Safety of PVAP and PVAP-T including a 90-day dietary toxicity study in rats and genotoxicity tests with polyvinyl acetate phthalate (PVAP)

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Contents lists available at ScienceDirect

Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox 5 6

Safety of PVAP and PVAP-T including a 90-day dietary toxicity study in rats and genotoxicity tests with polyvinyl acetate phthalate (PVAP)

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Q1

a

8 9

b

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Colorcon, Inc., 275 Ruth Road, Harleysville, PA 19438, USA School of Medicine, Virginia Commonwealth University, Richmond, VA 23229, USA

a r t i c l e

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C.C. DeMerlis a,⇑, D.R. Schoneker a, J.F. Borzelleca b

i n f o

Article history: Received 4 March 2014 Accepted 17 April 2014 Available online xxxx

Q3

Keywords: PVAP PVAP-T Polyvinyl acetate phthalate Polyvinyl acetate phthalate and titanium dioxide 90-Day study Subchronic study Genotoxicity tests Enteric coating Enteric polymer Pharmaceutical excipient Modified release coating

a b s t r a c t The safety of PVAP was evaluated in a 90-day subchronic toxicity study in rats. Sprague Dawley Crl:CD(SD) rats were administered a dietary concentration of 0.75%, 1.5% and 5.0% PVAP for a minimum of 90 days. There were no adverse effects reported. The no-observed-adverse-effect level (NOAEL) in the 90-day sub chronic study was the 5% dietary concentration, which corresponds to a dose of 3120 mg/kg/ day for males and 3640 mg/kg/day for females, the highest level tested. PVAP is co-processed with titanium dioxide to produce polyvinyl acetate phthalate and titanium dioxide (PVAP-T). The chemical composition, physiochemical properties and specifications of PVAP-T are unchanged during manufacturing process based on various analytical studies. Therefore, the toxicological data that support the safety of PVAP can be used to support the use of PVAP-T as a pharmaceutical excipient. An independent expert panel evaluated the safety of PVAP and PVAP-T. Based on the toxicology study results, safety assessment and the estimated exposure assessment for PVAP and PVAP-T, the expert panel concluded that PVAP and PVAP-T could safely be used in drug products up to 829 mg per day which was the estimated exposure provided to the expert panel for current applications of PVAP and PVAP-T. Ó 2014 Published by Elsevier Ltd.

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1. Introduction

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Polyvinyl acetate phthalate (PVAP) is a reaction product of phthalic anhydride, sodium acetate and partially hydrolyzed polyvinyl alcohol. It contains not less than 55% and not more than 62% of phthalyl (0-carboxybenzoyl C8H5O3) groups. The polyvinyl alcohol is a low molecular weight grade and is 87–89 mol percent hydrolyzed. Since the PVAP polymer is a partial esterification of partially hydrolyzed polyvinyl acetate, it may be represented as shown in Fig. 1. PVAP is also co-processed with titanium dioxide to produce polyvinyl acetate phthalate and titanium dioxide (PVAP-T). Titanium dioxide USP/NF (TiO2) is incorporated into the PVAP polymer matrix during polymer formation. The incorporation of the TiO2 inside the polymer matrix provides unique properties which differ from simple blending of the two materials. The co-processed product is then micronized to achieve a target particle size. Polyvinyl acetate phthalate (PVAP) and co-processed polyvinyl acetate phthalate and titanium dioxide (PVAP-T) are enteric

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Q2

⇑ Corresponding author. Tel.: +1 12156612766; fax: +1 215 661 2366.

coating polymers used as pharmaceutical excipients and have been used in approved prescription and over-the-counter drug products in the United States and globally for many years. PVAP and PVAP-T are typically used in fully formulated dry powder coating systems manufactured by Colorcon, such as SuretericÒ and CoatericÒ, for rapid reconstitution providing an efficient method of applying enteric film coatings. PVAP is listed in the FDA’s Inactive Ingredient Database (IID) and a maximum potency amount is not included. FDA Inactive Ingredient Database lists excipients used in approved drug products, their route of administration and the maximum dosage which has previously been approved by FDA (maximum potency per dosage unit). Colorcon has queried the FDA in the past regarding the listing for PVAP and found that a level of at least 43 mg per dosage unit was acceptable and under the maximum potency for PVAP that FDA had found for an approved drug. PVAP-T is not directly listed in the FDA Inactive Ingredient Database (IID). However, it is used in the Colorcon enteric coating system CoatericÒ which is listed in the IID with a current maximum potency of 26 mg. SuretericÒ, another Colorcon enteric coating system containing PVAP-T, is commonly used as an enteric

E-mail address: [email protected] (C.C. DeMerlis). http://dx.doi.org/10.1016/j.fct.2014.04.031 0278-6915/Ó 2014 Published by Elsevier Ltd.

Please cite this article in press as: DeMerlis, C.C., et al. Safety of PVAP and PVAP-T including a 90-day dietary toxicity study in rats and genotoxicity tests with polyvinyl acetate phthalate (PVAP). Food Chem. Toxicol. (2014), http://dx.doi.org/10.1016/j.fct.2014.04.031

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Fig. 1. PVAP chemical structure. Depending on the phthalyl content, (a) will vary with (b) in mole percent. The acetyl content (c) remains constant depending on the starting material.

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coating system in commercial OTC drug products in the U.S. for many years. A monograph with official standards for PVAP is included in the United States Pharmacopeia/National Formulary (USP/NF). PVAP-T cannot be considered NF; however, it meets all NF specifications for PVAP except residue on ignition (due to the presence of TiO2). Colorcon maintains a U.S. Drug Master File at the Center for Drug Evaluation and Research, Food and Drug Administration, which includes the toxicological data and chemistry, manufacturing and controls information to support the use of PVAP and PVAP-T. PVAP is Generally Recognized as Safe (GRAS) for use in printing inks in the United States for marking dietary supplements. Colorcon conducted a GRAS Expert Panel review of PVAP for this application. PVAP and PVAP-T have been used for many years in commercial drug products marketed in various countries in the European and the Asia Pacific Regions. However, PVAP and PVAPT are not listed for use in Japan at this time since a precedence of use has not yet been established. This status may change if a drug company decides to use PVAP in a drug product intended for the Japanese market in the future based on the current safety studies for PVAP. Extensive toxicological testing of PVAP was conducted by Merck with PVAP in the 1960s and Colorcon was able to secure the reports of these studies. The reports did not describe the characterization, specifications or manufacturing process for the test material used in the studies. The studies were conducted consistent with generally accepted guidelines at that time. The studies were critically evaluated by experts and their findings published in Food and Chemical Toxicology in 2003 (Schoneker et al., 2003). A series of safety studies with PVAP manufactured by Colorcon to NF specifications which included a definitive 90-day sub-chronic toxicity study, a developmental toxicity study and several genotoxicity tests consistent with current Good Laboratory Practice (GLP) regulations and internationally recognized guidelines were conducted to provide additional safety data to support current and new applications for PVAP and PVAP-T. The results of the 90-day sub-chronic dietary study in rats and two genotoxicity tests are reported herein.

