, and food efficiency ratios for female and male rats orally dosed with sodium periodate for 14-days. Females

Control Mean

Body weight (g) Day 0 224.3 Day 1 226.6 Day 3 232.7 Day 7 240.2 Day 13 259.1 Food consumption (g) Days 0e1 38.32 Days 1e3 77.74 Days 3e7 150.42 Days 7e13 239.82 Total 506.30 Food efficiency ratio Days 0e1 0.12 Days 1e3 0.15 Days 3e7 0.10 Days 7e13 0.16 Total 0.14 Males

20 mg/kg

Mean

80 mg/kg

159 mg/kg

318 mg/kg

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

13.95 13.66 14.58 16.61 16.62

222.6 225.8 229.0 233.9 246.9

12.63 12.00 12.06 12.59 13.15

227.8 231.7 235.3 243.6 255.1

15.96 16.09 16.73 19.08 16.74

222.0 225.0 226.5 232.1 244.9

10.06 9.51 10.72 11.88 12.72

228.9 230.8 233.8 240.1 245.0

15.84 16.79 20.53 23.29 22.82

221.5 224.0 218.7 224.6 228.7*

10.08 10.21 13.91 10.39 12.93

3.65 5.50 15.32 22.58 42.82

38.10 73.86 142.82 219.84 474.62

3.27 6.82 8.31 12.54 24.01

39.10 76.82 153.64 232.62 502.18

2.37 6.84 9.73 16.85 31.16

35.74 72.62 139.14 220.32 467.82

3.51 3.27 6.07 11.64 18.31

35.06 72.58 143.64 205.86 457.14

5.01 9.29 15.92 31.33 55.61

29.60 56.16 116.23 177.85 249.88***

5.28 10.65 16.14 6.29 141.45

0.13 0.06 0.05 0.05 0.02

0.16 0.08 0.07 0.12 0.10

0.16 0.09 0.05 0.02 0.02

0.20 0.09 0.11 0.10 0.11

0.21 0.05 0.04 0.04 0.01

0.15 0.04 0.08 0.12 0.10*

0.28 0.07 0.02 0.02 0.02

0.09 0.07 0.09 0.04 0.07***

0.15 0.12 0.05 0.04 0.02

0.14 0.21 0.06 0.05 0.04***

0.31 0.16 0.09 0.04 0.01

Control

Body weight (g) Day 0 292.2 Day 1 297.9 Day 3 312.3 Day 7 338.3 Day 13 375.6 Food consumption (g) Days 0e1 49.96 Days 1e3 104.38 Days 3e7 211.04 Days 7e13 325.46 Total 690.84 Food efficiency ratio Days 0e1 0.23 Days 1e3 0.28 Days 3e7 0.25 Days 7e13 0.23 Total 0.24

40 mg/kg

SD

46 mg/kg

93 mg/kg

185 mg/kg

370 mg/kg

741 mg/kg

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

16.10 18.42 20.23 27.50 33.57

290.7 296.2 306.4 330.4 363.7

16.83 18.27 17.98 20.13 23.39

290.0 296.7 305.4 328.5 356.5

22.50 22.45 23.76 27.80 29.65

285.3 287.6 293.7 306.5 321.5**

12.64 14.90 23.04 31.59 42.70

287.6 289.3 284.9*** 273.1***

18.70 20.07 20.53 17.35

281.5 280.8 263.5***

12.93 12.66 14.59

3.49 7.07 14.82 17.34 40.74

47.92 97.68 201.10 306.54 653.24

4.84 5.58 17.73 29.16 54.57

47.08 97.42 199.00 294.84 638.34

4.36 7.54 20.70 25.44 57.12

40.72 81.72 163.28 225.84 511.56*

8.23 18.91 39.03 63.87 125.79

33.94 61.26 115.17

1.64 9.84 12.23

27.82 41.93

4.20 9.96

0.10 0.04 0.02 0.02 0.01

0.22 0.21 0.24 0.22 0.22

0.09 0.05 0.03 0.02 0.02

0.29 0.18 0.23 0.19 0.21

0.12 0.05 0.02 0.01 0.01

0.09 0.11 0.13 0.11 0.12*

0.16 0.21 0.14 0.12 0.12

0.10 0.16 0.18

0.22 0.18 0.21

0.09 0.77

0.29 0.14

SD ¼ standard deviation; *Significantly different from control at p < 0.05; ** Significantly different from control at p < 0.01; *** Significantly different from control at p < 0.001.

in the 370 mg/kg-day group (p < 0.001). In the 185 mg/kg-day group, body weight was reduced 14.4%, compared to the control, at day 13 (p ¼ 0.006). Food consumption and food efficiency ratios did not differ between treated and control groups for either males or females when measured at time points within the study (i.e., measured on days 1, 3, 7, and 13). Total food consumption, however, was reduced, relative to the control, in females given 318 mg/kg-day sodium periodate (p < 0.001). In males, total food consumption was reduced in the 185 mg/kg-day group (p ¼ 0.013). Net food efficiency ratios (i.e., 14-day) were reduced in female rats given 80, 159, and 318 mg/kg-day sodium periodate (p ¼ 0.041, p < 0.001, p < 0.001, respectively) (Table 2). In male rats, net food efficiency ratios were reduced in the 185 mg/kg-day group (p ¼ 0.028) (Table 2).

3.2.1.2. Urinalysis. Urine volume and water intake tended to increase with dose while specific gravity decreased. In females, urine volume was increased and urine specific gravity decreased in the 159 mg/kg-day sodium periodate group (p ¼ 0.043 and p ¼ 0.012, respectively). Water intake was generally increased in the 80, 159, and 318 mg/kg-day groups; however, these increases were not significant. Urine pH was increased in females in the 80 and 159 mg/kg-day group (p < 0.001 and p < 0.001, respectively). In the 318 mg/kg-day group, increased amounts of blood were found in

the urine (p < 0.001). The remaining urine parameters: color, appearance, glucose (negative), bilirubin (negative), ketone (negative), protein (negative-30 mg/dl), urobilinogen (0.2 mg/dl), nitrites (negative), and leucocytes (negative) did not differ between sodium periodate treated and control groups for females. In males, the measured urine parameters were unaffected by sodium periodate treatment in the dose groups that survived to the time of urinalysis. Water intake and urine volume were, however, slightly increased, relative to the control, in the 185 mg/kg-day group as was observed in females. Blood was also noted in the urine of several males in the 185 mg/kg-day group. 3.2.1.3. Pathology. Gross pathology findings included: red areas in the stomach, raised white areas and cysts-like areas in the glandular stomach, sloughing of the lining of the stomach, clear mucous in the cecum and/or yellow liquid in the gastrointestinal tract, red mesenteric lymph nodes, red outer rim of medulla of kidney, pale and/or dark kidney, mottled and/or dark liver, and fluid-filled cyst surrounding adrenal gland. 3.2.1.4. Organ weight and ratios. Liver-to-body weight ratios were increased in females in the 159 and 318 mg/kg-day groups (p ¼ 0.001 and p < 0.001, respectively; Table 3). Spleen-to-body weight and spleen-to-brain weight ratios were reduced in

28

E.M. Lent et al. / Regulatory Toxicology and Pharmacology 83 (2017) 23e37

females in the 318 mg/kg-day group (p ¼ 0.042 and p ¼ 0.004, respectively) and males in the 370 (p < 0.001 and p < 0.001, respectively) and 741 mg/kg-day groups (p ¼ 0.001 and p < 0.001, respectively; Table 4). Mean absolute thymus weight was decreased, relative to the control, in females in the 318 mg/kg-day group (p ¼ 0.040, respectively). In males in the 185, 370, and 741 mg/kg-day groups, mean absolute thymus weight (p ¼ 0.001, p < 0.001, and p < 0.001, respectively), thymus-to-body weight ratios (p ¼ 0.009, p < 0.001, and p < 0.001, respectively), and thymus-to-brain weight ratios (p ¼ 0.001, p < 0.001, and p < 0.001, respectively) were decreased compared to controls. Ovary-to-brain weight ratio was reduced in the 318 mg/kg-day group (p ¼ 0.005). Mean absolute weight of the epididymides was reduced in the 370 and 741 mg/kg-day groups (p < 0.001 and p < 0.001, respectively). 3.2.1.5. Histopathology. Treatment-related changes were observed in the thymus, spleen, kidneys, stomach, intestines, epididymides, and testes (Tables 5 and 6). Treatment-related changes in the thymus consisted of atrophy, which was characterized by loss of cortical lymphocytes and apoptosis. Atrophy of the thymus was noted in all groups, including the control groups. The incidence and severity increased with dose and the minimal thymic atrophy in the control groups was interpreted to be consistent with aging and involution. Treatmentrelated changes in the spleen consisted of white pulp atrophy, which manifested as loss of lymphocytes in splenic nodules, and red pulp atrophy or loss of extramedullary hematopoietic elements (red and white cell precursors) and contracted sinuses. These histologic changes correlated with gross notations of small thymus or spleen. In the kidneys, acute tubular necrosis was noted in females in the 318 mg/kg-day group and in males in the 185, 370, and 741 mg/ kg-day groups. Accumulation of eosinophilic hyaline droplets

