Safety evaluation of an enzymatically-synthesized glycogen (ESG)

Regulatory Toxicology and Pharmacology 57 (2010) 210–219

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Safety evaluation of an enzymatically-synthesized glycogen (ESG) Shahrzad Tafazoli a,*, Andrea W. Wong a, Hideki Kajiura b,c, Ryo Kakutani b, Takashi Furuyashiki b, Hiroki Takata b, Takashi Kuriki b a

Cantox Health Sciences International, 2233 Argentia Road, Suite 308, Mississauga, Ontario, Canada L5N 2X7 Institute of Health Sciences, Ezaki Glico Co., Ltd., 4-6-5, Utajima, Nishiyodogawa-ku, Osaka 555-8502, Japan c Development Department, Food Material Division, Glico Foods Co., Ltd., 7-16 Kasuga-cho, Takatsuki, Osaka 569-0053, Japan b

a r t i c l e

i n f o

Article history: Received 30 November 2009 Available online 1 March 2010 Keywords: Enzymatically-synthesized glycogen Acute Subchronic Mutagenicity Dietary fiber

a b s t r a c t An enzymatically-synthesized glycogen (ESG), intended for use as a food ingredient, was investigated for potential toxicity. ESG is synthesized in vitro from short-chain amylose by the co-operative action of branching enzyme and amylomaltase. In an acute toxicity study, oral administration of ESG to Sprague–Dawley rats at a dose of 2000 mg/kg body weight did not result in any signs of toxicity. ESG did not exhibit mutagenic activity in an in vitro bacterial reverse mutation assay. In a subchronic toxicity study, increased cecal weights noted in the mid- (10%) and high-dose (30%) animals are common findings in rodents fed excess amounts of carbohydrates that increase osmotic value of the cecal contents, and thus were considered a physiological rather than toxicological response. The hematological and histopathological effects observed in the high-dose groups were of no toxicological concern as they were secondary to the physiological responses resulting from the high carbohydrate levels in the test diets. The noobserved-adverse-effect level for ESG in rats was therefore established to be 30% in the diet (equivalent to approximately 18 and 21 g/kg body weight/day for male and female rats, respectively). These results support the safety of ESG as a food ingredient for human consumption. Ó 2010 Elsevier Inc. All rights reserved.

1. Introduction Glycogen, the major storage polysaccharide in animals and microorganisms, is a highly branched (1 ? 4)(1 ? 6)-linked a-Dglucan with molecular weight ranging from 1000 to 1,000,000 kDa (Geddes, 1986). In animal tissues, glycogen is synthesized from uridine-diphosphate glucose via the action of glycogenin (EC 2.4.1.186), glycogen synthase (EC 2.4.1.11), and branching enzyme (BE, a-1,4-glucan:a-1,4-glucan-6-glycosyltransferase, EC 2.4.1.18; Roach, 2002). Glycogen also can be synthesized in vitro from glucose-1-phosphate using the co-operative action of a-glucan phosphorylase (GP, EC 2.4.1.1) and BE as catalysts (Cori and Cori, 1943; Fujii et al., 2003; Tolmasky and Krisman, 1987); this method is known as Cori’s method. In this method, GP elongates the a-(1–4)-glucan chain, and BE introduces branch points in the growing chain. Cori’s method was later modified by using sucrose as a starting material, and converting it to glucose-1-phosphate through the action of sucrose phosphorylase (Ohdan et al., 2006; Ryoyama et al., 2004). Recently, a novel enzymatic process of glycogen synthesis was developed, in which starch is used as a starting material. In this process, the branched linkages of starch are hydrolyzed using * Corresponding author. Fax: +1 905 542 1011. E-mail address: [email protected] (S. Tafazoli). 0273-2300/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.yrtph.2010.02.009

isoamylase (EC 3.2.1.68) to produce a mixture of short-chain amyloses, which are assembled into glycogen by the action of BE (EC 2.4.1.18) in the presence of amylomaltase. A large number of small oligosaccharides are produced as a by-product of the synthesis reaction catalyzed by BE. As these oligosaccharides are poor substrates for BE, elongation of the malto-oligosaccharide by amylomaltase increases the substrates available for BE, and consequently the yield of glycogen product is increased (Kajiura et al., 2008). Enzymatically-synthesized glycogen derived by this process, herein referred to as ESG, has a molecular weight in the range of 3000–30,000 kDa, and is soluble in water giving an opalescent solution resembling glycogen derived from natural sources such as animal tissues or shellfish (NSG) (Kajiura et al., 2008). Takata et al. (2009) examined the differences in the structural composition between ESG and NSG, and determined that despite their similarities in molecular size and shape, ESG has a narrower unit chain distribution, and more evenly distributed a-(1,6)-linkages compared to those in NSG. The digestion of NSG by porcine pancreatic a-amylase was reported to produce saccharides (G1– 3), branched oligosaccharides (G4–7) and a-macrodextrin molecules of approximately 10 kDa in molecular weight, whereas a-amylase digestion of ESG yielded saccharides (G1–3) and a large a-macrodextrin molecule with a molecular weight of approximately 1000–1600 kDa. Additionally, following treatment with a mixture of pancreatic a-amylase and glucoamylase to evaluate

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the dietary fiber content, a significant amount (i.e., approximately 16.9%) of fiber was detected from ESG, whereas none (i.e., 0.2%) was derived from NSG. Based on these results, the authors concluded that orally ingested ESG and NSG may be digested differently in the gastrointestinal tract (Takata et al., 2009). Thus far, animal studies on other enzymatically-synthesized glycogens have focused on determining the potential beneficial effects of synthesized glycogen in comparison to NSG or other commercially available polysaccharides. For instance, Kakutani et al. (2007) investigated the macrophage-stimulating activity of synthetic glycogens, including ESG (average molecular weight of 5000 kDa), and determined that the immunostimulatory effects of enzymatically-synthesized glycogens are dependent on their molecular weight rather than their structural properties, with the optimal size ranging from 5000 to 6500 kDa. Furthermore, Ryoyama et al. (2004) demonstrated that oral administration of synthesized glycogen to Meth A tumor-bearing BALB/c mice at a dose of 50 lg/mL in drinking water resulted in a significant increase in their survival time. Additionally, in vitro treatment of Meth A tumor cells (BALB/c mouse-derived fibrosarcoma cells) with synthesized glycogen significantly inhibited Meth A tumor growth following subcutaneous implantation into the back of BALB/c mice. The glycogen used in this study was synthesized from sucrose and maltotetraose using sucrose phosphorylase, a-glucan phosphorylase, and BE (EC 2.4.1.18). As traditional toxicological testing has not been previously conducted on ESG, such studies were undertaken to establish the safe use of ESG as a food ingredient for human consumption. The potential toxicity of ESG was evaluated in an acute oral rat toxicity study, a 13-week subchronic toxicity study in rats, and a bacterial reverse mutation assay. The findings of these studies support the safety of ESG for human consumption.

2. Materials and methods 2.1. Test article ESG was prepared from dextrin using three enzymatic processes, firstly with a debranching reaction using isoamylase to form short-chain amylose, followed by a BE and amylomaltase to synthesize glycogen from amylose. For the in vitro reverse mutation assay and acute toxicity study test, ESG was provided by Ezaki Glico Co., Ltd., as a white powder comprising 88.8% glycogen, where the minor constituents included moisture (3%) and malto-oligosaccharides with degree of polymerization ranging from 1 to 20. The test article was diluted in water prior to testing, and was stored between 17.7 and 22.3 °C in an airtight container prior to use. The stability of the test article was confirmed throughout the course of both studies. For the subchronic toxicity study, diets containing ESG were provided by Ezaki Glico Co., Ltd., and used without further modification. The test diets were composed of AIN93M feed and ESG at concentrations of 3%, 10%, and 30% (Table 1). All test diets were stored at room temperature (19.1–26.5 °C) in airtight containers prior to use. In all studies, the test article met appropriate food-grade specifications, as levels of potential contaminants, including heavy metals and microorganisms, were below the specified limit.