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2. Materials and methods

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The purpose of this study was to evaluate the palatability and potential toxicity and toxicokinetics of PVAP when administered in the diet to rats for a minimum of 90 days. The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight changes, food consumption, test article consumption, ophthalmology, full functional observational battery assessments, clinical pathology parameters (hematology, coagulation, clinical chemistry, and urinalysis), toxicokinetic parameters, gross necropsy findings, organ weights, and histopathologic examinations. The study was conducted in compliance with the Good Laboratory Practice (GLP) regulations as described by the FDA (21 CFR Part 58) and OECD [C(97)186/ final; effective 1997] with the following exception: Analyses conducted to support

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the information cited in the Certificate of Analysis for the test article were conducted in a laboratory that employs applicable Good Manufacturing Practices (GMP’s). The design of the study is based on the following regulatory guidelines:

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 FDA, Guidance for industry: Nonclinical Studies for Development of Pharmaceutical Excipients. www.fda.gov/cder/guidance/5544fnl.htm, 2002.  Redbook, 2000. Toxicological Principles for the Safety Assessment of Food Ingredients. IV.B.1 General Guidelines for the Designing and Conducting Toxicity Studies. Office of Food Additive Safety, FDA. November 2003.  OECD Testing Guideline No. 408. Repeated Dose 90-day Oral Toxicity Study in Rodents, 21 September 1998.  ICH Harmonised Tripartite Guideline M3. Nonclinical Safety Studies for the Conduct of Human Clinical Trials for Pharmaceuticals, 11 June 2009.

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2.1. Test material

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Commercial samples of PVAP (NF) CAS # 34481-48-6 were manufactured and provided by Colorcon, Inc. The test material was a white powder and was stored at room temperature up to 75% relative humidity with desiccant present in the storage container. A Certificate of Analysis for the test and control articles was maintained. A 1-g sample of each lot of the bulk test and control articles was collected as a reserve sample and maintained at room temperature by the testing facility.

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2.2. Preparation of dose formulations

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Test article diet formulations were prepared at appropriate concentrations (w/ w) to meet dosage level requirements and were formulated to contain the appropriate amount of test article. The test diet was prepared once per week, dispensed in labeled feed jars and stored at room temperature during the dosing period in labeled plastic containers covered with labeled plastic bags. Animals received the test diet ad libitum for the extent of the in-life portion of the study, except during the fasting period prior to scheduled clinical pathology determinations. Conformation analysis of concentration of PVAP in test diets was conducted during weeks 1, 2, 5, 8, 11 and 13. Samples were collected from the top, middle, and bottom of the Group 2 and 4 dosing formulations for homogeneity analysis for day 1. Concentration and homogeneity analysis of the formulated test article was determined using validated methods. Stability was previously determined for concentrations and storage conditions bracketing those used in the study.

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2.3. Animals and husbandry

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Male and female Sprague Dawley Crl:CD(SD) rats were received from Charles River Laboratories, Portage, Michigan. The animals were examined and weighed on the day following receipt, and all were allowed to acclimate to the laboratory environment for a minimum of 12 days prior to the first day of dosing. The Sprague Dawley rat was chosen as the animal model for this study as it is a preferred rodent species for preclinical toxicity testing by regulatory agencies. The dietary route of exposure was selected since this is the intended route of human exposure. The animals were housed individually in suspended stainless steel cages during acclimation and while on study. The animals were individually identified using metal ear tags and cage cards. Housing and care were as specified in the USDA Animal Welfare Act (9 CFR, Parts 1, 2, and 3) and as described in the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996). Targeted environmental conditions were as follows:

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Temperature Humidity Light cycle Air changes

64–79 °F (18–26 °C) 50 ± 20% 12-h light/12-h dark cycle Ten or more air changes per hour with 100% fresh air 201

Actual room temperature and relative humidity were recorded a minimum of once daily and ranged from 67 to 73 °F (19 to 23 °C) and 40% to 65%, respectively.

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PMI Nutrition International Certified Rodent ChowÒ #5002 was provided ad libitum throughout the study, except during fasting for clinical pathology determinations. Municipal tap water following treatment by reverse osmosis and ultraviolet irradiation was available ad libitum throughout the study. Veterinary care was available throughout the study and animals were examined by the veterinary staff as warranted by clinical signs or other changes.

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2.4. Experimental design

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On day-2 (females) and day-1 (males and toxicokinetic animals), the animals were weighed and examined for detailed clinical observations. Animals determined to be suitable as test subjects were divided into two main populations, those for possible assignment to the toxicity phase and those for possible assignment to the toxicokinetic phase. Animals in each population were then randomly assigned to groups by a stratified randomization scheme designed to achieve similar group

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Please cite this article in press as: DeMerlis, C.C., et al. Safety of PVAP and PVAP-T including a 90-day dietary toxicity study in rats and genotoxicity tests with polyvinyl acetate phthalate (PVAP). Food Chem. Toxicol. (2014), http://dx.doi.org/10.1016/j.fct.2014.04.031

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No. of animals Toxicity

1 2 3 4 a b

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Q4

20 20 20 20

a

Toxicokinetic 5 5 5 5

Dose material

Dietary concentration (%)

Control PVAP PVAP PVAP

0.00 0.75 1.50 5.00

b

mean body weights. The toxicity phase animals were approximately 8 weeks of age at the time of randomization with body weights ranging from 292 to 339 g for the males and 192 to 237 g for the females. The toxicokinetic phase animals were approximately 9 weeks of age at the time of randomization with body weights ranging from 306 to 342 g for the males and 219 to 239 g for the females. The experimental design was as follows (see Table 1): The dose levels were selected based on information provided by Colorcon (range-finding study with PVAP in rats) and in an attempt to produce graded responses to the test article. Control diet (Group 1) or test article-treated diet (Groups 2–4) was administered ad libitum (except on days of fasting for clinical pathology determinations) to the appropriate animals from days 1 to 90. New control and test article-treated diet was provided once at the beginning of each week. The test article in the diet was maintained at a constant concentration (%) for each group. Mean test article consumption for each sex/group was calculated daily, except on days of fasting for clinical pathology determinations. The first day of dosing was designated as study day 1. 2.5. In-life observations

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General health/mortality and morbidity checks were performed twice daily in the morning and afternoon. Detailed clinical observations were performed on days-2 (females)/-1 (males), 1, 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, 85, 90, and 91. Cage-side observations were performed once daily in the morning on days 1– 90, except on the days of detailed clinical observation. Individual body weights were recorded on days-5 (females)/-4 (males), -2 (females)/-1 (males), 1, 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, 85, and 90. A final fasted body weight was recorded on the day of scheduled euthanasia (day 91). Quantitative food consumption measurements were recorded once daily on days 1–90. Ophthalmological examinations were performed by a board-certified veterinary ophthalmologist prior to in-life initiation and during the last week of dosing. The ocular examinations were conducted using a hand-held slit lamp and indirect ophthalmoscope. A short-acting mydriatic solution was used to dilate the eyes and facilitate the indirect ocular examinations. Full functional observational battery (FOB) evaluations were performed prior to in-life initiation and during the last week of dosing. The full FOB assessment included home cage, removal from home cage, open field evaluation, manipulative tests, and motor activity. The evaluations were performed blind (animals were not identified by group) prior to dosing.