within renal tubular epithelium was noted in females in the 318 mg/kg-day group and in males in the 741 mg/kg-day group. Mineralization of necrotic cells was also noted in sodium periodate treated females and males. Scattered foci of mineralization, which is occasionally seen in untreated rats, were present in one female control. These changes corresponded to gross notations of kidney discoloration (red/brown/pale). In the stomach, treatment-related changes in females in the 318 mg/kg-day group and males in the 185, 370, and 741 mg/kg-day groups consisted of mucosal (epithelial cell) ulcer/erosion in the forestomach, areas of necrosis in the glandular stomach, hemorrhage and inflammation. These changes corresponded to gross notations of the stomach e blood, cyst, nodule, or raised area. Areas of inflammation, mononuclear cell infiltrate, and hemorrhage were noted in large intestinal sections from females in the 318 mg/kg-day group and from males in the 185 and 741 mg/kg-day groups. In females, mean follicle height score was 1.8, 2.0, 1.8, 1.7, 1.7, and 1.4 in control, 20, 40, 80, 159, and 318 mg/kg-day groups, respectively. In males, mean follicle height score was 2.4, 2.2, 2.4, 2.0, 1.9, and 1.8 in control, 46, 93, 185, 370, and 741 mg/kg-day groups, respectively. Follicle score did not differ between treated and control groups. Males in the 185, 370, and 741 mg/kg-day groups had minimal to mild alterations of their testes. Morphologically, this consisted of seminiferous tubules containing fewer germinal epithelial cells, often leaving a larger and empty tubular lumen with fewer mature spermatozoa. A similar morphologic appearance was noted in one control male and two males in the 46 mg/kg-day group. Therefore, increased incidence and severity compared to controls were interpreted as treatment-related changes. Epididymal granulomas were noted in one male in each of the 185 and 741 mg/kg-day groups.

Table 3 Absolute and relative organ weight for female rats orally dosed with sodium periodate for 14-days. Females

Adrenals Brain Heart Kidneys liver Ovaries Spleen Thymus Thyroid Uterus Adrenals-to-body weight Brain-to-body weight Heart-to-body weight Kidneys-to-body weight Liver-to-body weight Ovaries-to-body weight Spleen-to-body weight Thymus-to-body weight Thyroid-to-body weight Uterus-to-body weight Adrenals-brain weight Heart-to-brain weight Kidneys-to-brain weight Liver-to-brain weight Ovaries-to-brain weight Spleen-to-brain weight Thymus-to-brain weight Thyroid-to-brain weight Uterus-to-brain weight

Control

20 mg/kg-day

40 mg/kg-day

80 mg/kg-day

159 mg/kg-day

318 mg/kg-day^

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

0.08 1.88 1.02 1.82 8.24 0.15 0.55 0.42 0.01 0.52 0.0003 0.0078 0.0042 0.0075 0.0339 0.0006 0.0023 0.0017 0.0000 0.0022 0.041 0.543 0.966 4.381 0.081 0.295 0.227 0.006 0.278

0.01 0.07 0.10 0.13 0.94 0.02 0.07 0.15 0.00 0.15 0.0000 0.0005 0.0002 0.0004 0.0022 0.0001 0.0003 0.0005 0.0000 0.0007 0.005 0.058 0.065 0.534 0.011 0.038 0.081 0.002 0.082

0.07 1.91 1.00 1.86 8.01 0.15 0.55 0.48 0.01 0.54 0.0003 0.0081 0.0043 0.0079 0.0341 0.0006 0.0024 0.0020 0.0000 0.0023 0.039 0.524 0.974 4.200 0.076 0.289 0.251 0.006 0.282

0.01 0.08 0.05 0.23 0.79 0.02 0.06 0.11 0.00 0.11 0.0000 0.0005 0.0003 0.0008 0.0024 0.0001 0.0003 0.0004 0.0000 0.0005 0.005 0.043 0.109 0.390 0.011 0.030 0.050 0.001 0.052

0.08 1.87 1.02 1.85 8.82 0.15 0.54 0.54 0.01 0.49 0.0003 0.0078 0.0042 0.0077 0.0364 0.0006 0.0023 0.0020 0.0001 0.0020 0.044 0.546 0.990 4.714 0.081 0.290 0.258 0.007 0.262

0.01 0.07 0.10 0.13 1.12 0.02 0.08 0.10 0.00 0.10 0.0000 0.0006 0.0003 0.0004 0.0030 0.0001 0.0003 0.0008 0.0000 0.0004 0.003 0.054 0.059 0.599 0.008 0.042 0.102 0.002 0.051

0.08 1.92 1.02 1.77 8.64 0.14 0.52 0.48 0.01 0.51 0.0003 0.0084 0.0044 0.0077 0.0375 0.0006 0.0023 0.0021 0.0001 0.0022 0.040 0.533 0.924 4.501 0.073 0.274 0.251 0.007 0.264

0.01 0.06 0.08 0.12 0.45 0.01 0.07 0.13 0.00 0.09 0.0000 0.0005 0.0004 0.0004 0.0020 0.0001 0.0003 0.0005 0.0000 0.0005 0.005 0.051 0.075 0.309 0.007 0.040 0.067 0.002 0.043

0.07 1.88 0.96 1.76 9.18 0.15 0.57 0.45 0.01 0.51 0.0003 0.0082 0.0042 0.0076 0.0397** 0.0006 0.0025 0.0019 0.0001 0.0022 0.038 0.513 0.935 4.872 0.078 0.303 0.241 0.007 0.270

0.01 0.07 0.10 0.19 1.48 0.02 0.08 0.16 0.00 0.13 0.0000 0.0008 0.0003 0.0004 0.0042 0.0001 0.0002 0.0006 0.0000 0.0007 0.005 0.058 0.109 0.760 0.011 0.046 0.091 0.002 0.064

0.09 1.84 0.85* 1.60* 9.06 0.12* 0.41* 0.25* 0.01 0.56 0.0004* 0.0086 0.0039 0.0074 0.0421*** 0.0005 0.0019* 0.0012 0.0001 0.0025 0.048 0.462* 0.871 4.928 0.064** 0.224* 0.139 0.006 0.296

0.01 0.12 0.11 0.14 0.68 0.02 0.08 0.08 0.00 0.46 0.0000 0.0006 0.0004 0.0005 0.0031 0.0001 0.0003 0.0003 0.0000 0.0019 0.009 0.064 0.117 0.374 0.011 0.048 0.048 0.002 0.219

*Significantly different from control at p < 0.05; ** Significantly different from control at p < 0.01; *** Significantly different from control at p < 0.001; ^Data from animals dosed less than 14-days due to mortality and moribund sacrifices (see Table 1).

