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A subchronic toxicity study in rats and genotoxicity tests with an aqueous ethylcellulose dispersion
Food and Chemical Toxicology 43 (2005) 1355–1364 www.elsevier.com/locate/foodchemtox
A subchronic toxicity study in rats and genotoxicity tests with an aqueous ethylcellulose dispersion C.C. DeMerlis
a,*
, D.R. Schoneker a, J.F. Borzelleca
b
a
b
Colorcon, West Point, PA, USA Medical College of Virginia, Richmond, VA, USA Accepted 15 March 2005
Abstract SureleaseÒ Aqueous Ethylcellulose Dispersion is an excipient used as a modified release coating for beads, granules, non-pariels, drug crystals and tablets and for taste masking applications for drug products and dietary supplement products. A study was conducted to assess the toxicity of spray-dried Surelease when administered orally, via dietary admixture, to Sprague-Dawley CDÒ rats (20/sex/group) at dose levels of 0, 2000, 3500, and 5000 mg/kg/day for a period of at least 3 months. After 3 months of treatment, all rats scheduled for terminal sacrifice were killed and selected organs were weighed. Complete macroscopic examinations and histopathological evaluation of selected tissues were conducted on all animals. Neuropathological evaluations were performed on 5 animals/sex/group. No mortality occurred during the study. Clinical observations, ophthalmology, body weight and food consumption, hematology, coagulation, clinical chemistry, urinalysis, functional observational assessments, motor activity, organ weights and ratios and macroscopic and microscopic observations did not reveal any significant, consistent, dose-dependent test article-related adverse effects. The NOAEL (no-observed-adverse-effect-level) is 5000 mg/kg/day, the highest dose tested. A series of genotoxicity tests were conducted with Surelease. Surelease showed no evidence of mutagenic activity in the bacterial reverse mutation test with and without metabolic activation and in the in vitro cell mutation assay under the experimental conditions employed. Surelease did not show any evidence of causing chromosome damage or bone marrow cell toxicity when administered by gavage in the mouse micronucleus in vivo test procedure. These findings support the safety of Surelease for use as an excipient. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Ethylcellulose aqueous dispersion; Surelease; Ethylcellulose; Excipient; Modified release coating; Polymeric dispersion; Taste masking coating; Excipient safety; Sustained release coating
1. Introduction Surelease is an excipient used as a modified release coating for beads, granules, non-pariels, drug crystals and tablets as well as a modified release binder in matrix granulations. The US Food and Drug Administration defines modified release dosage forms as those whose drug-release characteristics of time course and/or loca-
*
Corresponding author. E-mail address: [email protected] (C.C. DeMerlis).
0278-6915/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2005.03.008
tion are chosen to accomplish therapeutic or convenience objectives not offered by conventional dosage forms such as a solution or an immediate release dosage form (US FDA Guidance, 1997). Surelease is also used for taste masking applications for drug products and dietary supplement products. Surelease is a complete plasticized aqueous polymeric dispersion using ethylcellulose as the rate controlling polymer. Surelease is referred to as a complete coating system because Surelease already contains the plasticizer which is premixed with the polymer during the manufacturing process.
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The administration of Surelease is typically by the oral route. Surelease has a long history of safe use in a number of products on a global basis. Surelease is used as a coating in drug products in the US, Europe, and other countries, and is also used in dietary supplement products regulated as foods in the US. Colorcon maintains Drug Master File number 9822 at the Center for Drug Evaluation and Research, US Food and Drug Administration to support the use of Surelease in drug products in the United States. Surelease is included in the FDA Inactive Ingredient Database for oral tablets (US FDA, 2004). The Surelease used in the studies described below is produced to meet global pharmaceutical standards using good manufacturing practices (GMPÕs). Surelease is characterized by proprietary specifications. The impurities are strictly monitored by process controls, stringent specifications and analytical methods developed by the manufacturer. The ingredients used in the manufacture of Surelease are commonly used food ingredients and pharmaceutical excipients. These ingredients meet the applicable compendial and regulatory requirements for the intended pharmaceutical uses in the United States, Europe and a number of other countries. These ingredients also meet the regulatory requirements for use as dietary supplement excipients in the United States. To appropriately characterize the toxicity of Surelease, a 90-day dietary study and three genotoxicity tests were conducted to provide current supportive information for Surelease.
2. Materials and methods This study was designed to meet or exceed the requirements of the following OECD (Organization for Economic Co-Operation and Development) Guidelines: 1. OECD Guidelines for Testing of Chemicals (1981) Subchronic Oral Toxicity—Rodent: 90-day Study, Guideline 408. 2. OECD: Guidelines for Testing of Chemicals (1997) Genetic Toxicology: Bacterial Reverse Mutation Test, Guideline 471. 3. OECD: Guidelines for Testing of Chemicals (1997) Genetic Toxicology: In vitro Mammalian Cell Gene Mutation Tests, Guideline 476. 4. OECD: Guidelines for Testing of Chemicals (1997) Genetic Toxicology: Mammalian Erythrocyte Micronucleus Test, Guideline 474. This study was also designed to meet or exceed the requirements of the FDA (United States Food and Drug Administration) 1993 ‘‘Redbook II’’ Toxicological Prin-
ciples for the Safety Assessment of Direct Food Additives and Color Additives Used in Food. This study was conducted at a contract research organization in compliance with: OECD Principles of Good Laboratory Practices ENV/MC/CHEM (98)17, EEC Good Laboratory Practices (90/18/EEC) and FDA Good Laboratory Practice Standards (Part 58 of 21 CFR). 2.1. Test article Surelease is a formulated product and contains the following ingredients: purified water, ethylcellulose, ammonium hydroxide, medium chain triglycerides, and oleic acid. Surelease is manufactured by Colorcon as a 25% solids aqueous ethylcellulose dispersion and was spray dried to a powder for use in this study. Surelease is available in several different formulations. Commercial samples of Surelease E-7-19010 from the same batch were provided by Colorcon (US) to the contract research organization. The test material was an off white to yellowish powder stored at room temperature. 2.2. Animals and husbandry Male and female 4 week old VAF/PlusÒ CDÒ (Sprague-Dawley derived) (Crl: CD (SD) IGS BR) rats were obtained from Charles River Laboratories (Kingston, NY, USA) and acclimated for 2 weeks prior to initiation of dosing. At initiation of treatment, animals were approximately 6 weeks old. The mean weight for the males was 193 g and the females 159 g. Individual weights of animals placed on test were within ±20% of the mean weight for each sex. All animals were examined during the acclimation period to confirm suitability for study and those considered unsuitable were eliminated. Animals were doubly housed in elevated, stainless steel, wire mesh cages during the first week of the acclimation period and individually housed thereafter. A 12-h light/dark cycle controlled via an automatic timer was provided. Temperature was monitored and maintained within the specified range of 19.9–25.1 °C. Relative humidity was monitored and maintained within the range of 24.75–85.63%. Water (Elizabethtown Water Company, Westfield, NJ, USA) was provided ad lib. Certified Rodent Diet, No. 5002 (meal) (PMI Nutrition International, St. Louis, MO) was available without restriction. Fresh feed was presented weekly. This study complied with all appropriate parts of the Animal Welfare Act Regulations: 9 CFR Parts 1 and 2 Final Rules, Federal Register, Volume 54, No. 168, August 31, 1989, pp. 36112–36163 effective October 30, 1989 and 9 CFR Part 3 Animal Welfare Standards; Final Rule, Federal Register, Volume 56, No. 32, February 15, 1991, pp. 6426–6505 effective March 18, 1991.