2.2. Acute toxicity study in rats This study was conducted in compliance with the Good Laboratory Practice Standard for Non-Clinical Safety Studies on Drugs (Ministry of Health and Welfare, Japan, 1997), and the Guidebook

Table 1 Composition of diets in the 90-day oral toxicity study in rats. AIN93M

AIN93M (3% ESG)

L-Cystine

14 0.18

14 0.18

14 0.18

14 0.18

Corn starch Pregelatinized corn starch Sucrose Soybean oil Cellulose powder AIN93M Minerals AIN93 Vitamins Choline bitartrate tert-Butylhydroquinone ESG

46.5692 15.5 10 4 5 3.5 1 0.25 0.0008 0

43.5692 15.5 10 4 5 3.5 1 0.25 0.0008 3

36.5692 15.5 10 4 5 3.5 1 0.25 0.0008 10

16.5692 15.5 10 4 5 3.5 1 0.25 0.0008 30

Casein from milk

Total

100

100

AIN93M (10% ESG)

100

AIN93M (30% ESG)

100

for Manufacturing and Selling Cosmetics and Quasi Drugs 2006 (Yakuji Nippo Limited, 2006). Five-week-old male and female Sprague–Dawley [Crl:CD(SD)] rats were obtained from Charles River Laboratories Japan, Inc. (Shiga, Japan). The animals were provided a pelleted diet (CE-2, Clea Japan, Inc.) and tap water ad libitum, and were housed under controlled conditions. Five animals of each sex were selected for the study. The ESG test article (88.8% glycogen content) was weighed and dissolved in water to prepare a solution of 200 mg/ mL. On the day of administration, the animals were 6 weeks of age with body weights ranging from 199 to 212 g in males, and 158 to 163 g in females. The animals were orally administered 10 mL/kg body weight/day of the ESG solution to achieve a dose of 2000 mg/kg body weight. The rats were evaluated for clinical signs twice per day during a 2-week observation period. Body weight was measured on days 1, 4, 7, 10, and 13 after administration. Following the 2-week observation period, all animals were killed by exsanguination under anesthesia, and macroscopically examined for changes in the skin surface, organs, and tissues. The heart, spleen, lungs, liver, and kidney were removed and fixed in 10% neutral buffered formalin. 2.3. 13-Week oral toxicity study This study was conducted in accordance with the Good Laboratory Practice Standard for Non-Clinical Safety Studies on Drugs (Ministry of Health and Welfare, Japan, 1997), and in compliance with the Organization of Economic Co-operation and Development (OECD) Guideline for the Testing of Chemicals Test No. 408 (OECD, 1998). 2.3.1. Animals and treatment Four-week-old male and 3.5-week-old female Sprague–Dawley [Crl:CD(SD)] rats were obtained from Charles River Laboratories Japan, Inc. (Shiga, Japan). The animals were provided a pelleted diet (AIN93M, Oriental Yeast Co., Ltd.) and tap water ad libitum, and were housed under controlled conditions: 21.7–22.7 °C, relative humidity of 46–54%, ventilation of 15 fresh air exchanges/h, and 12-h light/dark cycles. The animals were quarantined and allowed to acclimate to laboratory conditions for a period of 7 days, and female animals were acclimatized for an additional 3 days. During this period, signs of general health, including body weight, food consumption, and ophthalmology were evaluated. Based on the results of these observations and examinations during the acclimation period, 40 rats of each sex were selected and randomized to control and test groups by a stratified randomization method (MiTOX system, Ver. 2.0, Mitsui Zosen Systems Research Inc.) based on

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animal body weight gain. Animals were housed individually during the administration period. On day 1 of the administration period, the animals were 5 weeks of age with body weights in the range of 140–155 g for males and 97–119 g for females. The ESG-containing diets were fed to the animals ad libitum for a period of 13 weeks (Table 2).

2.3.2. Observations Animals were observed twice daily for clinical signs and mortality. In addition, animals in the control and high-dose groups were examined twice daily for general physical condition and behavior on day 0, day 48, and day 90. Home-cage observations, holding observations, open-field tests, and manipulation tests were conducted to evaluate a range of response parameters. Body weights were measured prior to treatment, weekly during treatment, and at study termination. Food consumption was determined prior to treatment, and twice weekly during treatment. Ophthalmological examinations also were conducted in all animals prior to treatment and on day 42 and day 84 of the administration period.

2.3.3. Hematology and clinical chemistry Prior to necropsy, blood samples were collected from the abdominal post-cava from animals anesthetized by intraperitoneal administration of sodium pentobarbital (Tokyo Chemical Industry Co., Ltd.). Blood samples were analyzed for hematology, coagulation, and serum chemistry parameters. Hematology parameters examined (Siemens Medical Solutions Diagnostics Manufacturing Ltd.) included: red blood cell count (RBC), white blood cell count (WBC), hematocrit, hemoglobin, platelet count, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC). A bone marrow smear was performed on the remaining blood samples, and smears of samples were fixed, stained, and examined for the following parameters: eosinophils, basophils, neutrophils, monocytes, lymphocytes, large unstained cells, and reticulocyte ratio. Prothrombin time (PT) and activated partial thromboplastin time (APTT) were assessed by a fully-automated coagulation analyzer (Sysmex Corporation). For serum chemistry analysis (JEOL Ltd. analyzer), the serum was separated from blood and examined for the following parameters: aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), c-glutamyl transpeptidase (c-GTP), creatine phosphokinase (CPK), total bilirubin, total protein, albumin, globulin, total cholesterol, triglyceride, glucose, blood urea nitrogen (BUN), creatinine, inorganic phosphate (P), calcium (Ca2+), sodium (Na+), potassium (K+), and chloride (Cl ). The following protein fractions were analyzed using an automated electrophoresis system (Olympus Corporation): albumin ratio, a1-globulin ratio, a2-globulin ratio, b-globulin ratio, c-globulin ratio, and albumin:globulin ratio.

Table 2 Dietary intake of ESG during 13-week toxicity study. Test groupa

Dietary treatment

Control Low-dose Mid-dose High-dose

AIN93M AIN93M + 3% ESG AIN93M + 10% ESG AIN93M + 30% ESG

Mean daily intake of ESG over 13-week study period (mg/kg bw/day) Males

Females

0 1822.2 6241.4 18,337.7

0 2206.6 7272.4 21,464.4

Abbreviations: bw, body weight; ESG, enzymatically-synthesized glycogen. a Ten animals/sex/group.

2.3.4. Urinalysis Urine was collected from 10 animals from each group (5/sex) for up to 4 h on days 43 and 85 of the administration period, and evaluated for the following parameters: specific gravity, color tone, pH, protein, sugar, ketone body, bilirubin, occult blood, urobilinogen, and urinary sediment. 2.3.5. Clinical pathology and histopathology All rats were anesthetized by sodium pentobarbital and following blood sample collection, were killed by exsanguination. A complete necropsy was performed. A standard list of organs were weighed, and both absolute and relative weights calculated. Tissues fixed for histopathological evaluation included: trachea, tongue, esophagus, stomach (proventriculus and glandular stomach), small intestine (duodenum, jejunum, and ileum), colon (cecum, colon, and rectum), pancreas, aorta, urinary bladder, vagina, spinal cord, sciatic nerve (right and left), sternum (right and left), femur (right and left), submandibular lymph node (right and left), eyeball/optic nerve (right and left), lacrimal gland (right and left), Harderian gland (right and left), skeletal muscle (thigh), mammary gland, and skin. All stored organs and tissues of each animal in the control and high-dose groups, as well as the ileum, cecum, and colon of every animal in the mid- and low-dose groups, were embedded, sectioned, stained with hematoxylin–eosin, and examined microscopically. In addition, macroscopic lesions observed at necropsy were microscopically examined from each animal in other dose groups. 2.3.6. Statistical analysis MUSCOT statistical software (Yukms Co., Ltd.) was used for all statistical tests performed (significance value of 0.05, two-tailed). Bartlett’s test was used to compare the mean values for body weight, food consumption, urinalysis, hematology, clinical chemistry, and absolute and relative organ weights between the treatment groups and control group. Dunnett’s test or a Dunnett-type test (Miller’s test) was used for multiple comparisons between the control group and each of the treatment groups. Wilcoxon rank sum test and Fisher’s exact test were performed to determine differences in graded data from urinalysis and for urine color, respectively, between the control and treatment groups. 2.4. In vitro bacterial reverse mutation assay (Ames test) This study was conducted in accordance with the Good Laboratory Practices (GLP) Standard for Non-Clinical Safety Studies on Drugs (Ministry of Health and Welfare, Japan, 1997), and in compliance with the Guidelines for Genotoxicity Studies of Drugs (Ministry of Health, Labor and Welfare, Japan, 1999). The bacterial reverse mutation assay, using the pre-incubation method as described by Gatehouse et al. (1994), was conducted in five strains of bacteria: Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537, and Escherichia coli WP2uvrA strain. The bacterial strains were mixed with dimethylsulfoxide (DMSO) and stored in an ultra-low temperature freezer set at 70 °C. Water for injection (Japanese Pharmacopeia, Otsuka Pharmaceutical Factory, Inc.) was used as a negative control (JP, 2006). The following compounds were used as positive controls in the absence of metabolic activation: 0.01, 0.02, and 0.1 lg/plate of 2(2furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) (Wako Pure Chemical Industries, Ltd.) for strains TA100, WP2uvrA, and TA98, respectively, 0.5 lg/plate of sodium azide (NaN3) (Wako) for TA1535, and 80 lg/plate of 9-aminoacridine hydrochloride hydrate (9-AA) for TA1537 (Sigma–Aldrich Co.). In the presence of metabolic activation, 1, 2, 10, 0.5, and 2 lg/plate of 2-aminoanthracene (2-AA) (Wako) was used as a positive control for strains TA100, TA1535, WP2uvrA, TA98, and TA1537, respectively. The concentrations of