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2.6. Clinical pathology

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Animals were fasted overnight prior to clinical pathology sample collections, but had ad libitum access to water. Blood samples for hematology, coagulation and clinical chemistry analyses were collected from the jugular vein or orbital plexus on days 30 and 58 and from the vena cava on day 91. Hematology parameters include red blood cell count, hemoglobin concentration, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin concentration (MCHC), mean corpuscular hemoglobin (MCH), reticulocyte count (absolute), platelet count, white blood cell count, neutrophil count, lymphocyte count, monocyte count, eosinophil count, basophil count and large unstained cells. Animals were individually housed in stainless steel urine collection cages containing water bottles with ball-bearing sipper tubes and urine was collected by cage pan drainage overnight. Urinalysis parameters evaluated were color, clarity, specific gravity, microscopic evaluation of urine sediment, total volume, pH, protein, glucose, bilirubin, ketones, nitrite, leukocytes, blood and urobilinogen. Coagulation parameters evaluated were activated partial thromboplastin time and prothrombin time. Clinical chemistry parameters evaluated were alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (Alk Phos’tase), gamma glutamyltransferase (GGT), total bilirubin, urea nitrogen, creatinine, calcium, phosphorus, total protein, albumin, globulin, albumin/globulin ratio (A/G Ratio), glucose, cholesterol, triglycerides, sodium, potassium and chloride.

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Males

Females

0.00 0.44 0.87 3.12

0.00 0.52 1.03 3.64

Number of animals for toxicity study: 20 males and 20 females, respectively. Number of animal for toxicokinetic study: 5 males and 5 females, respectively.

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Actual dose (g/kg/day)

2.7. Pathology Terminal procedures are summarized in the following table (see Table 2):

All toxicity phase animals were subjected to a complete gross necropsy examination. The necropsy examination included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues. A board-certified veterinary pathologist was present at the scheduled necropsies. The following organs were weighed for all toxicity phase animals at scheduled euthanasia: adrenal gland, brain, epididymis, heart, kidney, liver, lung, ovary, pituitary gland, prostate gland, salivary gland, seminal vesicle, spleen, testis, thymus, thyroid gland with parathyroid gland and uterus. The following tissues were collected from all toxicity phase animals and preserved in 10% neutral buffered formalin, unless otherwise indicated: adrenal gland (paired), animal identification, aorta, bone (femur), bone, sternum, bone marrow, sternum, brain (cerebrum, cerebellum, brain stem, medulla), cervix, epididymis (paired), esophagus, eye (paired), harderian gland (paired), heart, heart, intestine, cecum, intestine (colon) intestine(duodenum), intestine(ileum with Peyer’s patch), intestine (rectum), kidney (paired), liver, lung, lymph node, mandibular lymph node, mesenteric, mammary gland, optic nerve, sciatic nerve, ovary (paired), pancreas, parathyroid gland, pituitary gland, prostate gland, salivary gland (paired), seminal vesicle (paired), skeletal muscle (thigh), skin (mammary), spinal cord (cervical, thoracic, lumbar), spleen, stomach (nonglandular and glandular), testis (paired), thymus, thyroid gland (paired), tongue, trachea, urinary bladder, uterus, vagina, gross lesions/masses. All tissues and organs collected at necropsy from the Group 1 and 4 animals and all gross lesions from all animals in all groups were processed and examined microscopically. The rectum was identified as a target organ in the 5%-treated animals; therefore, it was processed and examined in the 0.75% and 1.5%-treated animals. The tissues were trimmed, embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin. Slides were examined microscopically by a board-certified veterinary pathologist.

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2.8. Statistical analysis

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Inferential statistical analyses were performed for the toxicity phase animals. The following parameters and end points were analyzed: body weights; body weight changes; food consumption; hematology, coagulation, clinical chemistry, and urinalysis (specific gravity, pH, and total volume); organ weights; and parametric, ranked, and count FOB data. Each data set was subjected to a statistical decision tree. Data sets for each interval were initially analyzed for homogeneity of variance using Levene’s test (Levene, 1960) followed by the Shapiro–Wilk test (Royston, 1995) for normality. A p < 0.001 level of significance was required for each test to reject the null hypothesis. If both Levene’s test and the Shapiro–Wilk test were not significant, a singlefactor parametric ANOVA (Gad and Weil, 1994) was applied, with animal grouping as the factor, using a p < 0.05 level of significance. If the parametric ANOVA was significant at p < 0.05, Dunnett’s test was used to identify statistically significant differences between the control group and each test article-treated group using a minimum significance level of p < 0.05. If either Levene’s test and/or the Shapiro–Wilk test were significant, then the Kruskal–Wallis non-parametric ANOVA (Siegel, 1956) was applied, with animal grouping as the factor, using a p < 0.05 level of significance. If the non-parametric Kruskal–Wallis ANOVA was significant at p < 0.05, Dunn’s test (Glantz, 1997) was used to identify statistically significant differences between the control group and each test article-treated group using a minimum significance level of p < 0.05. Descriptive (categorical) and quota functional observational battery (FOB) data was analyzed by Fisher’s Exact test. If significance was detected, a group by group comparison proceeded using the Chi-Square test.

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3. Results

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3.1. Dose formulation analysis

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All diet mixtures prepared were within acceptable limits of ±15% error. Mean concentration results from samples taken from

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Table 2 Terminal procedures. Group no.

No. of male/female rats

1 20/20 2 20/20 3 20/20 4 20/20 Unscheduled euthanasia animal

Scheduled euthanasia day

91 91 91 91

Terminal procedures

Histopathology

Gross necropsy

Tissue collection

Organ weights

x x x x x

x x x x x

x x x x –

Full Limited Limited Full Full

Note: x = procedure conducted.

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the top, middle, and bottom of the formulation were calculated and the homogeneity was calculated by determining the percent relative standard deviation (RSD) of the three mean values. Homogeneity was determined for the dose formulations prepared on 20 January 2009 and the samples were within the acceptable range of 65% RSD. Stability of formulations bracketing concentrations from 0.5% to 5.0% w/w and storage conditions (room temperature) was confirmed for 43 days.

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3.2. Mortality

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All animals survived until scheduled sacrifice except two toxicity animals: one 0%-treated male and one 5%-treated female. Both deaths were a result of accidental injuries and were not considered to be control or test article related. The 0%-treated male was euthanized moribund on day 56 due to a fractured rostrum. The 5%-treated female was euthanized moribund on day 84 due to a fractured tibia and fibula.

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3.3. Clinical observations

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PVAP-related soft stools were observed primarily in 5%-treated males throughout the dosing period. A few sporadic observations were also noted in 0.75% and 1.5%-treated males and P1.5%-treated females during the study.

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3.3.1. Body weights Male and female body weight growth curves are presented in the Figs. 2 and 3: There were no PVAP-related effects observed in mean body weights or body weight gain during the study. A few statistically significant differences were noted in body weight gain throughout the study; however, due to their sporadic nature, these differences were not considered toxicologically meaningful.