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2.3. Experimental design and test diet preparation Animals were randomly distributed into 4 groups of 20 animals per sex per group and received either untreated control diet or spray-dried Surelease mixed with control diet at dose levels 2000, 3500, and 5000 mg/kg/ day for a period of at least three months. Fresh diets were provided weekly and dietary concentrations were adjusted weekly based on body weight and feed consumption data from the preceding week. Prior to initiation of the study, analyses of homogeneity and stability of Surelease in low and high concentration diets were conducted. Confirmation analyses of all three dietary levels were assayed weekly for the first month, weeks 8, and 13 of the study. 2.4. In-life observations Cage-side observations for mortality and general condition were made at least twice daily (once in the morning and once in the afternoon). Cageside observations for clinical signs were made once daily, concurrent with one of the viability checks, for any signs of toxic or pharmacologic effects (e.g., abnormalities in general condition, appearance, activity, behavior, respiration). Animals were removed from their cages and examined twice pretest and once weekly during the study period. Examinations included observations of general condition, skin and fur, eyes, nose, oral cavity, abdomen and external genitalia, occurrence of secretions and excretions, and autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypy (e.g., excessive grooming, repetitive circling) or bizarre behavior (e.g., self-mutilation, walking backward) were recorded. An ophthalmoscopic examination was conducted pretest and at study termination. Lids, lacrimal apparatus and conjunctiva were examined grossly. The cornea, anterior chamber, lens, iris, vitreous humor, retina and optic disc were examined by indirect ophthalmoscopy. Mydriacyl 1% was used to induce mydriasis. Animals were removed from their cages and weighed twice pretest, weekly during treatment and terminally (after fasting). Terminal, fasted body weights were obtained just prior to necropsy. Feed was available without restriction 7 days/week. Animals were presented with full feeders of known weight. After 6 days, feeders were reweighed and the resulting weight was subtracted from the full feeder weight to obtain the grams consumed per animal over the 6-day period. Food consumption was measured (weighed) weekly, beginning one week prior to treatment. Neurobehavioral testing was staggered over four sessions with approximately equal numbers of animals per sex per dose group in each session. Noise level in the
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functional observational battery room was maintained within a level of 55–65 dB by a White Noise Generator, Model MDF 5001 (MDF Products, Inc. Danbury, Connecticut). Temperature, humidity, and illumination of each room were measured and recorded to ensure that variations in environmental conditions were minimal during all evaluations. The functional observational battery was performed for all animals before evaluations of motor activity. Using a modified version of SchulzeÕs procedures (Schulze, 1990), the locomotor activity of 10 animals/sex/group was monitored using an automated Photobeam Activity System (San Diego Instruments, Inc., San Diego, California). Sessions were 60 min in length; each session was divided into 12, 5-min intervals. The time of testing was balanced across treatment groups. The following evaluations were performed as part of the functional observational battery: home cage evaluations, handling evaluations, open field evaluations, reflex assessments, grip strength, landing foot splay, proprioception, air righting ability, and body weight. 2.5. Clinical pathology Hematology, clinical chemistry, and urinalysis evaluations were performed on days 30 and 60 and at study termination on 10 animals/sex/group. Coagulation parameters were performed on day 60 and at study termination on 10 animals/sex/group. Blood was obtained from the retrobulbar venous plexus of fasted rats under light CO2/O2 anesthesia. Hematology parameters included hemoglobin concentration, hematocrit, erythrocyte count, platelet count, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, total leukocyte count, reticulocyte count, differential leukocyte count and erythrocyte and platelet morphology. Coagulation parameters evaluated were prothrombin time and active partial thromboplastin time. Clinical chemistry determinations included aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, blood urea nitrogen, creatinine, glucose, cholesterol, triglycerides, total protein, albumin, total bilirubin, sodium, potassium, chloride, calcium, inorganic phosphorus, gamma-glutamyl transferase, globulin, and albumin/globulin ratio. Urinalysis included volume, appearance, and specific gravity and the following parameters were analyzed: protein, nitrites, glucose, ketones, pH, bilirubin, and urobilinogen. 2.6. Pathology Carbon dioxide inhalation was used as the method of euthanasia. At the end of the treatment period, all animals were subjected to a complete macroscopic postmortem examination, which included examination of
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the external surface and all orifices; the external surfaces of the brain and spinal cord; the organs and tissues of the cranial, thoracic, abdominal and pelvic cavities and neck; and the remainder of the carcass for the presence of macroscopic morphologic abnormalities. The following organs were weighed: adrenal glands, brain, heart, kidneys, liver, ovaries, pituitary, prostate, spleen, testes, thymus, thyroid, and uterus (with cervix). All tissues were preserved in 10% neutral buffered formalin. Eyes were placed in glutaraldehyde/paraformaldehyde initially and then retained in 10% formalin. Testes and epididymides were placed in Modified DavidsonÕs solution initially and then retained in 10% formalin. Lungs and urinary bladder were infused with formalin prior to their immersion into a larger volume of the same fixative. After fixation, the tissues and organs from all animals were routinely processed and embedded in paraffin; these tissues were sectioned and stained with hematoxylin and eosin and examined by light microscopy. The following tissues were examined: adrenal glands, aorta, bone marrow smear, bone, bone marrow, brain, epididymides, esophagus, eyes with optic nerve, head, heart, kidneys, lacrimal glands, large intestine, larynx, liver, lungs, lymph nodes, mammary glands, muscle, nerve, ovaries, pancreas, PeyerÕs patches, pharynx, pituitary, prostate, salivary glands, seminal vesicles, skin, small intestine, spinal cord, spleen, stomach, testes, thymus, thyroid, tongue, trachea, urinary bladder, uterus (with cervix), vagina, ZymbalÕs gland, and all gross lesions. The bones were decalcified in Decalcifier IITM. Slides of the indicated tissues were prepared and examined microscopically for all animals in the control and high-dose groups. Tissues with macroscopic lesions were examined in all groups. Any abnormalities not noted during macroscopic examinations which were seen during histology processing were recorded. 2.7. Neuropathology Five animals per sex per group (selected from animals used for neurobehavioral studies) were anesthetized with Acepromazine/Ketamine/Xylazine and transcardially perfused with phosphate buffered saline followed by 1% glutaraldehyde and 4% paraformaldehyde in the same buffer. Slides of the indicated tissues were prepared and examined microscopically for all animals in the control and high-dose groups. The following tissues were examined: brain (forebrain, cerebral cortex, hippocampus, basal ganglia, midbrain, cerebellum and pons, medulla), eye with optic nerve, spinal cord (cervical, thoracic, lumbar, cross and longitudinal sections), sciatic nerve (cross and longitudinal sections), tibial nerve (cross and longitudinal sections), sural nerve (cross and longitudinal sections), trigeminal ganglia, dorsal root ganglia (from C3 to C6 and L4 to L6), dorsal root fibers (from C3 to C6 and L4 to L6) and ventral root
fibers (from C3 to C6 and L4 to L6). Tissues with macroscopic lesions were examined in all groups. After fixation, the peripheral nerves of all neuropathology animals were processed, embedded in epoxy resin and cut at a microtome setting of 2 lm. All other tissues, including the brain and spinal cord, ganglia and dorsal and ventral root fibers were routinely processed and embedded in paraffin. Plastic sections of peripheral nerves were stained with toluidine blue and all other nervous tissue were stained with hematoxylin and eosin. The brain and spinal cord sections were stained with hematoxylin and eosin. 2.8. Statistical analyses The following parameters were analyzed statistically: body weight, body weight change from baseline, feed consumption, hematology, coagulation, clinical chemistry, organ weights, motor activity counts, forelimb and hindlimb grip strength measurements and landing foot splay measurements. Mean values of all dose groups were compared to the mean value for the control group at each time interval. Evaluation of equality of group means was made by the appropriate statistical method, followed by a multiple comparison test if needed. BartlettÕs test (Bartlett, 1937; Sokal and Rohlf, 1995) was performed to determine if groups had equal variances. For all parameters except organ weights, if the variances were equal, parametric procedures were used; if not, non-parametric procedures were used. Organ weight data was analyzed only by parametric methods. The parametric method was the standard one-way analysis of variance (ANOVA) using the F ratio to assess significance (Armitage, 1971; Dunlap and Duffy, 1975). If significant differences among the means were indicated, additional tests were used to determine which means were significantly different from the control: DunnettÕs (Dunlap et al., 1981; Dunnett, 1955, 1964), Williams (Williams, 1971, 1972), or Cochran and CoxÕs modified t-test (Cochran and Cox, 1959). The non-parametric method was the Kruskal–Wallis test (Kruskal and Wallis, 1952, 1953) and if differences were indicated, ShirleyÕs test (Shirley, 1977), SteelÕs test (Steel, 1959) or Pairwise Comparison with Bonferroni Correction (Games and Howell, 1976) were used to determine which means differed from control. BartlettÕs test for equality of variance was conducted at the 1% significance level; all other statistical tests were conducted at the 5% and 1% significance levels. Motor activity counts were collected in 12 consecutive 5-min intervals on each test day. The set of counts over these 12 intervals gave a ‘‘profile’’ of an animalÕs motor activity. These profiles were compared within each study period using a multivariate model that assessed whether the profiles for different groups had
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the same shape (the parallel hypothesis). The WilksÕ Lambda, a variant of the Hotelling T2 (Morrison, 1976) were used to test the parallel hypothesis; twoway ANOVA with interaction (OÕBrien and Kaiser, 1985) were used to test for the overall treatment effect with respect to the level hypothesis followed, if necessary, by DunnettÕs t-test to find groups that differed from control. The Shapiro–Wilk test (Shapiro and Wilk, 1965) were used to assess normality of the residuals from the multivariate model; when p < 0.01 for this test, the Blom inverse normal transformation (Blom, 1958) were applied to achieve normality of the residuals. Other tests were deemed significant at the p = 0.05 level. 2.9. Ames test In this in vitro assessment of the mutagenic potential of Spray-Dried Surelease Aqueous Ethylcellulose Dispersion, histidine dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan dependent mutant of Escherichia coli, strain WP2uvrA/pKM101 (CM891), were exposed to Surelease diluted in acetone. Acetone was also used as a negative control. Sodium azide, 9-aminoacridine, 2-nitrofluorene and 2-(2-furyl)-3-(5nitro-2-furyl)acrylamide were used as positive controls in the absence of the S9 fraction and 2-aminoanthracene and benzo[a]pyrene were used as positive controls in the presence of the S9 fraction. Two independent mutation tests were performed in the presence and absence of liver preparations from Aroclor 1254-treated rats (S9 mix). Both tests were standard plate incorporation assays.
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2.11. Mouse micronucleus test This study was designed to assess the potential induction of micronuclei by Spray-Dried Surelease Aqueous Ethylcellulose Dispersion in bone marrow cells of mice. Mice were treated with a single oral administration of the test substance at dose levels of 500, 1000 and 2000 mg/kg body weight. A preliminary toxicity test had previously shown that a dose of 2000 mg/kg (the standard limit dose for the micronucleus test) was tolerated; this level was therefore selected as an appropriate maximum for use in the micronucleus test. The test substance, negative and positive control substances were administered by gavage. The negative control group received the vehicle, corn oil and the positive control group received mitomycin C at 12 mg/kg body weight. Following the preliminary toxicity test, no substantial differences in toxicity were observed between the sexes, consistent with current guidelines. The micronucleus test was performed using male animals only. Bone marrow smears were obtained from seven male animals in the negative control, each of the test substance groups and five male animals in the positive control group 24 h after dosing. In addition, bone marrow smears were obtained from seven male animals in the negative control and high level treatment groups 48 h after dosing. One smear from each animal was examined for the presence of micronuclei in 2000 immature erythrocytes. The proportion of immature erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal. A record of the incidence of micronucleated mature erythrocytes was also kept.
2.10. Mouse lymphoma test
3. Results
Spray-Dried Surelease Aqueous Ethylcellulose Dispersion was tested for mutagenic potential in an in vitro mammalian cell mutation assay. This test system is based on detection and quantitation of forward mutation in the subline 3.7.2.c of mouse lymphoma L5178Y cells, from the heterozygous condition at the thymidine kinase locus (TK+/ ) to the thymidine kinase deficient genotype (TK / ). Two independent mutagenicity tests were carried out in both the absence and presence of S9 mix with a 3-h exposure, and one in the absence of S9 mix with a 24-h exposure. The maximum concentration of Surelease to which the cells were exposed was limited by solubility. The solvent chosen was ethanol, and the highest final concentration that was judged to be usable was 250 lg/ml. Methyl methanesulphonate was used as a positive control in the absence of the S9 fraction and 3-methylcholanthrene was used as a positive control in the presence of the S9 fraction.