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the positive controls were achieved by dissolution and dilution with DMSO for AF-2, 9-AA, and 2-AA, and with water for NaN3. In the preliminary dose-finding assay, ESG was added to each bacterial strain at final concentrations of 19.5, 78.1, 313, 1250, and 5000 lg/plate. Metabolic activation was achieved via incubation with an S9 microsomal fraction (S9-mix) (Oriental Yeast Co., Ltd). ESG did not inhibit bacterial growth in any strain or induce a dose-dependent increase in the number of revertant colonies compared to the negative control in the presence or the absence of S9-mix (data not shown). Based on the results of the dose-finding experiment, concentrations of 313, 625, 1250, 2500, and 5000 lg/plate were used for the bacterial reverse mutation assay. To prepare the bacterial cultures, frozen bacterial suspensions were rapidly thawed and 20 lL of each S. typhimurium TA strain or E. coli strain were seeded in L-shaped glass tubes containing 10 mL of 2.5% nutrient broth (Oxoid Ltd.). The tubes were incubated in a thermostatic shaking incubator (PI-301, AS ONE Corporation) at 37 °C for 12 h. ESG test solutions and positive or negative controls were added to 0.1 mL of a fully grown culture of each bacterial strain at a volume of 0.1 mL. For the test systems with metabolic activation, 0.5 mL of metabolic activation mix (S9-mix) was added, while for the systems without metabolic activation 0.5 mL of 0.1 M phosphate buffer (pH 7.4) was added instead to the bacterial culture. The test mixtures were then shaken in a thermostatic shaking incubator (Shaking Bath; BW201, Yamato Scientific Co., Ltd.) at 37 °C for 20 min. After shaking incubation, the test mixtures were mixed with 2 mL of soft agar (0.6% agar and 0.5% NaCl) supplemented with 0.5 mM D-biotin/0.5 mM L-histidine (1:10) for S. typhimurium strains or 0.5 mM D-biotin/0.5 mM L-tryptophan (1:10) for E. coli strain, and added to the minimal glucose agar plates. All samples were plated in duplicate and incubated at

37 °C for a period of 48 h. After incubation, the number of revertant colonies was examined microscopically using a stereo microscope (40, SPT-40L, Carton Optical Industries Ltd.). 3. Results 3.1. Acute toxicity study in rats No mortality occurred following the oral administration of a single dose of 2000 mg ESG/kg body weight to male and female Crl:CD(SD) rats, and no abnormalities in clinical signs, body weight, or gross necropsy were observed during the 2-week observation period. Based on these findings, the oral LD50 for ESG was determined to be greater than 2000 mg/kg body weight. 3.2. Subchronic toxicity study in rats 3.2.1. Observations No mortality, behavioral changes, or ophthalmological abnormalities were observed during the 13-week study period. White stools were intermittently observed in all males and females of the high-dose group (30% ESG diet) during the administration period. In the mid-dose group (10% ESG diet), white stools were intermittently observed in all males from days 2 to 50, and in seven females from days 1 to 20. In the low-dose group (3% ESG diet), white stools were intermittently or continuously observed in three males from days 16 to 23. Soft stools were observed in high-dose males on 1 or 2 days in week 7, and continuously or intermittently observed in seven high-dose females during weeks 6–13.

Table 3 Body weight and body weight gains in the 13-week study. Treatment groupa

Body weights Baseline Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8 Week 9 Week 10 Week 11 Week 12 Week 13 Body weight gain Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8 Week 9 Week 10 Week 11 Week 12 Week 13

Males

Females

Control

3% ESG

10% ESG

30% ESG

Control

3% ESG

10% ESG

30% ESG

147.9 ± 4.5b 219.8 ± 9 284.6 ± 12 352.3 ± 19.4 409.4 ± 25.4 454 ± 28.8 494.1 ± 35 522.9 ± 40 551.2 ± 44.3 575.3 ± 49.6 598.2 ± 50.9 617.8 ± 52.4 636.9 ± 53.6 650.2 ± 57.1

148.1 ± 4.3 220.7 ± 8.3 286.5 ± 11.1 354.7 ± 14.7 407.7 ± 19 450.5 ± 22.8 490.3 ± 27.4 517.1 ± 29.9 546.9 ± 36.3 567.3 ± 42.7 587.4 ± 48.4 603.9 ± 52.4 620.1 ± 56.8 632.2 ± 59.8

148.1 ± 4.1 219.7 ± 10.4 286.6 ± 17.9 347.4 ± 23.2 397.2 ± 30.8 435.1 ± 35.7 471.6 ± 38.6 498.5 ± 37.0 522.8 ± 41.0 545.4 ± 46.0 565 ± 46.2 581.1 ± 48.6 600.7 ± 50.1 609.9 ± 49.3

147.6 ± 4.1 211 ± 8.5 275.6 ± 13.8 338.4 ± 19.4 388.7 ± 25.2 428.9 ± 30.8 463.2 ± 37.0 487.5 ± 40.2 514.2 ± 44.9 535.9 ± 48.3 556.6 ± 52.3 572.6 ± 57.1 591.3 ± 60.8 602 ± 61.4

108.6 ± 5.9 150.7 ± 9.0 181.1 ± 12.2 215.1 ± 18.9 238.8 ± 23.6 259.1 ± 26.3 273.8 ± 26.0 283.2 ± 28.0 292.4 ± 31.9 301.5 ± 33.3 314.5 ± 35.0 323.9 ± 37.4 329.4 ± 40.3 334.2 ± 39.2

108.8 ± 5.3 146.7 ± 8.8 176.8 ± 12.0 205.6 ± 17.7 227.6 ± 19.2 249.1 ± 20.5 263.6 ± 23.4 274.3 ± 26.9 283.5 ± 27.4 296.8 ± 28.8 306.1 ± 33.8 310.3 ± 35.7 316 ± 36.3 322.6 ± 35.9

109.0 ± 6.4 149.1 ± 7.5 175 ± 11.3 203.9 ± 13.9 225.4 ± 15.6 238.7 ± 15.2 250.3 ± 16.3* 263.9 ± 19.6 272.2 ± 23.2 279.8 ± 22.4 285.7 ± 25.5 292 ± 27.7 297.8 ± 29.8 302.3 ± 30.4

108.7 ± 5.5 139.2 ± 10.2* 165.9 ± 10.0* 190.9 ± 13.1** 211.5 ± 15.1** 225.7 ± 14.0** 239 ± 15.0** 250.5 ± 18.7* 254.9 ± 21.4** 262.4 ± 21.1** 268.2 ± 21.4** 272.2 ± 22.6** 274.1 ± 22.9** 278.6 ± 22.8**

71.9 ± 6.8 64.8 ± 4.4 67.7 ± 8.9 57.1 ± 8.5 44.6 ± 5.3 40.1 ± 7.9 28.8 ± 7.6 28.3 ± 6.3 24.1 ± 7.3 22.9 ± 5.0 19.6 ± 4.4 19.1 ± 4.3 13.3 ± 6.4

72.6 ± 4.4 65.8 ± 4.7 68.2 ± 6.6 53.0 ± 7.0 42.8 ± 5.5 39.8 ± 7.3 26.8 ± 7.5 29.8 ± 6.6 20.4 ± 7.7 20.1 ± 7.5 16.5 ± 4.8 16.2 ± 7.5 12.1 ± 5.4

71.6 ± 7.0 66.9 ± 8.7 60.8 ± 7.4 49.8 ± 9.1 37.9 ± 7.0 36.5 ± 4.8 26.9 ± 4.7 24.3 ± 5.9 22.6 ± 6.3 19.6 ± 5.3 16.1 ± 4.4 19.6 ± 4.6 9.2 ± 5.9