Fig. 3. Female group mean body weights (g).

3.4. Food consumption

Q5

Statistically significant differences in mean food consumption were noted in males during week 9 and 13 in group 2, during week 3 in group 3, during weeks 1–13 in group 4 and in females during weeks 2–5 in group 4. PVAP-related increases in mean food consumption were observed in 5%-treated males and females during the dosing period. Statistically significant increases up to 15.9% and 10.2% were observed in males and females, respectively, compared to the controls. In the absence of correlative increases in mean body weights, these differences from control were not considered toxicologically meaningful and were likely related to the caloric dilution as a result of the high levels of PVAP incorporation within the diet. A few additional statistically significant differences were noted; however, due to their sporadic occurrence, these differences were not considered toxicologically meaningful.

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3.5. Test article consumption

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The average mean test article consumption during the treatment period for the toxicity males and females is listed in Table 3 and in Figs. 4 and 5.

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3.6. Ophthalmology

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There were no PVAP-related ocular findings noted during the study.

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Table 3 Mean test article consumption (g/kg/day, 90 Days).

Fig. 2. Male group mean body weights (g).

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Sex

Group 2 (0.75%)

Group 3 (1.50%)

Group 4 (5.00%)

Males Females

0.44 0.52

0.87 1.03

3.13 3.64

Please cite this article in press as: DeMerlis, C.C., et al. Safety of PVAP and PVAP-T including a 90-day dietary toxicity study in rats and genotoxicity tests with polyvinyl acetate phthalate (PVAP). Food Chem. Toxicol. (2014), http://dx.doi.org/10.1016/j.fct.2014.04.031

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3.7. Functional observational battery

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There were no PVAP-related effects in functional observational battery data during the study. A slight gait abnormality (feet markedly point outward from body) was noted in one 5%-treated female on Day 84. In the absence of any other significant changes in the battery, this was considered incidental.

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3.8. Hematology and coagulation

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Statistically significant differences were observed sporadically during the dosing period, but there were no PVAP-related effects in mean hematology and coagulation parameters during the study. These differences from control were not considered toxicologically meaningful because all values fell within normalized or actual historical ranges and/or because dose–response trends over time were absent (i.e., differences noted in interim data but were not present at termination).

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3.9. Clinical chemistry

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Statistically significant differences were observed sporadically throughout the dosing period, but there were no PVAP-related effects in mean clinical chemistry parameters during this study. All differences from control noted in the test article-treated groups were within or near normalized historical control ranges and were not considered to be toxicologically meaningful due to the absence of microscopic correlates and/or due to the absence of a dose response.

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3.10. Urinalysis

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There were no PVAP-related effects in macroscopic or microscopic urinalysis parameters during the treatment period. Statistically significant differences in mean urinalysis data for pH for the males were noted for group 4 at day 30, 56 and 91. Statistically significant differences were observed in mean pH in 5%-treated males on Days 30, 56, and 91; however, they were not considered to be PVAP related due to the absence of microscopic correlates and/or the small magnitude of the difference, no dose response, and the occurrence in only one sex.

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3.11. Gross necropsy

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PVAP-related gross findings were observed in 5%-treated males and females on Day 91 and consisted of the presence of soft and/or watery fecal material in the cecum and colon. All other findings noted at scheduled necropsy were considered incidental.

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Fig. 4. Test article consumption data: males.

5

Fig. 5. Test article consumption data: females.

3.12. Organ weights

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There were no PVAP-related effects on absolute or relative organ weights at scheduled termination (Tables 4–7). Statistically significant differences in mean organ weight data were noted below; however, in the absence of microscopic correlates, these differences were not considered relevant (see Tables 8 and 9).

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3.13. Histopathology

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PVAP-related histopathology findings were observed in the rectum and cecum.

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3.13.1. Rectum Lymphocytic infiltration was characterized by increased numbers of small lymphocytes in lymphatics, perivascularly, and among the interstitial connective tissues of the submucosal space. In many cases, there was also an infiltration of lymphocytes in the deep mucosa, muscularis mucosae, muscularis, and serosa. This dose-dependent finding was considered to be test article related in males and females at all dietary concentrations for 90 days. Macrophage accumulation was characterized by increased numbers of macrophages with a vacuolated (foamy) cytoplasm in the lamina propria of the mucosa and within the interstitial tissues of the submucosa. This finding was considered to be test article related in males and females at the 1.5% and 5% dietary concentrations for 90 days. The mesenteric lymph nodes were within normal limits for all male and female animals. Increased interstitial matrix was characterized by increased fibrillar, amorphous, basophilic (blue) material in the submucosa. This finding was considered to be test article related in males and females at the 5.0% dietary concentration for 90 days.

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3.13.2. Cecum Lymphocytic infiltration and macrophage accumulation, similar to that seen in the rectum, was observed in the cecum of one 5%treated female. The test article-related histopathology findings of lymphocytic infiltration, macrophage accumulation, and increased interstitial matrix are similar to those reported previously in rats and mice treated 90 days by gavage with another macromolecule (Nyska et al., 2002). Elmiron is a semisynthetic pentose polysaccharide of 659 M. In this study, Elmiron caused the infiltration of vacuolated histiocytes (macrophages) in multiple tissues including the lamina propria of the rectum. The authors proposed that the macromolecule was absorbed through disruptions in the rectal mucosa and was transported to other tissues via the lymphatics. These findings were considered to be nonadverse and of minimal toxicological significance.

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Table 4 Summary of absolute and relative organ weight data (Males). Group

Absolute organ weight (g)

Dose concentration (%) Final body weight (g) Adrenals (g) Pituitary (g) Thyroid glands (g) Thymus (g) Liver (g) Brain (g) Heart g Kidneys (g) Lungs (g) Salivary glands (g) Spleen (g) Epididymides (g) Prostate (g) Seminal vesicles (g) Testes (g) *

1

2

3

4

0.00 528.0 ± 38.4 0.0530 ± 0.0087 0.0140 ± 0.0017 0.0230 ± 0.0033 0.2420 ± 0.0622 12.760 ± 1.609 2.260 ± 0.084 1.60 ± 0.108 3.320 ± 0.215 1.640 ± 0.105 0.840 ± 0.082 0.830 ± 0.182 1.500 ± 0.150 1.230 ± 0.188 1.880 ± 0.366 3.660 ± 0.262

0.75 546.0 ± 38.2 0.0590 ± 0.0082 0.0130 ± 0.0017 0.0240 ± 0.0038 0.2290 ± 0.0516 13.080 ± 1.352 2.260 ± 0.089 1.570 ± 0.139 3.490 ± 0.279 1.710 ± 0.107 0.880 ± 0.110 0.810 ± 0.159 1.480 ± 0.165 1.220 ± 0.185 1.810 ± 0.305 3.640 ± 0.293

1.50 544.0 ± 43.9 0.0570 ± 0.0081 0.0130 ± 0.0015 0.0240 ± 0.0057 0.2240 ± 0.0586 13.150 ± 1.312 2.250 ± 0.119 1.550 ± 0.126 3.440 ± 0.285 1.690 ± 0.111 0.870 ± 0.082 0.830 ± 0.131 1.460 ± 0.111 1.240 ± 0.157 1.840 ± 0.344 3.640 ± 0.248

5.00 532.0 ± 38.3 0.0570 ± 0.0094 0.0140 ± 0.0014 0.025 ± 0.005 0.214 ± 0.057 13.140 ± 1.428 2.260 ± 0.090 1.580 ± 0.161 3.570* ± 0.264 1.660 ± 0.144 0.900 ± 0.101 0.800 ± 0.151 1.510 ± 0.115 1.170 ± 0.184 1.960 ± 0.319 3.680 ± 0.227

P < 0.05.

Table 5 Summary of absolute and relative organ weight data (organ-to-body weight ratios).

*

Group

1

2

3

4

Dose concentration (%) Final body weight (g) Adrenals ratio Pituitary ratio Thyroid glands ratio Thymus ratio Liver ratio Brain ratio Heart ratio Kidneys ratio Lungs ratio Salivary glands ratio Spleen ratio Epididymides ratio Prostate ratio Seminal vesicles ratio Testes ratio

0.00 528.0 ± 38.4 0.01010 ± 0.00179 0.00260 ± 0.00039 0.00430 ± 0.00066 0.04610 ± 0.01269 2.4080 ± 0.1524 0.4290 ± 0.029 0.3040 ± 0.0213 0.6300 ± 0.0388 0.3120 ± 0.0254 0.159 ± 0.013 0.1570 ± 0.0328 0.2850 ± 0.0388 0.2340 ± 0.0356 0.3580 ± 0.0767 0.6960 ± 0.0666

0.75 546.0 ± 38.2 0.01080 ± 0.00156 0.00240 ± 0.00034 0.00450 ± 0.00066 0.04170 ± 0.00809 2.3900 ± 0.1212 0.4150 ± 0.0320 0.2880 ± 0.0234 0.6390 ± 0.0441 0.3130 ± 0.0140 0.1620 ± 0.0195 0.1480 ± 0.0246 0.2710 ± 0.0299 0.2240 ± 0.0359 0.3330 ± 0.0539 0.6690 ± 0.0721

1.50 544.0 ± 43.9 0.01050 ± 0.00147 0.00240 ± 0.00029 0.00430 ± 0.00104 0.04110 ± 0.01031 2.4170 ± 0.1541 0.4160 ± 0.0409 0.2860* ± 0.0206 0.635 ± 0.0585 0.3120 ± 0.0219 0.16 ± 0.0135 0.152 ± 0.0207 0.2690 ± 0.0255 0.2280 ± 0.0294 0.3410 ± 0.071 0.6710 ± 0.0609

5.00 532.0 ± 38.3 0.01070 ± 0.00177 0.00270 ± 0.00024 0.00470 ± 0.00107 0.04020 ± 0.00973 2.4680 ± 0.1309 0.4270 ± 0.0360 0.2960 ± 0.0174 0.6720* ± 0.0433 0.3120 ± 0.0233 0.1700 ± 0.0199 0.1500 ± 0.0243 0.2850 ± 0.032 0.2210 ± 0.0376 0.3690 ± 0.0602 0.6980 ± 0.0767

P < 0.05.

Table 6 Summary of absolute and relative organ weight data (organ-to-brain weight ratios).

471 472 473 474

Group

1

2

3

4

Dose concentration (%) Adrenals ratio Pituitary ratio Thyroid glands ratio Thymus ratio Liver ratio Heart ratio Kidneys ratio Lungs ratio Salivary glands ratio Spleen ratio Epididymides ratio Prostate ratio Seminal vesicles ratio Testes ratio

0.00 2.35770 ± 0.39748 0.60090 ± 0.08333 1.00710 ± 0.1314 10.6970 ± 2.5946 565.500 ± 66.583 71.1660 ± 5.6358 147.1700 ± 8.8981 72.9410 ± 5.1701 37.2020 ± 3.1176 36.8530 ± 8.7039 66.5320 ± 7.6180 54.7720 ± 8.3031 83.586 ± 17.348 162.390 ± 12.177

0.75 2.61010 ± 0.37495 0.57860 ± 0.0799 1.07860 ± 0.17224 10.0960 ± 2.1197 579.170 ± 59.881 69.6670 ± 6.2365 154.5900 ± 13.6650 75.6280 ± 4.7310 39.0450 ± 4.9639 35.7010 ± 6.4450 65.5950 ± 7.9998 53.9600 ± 7.7048 80.488 ± 14.695 161.330 ± 14.077

1.50 2.54350 ± 0.36435 0.59030 ± 0.07562 1.05350 ± 0.27191 9.9563 ± 2.6228 585.820 ± 66.773 69.0460 ± 6.7091 153.3500 ± 14.3390 75.2050 ± 5.0122 38.7280 ± 4.1486 36.7830 ± 5.1414 64.8120 ± 5.3060 54.8800 ± 5.9526 81.713 ± 13.981 161.920 ± 12.989

5.00 2.52110 ± 0.42163 0.62720 ± 0.06508 1.08620 ± 0.20293 9.5001 ± 2.5066 583.090 ± 72.036 69.9170 ± 7.6540 158.0000 ± 10.8130 73.5120 ± 6.9918 40.0110 ± 4.4110 35.5240 ± 7.1387 66.7090 ± 4.8534 51.7830 ± 7.9628 86.616 ± 13.947 163.300 ± 10.425

All other histopathology findings were considered to be unrelated to PVAP treatment because they were infrequent, generally of low severity, and similarly distributed among treated and control groups.

3.14. Toxicokinetic phase

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All toxicokinetic (TK) animals survived until scheduled euthanasia. A method for the analysis of PVAP in plasma was not possi-

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Please cite this article in press as: DeMerlis, C.C., et al. Safety of PVAP and PVAP-T including a 90-day dietary toxicity study in rats and genotoxicity tests with polyvinyl acetate phthalate (PVAP). Food Chem. Toxicol. (2014), http://dx.doi.org/10.1016/j.fct.2014.04.031

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2

3

4

Dose concentration (%) Final body weight (g) Adrenals (g) Pituitary (g) Thyroid glands (g) Thymus (g) Ovaries (g) Liver (g) Brain (g) Heart (g) Kidneys (g) Lungs (g) Salivary glands (g) Spleen (g) Uterus (g)

0.00 280.0 ± 20.7 0.0620 ± 0.0092 0.0190 ± 0.0034 0.0180 ± 0.0038 0.2370 ± 0.0461 0.0810 ± 0.0177 6.830 ± 0.461 2.030 ± 0.070 0.940 ± 0.077 1.900 ± 0.115 1.190 ± 0.074 0.520 ± 0.057 0.490 ± 0.071 0.530 ± 0.155

0.75 281 ± 22.7 0.0660 ± 0.0098 0.0170 ± 0.0034 0.0160 ± 0.0028 0.2080 ± 0.0665 0.0910 ± 0.0380 6.800 ± 0.663 2.030 ± 0.070 0.960 ± 0.100 1.910 ± 0.145 1.220 ± 0.106 0.530 ± 0.058 0.500 ± 0.095 0.540 ± 0.136

1.50 286 ± 18.8 0.0650 ± 0.0106 0.0190 ± 0.0041 0.0100 ± 0.0030 0.2160 ± 0.0434 0.0850 ± 0.0155 6.840 ± 0.631 2.030 ± 0.087 0.990 ± 0.082 1.980 ± 0.139 1.200 ± 0.092 0.530 ± 0.059 0.490 ± 0.058 0.640 ± 0.206

5.00 276 ± 22.3 0.0650 ± 0.0110 0.0170 ± 0.0031 0.0170 ± 0.0029 0.2120 ± 0.0761 0.0840 ± 0.0129 6.900 ± 0.849 2.050 ± 0.104 0.940 ± 0.086 1.920 ± 0.199 1.180 ± 0.107 0.540 ± 0.052 0.500 ± 0.101 0.530 ± 0.131

Table 8 Summary of absolute and relative organ weight data (organ-to-body weight ratios). Group

1

2

3

4

Dose concentration (%) Final body weight (g) Adrenals ratio Pituitary ratio Thyroid glands ratio Thymus ratio Ovaries ratio Liver ratio Brain ratio Heart ratio Kidneys ratio Lungs ratio Salivary glands ratio Spleen ratio Uterus ratio

0

0.75 281.0 ± 22.7 0.02360 ± 0.00348 0.00620 ± 0.00121 0.00580 ± 0.00101 0.07370 ± 0.02235 0.03220 ± 0.01349 2.4160 ± 0.1316 0.7230 ± 0.0572 0.3420 ± 0.0167 0.6800 ± 0.0510 0.4350 ± 0.0320 0.1900 ± 0.0149 0.1780 ± 0.0360 0.1930 ± 0.0508

1.5 286.0 ± 18.8 0.02290 ± 0.00375 0.00670 ± 0.00144 0.00580 ± 0.00091 0.07580 ± 0.01568 0.02980 ± 0.00552 2.3910 ± 0.1618 0.7120 ± 0.0436 0.3470 ± 0.0273 0.6910 ± 0.0344 0.4190 ± 0.0239 0.1840 ± 0.0193 0.1730 ± 0.0192 0.2230 ± 0.0738

5

280.0 ± 20.7 0.02240 ± 0.00364 0.00670 ± 0.00137 0.00650 ± 0.00138 0.08470 ± 0.01604 0.02900 ± 0.00583 2.4450 ± 0.1620 0.7300 ± 0.0558 0.3380 ± 0.0294 0.6800 ± 0.0330 0.4260 ± 0.0190 0.1870 ± 0.0168 0.1750 ± 0.0263 0.1920 ± 0.0564

276.0 ± 22.3 0.02370 ± 0.0036 0.00620 ± 0.00111 0.00620 ± 0.00099 0.07590 ± 0.02100 0.03050 ± 0.00437 2.4980 ± 0.1774 0.7500 ± 0.0728 0.3400 ± 0.0233 0.6980 ± 0.0552 0.4300 ± 0.0275 0.1950 ± 0.0168 0.1810 ± 0.0255 0.1930 ± 0.0444

Table 9 Summary of absolute and relative organ weight data (organ-to-brain weight ratios). Group

1

2

3

4

Dose concentration (%) Adrenals ratio Pituitary ratio Thyroid glands ratio Thymus ratio Ovaries ratio Liver ratio Heart ratio Kidneys ratio Lungs ratio Salivary glands ratio Spleen ratio Uterus ratio

0 3.06700 ± 0.41552 0.91580 ± 0.17033 0.89570 ± 0.18662 11.6610 ± 2.2376 3.99230 ± 0.84276 336.210 ± 25.984 46.4730 ± 3.6318 93.4210 ± 5.7507 58.6260 ± 3.8775 25.7740 ± 2.7308 24.0230 ± 3.4650 26.2260 ± 7.3607

0.75 3.26980 ± 0.44890 0.86260 ± 0.16619 0.79640 ± 0.12485 10.2120 ± 3.0881 4.51140 ± 2.03880 336.060 ± 32.259 47.6490 ± 4.7723 94.2310 ± 6.7637 60.3720 ± 4.7609 26.3280 ± 2.4996 24.5900 ± 4.3819 26.7230 ± 7.2438

1.5 3.21520 ± 0.49782 0.93690 ± 0.20930 0.82320 ± 0.13365 10.6430 ± 2.0820 4.19760 ± 0.80934 337.030 ± 30.042 48.8910 ± 4.6947 97.3100 ± 5.6928 59.0490 ± 4.0011 25.9770 ± 2.9423 24.3660 ± 2.8276 31.3250 ± 9.9874

5 3.17070 ± 0.45678 0.82780 ± 0.16042 0.83280 ± 0.15046 10.4300 ± 4.1494 4.10550 ± 0.67424 336.660 ± 45.401 45.6700 ± 4.9541 93.9260 ± 11.5620 57.8410 ± 6.6602 26.2000 ± 2.8813 24.5600 ± 5.4375 26.0480 ± 6.5571

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ble using the instrumentation and technology currently available in the contract research laboratory. A suitable analytical method could not be established for analyzing PVAP in rat plasma and a memorandum with the explanation was included in the study report. Therefore, bioanalytical analysis of the samples collected within the study and TK interpretation were not performed. The TK samples were archived at the contract research organization.

486

4. Discussion and conclusion

487

The purpose of the 90-day dietary study was to evaluate the potential toxicity of PVAP when administered in the diet to Sprague

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488

Dawley CRL: CD (SD) rats (20/sex/group). The following evaluations were completed: clinical signs, body weights, body weight changes, food consumption, test article consumption, ophthalmology, full functional observational battery assessments, clinical pathology parameters (hematology, coagulation, clinical chemistry, and urinalysis), gross necropsy findings, organ weights, and histopathologic examinations. All animals survived until scheduled sacrifice except two toxicity animals: one 0%-treated male was euthanized moribund on Day 56 due to a fractured rostrum, and one 5%-treated female was euthanized moribund on Day 84 due to a fractured tibia and fibula. These deaths were accidental and were not considered to be control or test article related.

Please cite this article in press as: DeMerlis, C.C., et al. Safety of PVAP and PVAP-T including a 90-day dietary toxicity study in rats and genotoxicity tests with polyvinyl acetate phthalate (PVAP). Food Chem. Toxicol. (2014), http://dx.doi.org/10.1016/j.fct.2014.04.031

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PVAP-related soft stools were observed primarily in 5%-treated males throughout the dosing period. A few observations were also noted in 0.75% and 1.5%-treated males and P1.5%-treated females during the study. PVAP-related lymphocytic infiltration and macrophage accumulation were observed in the rectum of both sexes at P0.75% and P1.5%, respectively, and in the cecum of one 5%-treated female. Increased interstitial matrix was also observed in the rectum of both sexes at the 5% dietary concentration. These findings were considered to be nonadverse and of minimal toxicological significance. They are similar to those reported previously in rats and mice treated for 90 days by gavage with another macromolecule (a semisynthetic pentose polysaccharide of 659 M) (Nyska et al., 2002). This macromolecule caused the infiltration of vacuolated histiocytes (macrophages) in multiple tissues including the lamina propria of the rectum. The authors proposed that the macromolecule was absorbed through disruptions in the rectal mucosa and was transported to other tissues via the lymphatics. There were no consistent, dose-related, statistically significant PVAP-related adverse effects in body weight, body weight changes, ophthalmic examinations, functional observational data, hematology parameters, coagulation parameters, clinical chemistry parameters, urinalysis parameters (macroscopic and microscopic), or absolute or relative organ weights. Statistically significant PVAP-related increases up to 15.9% and 10.2% in mean food consumption were observed in 5%-treated males and females, respectively, compared to the controls, likely as a result to compensate for the dilution in calories from the incorporation of PVAP at 5% of the diet. PVAP-related gross findings were observed in 5%-treated males and females on day 91 and consisted of the presence of soft and/or watery fecal material in the cecum and colon. There were no toxicologically meaningful gross or microscopic changes noted during the study. The toxicokinetic phase could not be completed because an analytical method could not be developed for PVAP in blood plasma. In conclusion, daily administration of polyvinyl acetate phthalate (PVAP) in the diet was well tolerated in male and female rats up to a concentration of 5%. No PVAP mortality or toxicity was observed. Based on these results, the no-observed-adverse-effect level (NOAEL) was considered to be the 5% dietary concentration, which corresponds to a dose level of 3.12 g/kg/day for males and 3.64 g/kg/day for females.

544

5. Genotoxicity tests

545

5.1. Ames test PVAP

546

The purpose of this study was to evaluate the genotoxicity of PVAP using a bacterial mutation test. Salmonella typhimurium strains (TA1535, TA1537, TA98, TA100) and Escherichia coli strain WP2 uvrA were treated with the test article at a range of concentrations up to 5000 lg/plate (the standard limit dose for this assay) in the presence and absence of a supplemented liver fraction (S9 mix) using the plate incorporation and pre-incubation versions of the bacterial mutation test. The procedures described in the test comply with the recommendations of the ICH S2A, the OECD guideline 471 for the testing of chemicals (1997), the US EPA health effects test guidelines and the US FDA Redbook, 2000. Bacteria were incubated with standard positive control agents, and the response of the various bacterial strains to these agents confirmed the sensitivity of the test system and the activity of the S9 mix. The test article PVAP was formulated as a suspension in aqueous 0.5% w/v methylcellulose. Sterile water for irrigation USP or DMSO was used as the vehicle for the positive controls.

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The following dose levels were tested: 1.58 lg/plate, 5.0 lg/plate, 15.8 lg/plate, 50 lg/plate, 158 lg/plate, 500 lg/plate, 1581 lg/ plate and 5000 lg/plate. Test article was tested at levels up to 5000 lg/plate, which is the standard limit dose recommended by regulatory guidelines. No substantial increases in the revertant colony counts were obtained with any strain following exposure to the test article at any dose level in either the plate incorporation or pre-incubation assay in the absence or presence of S9 mix. No visible thinning of the background lawn of non-revertant bacteria was obtained following exposure to PVAP, indicating that the test article was non-toxic to the bacteria at the levels tested. No precipitation was observed. It is concluded that PVAP did not show any evidence of genotoxic activity in this in vitro mutagenicity assay when tested in accordance with regulatory guidelines.

565

5.2. Chromosome aberration test PVAP

581

The purpose of this study was to evaluate the genotoxicity of PVAP using an in vitro chromosome aberration test. Human peripheral blood lymphocytes were stimulated into division in culture then treated with the test article at a range of concentrations up to the standard limit of 5000 lg/mL. Cultures were treated for 4 hours in the absence and presence of rat S9 mix and for 21 hours in the absence; appropriate concurrent vehicle and positive controls were included for each treatment regime. The procedures described in this report comply with the recommendations of the ICH S2A, the OECD guideline 473 for the testing of chemicals (1997), the US EPA health effects test guidelines and the US FDA Redbook, 2000. The test article PVAP was formulated as a suspension in aqueous 0.5% w/v methylcellulose. Sterile water for irrigation USP was used as the vehicle for the positive controls. The following dose levels were tested: 10.0 lg/mL, 20.0 lg/mL, 40.0 lg/mL, 80.0 lg/mL, 160 lg/mL, 320 lg/mL, 640 lg/mL and 1280 lg/mL, 2560 lg/mL and 5000 lg/mL. The highest dose level tested was the maximum dose level recommended by the OECD (5000 lg/mL). At the highest one or two dose levels of PVAP, some toxicity was evident in all three treatment regimes, as indicated by a substantial reduction in mitotic index and a general reduction in the absolute number of readable metaphases. Metaphases from cultures treated with the three or four highest dose levels of test article not producing excessive toxicity (together with appropriate vehicle and selected positive control cultures) were subjected to detailed examination for the presence of chromosomal aberrations using light microscopy. Cultures treated with PVAP at levels at any experimental point did not show any statistically significant increases in the incidence of cells with aberrant metaphases. The positive control agents caused highly significant increases in the proportion of cells with chromosome damage, confirming the sensitivity of the system and the effectiveness of the S9 mix. It is concluded that PVAP did not show any evidence of genotoxic activity in this in vitro test for induction of chromosome damage when tested in accordance with regulatory guidelines.

582

5.3. PVAP-T toxicology study results

619

Two GLP studies were conducted to provide further supporting information for the safety of PVAP-T.

620

5.4. Acute oral toxicity PVAP-T

622

The acute oral toxicity of co-processed PVAP and titanium dioxide (PVAP-T) was assessed when administered by gavage as

623

Please cite this article in press as: DeMerlis, C.C., et al. Safety of PVAP and PVAP-T including a 90-day dietary toxicity study in rats and genotoxicity tests with polyvinyl acetate phthalate (PVAP). Food Chem. Toxicol. (2014), http://dx.doi.org/10.1016/j.fct.2014.04.031

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a single oral dose to Sprague Dawley male and female rats. There were no deaths and no signs of intoxication. The acute oral LD50 of PVAP-T was estimated to be greater than 5000 mg/kg in the rat, the highest dose tested and the recommended limit dose.

629

5.5. Ames test PVAP-T

630

667

A bacterial mutation test was performed to evaluate the potential genotoxicity of PVAP-T. The purpose of this study was to evaluate the genotoxicity of PVAP-T using a bacterial mutation test. Salmonella typhimurium strains (TA1535, TA1537, TA98, TA100) and Escherichia coli strain WP2 uvrA were treated with the test article at a range of concentrations up to 5000 lg/plate (the standard limit dose for this assay) in the presence and absence of a supplemented liver fraction (S9 mix) using the plate incorporation and pre-incubation versions of the bacterial mutation test. The procedures described in the test comply with the recommendations of the ICH S2A, the OECD guideline 471 for the testing of chemicals (1997), the US EPA health effects test guidelines and the US FDA Redbook, 2000. Bacteria were incubated with standard positive control agents, and the response of the various bacterial strains to these agents confirmed the sensitivity of the test system and the activity of the S9 mix. The test article PVAP-T was formulated as a suspension in aqueous 0.5% w/v methylcellulose. Sterile water for irrigation USP or DMSO was used as the vehicle for the positive controls. The following dose levels were tested: 1.58 lg/plate, 5.0 lg/plate, 15.8 lg/plate, 50 lg/plate, 158 lg/plate, 500 lg/plate, 1581 lg/ plate and 5000 lg/plate. Test article was tested at levels up to 5000 lg/plate, which is the standard limit dose recommended by regulatory guidelines. No substantial increases in the revertant colony counts were obtained with any strain following exposure to the test article at any dose level in either the plate incorporation or pre-incubation assay in the absence or presence of S9 mix. No visible thinning of the background lawn of non-revertant bacteria was obtained following exposure to PVAP-T, indicating that the test article was non-toxic to the bacteria at the levels tested. Insoluble material (presumably test article) was observed at the highest dose levels in the plate incorporation and the pre-incubation assay. It is concluded that PVAP-T did not show any evidence of genotoxic activity in this in vitro mutagenicity assay when tested in accordance with regulatory guidelines.

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5.6. PVAP-T analytical studies

669

The toxicological data for PVAP is used to support the safety of PVAP-T. In order to bridge PVAP-T to the PVAP data, several analytical studies were conducted to demonstrate that PVAP and TiO2 are not chemically altered during the manufacturing process or during transit through the gastrointestinal tract. PVAP-T is a co-processed excipient which is manufactured by combining PVAP and TiO2 using a physical process that results in an intimate mixture of the particles. No chemical modification occurs in the process. A series of studies were performed to determine if the PVAP-T co-processed excipient exhibits the same chemical composition as would exist for a standard physical blend of PVAP and TiO2. Three batches of a physical blend of PVAP and TiO2 and coprocessed PVAP-T were manufactured according to the standard operating procedures. The batches used the same lots of raw materials and were individually sampled and tested. Samples were evaluated using standard compendial release testing and other analytical techniques consisting of FTIR spectral analysis, size

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9

exclusion chromatography with photodiode array detection, reversed phase HPLC, differential scanning calorimetery, thermogravimetric analysis, powder X-ray diffraction, particle size analysis both wet and dry, headspace gas chromatography and biorelevant dissolution testing. The results of this study clearly demonstrate that there were no chemical differences between a physical blend or co-processed PVAP and titanium dioxide. The routine QC testing met the predetermined specifications and did not show any trends or differences between the blended or co-processed materials. Testing was performed in USP gastric and intestinal fluid, both with and without enzymes, to evaluate the effects of co-processing on the PVAP polymer with titanium dioxide when exposed to different pH and enzymatic activities. No difference in UV/Vis spectra was observed between the co-processed and blended PVAP-T samples in the four media tested. PVAP was not soluble in either of the fluids. There is no chemical difference between the PVAP and TiO2 physical blends and the PVAP-T co-processed product. No signs of any degradation products were found in the PVAP-T samples. This information helps to bridge to the toxicology studies conducted with PVAP to PVAP-T and supports the safety of PVAP-T.

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5.7. Independent expert safety evaluation of PVAP and PVAP-T

708

The International Pharmaceutical Excipients Council (IPEC) of the Americas developed a New Excipient Safety Evaluation Procedure. The goal of this process is to provide an independent evaluation of the safety and regulatory acceptance of a new excipient before a regulatory filing. The process is meant to mirror that of regulatory agencies, ideally providing confidence to pharmaceutical manufacturers that the excipient will be acceptable in their formulations. This procedure has been discussed with the U.S. FDA and they acknowledged that this type of review would be very beneficial when evaluating a new excipient. The New Excipient Evaluation Committee (NEEC) is the expert panel that conducts the excipient safety evaluation. The NEEC Expert Panel independently and collectively critically evaluated the data and information summarized for PVAP and PVAP-T and concluded that PVAP and PVAP-T are safe for their intended use as an excipient in film coating formulations and matrix tablet drug products. Based on the toxicology study results, safety assessment and the estimated exposure assessment in the NEEC’s report for PVAP and PVAP-T, the expert panel concluded that PVAP and PVAP-T could safely be used in drug products up to 829 mg per day which was the estimated exposure provided to the expert panel for current applications of PVAP and PVAP-T.

709

6. Summary and conclusion

731

The safety of PVAP and PVAP-T is based on the following supporting information and study results:

732

 PVAP and PVAP-T have a long history of safe use in drug products in many countries and are used commercially in Colorcon formulated products such as Opadry Enteric, Opacode, Sureteric and Coateric. An Expert Panel of toxicologists concluded that PVAP is Generally Recognized as Safe (GRAS) by scientific procedures for use in printing inks in the United States for marking dietary supplements.  The series of safety studies conducted by Colorcon with PVAP include a definitive 90-day subchronic toxicity study, a developmental toxicity study and several genotoxicity tests. There were no adverse effects reported in the 90-day subchronic toxicity study and the developmental toxicity study. The no-observedadverse-effect level (NOAEL) in a GLP 90-day sub chronic study

734

Please cite this article in press as: DeMerlis, C.C., et al. Safety of PVAP and PVAP-T including a 90-day dietary toxicity study in rats and genotoxicity tests with polyvinyl acetate phthalate (PVAP). Food Chem. Toxicol. (2014), http://dx.doi.org/10.1016/j.fct.2014.04.031

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was 5% dietary concentration, which corresponds to a dose of 3.12 g/kg/day for males and 3.64 g/kg/day for females, the highest level fed. PVAP is neither mutagenic nor genotoxic.  There were no treatment-related, dose-dependent, statistically significant adverse effects on any of the maternal and fetal parameters evaluated in the developmental study. Therefore, the maternal and developmental no-observable-adverse-effect level (NOAEL) of PVAP is the highest concentration tested, i.e., 3.0% (equivalent to 2324.6 mg PVAP/kg/day). The developmental study is being published in the Regulatory Toxicology and Pharmacology journal.  The available toxicological data support the commercial uses of PVAP and PVAP-T. The chemical composition, physiochemical properties and specifications are unchanged during manufacture of PVAP-T based on the analytical studies conducted by Colorcon. Therefore, the toxicological data that support the safety of PVAP can be used to support the use of PVAP-T as an excipient.  Based on the toxicology study results, safety assessment and the estimated exposure assessment in the NEEC’s report for PVAP and PVAP-T, the expert panel concluded that PVAP and PVAP-T could safely be used in drug products up to 829 mg per day.

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Conflict of Interest

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C.C. DeMerlis and D.R. Schoneker are employees of Colorcon, Inc. Colorcon retained J.F. Borzelleca as a consultant and compensated him for his services.

Transparency Document

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The Transparency document associated with this article can be found in the online version.

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Acknowledgment

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The authors thank Hua Deng and David Ferrizzi of Colorcon, Inc. for assistance in the preparation of the manuscript.

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Please cite this article in press as: DeMerlis, C.C., et al. Safety of PVAP and PVAP-T including a 90-day dietary toxicity study in rats and genotoxicity tests with polyvinyl acetate phthalate (PVAP). Food Chem. Toxicol. (2014), http://dx.doi.org/10.1016/j.fct.2014.04.031