E.M. Lent et al. / Regulatory Toxicology and Pharmacology 83 (2017) 23e37

29

Table 4 Absolute and relative organ weight for male rats orally dosed with sodium periodate for 14-days. Males

Adrenals Brain Heart Kidneys Epididymides Liver Spleen Testes Thymus Thyroid Adrenals-to-body weight Brain-to-body weight Heart-to-body weight Kidneys-to-body weight Epididymides-to-body weight Liver-to-body weight Spleen-to-body weight Testes-to-body weight Thymus-to-body weight Thyroid-to-body weight Adrenals-to-brain weight heart-to-brain weight Kidneys-to-brain weight Epididymides-to-brain weight Liver-to-brain weight Spleen-to-brain weight Testes-to-brain weight Thymus-to-brain weight Thyroid-to-brain weight

Control

46 mg/kg-day

93 mg/kg-day

185 mg/kg-day

370 mg/kg-day^

741 mg/kg-day^

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

0.07 2.04 1.50 2.73 0.96 13.41 0.82 3.24 0.57 0.01 0.0002 0.0058 0.0043 0.0078 0.0027 0.0380 0.0023 0.0092 0.0016 0.0000 0.033 0.738 1.337 0.475 6.577 0.401 1.589 0.276 0.007

0.01 0.17 0.13 0.28 0.07 1.79 0.15 0.30 0.17 0.00 0.0000 0.0005 0.0001 0.0005 0.0003 0.0022 0.0003 0.0009 0.0004 0.0000 0.007 0.052 0.076 0.065 0.746 0.060 0.129 0.065 0.002

0.07 1.99 1.31 2.66 0.91 12.86 0.76 3.20 0.49 0.02 0.0002 0.0059 0.0038 0.0078 0.0027 0.0376 0.0022 0.0094 0.0015 0.0000 0.034 0.655 1.335 0.458 6.449 0.382 1.611 0.248 0.008

0.01 0.12 0.13 0.31 0.09 1.74 0.14 0.21 0.11 0.00 0.0000 0.0003 0.0003 0.0005 0.0003 0.0034 0.0004 0.0010 0.0003 0.0000 0.006 0.052 0.150 0.055 0.725 0.073 0.148 0.056 0.001

0.06 2.02 1.36 2.72 0.97 13.12 0.78 3.20 0.48 0.01 0.0002 0.0060 0.0040 0.0080 0.0029 0.0388 0.0023 0.0095 0.0014 0.0000 0.030 0.671 1.344 0.480 6.504 0.386 1.585 0.237 0.007

0.01 0.12 0.14 0.31 0.09 1.72 0.18 0.18 0.10 0.00 0.0000 0.0004 0.0002 0.0005 0.0003 0.0033 0.0004 0.0006 0.0002 0.0000 0.005 0.056 0.140 0.054 0.914 0.089 0.079 0.045 0.001

0.07 1.95 1.11* 2.49 0.87 11.53 0.62* 3.14 0.34** 0.01 0.0002 0.0065 0.0036 0.0083 0.0029 0.0377 0.0020 0.0104 0.00108** 0.0000 0.035 0.569* 1.281 0.449 5.915 0.319 1.611 0.174** 0.007

0.01 0.05 0.23 0.31 0.13 2.51 0.18 0.22 0.14 0.00 0.0000 0.0009 0.0005 0.0014 0.0003 0.0049 0.0005 0.0013 0.0004 0.0000 0.007 0.120 0.169 0.066 1.296 0.095 0.130 0.073 0.002

0.07 1.91 1.14* 2.36 0.69*** 11.28 0.38* 3.33 0.22*** 0.01 0.0003* 0.0071* 0.0042 0.0087 0.0026 0.0417 0.0014*** 0.0123* 0.0008*** 0.0001 0.039 0.597 1.230 0.363* 5.901 0.198*** 1.745 0.117*** 0.007

0.02 0.08 0.26 0.68 0.07 2.22 0.08 0.26 0.08 0.00 0.0001 0.0005 0.0008 0.0025 0.0004 0.0077 0.0003 0.0010 0.0003 0.0000 0.008 0.147 0.315 0.034 1.066 0.036 0.158 0.039 0.001

0.06 1.87* 1.32 2.05* 0.62*** 10.69* 0.38* 3.10 0.22*** 0.01 0.0002 0.0075* 0.0053* 0.0082 0.0025 0.0425 0.0015** 0.0124* 0.0008*** 0.0001 0.032 0.707 1.098 0.330* 5.725 0.205*** 1.656 0.115*** 0.007

0.01 0.07 0.30 0.18 0.07 1.01 0.07 0.27 0.06 0.00 0.0000 0.0007 0.0012 0.0011 0.0005 0.0035 0.0002 0.0021 0.0002 0.0000 0.005 0.157 0.103 0.040 0.603 0.038 0.140 0.032 0.002

*Significantly different from control at p < 0.05; ** Significantly different from control at p < 0.01; *** Significantly different from control at p < 0.001; ^Data from animals dosed less than 14-days due to mortality and moribund sacrifices (see Table 1).

Other incidental changes noted in organs examined were interpreted to be background or not of biologic or toxicologic relevance.

p ¼ 0.027, respectively). Creatinine (CREA) levels were increased relative to the control group only in males in the 370 mg/kg-day group (p ¼ 0.033).

3.2.1.6. Clinical chemistry. Table 7 shows the clinical chemistry parameters in female and male rats. Cholesterol levels increased in a dose dependent manner in sodium periodate treatment groups in males and females. Cholesterol levels were increased, compared to the controls, in females in the 40, 80, 159, and 318 mg/kg-day groups (p ¼ 0.017, p ¼ 0.003, p ¼ 0.001, and p < 0.001, respectively) and males in the 46, 93, 185, 370, and 741 mg/kg-day groups (p ¼ 0.037, p ¼ 0.006, p ¼ 0.001, p ¼ 0.013, and p ¼ 0.002, respectively). Alkaline phosphatase (ALKP) levels were reduced, relative to the control group, in females in the 80, 159, and 318 mg/kg-day groups (p ¼ 0.037, p ¼ 0.045, and p ¼ 0.002, respectively) and in males in the 185 mg/kg-day group (p ¼ 0.008), but were elevated in males in the 741 mg/kg-day group (p ¼ 0.032). Alanine aminotransferase (ALT) levels were elevated in females in the 318 mg/kg-day group (p ¼ 0.005) and in males in the 370 mg/kg-day group (p ¼ 0.045). Amylase (AMYL) levels were elevated females in the 318 mg/kg-day group (p ¼ 0.014) but were decreased in males in the 185 mg/kgday group (p ¼ 0.013). Females demonstrated a slight decrease only in serum chloride levels in the 318 mg/kg-day group (p ¼ 0.008). While in males, serum sodium and chloride levels were decreased in the 370 (p ¼ 0.007 and p ¼ 0.002, respectively) and 741 mg/kg-day (p ¼ 0.037 and p ¼ 0.004, respectively) groups, and potassium levels were slightly decreased in all sodium periodate treatment groups (p ¼ 0.010, p ¼ 0.140, p ¼ 0.004, p ¼ 0.008, and p ¼ 0.016). Blood urea nitrogen (BUN) was elevated relative to the control in males in the 370 and 741 mg/kg-day groups (p ¼ 0.001, and

3.2.1.7. Hematology. Table 8 shows the hematology parameters in female and male rats. Neutrophil count was increased, relative to the control group, in females in the 318 mg/kg-day group (p < 0.001) and males in the 185, 370, and 741 mg/kg-day groups (p ¼ 0.034, p ¼ 0.001, and p ¼ 0.007, respectively). Lymphocyte counts were decreased, relative to the control group, in females in the 318 mg/kg-day group (p < 0.001) and males in the 185, 370, and 741 mg/kg-day groups (p ¼ 0.041, p ¼ 0.001, and p ¼ 0.002, respectively). Eosinophil count was decreased relative to the control group in females in the 318 mg/kg-day group (p ¼ 0.012). Red blood cell counts (RBC), hemoglobin (HGB), and percent hematocrit (HCT) were slightly reduced relative to the control in males in the 185 mg/kg-day group (p ¼ 0.002, p < 0.001, and p < 0.001, respectively). Additionally, RBC was reduced in the 93 mg/kg-day dose group (p ¼ 0.041). Mean cell hemoglobin concentration (MCHC) was slightly elevated in males in the 370 and 741 mg/kg-day groups (p ¼ 0.043 and p ¼ 0.003, respectively). In males in the 741 mg/kg-day group, mean cell volume (MCV) was slightly reduced (p ¼ 0.027), and red cell distribution width (RDW) and mean platelet volume (MPV) were slightly increased (p ¼ 0.020 and p ¼ 0.020, respectively). Red cell distribution width (RDW) was slightly increased in females in the 318 mg/kg-day group (p ¼ 0.001). 3.2.1.8. Thyroid hormone analyses. Serum T4 and TSH levels did not differ between the control group and sodium periodate treatment groups in female rats. Serum T3 levels were decreased compared to

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E.M. Lent et al. / Regulatory Toxicology and Pharmacology 83 (2017) 23e37

Table 5 Incidence summary of microscopic observations with average Grade score for female rats orally gavaged with sodium periodate for 14-days. Number Observed Per Group Tissue/Observation

Group (mg/kg-day)

0

20

40

80

159

318

Adrenal Glands

Number Examined:

10

10

10

10

10

10

Brain

Number Examined:

10

10

10

10

10

10

Heart Cardiomyopathy

Number Examined:

10 0 0.0

10 0 0.0

10 0 0.0

10 0 0.0

10 0 0.0

10 1 1.0

10 0 0.0 0 0.0

10 0 0.0 0 0.0

10 0 0.0 0 0.0

10 0 0.0 0 0.0

10 0 0.0 0 0.0

10 1 1.0 1 2.0

10 0 0.0

10 0 0.0

10 0 0.0

10 1 2.0

10 0 0.0

10 1 2.0

10 0 0.0 0 0.0 1 1.0 0 0.0 0 0.0

10 0 0.0 0 0.0 4 1.0 0 0.0 0 0.0

10 0 0.0 0 0.0 3 1.0 0 0.0 2 1.0

10 0 0.0 0 0.0 1 1.0 0 0.0 1 1.0

10 0 0.0 1 1.0 2 1.0 0 0.0 1 1.0

10 2 1.5 0 0.0 0 0.0 3 1.7 2 1.0

10 9 1.0 0 0.0 2 1.0

10 6 1.0 0 0.0 0 0.0

10 7 1.0 0 0.0 2 1.0

10 6 1.0 0 0.0 0 0.0

10 6 1.0 0 0.0 0 0.0

10 4 1.0 1 1.0 0 0.0

0 0 0.0 0 0.0

0 0 0.0 0 0.0

0 0 0.0 0 0.0

0 0 0.0 0 0.0

1 1 2.0 1 3.0

Average Grade: Intestine, Large Inflammation

Number Examined: Average Grade:

Mononuclear cell infiltrate, muscle wall Average Grade: Intestine, Small Mineralization, peyer's patch

Number Examined:

Kidneys Accumulation Hyaline Droplets

Number Examined:

Average Grade:

Average Grade: Dilated pelvis Average Grade: Mineralization Average Grade: Necrosis Average Grade: Nephropathy Average Grade: Liver Mononuclear cell infiltrate

Number Examined: Average Grade:

Necrosis Average Grade: Vacuolization Cytoplasm Average Grade: Lymph Nodes, Mesenteric Hemorrhage

Number Examined:

Average Grade:

0 0 0.0 0 0.0

Ovaries

Number Examined:

10

10

10

10

10

10

Spleen Deformity

Number Examined:

10 1 2.0 0 0.0 0 0.0

10 0 0.0 0 0.0 0 0.0

10 0 0.0 0 0.0 1 2.0

10 0 0.0 0 0.0 0 0.0

10 0 0.0 0 0.0 0 0.0

10 0 0.0 4 2.0 6 1.8

10 0 0.0 0 0.0 2 1.0 0 0.0 0 0.0

10 0 0.0 0 0.0 8 1.0 0 0.0 0 0.0

10 0 0.0 0 0.0 3 1.0 0 0.0 0 0.0

10 0 0.0 0 0.0 4 1.0 0 0.0 0 0.0

10 0 0.0 0 0.0 3 1.0 0 0.0 0 0.0

10 1 1.0 2 2.0 1 1.0 2 1.0 2 1.0

10 2 1.0 0 0.0

9 1 1.0 0 0.0

10 2 1.0 0 0.0

10 3 1.0 1 1.0

10 9 2.2 0 0.0

10

10

10

10

10

Average Grade: Infiltrate, histiocyte

Average Grade: Red pulp atrophy Average Grade: White pulp atrophy Average Grade: Stomach Erosion/ulcer, forestomach

Number Examined: Average Grade:

Hemorrhage Average Grade: Infiltrate, eosinophil Average Grade: Inflammation Average Grade: Necrosis, glandular stomach Average Grade: Thymus Atrophy

Number Examined:

Average Grade:

10 2 1.0 0 0.0

Thyroid Glands Follicular cell height

Number Examined:

10

Average Score:

1.8

2.0

1.8

1.7

1.7

1.4

Uterus Dilation

Number Examined:

10 4 1.0 0 0.0

10 4 1.0 1 1.0

10 3 1.0 1 1.0

10 2 1.0 2 1.0

10 4 1.0 1 1.0

10 3 1.0 0 0.0

Average Grade: Epithelial cell hyperplasia

Average Grade: Endometrial hyperplasia Average Grade:

E.M. Lent et al. / Regulatory Toxicology and Pharmacology 83 (2017) 23e37

the control in females in the 318 mg/kg-day group (p ¼ 0.002). Serum T4, T3, and TSH levels were decreased compared to the control in males in the 370 (p ¼ 0.001, p ¼ 0.005, and p ¼ 0.016, respectively) and 741 mg/kg-day groups (p ¼ 0.002, p ¼ 0.011, and p ¼ 0.005, respectively). Serum T3 levels were also reduced in the 185 mg/kg-day group (p ¼ 0.015) (Fig. 1). 3.2.1.9. Determination of BMD and BMDL10. Increased serum cholesterol was identified as the critical endpoint in this study based on the dose-related response in males and females as well as the association of this indicator of kidney toxicity and uremia (Keane et al., 2013; Moestrup and Nielsen, 2005). Hyperlipidemia associated with kidney toxicity and uremia may involve impairment of the lipid clearance capacity of the kidneys (Moestrup and Nielsen, 2005) and/or lipoprotein and hepatic lipase deficiencies mediated by hyperparathyroidism (Kraemer et al., 1982; Sato et al., 2002; Vaziri and Liang, 1996; Vaziri et al., 1997). Histopathologic evidence of kidney toxicity (i.e., necrosis) and other serum (e.g., increased BUN and CREA) and urine (e.g., urine volume) markers of uremia were present in high dose groups. These endpoints were not modeled as they did not demonstrate a clear dose-response and the effects were only evident in higher dose groups (Barnes and Dourson, 1988; USEPA, 2002b). Benchmark Dose Software (BMDS v.2.5) was used to fit mathematical models to the serum cholesterol data for females and males separately and calculate a lower-bound confidence limit on a dose corresponding to a 10 percent response rate (BMDL10) (USEPA, 1995; USEPA, 2012). The exponential 4, exponential 5, and Hill models were selected based on goodnessof-fit and statistical parameters (p > 0.1, lowest AIC values and residuals). Mean BMDs of 33.3 and 55.2 mg/kg-day were calculated for females and males, respectively, based on the results of these three models. This corresponded to a BMDL10 of 17.2 and 33.7 mg/ kg-day for females and males, respectively. 4. Discussion Sodium and potassium periodate have been identified as potential replacements for perchlorate for use as oxidizers in military pyrotechnic devices (Ball, 2012; Fields, 2012; Moretti et al., 2012). The use of perchlorate has been associated with environmental impacts and health hazards including thyroid dysfunction and developmental abnormalities. Based on structure activity relationships, munitions developers hypothesized sodium and potassium periodate to be less toxic than the oxidizers currently in use. The main objective of this study was to investigate the acute and subacute oral toxicity of sodium and potassium periodate in rats. Clinical signs of toxicity were observed in rats at doses of 560 mg/kg and greater of potassium periodate and at doses of 175 mg/kg and greater of sodium periodate in the acute phase of the study. These signs were similar to those observed following acute intraperitoneal or oral administration of potassium and sodium iodates (Webster et al., 1957). However, convulsions and paresis noted with iodates were not present with periodate salts. These clinical signs may have been cation-associated effects as the authors indicated that toxicity of the salts was apparent at higher doses. Oral doses of potassium large enough to overwhelm the renal excretory mechanisms can produce potassium toxicity which manifests with characteristic acute cardiovascular changes, general weakness, ascending paralysis, gastroenteritis, vomiting, paralytic ileus, local mucosal necrosis and potential perforation, polydipsia/ polyuria, early renal tubular necrosis, and convulsions (Saxena, 1989). The LD50s for the two periodate salts were very similar in males (685 and 741 mg/kg for potassium and sodium periodate, respectively) and in females for potassium periodate (732 mg/kg).