3.1. Mortality No mortality occurred during the study. 3.2. Physical observations and ophthalmoscopic examinations Weekly clinical observations recorded during the study were considered common findings in laboratory rats and unrelated to test article administration. Administration of Surelease did not result in any test article-related ocular findings. 3.3. Body weights and food consumption There were no statistically significant or treatment related adverse effects on body weights (Figs. 1 and 2). Feed consumption values of females administered 3500 and 5000 mg/kg/day of Surelease were slightly greater
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550 135
525
Group 1 - 0 mg/kg/day (Control) Group 2 - 2000 mg/kg/day
500
Group 3 - 3500 mg/kg/day
125
Group 4 - 5000 mg/kg/day
475 450
115
Feed Consumption (g/kg/day)
425
Body Weight (grams)
400 375 350 325 300 275
105
95
85
75
250 65
225 200
55
Group 1 - 0 mg/kg/day (Control) 175
Group 2 - 2000 mg/kg/day
150
Group 3 - 3500 mg/kg/day
45
Group 4 - 5000 mg/kg/day 125 35
100 -8 -3 2
-2
7 12 17 22 27 32 37 42 47 52 57 62 67 72 77 82 87 92
3
8 13 18 23 28 33 38 43 48 53 58 63 68 73 78 83 88 Days
Day
Fig. 3. Mean food consumption: males.
Fig. 1. Mean body weights: males.
325 Group 1 - 0 mg/kg/day (Control)
135
Group 2 - 2000 mg/kg/day Group 3 - 3500 mg/kg/day
300
Group 4 - 5000 mg/kg/day
125
275 115
Feed Consumption (g/kg/day)
Body Weight (grams)
250
225
200
175
150
105
95
85
75
65
Group 1 - 0 mg/kg/day (Control) Group 2 - 2000 mg/kg/day
125
Group 3 - 3500 mg/kg/day
55
Group 4 - 5000 mg/kg/day 100
45
-8 -3 2
7 12 17 22 27 32 37 42 47 52 57 62 67 72 77 82 87 92 Day
Fig. 2. Mean body weights: females.
-2
3
8
13 18 23 28 33 38 43 48 53 58 63 68 73 78 83 88 Day
Fig. 4. Mean food consumption: females.
C.C. DeMerlis et al. / Food and Chemical Toxicology 43 (2005) 1355–1364 Table 1 Mean test material intake values Group
2 3 4
Desired dose (mg/kg/day)
2000 3500 5000
Mean test article intake (mg/kg/day) Males
Females
1986 3497 5007
1985 3456 4948
and statistically significant when compared to control values during the study (Figs. 3 and 4). Feed consumption values of test article-treated males and females fed 2000 mg/kg/day were comparable to control values. Mean test material intake values are presented in Table 1 and indicate that animals received the appropriate dose. 3.4. Neurobehavioral observations No test article-related effects were evident in motor activity for either sex in any of the treatment groups. Test article administration did not affect the neurological condition of the animals as measured by a functional observational battery of assessments. Landing foot splay and grip strength for all test article-treated groups were comparable to control values or within the range of normal variation. 3.5. Clinical laboratory studies—hematology, coagulation and clinical chemistry No test article-related adverse effects were noted in the hematology parameters, clinical chemistry parameters, and urinalysis data evaluated on Day 30, 60 and at study termination. No test article-related adverse effects were noted in the coagulation parameters evaluated on Day 60 and at study termination. 3.6. Terminal organ weights, organ/body weight ratio and organ/brain weight ratio There were no statistically significant effects on terminal organ weights, organ/body weight ratio and organ/ brain weight ratio (Tables 2 and 3). 3.7. Pathology—macroscopic Macroscopic findings occurred with comparable incidence and severity in control or test article-treated groups or they occurred sporadically as commonly seen in rats of this strain and age at the test facility. 3.8. Pathology—microscopic There was no evidence of neurotoxicity in the subset of animals sampled for detailed neuropathological eval-
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uation. Occasional degenerate fibers were observed in the peripheral nerves from both control and Surelease treated animals, but the incidence was similar between test article-treated groups and control groups. The remaining protocol listed tissues from this subset of perfusion-fixed animals were also examined microscopically and no treatment related findings attributable to administration of Surelease were noted. There were no microscopic findings in other tissues examined that were attributable to Surelease. Mild inflammatory changes were present in the lungs from a proportion of animals from the control and Surelease treated groups and probably reflect a minor respiratory infection and are not toxicologically significant. Focal inflammatory changes were present in the Harderian glands in the control and Surelease treated animals. The findings were frequently unilateral and are the result of intraorbital bleeding procedures and not treatment related. All other microscopic findings were typical of the normal background pathology seen in this age and strain of rat. 3.9. Ames test Concentrations of Spray-Dried Surelease Aqueous Ethylcellulose Dispersion up to 5000 lg/plate were tested. This is the standard limit concentration recommended in the regulatory guidelines. Other concentrations used were a series of ca. half-log10 dilutions of the highest concentration. No signs of toxicity were observed towards the tester strains in either mutation test. No evidence of mutagenic activity was seen at any concentration of Surelease in either mutation test. The concurrent positive controls demonstrated the sensitivity of the assay and the metabolizing activity of the liver preparations. It is concluded that, under the test conditions employed, Surelease showed no evidence of mutagenic activity in this bacterial system. 3.10. Mouse lymphoma test Surelease was relatively non-toxic in this mammalian cell culture system in all tests, as measured by Day0 relative survival, either in the absence or presence of S9 mix. At least two precipitating concentrations were assessed in the main mutagenicity tests, with the effects of a maximum concentration of 250 lg/ml being tested on each occasion. No significant increases in mutant frequency were observed in the first main test in the presence of S9 mix or in the second main test in either the absence or presence of S9 mix.
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Table 2 Organ weights in male rats after being fed SureleaseÒ for 3 months Parameter
0 mg/kg/day
2000 mg/kg/day
3500 mg/kg/day
5000 mg/kg/day
Mean weight (g) Terminal body weight Adrenal glands Brain Heart Kidneys Liver Pituitary gland Prostate Spleen Testes Thymus Thyroid/parathyroids
505.10 ± 43.80 0.08 ± 0.02 2.31 ± 0.18 1.75 ± 0.17 4.42 ± 0.46 16.78 ± 2.89 0.02 ± 0.01 1.33 ± 0.41 0.94 ± 0.09 3.46 ± 0.21 0.37 ± 0.10 0.03 ± 0.01
499.60 ± 51.20 0.08 ± 0.01 2.40 ± 0.16 1.72 ± 0.27 4.45 ± 0.63 16.11 ± 2.82 0.01 ± 0.00 1.36 ± 0.36 0.89 ± 0.17 3.54 ± 0.28 0.35 ± 0.12 0.03 ± 0.00
520.50 ± 54.00 0.07 ± 0.01 2.39 ± 0.16 1.75 ± 0.23 4.39 ± 0.47 16.17 ± 3.02 0.01 ± 0.00 1.49 ± 0.35 0.91 ± 0.13 3.49 ± 0.28 0.40 ± 0.09 0.03 ± 0.01
512.00 ± 36.20 0.08 ± 0.01 2.38 ± 0.11 1.75 ± 0.22 4.34 ± 0.36 16.05 ± 2.56 0.01 ± 0.00 1.46 ± 0.30 0.99 ± 0.16 3.50 ± 0.29 0.39 ± 0.10 0.03 ± 0.01
Mean organ-to-terminal body weight ratios Terminal body weight 505.10 ± 43.80 Adrenal glands 0.02 ± 0.00 Brain 0.46 ± 0.06 Heart 0.35 ± 0.04 Kidneys 0.89 ± 0.09 Liver 3.32 ± 0.46 Pituitary gland 0.00 ± 0.00 Prostate 0.27 ± 0.09 Spleen 0.16 ± 0.02 Testes 0.69 ± 0.08 Thymus 0.07 ± 0.02 Thyroid/parathyroids 0.01 ± 0.00
499.60 ± 51.20 0.02 ± 0.00 0.48 ± 0.05 0.34 ± 0.04 0.89 ± 0.10 3.21 ± 0.36 0.00 ± 0.00 0.28 ± 0.06 0.18 ± 0.02 0.72 ± 0.09 0.07 ± 0.02 0.01 ± 0.00
520.50 ± 54.00 0.01 ± 0.00 0.46 ± 0.05 0.34 ± 0.03 0.85 ± 0.08 3.09 ± 0.37 0.00 ± 0.00 0.29 ± 0.07 0.17 ± 0.02 0.67 ± 0.06 0.08 ± 0.02 0.01 ± 0.00
512.00 ± 36.20 0.01 ± 0.00 0.47 ± 0.05 0.34 ± 0.05 0.85 ± 0.08 3.13 ± 0.44 0.00 ± 0.00 0.29 ± 0.06 0.18 ± 0.03 0.69 ± 0.08 0.08 ± 0.02 0.01 ± 0.00
Mean organ-to-brain weight ratios Terminal body weight 505.10 ± 43.80 Adrenal glands 3.52 ± 0.70 Brain 100.00 ± 0.00 Heart 75.75 ± 5.01 Kidneys 191.40 ± 15.69 Liver 730.60 ± 140.58 Pituitary gland 0.64 ± 0.19 Prostate 57.29 ± 15.48 Spleen 40.55 ± 3.71 Testes 150.57 ± 14.08 Thymus 15.93 ± 4.15 Thyroid/parathyroids 1.43 ± 0.28
499.60 ± 51.20 3.13 ± 0.50 100.00 ± 0.00 71.42 ± 8.61 185.12 ± 21.09 671.47 ± 110.53 0.60 ± 0.17 56.58 ± 13.02 37.07 ± 6.61 147.95 ± 12.24 14.66 ± 4.74 1.33 ± 0.19
520.50 ± 54.00 3.07 ± 0.59 100.00 ± 0.00 73.43 ± 8.77 184.38 ± 20.60 678.28 ± 124.98 0.60 ± 0.13 62.20 ± 13.83 37.93 ± 4.22 146.49 ± 12.43 16.80 ± 3.34 1.43 ± 0.28
512.00 ± 36.20 3.17 ± 0.55 100.00 ± 0.00 73.44 ± 8.16 182.95 ± 18.03 677.97 ± 127.51 0.58 ± 0.12 61.19 ± 12.05 38.58 ± 6.12 147.19 ± 12.17 16.46 ± 4.37 1.39 ± 0.27
In the absence of S9 mix in first main mutagenicity test, exposure to 250 lg/ml of Surelease for 3 h was associated with a statistically significant increase in mutant frequency, where Day0 relative survival was 78% of the solvent controls. However, the mutant frequency of 0.000268 lay within the upper 95% confidence limits for the negative historical control. Other concentrations tested, namely 12.5, 25, 50, 100 and 200 lg/ml, where Day0 relative survival values were 106–80% of solvent controls, did not cause any significant increases in mutant frequency. In all tests the positive control substances increased mutant frequency significantly. It was concluded that Surelease did not demonstrate mutagenic potential in this in vitro cell mutation assay under the experimental conditions described.