63.4 ± 5.9* 64.6 ± 7.2 62.8 ± 9.2 50.3 ± 8.0 40.2 ± 7.4 34.3 ± 6.9 24.3 ± 4.4 26.7 ± 6.4 21.7 ± 6.2 20.7 ± 6.4 16.0 ± 7.0 18.7 ± 6.8 10.7 ± 4.9

42.1 ± 4.5 30.4 ± 4.6 34.0 ± 9.3 23.7 ± 7.2 20.3 ± 5.0 14.7 ± 1.2 9.4 ± 5.1 9.2 ± 5.2 9.1 ± 5.7 13.0 ± 5.9 9.4 ± 4.6 5.5 ± 4.9 4.8 ± 5.0

37.9 ± 5.5 30.1 ± 5.1 28.8 ± 6.3 22.0 ± 6.9 21.5 ± 4.0 14.5 ± 4.8 10.7 ± 5.7 9.2 ± 5.5 13.3 ± 6.4 9.3 ± 6.2 4.2 ± 3.7* 5.7 ± 3.9 6.6 ± 3.3

40.1 ± 4.8 25.9 ± 6.3 28.9 ± 4.1 21.5 ± 5.7 13.3 ± 4.0** 11.6 ± 5.0 13.6 ± 5.1 8.3 ± 5.8 7.6 ± 4.2 5.9 ± 5.5* 6.3 ± 3.1 5.8 ± 4.6 4.5 ± 4.9

30.5 ± 7.2** 26.7 ± 5.2 25.0 ± 6.4* 20.6 ± 5.4 14.2 ± 4.1** 13.3 ± 3.9 11.5 ± 5.4 4.4 ± 5.3 7.5 ± 3.0 5.8 ± 4.6* 4.0 ± 3.9** 1.9 ± 3.5 4.5 ± 3.2

ESG, enzymatically-synthesized glycogen. Significantly different compared to control group, *P < 0.05, **P < 0.01. a Ten animals/sex/group. b Values are expressed as mean ± SD.

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Body weights were significantly decreased in high-dose females during the administration period compared to the control group, and a reduction in body weight gain was observed on weeks 1, 3, 5, 10, and 11 (Table 3). Mid-dose females also exhibited reduced body weights on week 6, while reduced body weight gains were observed in high-dose males during week 1, in mid-dose females during weeks 5 and 10, and low-dose females during week 11 compared to their respective control groups. Compared to control animals, food consumption was significantly decreased in high-dose females from weeks 1 to 6, week 8, and weeks 10 to 12 (Fig. 1). A reduction in food consumption also was observed in high-dose males during week 1 and in middose females during weeks 5 and 11 compared to their respective control groups (Fig. 2).

females. Decreased leukocyte, lymphocyte, and basophil counts, and increased neutrophil ratio were observed in the high-dose males. An increase in monocyte counts was observed in the middose males. Serum clinical chemistry analysis revealed a number of significant differences between the treated and control groups (Table 5). In females, decreased total protein, albumin, and triglyceride levels, and an elevated b-globulin ratio were observed in the high-dose group. Males in all treated groups exhibited decreased serum ALT levels. A significant increase in serum ALP levels and significant decrease in serum AST levels also was observed in high-dose males compared to the control group. Increased albumin levels, high albumin and albumin:globulin ratios, and low a2-globulin ratios were observed in mid-dose males, but not at the high-dose group.

3.2.2. Hematology and clinical chemistry Hematology data showed several significant differences between treated and control groups (Table 4). High-dose females exhibited elevated leukocyte, lymphocyte, and neutrophil counts, while elevated leukocyte counts were observed in mid-dose

3.2.3. Urinalysis No statistically significant differences between the treatment and control groups were observed for all urinalysis parameters, with the exception of decreased pH in high-dose females in week 13 (data not shown).

Food intake (g/rat/day)

22

20

18

* 16

*

*

*

* *

*

*

*

14

*

Control

*

3% ESG 10% ESG 30% ESG

*

12 0

5

10

15

Week of Administration Fig. 1. Food consumption of female rats during the 13-week toxicity study. Compared to control, food consumption was significantly decreased in high-dose females from weeks 1 to 6, week 8, and weeks 10 to 12, and in mid-dose females during weeks 5 and 11 (, P < 0.05). No significant differences in food consumption were noted between the low-dose treatment group and control.

Food intake (g/rat/day)

30

Control 3% ESG 10% ESG

25

30% ESG

* * 20

15 0

5

10

15

Week of Administration Fig. 2. Food consumption of male rats during the 13-week toxicity study. Overall, there were no significant differences between treated and control groups, with the exception of a significant decrease in food intake in high-dose males on week 1 of the study compared to controls (, P < 0.05).

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S. Tafazoli et al. / Regulatory Toxicology and Pharmacology 57 (2010) 210–219 Table 4 Hematology parameters following oral exposure to ESG for 13 weeks. Measured hematology parameters

Treatment groupa Males

RBC (106/mm3) WBC (103/mm3) Ht (%) Hb (g/dL) Platelet count (103/mm3) MCV (fL) MCH (pg) MCHC (g/dL) Reticulocyte (%) Eosinophil (103/mm3) Eosinophil (%) Basophil (103/mm3) Basophil (%) Monocyte (103/mm3) Monocyte (%) Lymphocyte (103/mm3) Lymphocyte (%) Neutrophil (103/mm3) Neutrophil (%) LUC (103/mm3) LUC (%) PT (s) APTT (s)

Females

Control

3% ESG

10% ESG

30% ESG

Control

3% ESG

10% ESG

30% ESG

8.24 ± 0.41b 8.14 ± 2.26 42.51 ± 1.51 15.16 ± 0.72 999.7 ± 101.5 51.64 ± 1.87 18.41 ± 0.95 35.67 ± 0.99 2.24 ± 0.31 0.14 ± 0.03 1.77 ± 0.65 0.022 ± 0.016 0.25 ± 0.13 0.18 ± 0.05 2.29 ± 0.71 6.61 ± 2.22 80.20 ± 5.65 1.05 ± 0.30 13.83 ± 5.45 0.14 ± 0.07 1.64 ± 0.59 9.63 ± 0.34 13.78 ± 0.64

8.14 ± 0.60 6.729 ± 1.402 42.42 ± 3.07 14.83 ± 1.06 952.5 ± 112.4 52.17 ± 1.22 18.25 ± 0.55 34.99 ± 1.12 2.29 ± 0.58 0.17 ± 0.12 2.45 ± 1.06 0.013 ± 0.005 0.20 ± 0.05 0.18 ± 0.08 2.86 ± 1.40 5.23 ± 1.11 77.68 ± 3.52 1.03 ± 0.22 15.54 ± 2.63 0.093 ± 0.075 1.31 ± 0.67 9.71 ± 0.35 13.98 ± 0.60

8.20 ± 0.39 7.64 ± 2.05 43.32 ± 1.40 15.20 ± 0.62 996.7 ± 101.5 52.85 ± 1.66 18.54 ± 0.90 35.06 ± 0.84 2.48 ± 0.36 0.15 ± 0.07 2.02 ± 0.80 0.018 ± 0.008 0.20 ± 0.09 0.17 ± 0.04 2.31 ± 0.64 5.94 ± 1.81 77.55 ± 4.71 1.24 ± 0.37 16.55 ± 3.58 0.10 ± 0.03 1.39 ± 0.54 9.45 ± 0.30 14.01 ± 0.80

8.16 ± 0.38 5.92 ± 1.47* 42.30 ± 1.38 14.99 ± 0.79 1016.2 ± 120.4 51.92 ± 1.09 18.38 ± 0.46 35.41 ± 0.83 2.20 ± 0.54 0.14 ± 0.04 2.36 ± 0.86 0.007 ± 0.007* 0.15 ± 0.10 0.12 ± 0.04 2.14 ± 0.86 4.51 ± 1.42* 75.14 ± 6.22 1.06 ± 0.18 18.69 ± 5.02 0.09 ± 0.04 1.51 ± 0.63 9.49 ± 0.24 13.57 ± 0.85

7.86 ± 0.41 4.99 ± 1.25 42.82 ± 1.73 14.75 ± 0.67 956.0 ± 79.2 54.58 ± 1.98 18.78 ± 0.43 34.44 ± 0.63 1.66 ± 0.55 0.08 ± 0.03 1.59 ± 0.54 0.008 ± 0.008 0.19 ± 0.10 0.12 ± 0.05 2.30 ± 0.83 4.07 ± 1.03 81.60 ± 5.50 0.64 ± 0.29 12.93 ± 5.04 0.07 ± 0.04 1.39 ± 0.62 8.77 ± 0.23 13.50 ± 0.81

7.889 ± 0.337 5.40 ± 1.83 42.43 ± 2.30 14.65 ± 0.76 968.6 ± 82.4 53.77 ± 1.21 18.57 ± 0.29 34.55 ± 0.48 1.83 ± 0.31 0.07 ± 0.03 1.33 ± 0.46 0.008 ± 0.006 0.14 ± 0.07 0.11 ± 0.15 2.38 ± 1.21 4.47 ± 1.72 82.02 ± 4.88 0.68 ± 0.25 13.13 ± 3.83 0.06 ± 0.03 1.00 ± 0.27 8.88 ± 0.41 12.96 ± 0.70

7.80 ± 0.03 7.14 ± 1.39* 42.29 ± 1.50 14.61 ± 0.52 959.8 ± 64.0 54.31 ± 1.83 18.77 ± 0.78 34.55 ± 0.64 1.79 ± 0.48 0.10 ± 0.03 1.47 ± 0.26 0.014 ± 0.008 0.18 ± 0.06 0.19 ± 0.06* 2.78 ± 0.86 5.72 ± 1.54 79.23 ± 8.05 0.99 ± 0.44 14.83 ± 8.02 0.109 ± 0.04 1.50 ± 0.44 8.91 ± 0.41 13.28 ± 1.38

7.77 ± 0.39 7.35 ± 2.6* 41.47 ± 1.40 14.44 ± 0.54 967.7 ± 83.0 53.42 ± 1.45 18.59 ± 0.66 34.8 ± 0.44 1.74 ± 0.38 0.13 ± 0.07 1.68 ± 0.52 0.015 ± 0.01 0.19 ± 0.07 0.15 ± 0.06 2.12 ± 0.58 5.92 ± 2.24* 80.36 ± 4.37 1.03 ± 0.39* 14.28 ± 4.52 0.10 ± 0.05 1.37 ± 0.46 8.85 ± 0.34 13.02 ± 0.76

Significantly different as compared to the control group, *P < 0.05. Abbreviations: APTT, activated partial thromboplastin time; Hb, hemoglobin; Ht, hematocrit; LUC, large unstained cell; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; PT, prothrombin time; RBC, red blood cell; WBC, white blood cell. a Ten animals/sex/group. b Values are expressed as mean ± SD.

Table 5 Clinical chemistry parameters following oral exposure to ESG for 13 weeks. Measured clinical chemistry parameters

AST (IU/L) ALT (IU/L) ALP (IU/L) LDH (IU/L) c-GTP (IU/L) CPK (IU/L) TBILL (mg/dL) TP (g/dL) ALB (g/dL) GLB (g/dL) CHO (mg/dL) TG (mg/dL) GLU (mg/dL) BUN (mg/dL) CRE (mg/dL) IP (mg/dL) Ca2+ (mg/dL) Na+ (mEq/L) K+ (mEq/L) Cl- (mEq/L) ALB (%) al-globulin (%) F2-globulin (%) b-globulin (%) c-globulin (%) A/G

Treatment groupa Males

Females

Control

3% ESG

10% ESG

30% ESG

Control

3% ESG

10% ESG

30% ESG

81.1 ± 10.4b 28.1 ± 6.3 297.4 ± 19.5 497.7 ± 162.1 1.4 ± 0.5 278.5 ± 74.9 0.08 ± 0.02 6.25 ± 0.44 3.21 ± 0.28 3.04 ± 0.25 89.9 ± 14.1 117.2 ± 39.4 167.4 ± 15.3 13.43 ± 1.34 0.28 ± 0.04 6.12 ± 1.10 10.15 ± 0.31 141.9 ± 0.7 4.33 ± 0.22 106.6 ± 1.7 51.20 ± 2.21 20.71 ± 2.06 6.89 ± 0.54 16.57 ± 0.84 4.63 ± 0.57 1.05 ± 0.10

73.9 ± 12.3 21.3 ± 3.2* 328.2 ± 60.3 604.9 ± 279.1 1.3 ± 0.5 313.4 ± 159.4 0.07 ± 0.02 6.25 ± 0.37 3.26 ± 0.18 2.99 ± 0.24 88.8 ± 17.3 122.90 ± 32.1 166.1 ± 12.7 14.31 ± 1.52 0.27 ± 0.03 7.18 ± 0.97 10.15 ± 0.26 141.5 ± 0.8 4.23 ± 0.19 107.1 ± 1.9 52.38 ± 1.59 19.94 ± 1.39 6.59 ± 0.56 16.60 ± 0.60 4.49 ± 0.58 1.10 ± 0.07

72.5 ± 11.7 22.1 ± 4.5* 285.9 ± 42.2 607.7 ± 326.9 1.4 ± 0.5 293.3 ± 126.8 0.09 ± 0.012 6.48 ± 0.34 3.45 ± 0.16* 3.03 ± 0.22 97.9 ± 24.3 100.5 ± 28.5 170.8 ± 16.5 14.43 ± 2.72 0.26 ± 0.04 6.64 ± 0.99 10.37 ± 0.24 142.7 ± 0.9 4.14 ± 0.16 107.1 ± 0.9 53.35 ± 1.65* 19.74 ± 1.53 6.37 ± 0.36* 16.10 ± 1.07 4.44 ± 0.72 1.15 ± 0.08*

66.3 ± 11.7* 21.2 ± 4.0* 353.4 ± 42.0* 437.2 ± 288.1 1.5 ± 0.5 239.0 ± 126.9 0.07 ± 0.02 6.22 ± 0.31 3.25 ± 0.20 2.97 ± 0.18 81.0 ± 15.8 95.7 ± 51.2 174.6 ± 34.6 13.36 ± 1.50 0.27 ± 0.04 6.44 ± 1.12 10.11 ± 0.24 142.1 ± 1.5 4.27 ± 0.19 107.7 ± 1.9 52.41 ± 1.85 20.10 ± 1.66 6.52 ± 0.36 16.36 ± 1.11 4.61 ± 0.70 1.10 ± 0.08

69.6 ± 9.2 22.5 ± 5.3 239.4 ± 58.8 516.7 ± 203.9 0.4 ± 0.5 271.0 ± 110.4 0.10 ± 0.03 6.74 ± 0.44 4.04 ± 0.36 2.70 ± 0.14 82.6 ± 12.9 116.8 ± 62.2 164.0 ± 45 15.29 ± 2.44 0.32 ± 0.03 6.48 ± 1.91 10.12 ± 0.40 139.6 ± 0.7 4.17 ± 0.62 108.7 ± 1.9 59.83 ± 1.86 15.66 ± 1.66 5.56 ± 0.42 13.56 ± 1.23 5.39 ± 1.24 1.49 ± 0.12

72.1 ± 13.7 22.4 ± 3.4 211.0 ± 64.7 517.2 ± 295 0.6 ± 0.5 263.9 ± 133.5 0.09 ± 0.04 6.64 ± 0.43 3.86 ± 0.37 2.78 ± 0.12 86.3 ± 15.6 140.1 ± 120.7 150.4 ± 11.9 16.44 ± 2.72 0.32 ± 0.03 6.09 ± 1.97 10.05 ± 0.34 139.4 ± 0.8 4.09 ± 0.20 107.8 ± 2.1 58.17 ± 2.07 16.15 ± 1.99 5.69 ± 0.38 14.35 ± 0.61 5.64 ± 1.15 1.40 ± 0.12

76.9 ± 9.6 24.2 ± 6.0 282.8 ± 125.1 591.2 ± 126 0.3 ± 0.5 299.7 ± 67.3 0.09 ± 0.02 6.58 ± 0.44 3.94 ± 0.31 2.64 ± 0.14 77.7 ± 12.5 118.4 ± 66.1 143.1 ± 8.9 16.68 ± 1.66 0.338 ± 0.026 5.669 ± 1.107 10.14 ± 0.31 139.5 ± 0.8 4.04 ± 0.17 107.7 ± 1.6 59.61 ± 1.03 15.05 ± 1.03 5.66 ± 0.31 13.93 ± 0.70 5.75 ± 0.86 1.48 ± 0.06

82.9 ± 15.3 22.3 ± 3.3 252.4 ± 101.1 710.3 ± 360.7 0.7 ± 0.5 349.6 ± 149.0 0.08 ± 0.03 6.19 ± 0.40 3.62 ± 0.32* 2.57 ± 0.14 72.3 ± 14.5 94.2 ± 23.5* 144.8 ± 16.0 16.81 ± 2.02 0.35 ± 0.05 6.39 ± 1.25 9.89 ± 0.33 139.6 ± 0.5 4.12 ± 0.39 108.4 ± 1.3 58.67 ± 2.11 14.79 ± 1.71 5.58 ± 0.29 14.97 ± 0.97* 5.99 ± 0.95 1.42 ± 0.13

Significantly different as compared to the control group, *P < 0.05. Abbreviations: A/G, albumin/globulin ratio; ALB, albumin; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; CHO, total cholesterol; CPK, creatine phosphokinase; CRE, creatinine; GLB, globulin; GLU, glucose; c-GTP, c-glutamyltranspeptidase; IP, inorganic phosphorus; LDH, lactate dehydrogenase; TBILL, total bilirubin; TG, triglycerides; TP, total protein. a Ten animals/sex/group. b Values are expressed as mean ± SD.

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3.2.4. Clinical pathology and histopathology Statistically significant increases in absolute and relative cecum weights in both males and females of the high-dose group compared to control animals were observed (Table 6). In addition, high-dose males also exhibited decreased absolute and relative thymus weights. Decreased absolute thyroid, heart, and kidney weights, as well as increased relative ovary, submaxillary gland, brain, and lung weights were observed in high-dose females. A significant decrease in absolute pituitary weights was observed in females at the mid-dose, and in males, decreased lung weights were observed at the mid-dose. At necropsy, reddening in the middle and accessory lobe of the right lung was observed in 1 control male. Increased cecal content was observed in all males and seven females of the high-dose group, and in four males and two females of the mid-dose group. Lightening of liver color and an enlarged liver were observed in two control males, one high-dose male, two mid-dose males, and one low-dose male. In the low-dose group, separation of the cervix from the uterine corpus was observed and shrinkage of the left thyroid gland was observed in two separate females. Several histopathological changes in the cecum of animals fed ESG were revealed upon microscopic examination of the organs and tissues (Table 7). Decreased goblet cell counts were observed in all high-dose animals, and two males and one female of the mid-dose group. Mononuclear cell infiltration of the lamina propria was observed in five high-dose females, one male and one female of the mid-dose group, and two females in the low-dose group. Other changes observed in the cecum of high-dose females included basophilia in the mucosal epithelium (5/10 animals), elevated basal granular cell counts (2/10 animals), and mucosal mineralization (1/10 animals). Decreased goblet cell counts and basophilia in the mucosal epithelium of the ileum were observed in one high-dose female. The same animal also exhibited decreased goblet cell counts, basophilia in the mucosal epithelium, and mononuclear cell infiltration of the lamina propria of the colon. The incidence and severity of other histopathological findings in other organs were comparable to the control group or were only observed in control animals (data not shown). 3.3. In vitro bacterial reverse mutation assay (Ames test) Using the pre-incubation method, all concentrations of ESG (19.5–5000 lg/plate) did not inhibit bacterial growth or induce a 2-fold increase in the number of revertant colonies in all strains tested in the presence or absence of metabolic activation, as compared to the negative control (data not shown). In addition, the concurrent positive controls induced a 2-fold increase in the number of revertant colonies, while the negative controls were within the range of historical laboratory values, thus confirming the sensitivity of the test and the activity of the S9-mix (data not shown). ESG, therefore, was determined to have no mutagenic activity, with or without metabolic activation, in the bacterial reverse mutation assay. 4. Discussion While several studies in animals were conducted to determine the potential beneficial effects of consuming synthesized glycogen as compared to NSG or other commercially available polysaccharides, the safety of ESG has not previously been evaluated in traditional toxicological testing. In the current series of studies, we report on the safety of ESG. Acute toxicity testing in male and female Crl:CD(SD) rats demonstrated that ESG did not induce adverse effects following oral administration at a dose of 2000 mg/kg body weight/day. Based

on these results, it was determined that ESG has a very low order of toxicity, with an oral LD50 value of greater than 2000 mg/kg body weight/day. In addition, in the standard bacterial reverse mutation assay, ESG failed to induce increases in the incidence of reverse mutations above negative control values in all the S. typhimurium and E. coli strains tested, and thus, was shown to be non-mutagenic in either the presence or absence of metabolic activation. In the 13-week oral toxicity study, all treated and control animals survived the experimental period in good general health. The white stools noted in both the mid-dose and high-dose male and female groups were likely attributed to the white color of the test article mixed in feed, which was being excreted undigested along with the stools. Additionally, water retention of stools due to the dietary fiber-like action of ESG (Furuyashiki et al., 2007; Takata et al., 2009), is suggested to be responsible for the soft stools noted in the high-dose male and female groups. The significant decrease in body weights observed in the highdose female rats was correlated with decreased food consumption. Similarly, a non-significant trend toward decreased body weight and food consumption was noted in male rats. As shown in Table 1, ESG replaced corn starch in the test diets, resulting in a decrease in the caloric values of the diets. The reduced caloric value, along with the dietary fiber-like effects of ESG (Furuyashiki et al., 2007), contributed to the lower body weights observed in ESGfed animals. Additionally, reduced food consumption in animals fed the high-dose diet may have resulted from the hardness of the test diet. Each of the diets used in subchronic study was tested for hardness, and it was shown that the 10% and 30% ESG-containing diets were at least 70% harder than the control and the 3% ESG diet (data not shown). Collectively, decreased food consumption in conjunction with the decreased nutrient value of the test article in relation to the diet, accounts for the decreased body weight gain and decreased body weights compared to the controls (Flamm et al., 2003), and as a result is not considered to be toxicologically significant (WHO, 1987). Low pH levels observed in the urine of the high-dose females were not considered to be test article-related, as no dose–response relationship was observed, no other significant findings in urinalysis parameters were reported, no significant differences in kidney function parameters were noted in clinical chemistry analysis, and no abnormalities in the kidneys were reported upon gross and histopathological examinations. The variations observed in the clinical chemistry parameters between treated and control animals could not be attributed to consumption of the test article, as they generally were not dosedependent. Moreover, none of these findings were accompanied by any macroscopic or histopathological changes. The differences were of small magnitude, and values did not exceed historical control ranges. The relationship to treatment of statistically significant hematological changes, including elevated leukocyte counts observed in females of the mid- and high-dose groups, and elevated lymphocyte and neutrophil counts in females of the high-dose group, is uncertain given that the magnitude of the changes was not large and the direction of the changes were not consistent across sex (i.e., general increase in WBC and lymphocytes in mid- and highdose females vs. decreases in high-dose males). It also is possible that the alterations in hematological parameters, despite the lack of consistency across sex, may have in part been associated with the histopathological changes noted in the cecum; changes which were indicative of ongoing inflammatory processes. It is known that active inflammation can be associated with changes in WBC, lymphocytes and neutrophils (Evans, 2009; Germolec et al., 2009; Hall and Everds, 2007). These parameters also are elevated in rats in which cecal inflammation is deliberately induced

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S. Tafazoli et al. / Regulatory Toxicology and Pharmacology 57 (2010) 210–219 Table 6 Absolute and relative organ weights following oral exposure to ESG for 13 weeks. Organ weights

Treatment groupa Males Control

Absolute organ weight (mg, Pituitary Thyroid (R) Thyroid (L) Thyroid (R + L) Adrenal (R) Adrenal (L) Adrenal (R + L) Testes (R) Testes (L) Testes (R + L) Ovary (R) Ovary (L) Ovary (R + L) Thymus Submaxillary gland (R) Submaxillary gland (L) Submaxillary gland (R + L) Spleen Brain Heart Lung Liver (g) Kidney (R) Kidney (L) Kidney (R + L) Cecum Epidydimis (R) Epidydimis (L) Epidydimis (R + L) Seminal Vesicle Prostate Uterus

unless otherwise 13.56 ± 1.97b 13.75 ± 3.56 12.71 ± 3.33 26.46 ± 6.53 27.35 ± 1.87 28.78 ± 1.67 56.13 ± 3.17 1799.3 ± 164.3 1781.8 ± 182.5 3581.1 ± 338.5 N/A N/A N/A 522.7 ± 172.6 430.3 ± 32.3 430.7 ± 36.1 861 ± 67.2 1073.8 ± 169.8 2252.7 ± 98.2 1759.5 ± 99 1632.2 ± 80.3 20.37 ± 3.19 1715.8 ± 115.1 1714.8 ± 145.3 3430.6 ± 256 3.172 ± 0.64 605.7 ± 119.8 625.4 ± 43.9 1231.1 ± 125.6 1856.4 ± 301.8 1780.2 ± 422.8 N/A