31

This suggests that the toxicity of these compounds was largely due to the periodate ion. In females, however, the LD50 for sodium periodate was considerably lower than that of males and that of potassium periodate. As female sensitivity to sodium periodate was not observed in the subacute study (i.e., toxicity was observed in males and females at approximately equivalent doses), female sensitivity in the acute study may have been due to their prandial state. Because periodate is reduced to iodate and subsequently to the less toxic iodide (Anghileri, 1965; Taurog et al., 1966), the presence of food in the intestinal tract to serve as a reducing substrate may decrease the toxicity of periodate and iodate when exposure occurs orally (Webster et al., 1957). Additionally, iodate associated increases in stomach pH are lessened in the presence of food (Webster et al., 1957). Although both males and females were fasted prior to the acute study, overnight fasting is typically more effective in emptying the gastric contents in female than male rats. This reduction in toxicity in the presence of food was apparent in the difference in sodium periodate toxicity between fasted rats (LD50 318 mg/kg) and fed rats (ALD 1431 mg/kg). However, a similar gender-related difference in toxicity would be expected in the potassium periodate acute study if the higher acute toxicity of sodium periodate in females were due to the periodate ion. Although the oral toxicity of iodates and periodates is reduced in the presence of food and the toxicity of iodates administered in drinking water (i.e., in divided doses) is greatly reduced (Webster et al., 1959), there is a point beyond which food cannot offer protection. Repeated oral exposure to sodium periodate resulted in mortalities in females at 318 mg/kg-day after 8 doses and in males at 185, 370 and 741 mg/kg-day after 13, 8, and 4 doses, respectively. As with iodate salts, these mortalities were likely attributable to kidney toxicity, uremia, and associated secondary effects (Webster et al., 1957). Subacute exposure to sodium periodate resulted in reduced body weight in female and male rats in the highest dose groups (80, 159, and 318 mg/kg-day and 185, 370, and 741 mg/kgday, respectively). The reduced body weight may have been due in part to hypophagia as total food consumption was reduced in the high dose females and the 185 mg/kg-day males. However, food conversion efficiency was also reduced in female and male dose groups exhibiting reduced body weight. The reduced food conversion efficiency was likely due to the gastrointestinal effects associated with sodium periodate exposure. In mice orally exposed to iodate salts, damage to parietal cells, suppression of hydrochloric acid secretion, increased alkaline mucous secretion, increased stomach pH, and delayed gastric emptying were observed. Fatty visceral changes in iodate exposed mice were attributed to reduced nourishment due to anorexia or delayed gastric emptying (Webster et al., 1957). Similar effects were observed with periodates such that thick mucus secretions were often noted in the stomach and the intestinal tracts were frequently empty or contained only yellow fluid. Additionally, histopathology demonstrated epithelial cell ulcer/erosion, necrosis, hemorrhage, and inflammation in the stomach and inflammation and hemorrhage in the small and large intestines of exposed rats, suggesting malabsorption may have contributed to the reduced food efficiency ratios. Subacute exposure to sodium periodate resulted in decreased weight of the thymus and spleen, thymocyte depletion in the thymus and spleen, altered leukocyte differentials, and possible altered reproductive function (decreased weight of the epididymides). Whether these effects are due solely to stress, are a secondary stress response to primary test-article related effects, or are direct test article effects is difficult to discern (Herzyk and Bussiere, 2008). Leukocyte differentials and weight of the adrenals and thymus are generally considered the most sensitive indicators of stress (Everds et al., 2013). Both females and males exhibited a

32

E.M. Lent et al. / Regulatory Toxicology and Pharmacology 83 (2017) 23e37

Table 6 Incidence summary of microscopic observations with average Grade score for male rats orally dosed with sodium periodate for 14-days. Number Observed Per Group Tissue/Observation

Group (mg/kg-day)

0

46

93

185

370

741

Adrenal Glands Hyperplasia

Number Examined:

10 0 0.0 0 0.0

10 0 0.0 1 2.0

10 0 0.0 0 0.0

10 1 1.0 1 2.0

9 0 0.0 0 0.0

8 0 0.0 0 0.0

10 0 0.0

10 0 0.0

10 0 0.0

10 0 0.0

9 0 0.0

8 1 2.0

10 0 0.0 0 0.0

10 0 0.0 0 0.0

10 0 0.0 0 0.0

10 1 4.0 1 3.0

9 0 0.0 0 0.0

8 1 2.0 0 0.0

10 1 1.0

10 0 0.0

10 0 0.0

10 1 1.0

9 0 0.0

8 0 0.0

10 0 0.0 0 0.0 0 0.0

10 0 0.0 0 0.0 0 0.0

10 0 0.0 0 0.0 0 0.0

10 0 0.0 1 1.0 1 2.0

9 0 0.0 0 0.0 0 0.0

8 1 1.0 1 1.0 0 0.0

10 1 1.0 0 0.0

10 0 0.0 0 0.0

10 0 0.0 0 0.0

10 0 0.0 1 2.0

9 0 0.0 0 0.0

8 0 0.0 0 0.0

10 0 0.0 0 0.0 0 0.0 0 0.0 1 1.0 0 0.0 0 0.0 4 1.0

10 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 6 1.0

10 0 0.0 1 1.0 1 1.0 0 0.0 0 0.0 0 0.0 0 0.0 6 1.0

10 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 1 1.0 1 3.0 7 1.0

9 0 0.0 0 0.0 0 0.0 1 1.0 0 0.0 3 1.7 7 2.0 3 1.0

8 2 2.0 0 0.0 0 0.0 0 0.0 0 0.0 3 1.0 6 1.5 1 1.0

10 10 1.0 0 0.0 10 1.0

10 9 1.0 1 1.0 6 1.0

10 9 1.0 0 0.0 8 1.0

10 7 1.0 0 0.0 0 0.0

9 2 1.5 1 4.0 2 1.5

8 2 1.0 1 2.0 0 0.0

0 0 0 0 0

0 0 0 0 0

0 0 0 0 0

0 0 0 0 0

4 2 2 4 3.5

6 4 1.3 6 3.5

10 1 2.0 0 0.0 0 0.0

10 0 0.0 0 0.0 1 1.0

10 0 0.0 0 0.0 0 0.0

10 1 2.0 1 3.0 3 1.7

9 0 0.0 8 1.5 8 2.0

8 0 0.0 8 2.0 8 2.5

10 0 0.0

10 0 0.0

10 0 0.0

10 1 2.0

9 2 2.5

8 2 3.0

Average Grade: Vacuolization, zona fasciculata Average Grade: Brain Hemorrhage

Number Examined:

Epididymis Granuloma

Number Examined:

Average Grade:

Average Grade: Hypospermia Average Grade: Heart Cardiomyopathy

Number Examined:

Intestine, Large Hemorrhage

Number Examined:

Average Grade:

Average Grade: Inflammation Average Grade: Mononuclear cell infiltrate, muscle wall Average Grade: Intestine, Small Granuloma, peyer's patch

Number Examined: Average Grade:

Inflammation Average Grade: Kidneys Accumulation Hyaline Droplets

Number Examined: Average Grade:

Dilatation Average Grade: Dilated pelvis Average Grade: Inflammation Average Grade: Lymphocytic infiltrate Average Grade: Mineralization Average Grade: Necrosis Average Grade: Nephropathy Average Grade: Liver Mononuclear cell infiltrate

Number Examined: Average Grade:

Necrosis Average Grade: Vacuolization Cytoplasm Average Grade: Lymph Nodes, Mesenteric Atrophy

Number Examined: Average Grade:

Hemorrhage Average Grade: Spleen Extramedullary hematopoiesis, increased

Number Examined: Average Grade:

Red pulp atrophy Average Grade: White pulp atrophy Average Grade: Stomach Erosion/ulcer, forestomach

Number Examined: Average Grade:

E.M. Lent et al. / Regulatory Toxicology and Pharmacology 83 (2017) 23e37

33

Table 6 (continued ) Number Observed Per Group Tissue/Observation

Group (mg/kg-day)

Hemorrhage Average Grade: Infiltrate, eosinophil Average Grade: Inflammation Average Grade: Necrosis, glandular stomach Average Grade:

0

46

93

185

370

741

0 0.0 1 1.0 0 0.0 0 0.0

0 0.0 2 1.0 0 0.0 0 0.0

0 0.0 2 1.0 0 0.0 0 0.0

0 0.0 0 0.0 1 3.0 1 1.0

3 1.0 2 1.0 4 1.8 4 1.0

2 1.0 4 1.3 3 1.7 5 1.0

10 1 1.0

10 2 1.0

10 0 0.0

10 4 1.3

9 9 1.3

8 6 1.0

10 1 3.0

10 2 1.0

10 5 1.0

9 9 2.8

8 8 3.1

Testes Degeneration

Number Examined:

Thymus Atrophy

Number Examined: Average Grade:

10 2 1.0

Thyroid Glands Follicular cell height

Number Examined:

10

10

10

10

9

8

Average Score:

2.4

2.2

2.4

2.0

1.9

1.8

Average Grade:

Table 7 Effects of subacute sodium periodate exposure on clinical chemistry parameters in Sprague Dawley rats. Females

Control

20 mg/kg

40 mg/kg

80 mg/kg

159 mg/kg

318 mg/kg

N examined ALKP, (U/L) ALT, (U/L) AMYL, (U/L) TBIL, (mg/dL) CHOL, (mg/dL) GLU, (mg/dL) ALB, (g/dL) GLOB, (mg/dL) TP, (g/dL) BUN, (mg/dL) CREA, (mg/dL) CA, (mg/dL) PHOS, (mg/dL) Na, (mmol/L) K, (mmol/L) Cl, (mmol/L)

10 134 ± 33.32 47.1 ± 6.69 615.7 ± 127.46 0.39 ± 0.15 70.3 ± 14.06 197.2 ± 80.88 3.18 ± 0.36 3.19 ± 0.26 6.35 ± 0.26 23.5 ± 4.67 0.78 ± 0.1 11.82 ± 0.45 13.62 ± 1.75 151.1 ± 1.45 9.8 ± 1.52 105 ± 1.63

10 126.7 ± 23.75 56.9 ± 13.27 794.3 ± 251.29 0.35 ± 0.08 78.1 ± 16.78 237.5 ± 42.24 3.36 ± 0.42 2.99 ± 0.3 6.32 ± 0.28 22.8 ± 6.14 0.7 ± 0.09 11.91 ± 0.41 13.1 ± 1.55 151.2 ± 1.23 9.52 ± 2.22 105.6 ± 1.71

10 108.5 ± 29.62 51.2 ± 6.46 705.7 ± 197.3 0.41 ± 0.06 84.2 ± 9.14* 192.6 ± 82.44 3.45 ± 0.32 3.16 ± 0.37 6.6 ± 0.49 26 ± 6.46 0.74 ± 0.12 11.76 ± 0.43 13.32 ± 1.12 151.9 ± 1.91 9.57 ± 1.83 106 ± 1.25

9 104.2 ± 23.32* 47 ± 7.19 736 ± 176.99 0.42 ± 0.08 99.3 ± 15.65** 209.56 ± 49.7 3.46 ± 0.26 3.19 ± 0.21 6.64 ± 0.21 18.89 ± 6.11 0.7 ± 0.07 11.6 ± 0.19 12.56 ± 1.44 151.56 ± 0.88 8.58 ± 1.47 104.22 ± 1.56

10 107.7 ± 24.06* 49.9 ± 8.44 795.7 ± 155.09 0.44 ± 0.05 102.1 ± 12.81** 213.7 ± 74.23 3.53 ± 0.21 3.02 ± 0.16 6.55 ± 0.26 22.1 ± 2.92 0.66 ± 0.13 11.62 ± 0.59 12.36 ± 1.1 151.9 ± 0.88 9.79 ± 1.18 105.6 ± 0.7

9^ 83 ± 18.93** 61.4 ± 10.43** 1085.7 ± 599.59* 0.46 ± 0.2 112.3 ± 16.65*** 240.33 ± 52.06* 3.21 ± 0.5 3.33 ± 0.24 6.54 ± 0.59 28.78 ± 21.25 0.66 ± 0.12 11.69 ± 0.54 12.6 ± 1.95 150.25 ± 3.01 8.68 ± 1.04 100.9 ± 4.76**

Males

Control

46 mg/kg

93 mg/kg

185 mg/kg

370 mg/kg

741 mg/kg

N examined ALKP, (U/L) ALT, (U/L) AMYL, (U/L) TBIL, (mg/dL) CHOL, (mg/dL) GLU, (mg/dL) ALB, (g/dL) GLOB, (mg/dL) TP, (g/dL) BUN, (mg/dL) CREA, (mg/dL) CA, (mg/dL) PHOS, (mg/dL) Na, (mmol/L) K, (mmol/L) Cl, (mmol/L)

10 218.8 ± 52.65 61.2 ± 26.55 1153.4 ± 170.54 0.35 ± 0.08 66 ± 18.46 220.7 ± 147.5 3.22 ± 0.16 3.07 ± 0.12 6.29 ± 0.14 16.6 ± 5.78 0.71 ± 0.14 11.89 ± 0.48 14.44 ± 0.87 152.9 ± 1.52 10.85 ± 1.03 105.2 ± 1.93

9 201.78 ± 33.88 50 ± 11.61 1093.33 ± 176.76 0.32 ± 0.12 86.2 ± 17.07* 208.78 ± 101.1 2.99 ± 0.2* 3.1 ± 0.12 6.1 ± 0.17 14.89 ± 2.85 0.63 ± 0.14 11.47 ± 0.38 13.22 ± 1.84 153.13 ± 1.89 8.88 ± 1.33* 105.5 ± 1.69

10 185.1 ± 32.57 70.2 ± 24.94 1134.4 ± 126.17 0.37 ± 0.13 91.5 ± 17.17** 225.5 ± 102.45 3.04 ± 0.3* 2.85 ± 0.37 5.91 ± 0.24 15.1 ± 3.75 0.56 ± 0.13 11.42 ± 0.3 14.14 ± 3.19 152.9 ± 1.97 9.8 ± 1.68 106.3 ± 1.16

10 159.8 ± 30.36** 67.8 ± 16.61 934 ± 324.68* 0.36 ± 0.13 104.5 ± 16.74** 232.4 ± 99.06 2.78 ± 0.25*** 3.03 ± 0.21 5.82 ± 0.33 23 ± 31.04 0.62 ± 0.12 11.23 ± 0.42 12.57 ± 2.17 151.11 ± 5.09 8.51 ± 1.6** 103.67 ± 5.2

8** 196.63 ± 78.59 78.4 ± 27.26* 1216 ± 684.44 0.86 ± 1.09 131.1 ± 47.3* 259.13 ± 125.5 2.89 ± 0.76 3.39 ± 0.45 6.3 ± 1.11 85.1 ± 43.26** 1.21 ± 0.51* 10.68 ± 1.87 15.51 ± 1.54 145.5 ± 5.65** 8.53 ± 1.31** 96 ± 5.97**

5** 302.4 ± 66.36* 60 ± 25.17 1492.4 ± 515.86 0.48 ± 0.18 142.4 ± 16.49** 428.6 ± 151.86 2.74 ± 0.21*** 3.24 ± 0.11* 5.98 ± 0.19 36 ± 21.15* 0.74 ± 0.27 11.7 ± 0.35 14.9 ± 1.26 148.8 ± 2.99* 8.73 ± 1.07* 99.3 ± 2.36**

ALB: albumin; ALKP: alkaline phosphatase; ALT: alanine aminotransferase; AMYL: amylase; BUN: blood urea nitrogen; CA: calcium; CHOL: cholesterol; CREA: creatinine; GLOB: globulin; GLU: glucose; PHOS: phosphorus; TBIL: total bilirubin; TP: total protein; Na: sodium; K: potassium; Cl: chloride. Mean ± SD; *Significantly different from control at p < 0.05; ** Significantly different from control at p < 0.01; *** Significantly different from control at p < 0.001; ^Data from animals dosed less than 14-days due to mortality and moribund sacrifices (see Table 1).

stress leukogram (neutrophilia, lymphopenia, and monocytosis); however, the decrease in thymus weight was only significant for males and neither sex showed an increase in adrenal weight or adrenal hyperplasia. In the absence of evidence of effects on the

adrenal gland, the role of stress in the effects on the immune, hematologic, and reproductive systems is unclear. However, as changes in leukocyte differentials were largely restricted to high dose groups with moribund animals, stress likely played a

34

E.M. Lent et al. / Regulatory Toxicology and Pharmacology 83 (2017) 23e37

Table 8 Effects of subacute sodium periodate exposure on hematology parameters in Sprague Dawley rats. Females

Control

20 mg/kg

40 mg/kg

80 mg/kg

159 mg/kg

318 mg/kg

N animals WBC:(K/uL) NEU:(K/uL) NEU:(N) LYM:(K/uL) LYM:(L) MONO:(K/uL) MONO:(M) EOS:(K/uL) EOS:(E) BASO:(K/uL) BASO:(B) RBC:(M/uL) HGB:(g/dL) HCT:(%) MCV:(fL) MCH:(pg) MCHC:(g/dL) RDW:(%) MPV:(fL)

9 10.75 ± 2.31 1.46 ± 0.55 13.78 ± 5.29 8.31 ± 2.33 76.44 ± 6.96 0.52 ± 0.22 5.13 ± 2.64 0.09 ± 0.03 0.8 ± 0.22 0.4 ± 0.16 3.86 ± 1.58 7.36 ± 0.5 15.2 ± 0.94 40.83 ± 2.78 55.48 ± 1.03 20.69 ± 0.61 37.26 ± 0.58 15.07 ± 0.7 5.58 ± 0.46

10 11.45 ± 2.84 1.11 ± 0.36 9.93 ± 2.68 9.65 ± 2.68 83.84 ± 4.14 0.31 ± 0.25 2.89 ± 2.41 0.12 ± 0.05 1.04 ± 0.46 0.25 ± 0.15 2.31 ± 1.28 7.02 ± 0.48 14.82 ± 0.64 39.35 ± 1.87 56.2 ± 1.55 21.16 ± 0.98 37.62 ± 0.89 14.84 ± 0.57 5.41 ± 0.5

9 10.13 ± 2.14 1.22 ± 0.42 12.16 ± 3.2 7.7 ± 1.83 75.73 ± 6.8 0.59 ± 0.21 5.95 ± 2.06 0.14 ± 0.06 1.35 ± 0.52* 0.47 ± 0.26 4.81 ± 3.03 7.2 ± 0.83 15.21 ± 1.37 40.59 ± 3.8 56.56 ± 1.9 21.3 ± 1.11 37.61 ± 0.84 14.76 ± 0.51 5.58 ± 0.64

10 11.08 ± 3.99 1.01 ± 0.29 9.77 ± 3.7 9.13 ± 3.61 81.58 ± 5 0.44 ± 0.23 3.98 ± 1.85 0.13 ± 0.05 1.15 ± 0.43 0.38 ± 0.19 3.52 ± 1.38 7.05 ± 0.45 14.76 ± 0.86 39.56 ± 2.64 56.16 ± 1.54 20.96 ± 0.67 37.3 ± 0.52 15.29 ± 0.71 5.23 ± 0.46

10 9.53 ± 2.39 1.61 ± 0.87 16.45 ± 6.62 6.77 ± 1.89 71.49 ± 10.74 0.55 ± 0.31 5.68 ± 2.63 0.13 ± 0.12 0.91 ± 0.25 0.52 ± 0.24 5.47 ± 2.14 6.89 ± 0.55 14.27 ± 0.82 38.4 ± 2.23 55.83 ± 1.85 20.73 ± 0.76 37.14 ± 0.37 15.21 ± 0.64 5.1 ± 0.26

9^ 9.6 ± 3.05 5.98 ± 3.49*** 59.12 ± 21.59* 2.84 ± 1.61*** 31.84 ± 17.29* 0.48 ± 0.4 5.4 ± 4.79 0.06 ± 0.06* 0.38 ± 0.2* 0.28 ± 0.23 3.27 ± 2.84 6.73 ± 0.9 13.7 ± 2.06 36.51 ± 5.09 54.27 ± 1.74 20.32 ± 0.82 37.46 ± 0.68 16.8 ± 0.79** 5.41 ± 0.46

Males

Control

46 mg/kg

93 mg/kg

185 mg/kg

370 mg/kg

741 mg/kg

N animals WBC:(K/uL) NEU:(K/uL) NEU:(N) LYM:(K/uL) LYM:(L) MONO:(K/uL) MONO:(M) EOS:(K/uL) EOS:(E) BASO:(K/uL) BASO:(B) RBC:(M/uL) HGB:(g/dL) HCT:(%) MCV:(fL) MCH:(pg) MCHC:(g/dL) RDW:(%) MPV:(fL)

10 11.68 ± 2.3 1.75 ± 0.44 15.06 ± 2.99 8.59 ± 1.87 73.33 ± 3.44 0.61 ± 0.13 5.28 ± 0.93 0.07 ± 0.03 0.62 ± 0.32 0.66 ± 0.19 5.7 ± 1.41 8.03 ± 0.29 16.2 ± 0.42 45.39 ± 0.99 56.55 ± 1.51 20.2 ± 0.53 35.67 ± 0.58 16.14 ± 0.76 5.04 ± 0.5

10 10.38 ± 3.66 1.62 ± 0.57 16.19 ± 4.72 7.61 ± 2.92 73.01 ± 7.04 0.54 ± 0.38 5.02 ± 2.87 0.07 ± 0.04 0.64 ± 0.26 0.53 ± 0.27 5.13 ± 2.08 7.78 ± 0.47 16.22 ± 0.99 45.35 ± 2.65 58.38 ± 2.42 20.88 ± 1.05 35.76 ± 0.52 15.93 ± 0.64 5.28 ± 0.33

9 10.17 ± 1.43 1.76 ± 0.62 17.01 ± 4.11 7.33 ± 0.98 72.32 ± 6.02 0.5 ± 0.31 4.89 ± 2.78 0.08 ± 0.03 0.83 ± 0.43 0.5 ± 0.23 4.93 ± 1.99 7.59 ± 0.62* 15.42 ± 1.3 43.78 ± 3.6 57.71 ± 2.06 20.36 ± 0.68 35.29 ± 0.49 15.82 ± 0.82 5.18 ± 0.24

10 10.98 ± 2.99 3.7 ± 2.77* 32.16 ± 19.33* 6.02 ± 2.57* 56.29 ± 19.08* 0.65 ± 0.28 6.01 ± 2.02 0.07 ± 0.05 0.65 ± 0.4 0.53 ± 0.16 4.89 ± 0.95 7.23 ± 0.66** 14.49 ± 1.06*** 40.07 ± 2.54*** 55.63 ± 2.49 20.07 ± 0.76 36.09 ± 0.82 16.89 ± 0.92 4.88 ± 0.24

7** 15.47 ± 4.67 11.38 ± 4.02** 73.37 ± 14.2* 2.4 ± 1.09** 15.87 ± 6.98* 1.33 ± 1.22 8.65 ± 8.21 0.04 ± 0.03 0.29 ± 0.26* 0.31 ± 0.33 1.83 ± 1.89* 7.89 ± 1.09 15.73 ± 1.98 43.13 ± 5.72 54.71 ± 1.93 19.97 ± 0.71 36.53 ± 0.75* 17.04 ± 1.34 5.19 ± 0.36

5** 14.97 ± 5.36 9.68 ± 6.29** 58.58 ± 22.94* 2.68 ± 0.73** 22 ± 14.76* 1.6 ± 1.21 11.53 ± 6.8 0.04 ± 0.02 0.27 ± 0.08* 0.96 ± 0.57 7.63 ± 4.68 8.67 ± 1.31 17.26 ± 2.1 45.82 ± 5.87 53.04 ± 2.43* 20 ± 1.04 37.72 ± 1.01** 18.16 ± 1.42* 5.84 ± 0.93*

WBC: white blood cell count; NEU: neutrophils; LYM: lymphocytes; MONO: monocytes; EOS: eosinophils; BASO: basophils; RBC: red blood cell count; HGB: hemoglobin; HCT: hematocrit; MCV: mean cell volume; MCH: mean cell hemoglobin; MCHC: mean cell hemoglobin concentration; RDW: red blood cell distribution width. Mean ± SD; *Significantly different from control at p < 0.05; ** Significantly different from control at p < 0.01; *** Significantly different from control at p < 0.001; ^Data from animals dosed less than 14-days due to mortality and moribund sacrifices (see Table 1).

significant role in this effect. The lack of an increase in adrenal weight may have resulted from hypothyroid-associated adrenal weight decreases (Tohei, 2004; Tohei et al., 1997, 1998). Sodium periodate exposure caused decreased peripheral T3 (females and males) and T4 levels (males). While chronic stress is often associated with decreased T3 and, in severe cases, decreased T4, stress induced hypothyroidism is present without altered TSH levels (Everds et al., 2013). Contrary to this and the expected pattern for hypothyroidism, the sodium periodate induced hypothyroidism was present with reduced TSH levels. This is also in contrast to the legacy incendiary oxidizer, ammonium perchlorate. Ammonium perchlorate administered to male and female rats at doses as low as 10 mg/kg-day for 14 days caused increased thyroid weight (Siglin et al., 2000; Yu et al., 2002) and follicular cell hypertrophy with microfollicle formation and colloid depletion (Siglin et al., 2000). The effects of ammonium perchlorate on the thyroid are mediated by inhibition of uptake of iodide as perchlorate competes for uptake into the thyroid by the NIS (Yu et al., 2002). Serum T3 and T4 are decreased and TSH levels increased in response to the decreased iodide uptake. Increased TSH levels result in shrinkage of thyroid follicle colloid area and increased cell height. If

sustained, high TSH levels result in follicular cell hypertrophy/hyperplasia (Capen, 1997; Capen and Martin, 1989). Although T3/T4 were reduced in sodium periodate treated rats, TSH was decreased rather than increased and follicle cell height did not differ with sodium periodate treatment. The condition of decreased T3 and T4 levels with normal or reduced TSH levels has been documented in the uremic rat model (Lim et al., 1980). In the present study, both male and female rats in the sodium periodate dose groups with decreased thyroid hormone levels also exhibited indications of uremia or kidney failure (e.g., increased BUN and creatinine, clinical observations of blood in the urine, polydipsia, polyuria, and acute tubular necrosis and hyaline droplets in renal tubular epithelium). The reason for the decrease in TSH with uremia is unclear, but may be due to pituitary disturbances including impaired response to thyrotropin-releasing hormone (TRH) and altered feedback regulation (Iglesias and Diez, 2009; Lim et al., 1980). Although thyroid enlargement, nodules, and carcinoma are more common in patients with uremia/chronic kidney disease (Iglesias and Diez, 2009; Mariani and Berns, 2012), sodium periodate-induced uremia did not lead to increased thyroid weight or altered thyroid histology. However, longer duration exposures might produce these effects.

E.M. Lent et al. / Regulatory Toxicology and Pharmacology 83 (2017) 23e37

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Fig. 1. Effects of subacute sodium periodate on total thyroxine (T4), total triiodothyronine (T3), and thyroid stimulating hormone (TSH) in female (a) and male (b) Sprague Dawley rats. Data are means ± SEM. *Significantly different from deionized water control group. The 318 (female), 370 (male), and 741 (male) mg/kg-day groups include samples from animals sacrificed early due to moribund status (see Table 1).

Many of the effects on endpoints measured in the subacute study may well be secondary to kidney toxicity and uremia. In the presence of renal failure, kidney functions including hormone production and secretion, acid-base homeostasis, fluid and electrolyte regulation, and waste-product elimination are impaired and abnormalities, such as anemia, acidemia, electrolyte imbalance, malnutrition, cardiovascular disease, endocrine abnormalities, and

immune impairment can occur. The electrolyte imbalance present in high dose groups consisted of reductions in sodium, potassium, and chloride levels in males and reductions in chloride levels in females. As these changes were largely restricted to the high dose groups, these effects cannot be definitively attributed to kidney toxicity as moribundity likely contributed to these effects. However, degenerative changes in the intestinal tract may also have

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E.M. Lent et al. / Regulatory Toxicology and Pharmacology 83 (2017) 23e37

contributed to the electrolyte imbalance as impaired salt and water absorption have been described in the diseased colon (Sandle, 1998). Inflamed intestinal mucosa have increased electrical conductance and permeability to monovalent ions and decreased Naþ, Kþ-ATPase activity, resulting in a loss of the lumen negative potential difference and impaired sodium and chloride absorption (Sandle, 1998). The malnutrition, as evidenced by reductions in body weight, associated with sodium periodate were more likely due to direct compound-related effects on the gastrointestinal system as described previously. Elevated serum cholesterol levels in nearly all dose groups may be due to altered lipid metabolism and clearance secondary to kidney disease (Keane et al., 2013; Quaschning et al., 2001; Shoji et al., 2001). There was no evidence of effects on the heart (e.g., heart weight with body weight covariate and histopathology). The gastrointestinal tract demonstrated compound-toxicity that was unrelated to uremia. In the stomach, treatment-related changes consisted of mucosal (epithelial cell) ulcer/erosion, areas of necrosis, hemorrhage, or inflammation. In the small and large intestines, inflammation, hemorrhage, or mononuclear cell infiltrate were present in higher dose groups. These effects may be attributable to the direct oxidation damage of sodium periodate on the mucous membranes and epithelium. Minor effects on red blood cell counts and hemoglobin levels that occurred in the high dose groups likely occurred secondarily as a result of blood loss from the gastrointestinal lesions. In contrast to the direct effects on the gastrointestinal tract and kidney toxicity observed with periodate dosing in this study; perchlorate exposure of rats is largely associated with thyroid toxicity. There was no evidence of gross or histopathological alterations of the gastrointestinal tract in rats given ammonium perchlorate in drinking water at 8.5 mg/kg-day (Siglin et al., 2000). However, irritation of the gastric mucosa was noted in dogs given 3811 mg/kg-day ammonium perchlorate via oral gavage (ATSDR, 2008). No effects on liver weight, histopathology or liver enzymes were noted in rats given ammonium perchlorate in drinking water at 8.5 mg/kg-day for 90 days (Siglin et al., 2000) or in rats given 0.1% potassium perchlorate in the diet for 19 weeks (Hiasa et al., 1987). No effects on kidney weight, histopathology or function have been noted in rats given perchlorate in the drinking water at doses up to 8.5 mg/kg-day for 14- and 90-days (Siglin et al., 2000). Effects on the thyroid have included decreased serum T4 and T3, increased TSH and thyroid weight, hypertrophy and hyperplasia of thyroid cells, inhibition of radioiodine uptake, colloid depletion, fibrosis, and papillary and/or follicular adenomas and carcinomas (Fernandez Rodriguez et al., 1991; Hiasa et al., 1987; Kapitola et al., 1971; Mannisto et al., 1979; Matsuzaki and Suzuki, 1981; Ortiz-Caro et al., 1983). In subacute studies of durations similar to those used in the current study, decreased absolute and relative thyroid weight and histological alterations of the thyroid including follicular cell hypertrophy with microfollicle formation and colloid depletion were observed in rats given perchlorate in drinking water at 8.5 mg/kg-day (Siglin et al., 2000). Increased serum TSH and/or decreased T3 and T4 were observed in rats at doses as low as 0.09 mg/kg-day (Yu et al., 2002) and 0.009 and 0.04 mg/kg-day in males and females, respectively (Siglin et al., 2000). In the current study, sodium periodate did not produce any histopathological effects in the thyroid. Changes in thyroid hormones occurred in males at doses greater than 185 mg/kg-day and in females at the top dose of 318 mg/kg-day; however, it could not be definitively concluded that such effects were test article related due to significant mortality and moribundity in the animals at those doses. Although the potential effects of sodium periodate on thyroid function would require a longer term study at lower doses, periodates can be concluded to be significantly less toxic to the thyroid than

perchlorate salts. In conclusion, a cascade of effects that was likely secondary to kidney toxicity and uremia was observed in female rats in the 318 mg/kg-day group and male rats in the 185, 370, and 741 mg/kgday groups. Increased cholesterol was identified as the critical endpoint in this study based on the dose-related response in males and females and was used to derive the BMDL10s of 17.2 and 33.7 mg/kg-day for females and males, respectively. Comparison with the lowest NOAEL for perchlorate-induced thyroid toxicity in rats (0.009 mg/kg-day) suggests that sodium periodate is less toxic than perchlorate on a subacute basis. Conflict of interest The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Funding The author(s) disclose receipt of the following financial support for the research, authorship, and/or publication of this article: Funding for this effort was provided by the US Army Environmental Quality Technology Program, Pollution Prevention pillar through the Army Research Development and Engineering Command, Environmental Acquisition Sustainment and Logistics Program (MIPR number MIPR2GDATHR117). Disclaimer The views expressed in this article are the views of the author(s) and do not reflect the official policy of the Department of the Army, the Department of Defense, or the U.S. government. Acknowledgements The authors gratefully acknowledge the following people for their assistance with this project: Allison M. Jackovitz, Theresa L. Hanna, Matthew A. Bazar, Mark R. Way, Wilfred C. McCain, Michael J. Quinn, Alicia A. Shiflett, and Karl P. Kroeck. We thank Anthony Skowronek (Battelle, Columbus, OH) for expert completion of the histopathological analyses for this study. We also thank Marc Williams for critical review of this manuscript. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.yrtph.2016.11.014. References Anghileri, L.J., 1965. Fate of intravenously injected iodate and periodate. Biochem. Pharmacol. 14, 1930. ASTM, 2010. Standard Test Method for Estimating Acute Oral Toxicity in Rats, Vol. E1163-10. Conshohocken, PA. ATSDR, 2008. Toxicological Profile for Perchlorates. US Department of Health and Human Services, Public Health Service, Agency for Toxic Substances and Disease Registry. Bailey, S.A., et al., 2004. Relationships between organ weight and body/brain weight in the rat: what is the best analytical endpoint? Toxicol. Pathol. 32, 448e466. Ball, P., 2012. Greener, Cleaner Fireworks? BBCFuture. Barnes, D.G., Dourson, M., 1988. Reference dose (RfD): Description and use in health risk assessment. Regul. Toxicol. Pharmacol. 8, 471486. Brechner, R.J., et al., 2000. Ammonium perchlorate contamination of Colorado River drinking water is associated with abnormal thyroid function in newborns in Arizona. J. Occup. Environ. Med. 42, 777e782. Buffler, P.A., et al., 2006. Thyroid function and perchlorate in drinking water: an evaluation among California Newborns, 1998. Environ. Health Perspect. 114, 798e804.

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