3.11. Mouse micronucleus test No statistically significant increases in the frequency of micronucleated immature erythrocytes and no substantial decreases in the proportion of immature erythrocytes were observed in mice treated with SprayDried Surelease Aqueous Ethylcellulose Dispersion and killed 24 or 48 h later, compared to vehicle control values (P > 0.01 in each case). The positive control compound, mitomycin C, produced significant increases in the frequency of micronucleated immature erythrocytes (P < 0.01). It is concluded that Surelease did not show any evidence of causing chromosome damage or bone marrow cell toxicity when administered orally by gavage in this in vivo test procedure.
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Table 3 Organ weights in female rats after being fed SureleaseÒ for 3 months Parameter
0 mg/kg/day
2000 mg/kg/day
3500 mg/kg/day
5000 mg/kg/day
Mean weight (g) Terminal body weight Adrenal glands Brain Heart Kidneys Liver Pituitary gland Ovaries Spleen Uterus Thymus Thyroid/parathyroids
282.00 ± 22.30 0.08 ± 0.01 2.10 ± 0.11 1.11 ± 0.16 2.47 ± 0.29 9.15 ± 1.34 0.02 ± 0.00 0.10 ± 0.02 0.64 ± 0.07 0.76 ± 0.10 0.34 ± 0.07 0.03 ± 0.00
280.40 ± 29.10 0.07 ± 0.01 2.08 ± 0.14 1.07 ± 0.14 2.38 ± 0.18 8.64 ± 1.23 0.02 ± 0.00 0.10 ± 0.01 0.64 ± 0.10 0.97 ± 0.47 0.32 ± 0.10 0.03 ± 0.00
289.90 ± 29.20 0.08 ± 0.00 2.15 ± 0.19 1.16 ± 0.17 2.62 ± 0.39 9.72 ± 1.45 0.02 ± 0.01 0.11 ± 0.02 0.69 ± 0.12 0.87 ± 0.35 0.37 ± 0.09 0.03 ± 0.01
274.50 ± 25.70 0.08 ± 0.01 2.09 ± 0.17 1.15 ± 0.14 2.47 ± 0.20 9.17 ± 0.85 0.02 ± 0.00 0.10 ± 0.02 0.68 ± 0.13 0.79 ± 0.22 0.35 ± 0.11 0.03 ± 0.00
Mean organ-to-terminal body weight ratios Terminal body weight 282.00 ± 22.30 Adrenal glands 0.027 ± 0.00 Brain 0.75 ± 0.07 Heart 0.39 ± 0.05 Kidneys 0.87 ± 0.08 Liver 3.24 ± 0.39 Pituitary gland 0.01 ± 0.00 Ovaries 0.03 ± 0.00 Spleen 0.23 ± 0.03 Uterus 0.27 ± 0.05 Thymus 0.12 ± 0.02 Thyroid/parathyroids 0.01 ± 0.00
280.40 ± 29.10 0.03 ± 0.00 0.75 ± 0.07 0.38 ± 0.05 0.85 ± 0.06 3.08 ± 0.23 0.01 ± 0.00 0.04 ± 0.00 0.23 ± 0.02 0.35 ± 0.17 0.11 ± 0.03 0.01 ± 0.00
289.90 ± 29.20 0.03 ± 0.00 0.75 ± 0.08 0.40 ± 0.05 0.90 ± 0.09 3.36 ± 0.41 0.01 ± 0.00 0.04 ± 0.01 0.24 ± 0.03 0.30 ± 0.12 0.13 ± 0.03 0.01 ± 0.00
274.50 ± 25.70 0.03 ± 0.00 0.77 ± 0.08 0.42 ± 0.05 0.90 ± 0.08 3.36 ± 0.41 0.01 ± 0.00 0.04 ± 0.01 0.25 ± 0.05 0.29 ± 0.07 0.13 ± 0.03 0.01 ± 0.00
Mean organ-to-brain weight ratios Terminal body weight 282.00 ± 22.30 Adrenal glands 3.61 ± 0.57 Brain 100.00 ± 0.00 Heart 52.55 ± 6.38 Kidneys 117.31 ± 11.69 Liver 436.44 ± 65.00 Pituitary gland 0.79 ± 0.23 Ovaries 4.52 ± 1.00 Uterus 35.97 ± 4.36 Spleen 30.38 ± 3.03 Thymus 16.11 ± 3.58 Thyroid/parathyroids 1.30 ± 0.24
280.40 ± 29.10 3.56 ± 0.66 100.00 ± 0.00 51.66 ± 5.34 114.80 ± 8.20 416.14 ± 53.25 0.85 ± 0.12 4.88 ± 0.60 46.30 ± 21.17 30.67 ± 4.21 15.41 ± 4.88 1.39 ± 0.20
289.90 ± 29.20 3.53 ± 0.32 100.00 ± 0.00 53.91 ± 5.21 121.32 ± 12.08 452.79 ± 69.59 0.84 ± 0.27 4.95 ± 1.05 40.49 ± 16.14 32.10 ± 4.04 17.27 ± 4.21 1.32 ± 0.27
274.50 ± 25.70 3.65 ± 0.36 100.00 ± 0.00 54.74 ± 4.09 118.11 ± 5.82 439.46 ± 43.94 0.88 ± 0.16 4.98 ± 0.73 37.95 ± 10.64 32.62 ± 5.55 16.59 ± 4.91 1.30 ± 0.29
4. Discussion Surelease is widely used and applications are expanding into dietary supplements and additional drug product uses. Colorcon has developed additional information and data to support the expanded uses. Feed consumption values of females administered 3500 and 5000 mg/kg/day of Surelease were slightly greater and statistically significant when compared to control values during the study. This was attributed to the need to maintain caloric intake necessary for normal growth as a result of the large amounts of Surelease in the diets. Feed consumption values of test article-treated males and females fed 2000 mg/kg/day were comparable to control values.
The test results indicate that Surelease is not genotoxic. There were occasional statistically significant and apparently dose related effects noted (for example, sodium, chloride and hematology), but in the absence of functional changes and histopathological findings these effects were, therefore, not considered toxicologically or biologically significant. Surelease administered in the diet to male and female Sprague-Dawley rats at doses of 2000, 3500 and 5000 mg/kg/day for 90 days did not result in statistically significant, consistent, dose-dependent and treatment related adverse effects on mortality, body weights, hematology, clinical chemistry, urinalysis data, functional observational assessments, motor activity, organ weight
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data, and macroscopic and microscopic observations. Therefore, the No-Observed-Adverse-Effect-Level (NOAEL) is 5000 mg/kg/day. In conclusion, the data and information summarized in this paper demonstrate that Surelease, meeting appropriate specifications, as intended for use in film-coating applications in pharmaceutical and dietary supplement products, does not present a safety concern for humans. References Armitage, P., 1971. Statistical Methods in Medical Research. Blackwell Scientific Publications, Oxford, UK. Bartlett, M.S., 1937. Properties of sufficiency and statistical tests. Proceedings of the Royal Society, Series A 160, 268–282. Blom, G., 1958. Statistical Estimates and Transformed Beta Variables. John Wiley and Sons, New York. Cochran, W.G., Cox, G.M., 1959. Experimental Designs. John Wiley, New York. Dunlap, W.P., Duffy, J.A., 1975. Fortran IV functions for calculating exact probabilities associated with z, chi-square, t and f values. Behavior Research Methods & Instrumentations 7, 59–60. Dunlap, W.P., Marx, M.S., Agamy, G.G., 1981. Fortran IV functions for calculating probabilities associated with DunnettÕs test. Behavior Research Methods & Instrumentation 13, 363–366. Dunnett, C.W., 1955. A multiple comparison procedure for comparing several treatments with a control. Journal of the American Statistical Association 50, 1096–1121. Dunnett, C.W., 1964. New tables for multiple comparisons with a control. Biometrics 20 (3), 482–491. Games, P.A., Howell, J.F., 1976. Pairwise multiple comparison procedures with unequal nÕs and/or variances: a Monte-Carlo study. Journal of Educational Statistics 1, 113–125.
Kruskal, W.H., Wallis, W.A., 1952. Use of ranks in one-criterion variance analysis. Journal of the American Statistical Association 47, 583–621. Kruskal, W.H, Wallis, W.A., 1953. Errata for Kruskal–Wallis (1952). Journal of the American Statistical Association 48, 907–911. Morrison, D.F., 1976. Multivariate Statistical Methods. McGraw-Hill Book Company, New York. OÕBrien, R.G., Kaiser, M.K., 1985. MANOVA method for analyzing repeated measures designs: an extensive primer. Psychological Bulletin 97 (2), 316–333. Shapiro, S.S, Wilk, M.B., 1965. An analysis of variance test for normality (complete samples). Biometrika 52, 591–611. Shirley, E.A.C., 1977. A non-parametric equivalent of WilliamsÕ test for contrasting increasing dose levels of a treatment. Biometrics 33, 386–389. Schulze, Gene E., 1990. Large-scale assessment of motor activity in rodents: procedures for routine use in toxicology studies. Journal of American College of Toxicology 9 (4), 455–463. Sokal, R.R., Rohlf, F.J., 1995. Biometry, third ed. W.H. Freeman, San Francisco. Steel, R.G.D., 1959. A multiple comparison rank sum test: treatments versus control. Biometrics 15, 560–572. United States Food and Drug Administration, Center for Drug Evaluation and Research, 1997. Guidance Document: SUPACMR Modified Release Solid Oral Dosage Forms, Scale-Up and Postapproval Changes: Chemistry, Manufacturing, and Controls; In Vitro Dissolution Testing and In Vivo Bioequivalence Documentation, September 1997. United States Food and Drug Administration, Center for Drug Evaluation and Research, 2004. Inactive Ingredient Database. Available from:. Williams, D.A., 1971. A test for differences between treatment means when several dose levels are compared with a zero dose control. Biometrics 27, 103–117. Williams, D.A., 1972. The comparison of several dose levels with a zero dose control. Biometrics 28, 519–531.