Females 3% ESG

10% ESG

30% ESG

Control

3% ESG

10% ESG

30% ESG

indicated) 12.46 ± 1.66 13.20 ± 3.37 13.00 ± 3.34 26.20 ± 6.01 24.23 ± 3.44 24.23 ± 3.44 50.74 ± 6.89 1750 ± 141.4 1728.3 ± 158 3478.3 ± 293.9 N/A N/A N/A 402.3 ± 81.4 395.2 ± 28.9 396.9 ± 35.5 792.1 ± 63.6

12.35 ± 1.19 12.52 ± 3.47 10.70 ± 2.62 23.22 ± 5.87 26.51 ± 3.76 30.29 ± 5.44 58.35 ± 10.26 1700 ± 117.9 1701.4 ± 120.2 3401.4 ± 232 N/A N/A N/A 399.9 ± 56 409 ± 50.5 404.8 ± 47.9 813.8 ± 97.6

12.62 ± 1.87 12.31 ± 2.93 10.92 ± 3.39 23.23 ± 5.82 26.60 ± 2.92 28.27 ± 2.90 54.87 ± 5.04 1739.6 ± 136.2 1735.3 ± 129.5 3474.9 ± 263.9 N/A N/A N/A 349.6 ± 56.9* 414.5 ± 48.2 410.6 ± 48.7 825.1 ± 95.7

16.07 ± 2.40 7.60 ± 2.23 8.40 ± 2.00 16.00 ± 3.72 27.94 ± 3.64 30.21 ± 3.95 58.15 ± 7.34 N/A N/A N/A 34.29 ± 5.64 36.41 ± 7.75 70.70 ± 12.39 361.8 ± 81.1 241.1 ± 26.6 237.8 ± 26.7 478.9 ± 52.4

13.98 ± 1.84 8.13 ± 1.50 6.67 ± 1.82 14.80 ± 2.31 28.84 ± 2.68 30.00 ± 3.56 58.84 ± 5.89 N/A N/A N/A 37.12 ± 5.66 34.08 ± 5.45 71.20 ± 7.26 343.9 ± 84.8 243.7 ± 14.2 242.7 ± 9.6 486.4 ± 20.1

13.85 ± 1.71* 8.19 ± 1.54 7.76 ± 1.27 15.95 ± 2.37 28.20 ± 4.69 30.06 ± 4.62 58.26 ± 9.17 N/A N/A N/A 33.04 ± 8.10 34.75 ± 6.88 67.79 ± 13.93 329.7 ± 49.9 244.7 ± 23.2 238 ± 26.9 482.7 ± 49.7

14.25 ± 1.72 6.88 ± 1.05 5.76 ± 1.61* 12.64 ± 2.09* 27.84 ± 3.06 27.97 ± 2.60 55.81 ± 5.51 N/A N/A N/A 37.60 ± 5.68 35.97 ± 3.79 73.57 ± 7.42 305.4 ± 112.8 230.3 ± 24.3 227.9 ± 20.8 458.2 ± 44.3

979.4 ± 115.1 2228.4 ± 124.7 1681.1 ± 184.3 1559.4 ± 86.3 18.73 ± 2.26 1684.4 ± 188.9 1651.5 ± 155.2 3335.9 ± 338.7 3.774 ± 0.776 602 ± 29.5 585.8 ± 31 1187.8 ± 57.6 1805.7 ± 220.7 1624.2 ± 232.5 N/A

956.1 ± 177.1 2215.7 ± 109.6 1660.3 ± 17.8 1495.8 ± 94.2* 18.48 ± 2.74 1672.9 ± 92.4 1636.2 ± 93.8 3309.1 ± 180.5 5.006 ± 1.88 591.8 ± 38.7 580.8 ± 50.3 1172.6 ± 83.5 1753.9 ± 222.7 1483.1 ± 229.8 N/A

957.7 ± 135.9 2257 ± 67.9 1699.2 ± 137 1582.4 ± 100.7 18.87 ± 4.37 1669.2 ± 214.4 1623.7 ± 196.3 3292.9 ± 402.6 9.916 ± 2.254* 645.5 ± 43.5 630.5 ± 39.1 1276 ± 81 1982.1 ± 219.5 1577 ± 237.7 N/A

643.6 ± 155 1977.5 ± 85.1 1058.5 ± 93.8 1180.7 ± 102.3 9.54 ± 1.20 896.4 ± 74.4 881.5 ± 72.8 1777.9 ± 140.1 2.403 ± 0.661 N/A N/A N/A N/A N/A 564.8 ± 138.8

629.1 ± 131.8 1952.1 ± 59.5 998.1 ± 79.8 1137.6 ± 92 9.57 ± 1.40 841.8 ± 69.6 824.9 ± 81.4 1666.7 ± 146.3 2.442 ± 0.724 N/A N/A N/A N/A N/A 838.1 ± 508.4

608.1 ± 117.5 1953.5 ± 47.1 975.2 ± 72.6 1100.4 ± 69.8 9.46 ± 1.28 853.4 ± 73.8 844.5 ± 85.5 1697.9 ± 157.1 3.474 ± 0.882 N/A N/A N/A N/A N/A 637 ± 321.5

602.1 ± 94.2 1953.1 ± 71.8 938.6 ± 96.4* 1122.5 ± 99.6 8.36 ± 0.62 785.2 ± 54* 766.6 ± 66.8* 1551.8 ± 116.6* 5.56 ± 2.021* N/A N/A N/A N/A N/A 499 ± 105.8

2.09 ± 0.37 2.06 ± 0.61 1.85 ± 0.68 3.92 ± 1.21 4.43 ± 0.51 4.71 ± 0.77 9.16 ± 1.19 290.9 ± 40.1 290.2 ± 39.2 581.2 ± 78.8 N/A N/A N/A 57.7 ± 6.1* 69 ± 8.5 68.3 ± 7.9 137 ± 16.3

4.91 ± 0.81 2.32 ± 0.72 2.54 ± 0.54 4.86 ± 1.14 8.58 ± 1.66 9.25 ± 1.56 17.84 ± 3.18 N/A N/A N/A 10.49 ± 2.06 11.13 ± 2.61 21.61 ± 4.40 110.6 ± 25.9 73.5 ± 8.5 72.4 ± 9.4 146.1 ± 17.5

4.46 ± 0.98 2.59 ± 0.67 2.16 ± 0.77 4.73 ± 1.26 9.11 ± 1.18 9.44 ± 1.15 18.56 ± 2.21 N/A N/A N/A 11.75 ± 2.32 10.88 ± 2.58 22.64 ± 4.18 107.8 ± 24.4 77.2 ± 10.2 77.1 ± 10.6 154.2 ± 20.5

4.60 ± 0.67 2.75 ± 0.62 2.58 ± 0.47 5.32 ± 0.99 9.32 ± 1.33 9.96 ± 1.46 19.30 ± 2.78 N/A N/A N/A 10.94 ± 2.49 11.51 ± 2.07 22.46 ± 4.14 109.4 ± 17.6 81.1 ± 5.9 78.7 ± 6.5 159.9 ± 11.9

5.23 ± 1.06 2.49 ± 0.52 2.11 ± 0.63 4.60 ± 0.99 10.12 ± 1.60 10.14 ± 1.33 20.27 ± 2.89 N/A N/A N/A 13.64 ± 2.30* 13.05 ± 1.81 26.67 ± 3.53* 108.4 ± 32.2 83.2 ± 9.4* 82.4 ± 7.4* 165.4 ± 16.5*

158.2 ± 12.2 376.5 ± 36.3 282.2 ± 20.6 263.0 ± 15.0 3.10 ± 0.453 277.4 ± 33.1 269.3 ± 25.3 546.7 ± 57.2 1.67 ± 0.45* 108.2 ± 15.9 105.7 ± 15.3 213.9 ± 31.4 331.7 ± 54.7 264.4 ± 54.6 N/A

195.7 ± 51.1 606 ± 76.8 322.2 ± 29.9 360 ± 38.1 2.89 ± 0.214 272.4 ± 17.7 268.8 ± 27.8 541.4 ± 44.4 0.74 ± 0.22 N/A N/A N/A N/A N/A 170.9 ± 34.4

198.3 ± 42.1 619.7 ± 85.9 314.4 ± 26.3 359.5 ± 42.5 3.00 ± 0.29 265.2 ± 21.2 259.6 ± 22.5 524.6 ± 42 0.78 ± 0.28 N/A N/A N/A N/A N/A 278.3 ± 219.4

200.3 ± 26.8 651.3 ± 65.5 323.9 ± 28.8 365.9 ± 32.8 3.12 ± 0.19 282.9 ± 18.1 279.5 ± 18.9 562.3 ± 35.8 1.15 ± 0.27 N/A N/A N/A N/A N/A 210.2 ± 103.9

217.7 ± 33.7 709.7 ± 86.8* 339.1 ± 33.9 404.8 ± 25* 3.02 ± 0.14 283.8 ± 21.2 276.8 ± 18.7 560.4 ± 38.3 1.98 ± 0.63* N/A N/A N/A N/A N/A 182 ± 46.9

Relative organ weight (mg/100 g bw, unless otherwise indicated) Pituitary 2.11 ± 0.37 1.95 ± 0.08 2.04 ± 0.16 Thyroid (R) 2.10 ± 0.51 2.08 ± 0.51 2.07 ± 0.60 Thyroid (L) 1.98 ± 0.52 2.06 ± 0.55 1.77 ± 0.45 Thyroid (R + L) 4.08 ± 0.97 4.17 ± 0.98 3.84 ± 1.01 Adrenal (R) 4.25 ± 0.54 3.85 ± 0.65 4.64 ± 0.93 Adrenal (L) 4.45 ± 0.56 4.23 ± 0.64 5.00 ± 1.00 Adrenal (R + L) 8.72 ± 1.06 8.06 ± 1.22 9.63 ± 190 Testes (R) 277.6 ± 26.2 279.1 ± 35.8 279.4 ± 15.1 Testes (L) 275.2 ± 31.5 275.4 ± 34.1 279.7 ± 15.5 Testes (R + L) 552.9 ± 56.9 554.3 ± 69.2 559.1 ± 29 Ovary (R) N/A N/A N/A Ovary (L) N/A N/A N/A Ovary (R + L) N/A N/A N/A Thymus 80.1 ± 22.8 64 ± 14.3 66.1 ± 10.9 Submaxillary gland (R) 66.5 ± 6.7 62.9 ± 7.0 67.8 ± 11.2 Submaxillary gland (L) 66.5 ± 6.8 63.3 ± 7.8 66.9 ± 10.7 Submaxillary 133.2 ± 13.4 126.2 ± 15 134.6 ± 21.8 gland(R + L) Spleen 165.6 ± 26.2 155.6 ± 19.4 156.5 ± 23.3 Brain 348.3 ± 31.6 354.5 ± 29.6 366 ± 37.1 Heart 271.5 ± 20.1 265.8 ± 12.6 272.4 ± 15.7 Lung 252.7 ± 25.9 247.9 ± 20.4 246.9 ± 26.4 Liver (g/100 g bw) 3.12 ± 0.27 2.96 ± 0.175 3.02 ± 0.254 Kidney (R) 265.1 ± 23.4 266.4 ± 17.2 275.4 ± 18.3 Kidney (L) 265 ± 29.1 261.4 ± 11.6 269.2 ± 15.6 Kidney (R + L) 529.9 ± 52.1 528 ± 27 544.6 ± 33 Cecum 0.49 ± 0.10 0.60 ± 0.14 0.83 ± 0.33 Epidydimis (R) 93.6 ± 19.2 96 ± 8.8 97.9 ± 11.8 Epidydimis (L) 96.5 ± 8.3 93.1 ± 8.4 96 ± 12.9 Epidydimis (R + L) 190.1 ± 21.9 189.0 ± 16.8 194.1 ± 23.9 Seminal Vesicle 287.2 ± 53.2 287.5 ± 43.8 289.7 ± 44.1 Prostate 274 ± 63 260.4 ± 51.3 245.6 ± 46.4 Uterus N/A N/A N/A

Significantly different as compared to the control group, *P < 0.05. Abbreviations: bw, body weight; ESG, enzymatically-synthesized glycogen; L, left; N/A, not applicable; R, right. a 10 animals/sex/group. b All values are reported as mean ± SD.

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Table 7 Histopathological findings in the cecum and ileum following oral exposure to ESG for 13 weeks. Measured parameters

Number of animals Male

Female

Control

3% ESG

10% ESG

30% ESG

Control

3% ESG

10% ESG

30% ESG

Cecum Decreased goblet count Basophilia in mucosal epithelium Mononuclear cell infiltration of lamina propria Elevated basal granular cell count Mucosal mineralization

0 0 0 0 0

0 0 0 0 0

2 0 1 0 0

10 0 0 0 0

0 0 0 0 0

0 0 2 0 0

1 0 1 0 0

10 5 5 2 1

Ileum Decreased goblet count Basophilia in mucosal epithelium Mononuclear cell infiltration of lamina propria

0 0 0

0 0 0

0 0 0

0 0 0

0 0 0

0 0 0

0 0 0

1 1 1

(Al-Gindan et al., 2009). Direct (systemic) test article effects on lymphocytes and neutrophils are less common than secondary effects (e.g., inflammation) (Weiss, 1993). A direct effect of ESG is particularly unlikely given that it is not likely to be absorbed to any significant extent. Significant changes in cecal weights and cecal contents in the high-dose male and female groups were attributed to the dietary fiber-like effects of ESG. These results also are in agreement with previous findings on the other dietary fibers such as soybean fiber (Levrat et al., 1991), potato starch (Mallett et al., 1988), pectin (Sacquet et al., 1982), and guar gum (Okazaki et al., 2002), which were attributed to an increase in the production of short-chain fatty acids, such as acetate, propionate or butyrate. Cecal weight increases are common findings in rodents fed excess amounts of materials such as carbohydrates, and are a well-known physiological adaptive response to high dietary concentrations of certain substances that alter the osmotic loading of the lower gut, and as such are not considered adverse (Adam et al., 2001; de Groot et al., 1974; Lynch et al., 1996; Scheppach, 1994; WHO, 1987). The observed reduction in thymus weight of the high-dose males was not considered to be of toxicological significance, as it was not accompanied by any gross or histopathological changes. Additionally, the significant reduction in absolute pituitary weights in females of the mid-dose group, and decreased absolute lung weights in the mid-dose males were considered to be incidental, due to the lack of dose-dependency. Decreased absolute thyroid, heart, and kidney weights were associated with decreased body weights in comparison to controls. The increased relative ovary, submaxillary gland, brain, spleen, and lung weights observed in the high-dose females were not considered to be toxicologically significant, as they were associated with differences in body weights, and were not accompanied by macroscopic or histopathological findings. The histopathological changes observed in the cecum, ileum, and colon were characterized as inflammatory responses. These changes were considered as a result of the prolonged presence of high concentrations of relatively hard and viscous undigested ESG in the intestinal tract. The presence of such material, when fed at 30% in the diet, would be expected to cause mechanical pressure and irritation to the intestinal epithelium. The greater susceptibility of female rats to these histopathological effects may have been due to the higher ESG consumption of females on a body weight basis (Table 2), resulting in larger amounts of unabsorbed ESG present in the cecum. Further support for a physical action of undigested ESG on the intestinal epithelium is the apparent lack of absorption of the test article to any significant degree. The results of an in vitro assay demonstrated that ESG was digested by porcine pancreatic a-amylase to form large molecules of a-macrodextrins with molecular weights greater than 1000 kDa (Takata

et al., 2009). As the large size of the macrodextrin would likely prohibit its absorption, systemic exposure to ESG or macrodextrin is expected to minimal in the current study. Based on the foregoing, the observations of inflammation in the ileum/cecum are considered of no toxicological concern as they are secondary to the physiological responses resulting from high carbohydrate levels in the ESG-containing diets fed to rats. All other histopathological changes were observed to occur at a similar frequency in treatment and control groups, or only occurred in the control group. Based on the results of the 13-week oral toxicity study, the noobserved-adverse-effect (NOAEL) level for ESG in rats was established to be 30% in the diet (equivalent to approximately 18,337 and 21,464 mg/kg body weight/day for male and female rats, respectively). In summary, an acute toxicity study indicated that ESG exhibits a very low order of acute toxicity in experimental rats by the oral route of exposure. ESG also did not display any mutagenic properties in an in vitro bacterial mutagenic assay. The effects noted in the 13-week subchronic study were not considered to be of toxicological concern, and therefore the NOAEL was established to be the highest concentration tested. Taken together, these data indicate that ESG would not pose a safety concern when used as a food ingredient.

Conflict of Interest statement The authors declare that there are no conflicts of interest.

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