A subchronic toxicity study in rats and genotoxicity tests with an aqueous ethylcellulose dispersion C.C. DeMerlis
a,*
, D.R. Schoneker a, J.F. Borzelleca
b
a
b
Colorcon, West Point, PA, USA Medical College of Virginia, Richmond, VA, USA Accepted 15 March 2005
Abstract SureleaseÒ Aqueous Ethylcellulose Dispersion is an excipient used as a modified release coating for beads, granules, non-pariels, drug crystals and tablets and for taste masking applications for drug products and dietary supplement products. A study was conducted to assess the toxicity of spray-dried Surelease when administered orally, via dietary admixture, to Sprague-Dawley CDÒ rats (20/sex/group) at dose levels of 0, 2000, 3500, and 5000 mg/kg/day for a period of at least 3 months. After 3 months of treatment, all rats scheduled for terminal sacrifice were killed and selected organs were weighed. Complete macroscopic examinations and histopathological evaluation of selected tissues were conducted on all animals. Neuropathological evaluations were performed on 5 animals/sex/group. No mortality occurred during the study. Clinical observations, ophthalmology, body weight and food consumption, hematology, coagulation, clinical chemistry, urinalysis, functional observational assessments, motor activity, organ weights and ratios and macroscopic and microscopic observations did not reveal any significant, consistent, dose-dependent test article-related adverse effects. The NOAEL (no-observed-adverse-effect-level) is 5000 mg/kg/day, the highest dose tested. A series of genotoxicity tests were conducted with Surelease. Surelease showed no evidence of mutagenic activity in the bacterial reverse mutation test with and without metabolic activation and in the in vitro cell mutation assay under the experimental conditions employed. Surelease did not show any evidence of causing chromosome damage or bone marrow cell toxicity when administered by gavage in the mouse micronucleus in vivo test procedure. These findings support the safety of Surelease for use as an excipient. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Ethylcellulose aqueous dispersion; Surelease; Ethylcellulose; Excipient; Modified release coating; Polymeric dispersion; Taste masking coating; Excipient safety; Sustained release coating
1. Introduction Surelease is an excipient used as a modified release coating for beads, granules, non-pariels, drug crystals and tablets as well as a modified release binder in matrix granulations. The US Food and Drug Administration defines modified release dosage forms as those whose drug-release characteristics of time course and/or loca-
*
Corresponding author. E-mail address: [email protected] (C.C. DeMerlis).
0278-6915/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2005.03.008
tion are chosen to accomplish therapeutic or convenience objectives not offered by conventional dosage forms such as a solution or an immediate release dosage form (US FDA Guidance, 1997). Surelease is also used for taste masking applications for drug products and dietary supplement products. Surelease is a complete plasticized aqueous polymeric dispersion using ethylcellulose as the rate controlling polymer. Surelease is referred to as a complete coating system because Surelease already contains the plasticizer which is premixed with the polymer during the manufacturing process.
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The administration of Surelease is typically by the oral route. Surelease has a long history of safe use in a number of products on a global basis. Surelease is used as a coating in drug products in the US, Europe, and other countries, and is also used in dietary supplement products regulated as foods in the US. Colorcon maintains Drug Master File number 9822 at the Center for Drug Evaluation and Research, US Food and Drug Administration to support the use of Surelease in drug products in the United States. Surelease is included in the FDA Inactive Ingredient Database for oral tablets (US FDA, 2004). The Surelease used in the studies described below is produced to meet global pharmaceutical standards using good manufacturing practices (GMPÕs). Surelease is characterized by proprietary specifications. The impurities are strictly monitored by process controls, stringent specifications and analytical methods developed by the manufacturer. The ingredients used in the manufacture of Surelease are commonly used food ingredients and pharmaceutical excipients. These ingredients meet the applicable compendial and regulatory requirements for the intended pharmaceutical uses in the United States, Europe and a number of other countries. These ingredients also meet the regulatory requirements for use as dietary supplement excipients in the United States. To appropriately characterize the toxicity of Surelease, a 90-day dietary study and three genotoxicity tests were conducted to provide current supportive information for Surelease.
2. Materials and methods This study was designed to meet or exceed the requirements of the following OECD (Organization for Economic Co-Operation and Development) Guidelines: 1. OECD Guidelines for Testing of Chemicals (1981) Subchronic Oral Toxicity—Rodent: 90-day Study, Guideline 408. 2. OECD: Guidelines for Testing of Chemicals (1997) Genetic Toxicology: Bacterial Reverse Mutation Test, Guideline 471. 3. OECD: Guidelines for Testing of Chemicals (1997) Genetic Toxicology: In vitro Mammalian Cell Gene Mutation Tests, Guideline 476. 4. OECD: Guidelines for Testing of Chemicals (1997) Genetic Toxicology: Mammalian Erythrocyte Micronucleus Test, Guideline 474. This study was also designed to meet or exceed the requirements of the FDA (United States Food and Drug Administration) 1993 ‘‘Redbook II’’ Toxicological Prin-
ciples for the Safety Assessment of Direct Food Additives and Color Additives Used in Food. This study was conducted at a contract research organization in compliance with: OECD Principles of Good Laboratory Practices ENV/MC/CHEM (98)17, EEC Good Laboratory Practices (90/18/EEC) and FDA Good Laboratory Practice Standards (Part 58 of 21 CFR). 2.1. Test article Surelease is a formulated product and contains the following ingredients: purified water, ethylcellulose, ammonium hydroxide, medium chain triglycerides, and oleic acid. Surelease is manufactured by Colorcon as a 25% solids aqueous ethylcellulose dispersion and was spray dried to a powder for use in this study. Surelease is available in several different formulations. Commercial samples of Surelease E-7-19010 from the same batch were provided by Colorcon (US) to the contract research organization. The test material was an off white to yellowish powder stored at room temperature. 2.2. Animals and husbandry Male and female 4 week old VAF/PlusÒ CDÒ (Sprague-Dawley derived) (Crl: CD (SD) IGS BR) rats were obtained from Charles River Laboratories (Kingston, NY, USA) and acclimated for 2 weeks prior to initiation of dosing. At initiation of treatment, animals were approximately 6 weeks old. The mean weight for the males was 193 g and the females 159 g. Individual weights of animals placed on test were within ±20% of the mean weight for each sex. All animals were examined during the acclimation period to confirm suitability for study and those considered unsuitable were eliminated. Animals were doubly housed in elevated, stainless steel, wire mesh cages during the first week of the acclimation period and individually housed thereafter. A 12-h light/dark cycle controlled via an automatic timer was provided. Temperature was monitored and maintained within the specified range of 19.9–25.1 °C. Relative humidity was monitored and maintained within the range of 24.75–85.63%. Water (Elizabethtown Water Company, Westfield, NJ, USA) was provided ad lib. Certified Rodent Diet, No. 5002 (meal) (PMI Nutrition International, St. Louis, MO) was available without restriction. Fresh feed was presented weekly. This study complied with all appropriate parts of the Animal Welfare Act Regulations: 9 CFR Parts 1 and 2 Final Rules, Federal Register, Volume 54, No. 168, August 31, 1989, pp. 36112–36163 effective October 30, 1989 and 9 CFR Part 3 Animal Welfare Standards; Final Rule, Federal Register, Volume 56, No. 32, February 15, 1991, pp. 6426–6505 effective March 18, 1991.
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2.3. Experimental design and test diet preparation Animals were randomly distributed into 4 groups of 20 animals per sex per group and received either untreated control diet or spray-dried Surelease mixed with control diet at dose levels 2000, 3500, and 5000 mg/kg/ day for a period of at least three months. Fresh diets were provided weekly and dietary concentrations were adjusted weekly based on body weight and feed consumption data from the preceding week. Prior to initiation of the study, analyses of homogeneity and stability of Surelease in low and high concentration diets were conducted. Confirmation analyses of all three dietary levels were assayed weekly for the first month, weeks 8, and 13 of the study. 2.4. In-life observations Cage-side observations for mortality and general condition were made at least twice daily (once in the morning and once in the afternoon). Cageside observations for clinical signs were made once daily, concurrent with one of the viability checks, for any signs of toxic or pharmacologic effects (e.g., abnormalities in general condition, appearance, activity, behavior, respiration). Animals were removed from their cages and examined twice pretest and once weekly during the study period. Examinations included observations of general condition, skin and fur, eyes, nose, oral cavity, abdomen and external genitalia, occurrence of secretions and excretions, and autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypy (e.g., excessive grooming, repetitive circling) or bizarre behavior (e.g., self-mutilation, walking backward) were recorded. An ophthalmoscopic examination was conducted pretest and at study termination. Lids, lacrimal apparatus and conjunctiva were examined grossly. The cornea, anterior chamber, lens, iris, vitreous humor, retina and optic disc were examined by indirect ophthalmoscopy. Mydriacyl 1% was used to induce mydriasis. Animals were removed from their cages and weighed twice pretest, weekly during treatment and terminally (after fasting). Terminal, fasted body weights were obtained just prior to necropsy. Feed was available without restriction 7 days/week. Animals were presented with full feeders of known weight. After 6 days, feeders were reweighed and the resulting weight was subtracted from the full feeder weight to obtain the grams consumed per animal over the 6-day period. Food consumption was measured (weighed) weekly, beginning one week prior to treatment. Neurobehavioral testing was staggered over four sessions with approximately equal numbers of animals per sex per dose group in each session. Noise level in the
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functional observational battery room was maintained within a level of 55–65 dB by a White Noise Generator, Model MDF 5001 (MDF Products, Inc. Danbury, Connecticut). Temperature, humidity, and illumination of each room were measured and recorded to ensure that variations in environmental conditions were minimal during all evaluations. The functional observational battery was performed for all animals before evaluations of motor activity. Using a modified version of SchulzeÕs procedures (Schulze, 1990), the locomotor activity of 10 animals/sex/group was monitored using an automated Photobeam Activity System (San Diego Instruments, Inc., San Diego, California). Sessions were 60 min in length; each session was divided into 12, 5-min intervals. The time of testing was balanced across treatment groups. The following evaluations were performed as part of the functional observational battery: home cage evaluations, handling evaluations, open field evaluations, reflex assessments, grip strength, landing foot splay, proprioception, air righting ability, and body weight. 2.5. Clinical pathology Hematology, clinical chemistry, and urinalysis evaluations were performed on days 30 and 60 and at study termination on 10 animals/sex/group. Coagulation parameters were performed on day 60 and at study termination on 10 animals/sex/group. Blood was obtained from the retrobulbar venous plexus of fasted rats under light CO2/O2 anesthesia. Hematology parameters included hemoglobin concentration, hematocrit, erythrocyte count, platelet count, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, total leukocyte count, reticulocyte count, differential leukocyte count and erythrocyte and platelet morphology. Coagulation parameters evaluated were prothrombin time and active partial thromboplastin time. Clinical chemistry determinations included aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, blood urea nitrogen, creatinine, glucose, cholesterol, triglycerides, total protein, albumin, total bilirubin, sodium, potassium, chloride, calcium, inorganic phosphorus, gamma-glutamyl transferase, globulin, and albumin/globulin ratio. Urinalysis included volume, appearance, and specific gravity and the following parameters were analyzed: protein, nitrites, glucose, ketones, pH, bilirubin, and urobilinogen. 2.6. Pathology Carbon dioxide inhalation was used as the method of euthanasia. At the end of the treatment period, all animals were subjected to a complete macroscopic postmortem examination, which included examination of
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the external surface and all orifices; the external surfaces of the brain and spinal cord; the organs and tissues of the cranial, thoracic, abdominal and pelvic cavities and neck; and the remainder of the carcass for the presence of macroscopic morphologic abnormalities. The following organs were weighed: adrenal glands, brain, heart, kidneys, liver, ovaries, pituitary, prostate, spleen, testes, thymus, thyroid, and uterus (with cervix). All tissues were preserved in 10% neutral buffered formalin. Eyes were placed in glutaraldehyde/paraformaldehyde initially and then retained in 10% formalin. Testes and epididymides were placed in Modified DavidsonÕs solution initially and then retained in 10% formalin. Lungs and urinary bladder were infused with formalin prior to their immersion into a larger volume of the same fixative. After fixation, the tissues and organs from all animals were routinely processed and embedded in paraffin; these tissues were sectioned and stained with hematoxylin and eosin and examined by light microscopy. The following tissues were examined: adrenal glands, aorta, bone marrow smear, bone, bone marrow, brain, epididymides, esophagus, eyes with optic nerve, head, heart, kidneys, lacrimal glands, large intestine, larynx, liver, lungs, lymph nodes, mammary glands, muscle, nerve, ovaries, pancreas, PeyerÕs patches, pharynx, pituitary, prostate, salivary glands, seminal vesicles, skin, small intestine, spinal cord, spleen, stomach, testes, thymus, thyroid, tongue, trachea, urinary bladder, uterus (with cervix), vagina, ZymbalÕs gland, and all gross lesions. The bones were decalcified in Decalcifier IITM. Slides of the indicated tissues were prepared and examined microscopically for all animals in the control and high-dose groups. Tissues with macroscopic lesions were examined in all groups. Any abnormalities not noted during macroscopic examinations which were seen during histology processing were recorded. 2.7. Neuropathology Five animals per sex per group (selected from animals used for neurobehavioral studies) were anesthetized with Acepromazine/Ketamine/Xylazine and transcardially perfused with phosphate buffered saline followed by 1% glutaraldehyde and 4% paraformaldehyde in the same buffer. Slides of the indicated tissues were prepared and examined microscopically for all animals in the control and high-dose groups. The following tissues were examined: brain (forebrain, cerebral cortex, hippocampus, basal ganglia, midbrain, cerebellum and pons, medulla), eye with optic nerve, spinal cord (cervical, thoracic, lumbar, cross and longitudinal sections), sciatic nerve (cross and longitudinal sections), tibial nerve (cross and longitudinal sections), sural nerve (cross and longitudinal sections), trigeminal ganglia, dorsal root ganglia (from C3 to C6 and L4 to L6), dorsal root fibers (from C3 to C6 and L4 to L6) and ventral root
fibers (from C3 to C6 and L4 to L6). Tissues with macroscopic lesions were examined in all groups. After fixation, the peripheral nerves of all neuropathology animals were processed, embedded in epoxy resin and cut at a microtome setting of 2 lm. All other tissues, including the brain and spinal cord, ganglia and dorsal and ventral root fibers were routinely processed and embedded in paraffin. Plastic sections of peripheral nerves were stained with toluidine blue and all other nervous tissue were stained with hematoxylin and eosin. The brain and spinal cord sections were stained with hematoxylin and eosin. 2.8. Statistical analyses The following parameters were analyzed statistically: body weight, body weight change from baseline, feed consumption, hematology, coagulation, clinical chemistry, organ weights, motor activity counts, forelimb and hindlimb grip strength measurements and landing foot splay measurements. Mean values of all dose groups were compared to the mean value for the control group at each time interval. Evaluation of equality of group means was made by the appropriate statistical method, followed by a multiple comparison test if needed. BartlettÕs test (Bartlett, 1937; Sokal and Rohlf, 1995) was performed to determine if groups had equal variances. For all parameters except organ weights, if the variances were equal, parametric procedures were used; if not, non-parametric procedures were used. Organ weight data was analyzed only by parametric methods. The parametric method was the standard one-way analysis of variance (ANOVA) using the F ratio to assess significance (Armitage, 1971; Dunlap and Duffy, 1975). If significant differences among the means were indicated, additional tests were used to determine which means were significantly different from the control: DunnettÕs (Dunlap et al., 1981; Dunnett, 1955, 1964), Williams (Williams, 1971, 1972), or Cochran and CoxÕs modified t-test (Cochran and Cox, 1959). The non-parametric method was the Kruskal–Wallis test (Kruskal and Wallis, 1952, 1953) and if differences were indicated, ShirleyÕs test (Shirley, 1977), SteelÕs test (Steel, 1959) or Pairwise Comparison with Bonferroni Correction (Games and Howell, 1976) were used to determine which means differed from control. BartlettÕs test for equality of variance was conducted at the 1% significance level; all other statistical tests were conducted at the 5% and 1% significance levels. Motor activity counts were collected in 12 consecutive 5-min intervals on each test day. The set of counts over these 12 intervals gave a ‘‘profile’’ of an animalÕs motor activity. These profiles were compared within each study period using a multivariate model that assessed whether the profiles for different groups had
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the same shape (the parallel hypothesis). The WilksÕ Lambda, a variant of the Hotelling T2 (Morrison, 1976) were used to test the parallel hypothesis; twoway ANOVA with interaction (OÕBrien and Kaiser, 1985) were used to test for the overall treatment effect with respect to the level hypothesis followed, if necessary, by DunnettÕs t-test to find groups that differed from control. The Shapiro–Wilk test (Shapiro and Wilk, 1965) were used to assess normality of the residuals from the multivariate model; when p < 0.01 for this test, the Blom inverse normal transformation (Blom, 1958) were applied to achieve normality of the residuals. Other tests were deemed significant at the p = 0.05 level. 2.9. Ames test In this in vitro assessment of the mutagenic potential of Spray-Dried Surelease Aqueous Ethylcellulose Dispersion, histidine dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan dependent mutant of Escherichia coli, strain WP2uvrA/pKM101 (CM891), were exposed to Surelease diluted in acetone. Acetone was also used as a negative control. Sodium azide, 9-aminoacridine, 2-nitrofluorene and 2-(2-furyl)-3-(5nitro-2-furyl)acrylamide were used as positive controls in the absence of the S9 fraction and 2-aminoanthracene and benzo[a]pyrene were used as positive controls in the presence of the S9 fraction. Two independent mutation tests were performed in the presence and absence of liver preparations from Aroclor 1254-treated rats (S9 mix). Both tests were standard plate incorporation assays.
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2.11. Mouse micronucleus test This study was designed to assess the potential induction of micronuclei by Spray-Dried Surelease Aqueous Ethylcellulose Dispersion in bone marrow cells of mice. Mice were treated with a single oral administration of the test substance at dose levels of 500, 1000 and 2000 mg/kg body weight. A preliminary toxicity test had previously shown that a dose of 2000 mg/kg (the standard limit dose for the micronucleus test) was tolerated; this level was therefore selected as an appropriate maximum for use in the micronucleus test. The test substance, negative and positive control substances were administered by gavage. The negative control group received the vehicle, corn oil and the positive control group received mitomycin C at 12 mg/kg body weight. Following the preliminary toxicity test, no substantial differences in toxicity were observed between the sexes, consistent with current guidelines. The micronucleus test was performed using male animals only. Bone marrow smears were obtained from seven male animals in the negative control, each of the test substance groups and five male animals in the positive control group 24 h after dosing. In addition, bone marrow smears were obtained from seven male animals in the negative control and high level treatment groups 48 h after dosing. One smear from each animal was examined for the presence of micronuclei in 2000 immature erythrocytes. The proportion of immature erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal. A record of the incidence of micronucleated mature erythrocytes was also kept.
2.10. Mouse lymphoma test
3. Results
Spray-Dried Surelease Aqueous Ethylcellulose Dispersion was tested for mutagenic potential in an in vitro mammalian cell mutation assay. This test system is based on detection and quantitation of forward mutation in the subline 3.7.2.c of mouse lymphoma L5178Y cells, from the heterozygous condition at the thymidine kinase locus (TK+/ ) to the thymidine kinase deficient genotype (TK / ). Two independent mutagenicity tests were carried out in both the absence and presence of S9 mix with a 3-h exposure, and one in the absence of S9 mix with a 24-h exposure. The maximum concentration of Surelease to which the cells were exposed was limited by solubility. The solvent chosen was ethanol, and the highest final concentration that was judged to be usable was 250 lg/ml. Methyl methanesulphonate was used as a positive control in the absence of the S9 fraction and 3-methylcholanthrene was used as a positive control in the presence of the S9 fraction.
3.1. Mortality No mortality occurred during the study. 3.2. Physical observations and ophthalmoscopic examinations Weekly clinical observations recorded during the study were considered common findings in laboratory rats and unrelated to test article administration. Administration of Surelease did not result in any test article-related ocular findings. 3.3. Body weights and food consumption There were no statistically significant or treatment related adverse effects on body weights (Figs. 1 and 2). Feed consumption values of females administered 3500 and 5000 mg/kg/day of Surelease were slightly greater
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550 135
525
Group 1 - 0 mg/kg/day (Control) Group 2 - 2000 mg/kg/day
500
Group 3 - 3500 mg/kg/day
125
Group 4 - 5000 mg/kg/day
475 450
115
Feed Consumption (g/kg/day)
425
Body Weight (grams)
400 375 350 325 300 275
105
95
85
75
250 65
225 200
55
Group 1 - 0 mg/kg/day (Control) 175
Group 2 - 2000 mg/kg/day
150
Group 3 - 3500 mg/kg/day
45
Group 4 - 5000 mg/kg/day 125 35
100 -8 -3 2
-2
7 12 17 22 27 32 37 42 47 52 57 62 67 72 77 82 87 92
3
8 13 18 23 28 33 38 43 48 53 58 63 68 73 78 83 88 Days
Day
Fig. 3. Mean food consumption: males.
Fig. 1. Mean body weights: males.
325 Group 1 - 0 mg/kg/day (Control)
135
Group 2 - 2000 mg/kg/day Group 3 - 3500 mg/kg/day
300
Group 4 - 5000 mg/kg/day
125
275 115
Feed Consumption (g/kg/day)
Body Weight (grams)
250
225
200
175
150
105
95
85
75
65
Group 1 - 0 mg/kg/day (Control) Group 2 - 2000 mg/kg/day
125
Group 3 - 3500 mg/kg/day
55
Group 4 - 5000 mg/kg/day 100
45
-8 -3 2
7 12 17 22 27 32 37 42 47 52 57 62 67 72 77 82 87 92 Day
Fig. 2. Mean body weights: females.
-2
3
8
13 18 23 28 33 38 43 48 53 58 63 68 73 78 83 88 Day
Fig. 4. Mean food consumption: females.
C.C. DeMerlis et al. / Food and Chemical Toxicology 43 (2005) 1355–1364 Table 1 Mean test material intake values Group
2 3 4
Desired dose (mg/kg/day)
2000 3500 5000
Mean test article intake (mg/kg/day) Males
Females
1986 3497 5007
1985 3456 4948
and statistically significant when compared to control values during the study (Figs. 3 and 4). Feed consumption values of test article-treated males and females fed 2000 mg/kg/day were comparable to control values. Mean test material intake values are presented in Table 1 and indicate that animals received the appropriate dose. 3.4. Neurobehavioral observations No test article-related effects were evident in motor activity for either sex in any of the treatment groups. Test article administration did not affect the neurological condition of the animals as measured by a functional observational battery of assessments. Landing foot splay and grip strength for all test article-treated groups were comparable to control values or within the range of normal variation. 3.5. Clinical laboratory studies—hematology, coagulation and clinical chemistry No test article-related adverse effects were noted in the hematology parameters, clinical chemistry parameters, and urinalysis data evaluated on Day 30, 60 and at study termination. No test article-related adverse effects were noted in the coagulation parameters evaluated on Day 60 and at study termination. 3.6. Terminal organ weights, organ/body weight ratio and organ/brain weight ratio There were no statistically significant effects on terminal organ weights, organ/body weight ratio and organ/ brain weight ratio (Tables 2 and 3). 3.7. Pathology—macroscopic Macroscopic findings occurred with comparable incidence and severity in control or test article-treated groups or they occurred sporadically as commonly seen in rats of this strain and age at the test facility. 3.8. Pathology—microscopic There was no evidence of neurotoxicity in the subset of animals sampled for detailed neuropathological eval-
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uation. Occasional degenerate fibers were observed in the peripheral nerves from both control and Surelease treated animals, but the incidence was similar between test article-treated groups and control groups. The remaining protocol listed tissues from this subset of perfusion-fixed animals were also examined microscopically and no treatment related findings attributable to administration of Surelease were noted. There were no microscopic findings in other tissues examined that were attributable to Surelease. Mild inflammatory changes were present in the lungs from a proportion of animals from the control and Surelease treated groups and probably reflect a minor respiratory infection and are not toxicologically significant. Focal inflammatory changes were present in the Harderian glands in the control and Surelease treated animals. The findings were frequently unilateral and are the result of intraorbital bleeding procedures and not treatment related. All other microscopic findings were typical of the normal background pathology seen in this age and strain of rat. 3.9. Ames test Concentrations of Spray-Dried Surelease Aqueous Ethylcellulose Dispersion up to 5000 lg/plate were tested. This is the standard limit concentration recommended in the regulatory guidelines. Other concentrations used were a series of ca. half-log10 dilutions of the highest concentration. No signs of toxicity were observed towards the tester strains in either mutation test. No evidence of mutagenic activity was seen at any concentration of Surelease in either mutation test. The concurrent positive controls demonstrated the sensitivity of the assay and the metabolizing activity of the liver preparations. It is concluded that, under the test conditions employed, Surelease showed no evidence of mutagenic activity in this bacterial system. 3.10. Mouse lymphoma test Surelease was relatively non-toxic in this mammalian cell culture system in all tests, as measured by Day0 relative survival, either in the absence or presence of S9 mix. At least two precipitating concentrations were assessed in the main mutagenicity tests, with the effects of a maximum concentration of 250 lg/ml being tested on each occasion. No significant increases in mutant frequency were observed in the first main test in the presence of S9 mix or in the second main test in either the absence or presence of S9 mix.
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Table 2 Organ weights in male rats after being fed SureleaseÒ for 3 months Parameter
0 mg/kg/day
2000 mg/kg/day
3500 mg/kg/day
5000 mg/kg/day
Mean weight (g) Terminal body weight Adrenal glands Brain Heart Kidneys Liver Pituitary gland Prostate Spleen Testes Thymus Thyroid/parathyroids
505.10 ± 43.80 0.08 ± 0.02 2.31 ± 0.18 1.75 ± 0.17 4.42 ± 0.46 16.78 ± 2.89 0.02 ± 0.01 1.33 ± 0.41 0.94 ± 0.09 3.46 ± 0.21 0.37 ± 0.10 0.03 ± 0.01
499.60 ± 51.20 0.08 ± 0.01 2.40 ± 0.16 1.72 ± 0.27 4.45 ± 0.63 16.11 ± 2.82 0.01 ± 0.00 1.36 ± 0.36 0.89 ± 0.17 3.54 ± 0.28 0.35 ± 0.12 0.03 ± 0.00
520.50 ± 54.00 0.07 ± 0.01 2.39 ± 0.16 1.75 ± 0.23 4.39 ± 0.47 16.17 ± 3.02 0.01 ± 0.00 1.49 ± 0.35 0.91 ± 0.13 3.49 ± 0.28 0.40 ± 0.09 0.03 ± 0.01
512.00 ± 36.20 0.08 ± 0.01 2.38 ± 0.11 1.75 ± 0.22 4.34 ± 0.36 16.05 ± 2.56 0.01 ± 0.00 1.46 ± 0.30 0.99 ± 0.16 3.50 ± 0.29 0.39 ± 0.10 0.03 ± 0.01
Mean organ-to-terminal body weight ratios Terminal body weight 505.10 ± 43.80 Adrenal glands 0.02 ± 0.00 Brain 0.46 ± 0.06 Heart 0.35 ± 0.04 Kidneys 0.89 ± 0.09 Liver 3.32 ± 0.46 Pituitary gland 0.00 ± 0.00 Prostate 0.27 ± 0.09 Spleen 0.16 ± 0.02 Testes 0.69 ± 0.08 Thymus 0.07 ± 0.02 Thyroid/parathyroids 0.01 ± 0.00
499.60 ± 51.20 0.02 ± 0.00 0.48 ± 0.05 0.34 ± 0.04 0.89 ± 0.10 3.21 ± 0.36 0.00 ± 0.00 0.28 ± 0.06 0.18 ± 0.02 0.72 ± 0.09 0.07 ± 0.02 0.01 ± 0.00
520.50 ± 54.00 0.01 ± 0.00 0.46 ± 0.05 0.34 ± 0.03 0.85 ± 0.08 3.09 ± 0.37 0.00 ± 0.00 0.29 ± 0.07 0.17 ± 0.02 0.67 ± 0.06 0.08 ± 0.02 0.01 ± 0.00
512.00 ± 36.20 0.01 ± 0.00 0.47 ± 0.05 0.34 ± 0.05 0.85 ± 0.08 3.13 ± 0.44 0.00 ± 0.00 0.29 ± 0.06 0.18 ± 0.03 0.69 ± 0.08 0.08 ± 0.02 0.01 ± 0.00
Mean organ-to-brain weight ratios Terminal body weight 505.10 ± 43.80 Adrenal glands 3.52 ± 0.70 Brain 100.00 ± 0.00 Heart 75.75 ± 5.01 Kidneys 191.40 ± 15.69 Liver 730.60 ± 140.58 Pituitary gland 0.64 ± 0.19 Prostate 57.29 ± 15.48 Spleen 40.55 ± 3.71 Testes 150.57 ± 14.08 Thymus 15.93 ± 4.15 Thyroid/parathyroids 1.43 ± 0.28
499.60 ± 51.20 3.13 ± 0.50 100.00 ± 0.00 71.42 ± 8.61 185.12 ± 21.09 671.47 ± 110.53 0.60 ± 0.17 56.58 ± 13.02 37.07 ± 6.61 147.95 ± 12.24 14.66 ± 4.74 1.33 ± 0.19
520.50 ± 54.00 3.07 ± 0.59 100.00 ± 0.00 73.43 ± 8.77 184.38 ± 20.60 678.28 ± 124.98 0.60 ± 0.13 62.20 ± 13.83 37.93 ± 4.22 146.49 ± 12.43 16.80 ± 3.34 1.43 ± 0.28
512.00 ± 36.20 3.17 ± 0.55 100.00 ± 0.00 73.44 ± 8.16 182.95 ± 18.03 677.97 ± 127.51 0.58 ± 0.12 61.19 ± 12.05 38.58 ± 6.12 147.19 ± 12.17 16.46 ± 4.37 1.39 ± 0.27
In the absence of S9 mix in first main mutagenicity test, exposure to 250 lg/ml of Surelease for 3 h was associated with a statistically significant increase in mutant frequency, where Day0 relative survival was 78% of the solvent controls. However, the mutant frequency of 0.000268 lay within the upper 95% confidence limits for the negative historical control. Other concentrations tested, namely 12.5, 25, 50, 100 and 200 lg/ml, where Day0 relative survival values were 106–80% of solvent controls, did not cause any significant increases in mutant frequency. In all tests the positive control substances increased mutant frequency significantly. It was concluded that Surelease did not demonstrate mutagenic potential in this in vitro cell mutation assay under the experimental conditions described.
3.11. Mouse micronucleus test No statistically significant increases in the frequency of micronucleated immature erythrocytes and no substantial decreases in the proportion of immature erythrocytes were observed in mice treated with SprayDried Surelease Aqueous Ethylcellulose Dispersion and killed 24 or 48 h later, compared to vehicle control values (P > 0.01 in each case). The positive control compound, mitomycin C, produced significant increases in the frequency of micronucleated immature erythrocytes (P < 0.01). It is concluded that Surelease did not show any evidence of causing chromosome damage or bone marrow cell toxicity when administered orally by gavage in this in vivo test procedure.
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Table 3 Organ weights in female rats after being fed SureleaseÒ for 3 months Parameter
0 mg/kg/day
2000 mg/kg/day
3500 mg/kg/day
5000 mg/kg/day
Mean weight (g) Terminal body weight Adrenal glands Brain Heart Kidneys Liver Pituitary gland Ovaries Spleen Uterus Thymus Thyroid/parathyroids
282.00 ± 22.30 0.08 ± 0.01 2.10 ± 0.11 1.11 ± 0.16 2.47 ± 0.29 9.15 ± 1.34 0.02 ± 0.00 0.10 ± 0.02 0.64 ± 0.07 0.76 ± 0.10 0.34 ± 0.07 0.03 ± 0.00
280.40 ± 29.10 0.07 ± 0.01 2.08 ± 0.14 1.07 ± 0.14 2.38 ± 0.18 8.64 ± 1.23 0.02 ± 0.00 0.10 ± 0.01 0.64 ± 0.10 0.97 ± 0.47 0.32 ± 0.10 0.03 ± 0.00
289.90 ± 29.20 0.08 ± 0.00 2.15 ± 0.19 1.16 ± 0.17 2.62 ± 0.39 9.72 ± 1.45 0.02 ± 0.01 0.11 ± 0.02 0.69 ± 0.12 0.87 ± 0.35 0.37 ± 0.09 0.03 ± 0.01
274.50 ± 25.70 0.08 ± 0.01 2.09 ± 0.17 1.15 ± 0.14 2.47 ± 0.20 9.17 ± 0.85 0.02 ± 0.00 0.10 ± 0.02 0.68 ± 0.13 0.79 ± 0.22 0.35 ± 0.11 0.03 ± 0.00
Mean organ-to-terminal body weight ratios Terminal body weight 282.00 ± 22.30 Adrenal glands 0.027 ± 0.00 Brain 0.75 ± 0.07 Heart 0.39 ± 0.05 Kidneys 0.87 ± 0.08 Liver 3.24 ± 0.39 Pituitary gland 0.01 ± 0.00 Ovaries 0.03 ± 0.00 Spleen 0.23 ± 0.03 Uterus 0.27 ± 0.05 Thymus 0.12 ± 0.02 Thyroid/parathyroids 0.01 ± 0.00
280.40 ± 29.10 0.03 ± 0.00 0.75 ± 0.07 0.38 ± 0.05 0.85 ± 0.06 3.08 ± 0.23 0.01 ± 0.00 0.04 ± 0.00 0.23 ± 0.02 0.35 ± 0.17 0.11 ± 0.03 0.01 ± 0.00
289.90 ± 29.20 0.03 ± 0.00 0.75 ± 0.08 0.40 ± 0.05 0.90 ± 0.09 3.36 ± 0.41 0.01 ± 0.00 0.04 ± 0.01 0.24 ± 0.03 0.30 ± 0.12 0.13 ± 0.03 0.01 ± 0.00
274.50 ± 25.70 0.03 ± 0.00 0.77 ± 0.08 0.42 ± 0.05 0.90 ± 0.08 3.36 ± 0.41 0.01 ± 0.00 0.04 ± 0.01 0.25 ± 0.05 0.29 ± 0.07 0.13 ± 0.03 0.01 ± 0.00
Mean organ-to-brain weight ratios Terminal body weight 282.00 ± 22.30 Adrenal glands 3.61 ± 0.57 Brain 100.00 ± 0.00 Heart 52.55 ± 6.38 Kidneys 117.31 ± 11.69 Liver 436.44 ± 65.00 Pituitary gland 0.79 ± 0.23 Ovaries 4.52 ± 1.00 Uterus 35.97 ± 4.36 Spleen 30.38 ± 3.03 Thymus 16.11 ± 3.58 Thyroid/parathyroids 1.30 ± 0.24
280.40 ± 29.10 3.56 ± 0.66 100.00 ± 0.00 51.66 ± 5.34 114.80 ± 8.20 416.14 ± 53.25 0.85 ± 0.12 4.88 ± 0.60 46.30 ± 21.17 30.67 ± 4.21 15.41 ± 4.88 1.39 ± 0.20
289.90 ± 29.20 3.53 ± 0.32 100.00 ± 0.00 53.91 ± 5.21 121.32 ± 12.08 452.79 ± 69.59 0.84 ± 0.27 4.95 ± 1.05 40.49 ± 16.14 32.10 ± 4.04 17.27 ± 4.21 1.32 ± 0.27
274.50 ± 25.70 3.65 ± 0.36 100.00 ± 0.00 54.74 ± 4.09 118.11 ± 5.82 439.46 ± 43.94 0.88 ± 0.16 4.98 ± 0.73 37.95 ± 10.64 32.62 ± 5.55 16.59 ± 4.91 1.30 ± 0.29
4. Discussion Surelease is widely used and applications are expanding into dietary supplements and additional drug product uses. Colorcon has developed additional information and data to support the expanded uses. Feed consumption values of females administered 3500 and 5000 mg/kg/day of Surelease were slightly greater and statistically significant when compared to control values during the study. This was attributed to the need to maintain caloric intake necessary for normal growth as a result of the large amounts of Surelease in the diets. Feed consumption values of test article-treated males and females fed 2000 mg/kg/day were comparable to control values.
The test results indicate that Surelease is not genotoxic. There were occasional statistically significant and apparently dose related effects noted (for example, sodium, chloride and hematology), but in the absence of functional changes and histopathological findings these effects were, therefore, not considered toxicologically or biologically significant. Surelease administered in the diet to male and female Sprague-Dawley rats at doses of 2000, 3500 and 5000 mg/kg/day for 90 days did not result in statistically significant, consistent, dose-dependent and treatment related adverse effects on mortality, body weights, hematology, clinical chemistry, urinalysis data, functional observational assessments, motor activity, organ weight
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data, and macroscopic and microscopic observations. Therefore, the No-Observed-Adverse-Effect-Level (NOAEL) is 5000 mg/kg/day. In conclusion, the data and information summarized in this paper demonstrate that Surelease, meeting appropriate specifications, as intended for use in film-coating applications in pharmaceutical and dietary supplement products, does not present a safety concern for humans. References Armitage, P., 1971. Statistical Methods in Medical Research. Blackwell Scientific Publications, Oxford, UK. Bartlett, M.S., 1937. Properties of sufficiency and statistical tests. Proceedings of the Royal Society, Series A 160, 268–282. Blom, G., 1958. Statistical Estimates and Transformed Beta Variables. John Wiley and Sons, New York. Cochran, W.G., Cox, G.M., 1959. Experimental Designs. John Wiley, New York. Dunlap, W.P., Duffy, J.A., 1975. Fortran IV functions for calculating exact probabilities associated with z, chi-square, t and f values. Behavior Research Methods & Instrumentations 7, 59–60. Dunlap, W.P., Marx, M.S., Agamy, G.G., 1981. Fortran IV functions for calculating probabilities associated with DunnettÕs test. Behavior Research Methods & Instrumentation 13, 363–366. Dunnett, C.W., 1955. A multiple comparison procedure for comparing several treatments with a control. Journal of the American Statistical Association 50, 1096–1121. Dunnett, C.W., 1964. New tables for multiple comparisons with a control. Biometrics 20 (3), 482–491. Games, P.A., Howell, J.F., 1976. Pairwise multiple comparison procedures with unequal nÕs and/or variances: a Monte-Carlo study. Journal of Educational Statistics 1, 113–125.
Kruskal, W.H., Wallis, W.A., 1952. Use of ranks in one-criterion variance analysis. Journal of the American Statistical Association 47, 583–621. Kruskal, W.H, Wallis, W.A., 1953. Errata for Kruskal–Wallis (1952). Journal of the American Statistical Association 48, 907–911. Morrison, D.F., 1976. Multivariate Statistical Methods. McGraw-Hill Book Company, New York. OÕBrien, R.G., Kaiser, M.K., 1985. MANOVA method for analyzing repeated measures designs: an extensive primer. Psychological Bulletin 97 (2), 316–333. Shapiro, S.S, Wilk, M.B., 1965. An analysis of variance test for normality (complete samples). Biometrika 52, 591–611. Shirley, E.A.C., 1977. A non-parametric equivalent of WilliamsÕ test for contrasting increasing dose levels of a treatment. Biometrics 33, 386–389. Schulze, Gene E., 1990. Large-scale assessment of motor activity in rodents: procedures for routine use in toxicology studies. Journal of American College of Toxicology 9 (4), 455–463. Sokal, R.R., Rohlf, F.J., 1995. Biometry, third ed. W.H. Freeman, San Francisco. Steel, R.G.D., 1959. A multiple comparison rank sum test: treatments versus control. Biometrics 15, 560–572. United States Food and Drug Administration, Center for Drug Evaluation and Research, 1997. Guidance Document: SUPACMR Modified Release Solid Oral Dosage Forms, Scale-Up and Postapproval Changes: Chemistry, Manufacturing, and Controls; In Vitro Dissolution Testing and In Vivo Bioequivalence Documentation, September 1997. United States Food and Drug Administration, Center for Drug Evaluation and Research, 2004. Inactive Ingredient Database. Available from: