Subchronic toxicity evaluation of potato protein isolates

Food and Chemical Toxicology 50 (2012) 373–384

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Food and Chemical Toxicology

journal homepage: www.elsevier .com/locate / foodchemtox

Subchronic toxicity evaluation of potato protein isolates

B. Lynch a,⇑, R.R. Simon a, F.M. van Otterdijk b, H.H. Emmen b,M.L.F. Giuseppin c, C. Kemme-Kroonsberg c

a Cantox Health Sciences International, An Intertek Company, 2233 Argentia Road, Suite 308, Mississauga, ON, Canada L5N 2X7b NOTOX B.V., Hambakenwetering 7, 5231 DD, ‘s-Hertogenbosch, The Netherlandsc AVEBE U.A., AVEBE-weg 1, 9607 PT Foxhol, The Netherlands

a r t i c l e i n f o a b s t r a c t

Article history:Received 18 April 2011Accepted 30 September 2011Available online 7 October 2011

Keywords:Potato proteinProtease inhibitorTrypsin inhibitora-Solaninea-ChaconineToxicity

0278-6915/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.fct.2011.09.039

Abbreviations: HMW, high molecular weight; L-B4-nitroanilide hydrochloride; LMW, low molecular wwhite blood cell count.⇑ Corresponding author. Tel.: +1 905 542 2900; fax

E-mail address: [email protected] (B. Lynch).

The protein content of potatoes has a high nutritional value on par with eggs and soybeans. As a result,processed potato protein isolates may have commercial value for addition to other food products toincrease protein content. A manufacturing process has been developed to produce total potato (TP), aswell as low (LMW) and high molecular (HMW) weight, protein isolates as food ingredients. To assessthe safety of these isolates, groups of 10 Wistar rats/sex were administered dietary admixtures contain-ing 15% HMW, 7.5% LMW or 15% TP protein isolates for a period of 90 days. There was no effect of treat-ment on clinical signs, mortality, body weight and body weight gain. No biologically significant changesoccurred in hematological and clinical chemistry parameters. No statistically significant changes in organweights were recorded. Histopathological analyses revealed no clear, treatment-related changes. A slightincrease in the incidence, but not severity, of vacuolation of the zona fasciculate of the adrenal gland wasnoted in males of the 15% HMW and 7.5% LMW groups. The finding was not considered adverse orascribed any toxicological significance. Overall, HMW, LMW, and TP protein isolates were well-toleratedand without adverse effect. These data support the safety of potato protein isolates.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction Medina, 2007). While these values are low in relation to many other

The cultivated potato, originating from South America, has beenin domesticated use for more than 8000 years (CIP, 2008). Potatoes,prepared by numerous means, are consumed on a nearly world-wide basis (Singh and Kaur, 2009). While traditionally a food cropof Europe and the Americas, almost half of the global supply of pota-toes and potato-based products is now consumed in Asia (Camireet al., 2009). Relative to other food crops, potatoes have a very highyield, an agronomic trait that places the vegetable with rice andwheat as one the top three food commodities of nutritional impor-tance globally (Camire et al., 2009; CIP, 2008; Karim et al., 1997).

The nutritional value and potential health benefits of potatoeshas been of interest in recent years (Andre et al., 2007; Bártováand Bárta, 2009; Burrowes and Ramer, 2008; Camire et al., 2009;Henry et al., 2005; Leeman et al., 2008; Robert et al., 2008; Shakyaand Navarre, 2008; Singh and Kaur, 2009). In its most commonform, the potato tuber is usually eaten following boiling, eitherpeeled or unpeeled. The protein content of potatoes is reported torange from 1.0% to 2.0% of fresh tuber weight (OECD, 2002; Ortiz-

ll rights reserved.

APA, L-Na-benzoyl-L-arginineeight; TP, total potato; WBC,

: +1 905 542 1011.

vegetable sources of dietary protein (Singh and Kaur, 2009), potatoproteins have been noted to have high protein biological values, inthe range of 90–100 (Camire et al., 2009), which compares well toother foodstuffs commonly associated with having high qualityprotein content such as whole eggs, soybean, and beans, where cor-responding protein biological values of 100, 84, and 73, respec-tively, have been reported. Patatin is the major protein present inpotato tubers, making up about 40% of the water soluble protein(Mignery et al., 1988; Shewry, 2003), and has been reported to haveallergenic properties in sensitive individuals (Camire et al., 2009;Seppälä et al., 1999, 2000). However, this allergenicity is greatly re-duced upon heating (Koppelman et al., 2002).

Given the biological value of potato proteins, and the high yield atwhich potatoes can be produced, there is increasing interest in theisolation of these proteins from potato juice, produced during themanufacture of starches from raw potatoes, for inclusion/use inother foodstuffs to increase protein content and nutritional value.Potato proteins also display unique physicochemical properties re-lated to their ability to form highly stable foams, a technical applica-tion that is in demand as the food industry is continually seekingnatural alternatives to chemical additives. In this regard, a methodhas been developed to isolate potato proteins, in a minimally pro-cessed form, from raw potato tubers. The isolation process involveschromatography and ultrafiltration techniques, and does not rely

374 B. Lynch et al. / Food and Chemical Toxicology 50 (2012) 373–384

upon any chemical processing that would alter the amino acid com-position. Furthermore, the processing steps are not intended tointroduce any changes in the primary structure of the protein. Thefractionation procedure developed produces three distinct proteinfractions: a high molecular weight (HMW) fraction (>35 kDa) anda low molecular weight (LMW) fraction (>4 kDa, but <35 kDa). Theproduct is a total protein isolate (TP) (>4 kDa), which comprises boththe HMW and LMW fractions and can be isolated directly as such orprepared as a blend of the two fractions. The HMW fraction consistsprimarily of patatin, the main potato storage protein, whereas theLMW fraction comprises a group of protease inhibitor proteins.The HMW fraction represents 50% of the total protein isolate.

Protein isolates from potato specifically are currently not listed inthe Code of Federal Regulations as an approved food additive in theUS. However, in 2002 the United States Food and Drug Administra-tion issued a letter of no objection in response to a Notice of Gener-ally Recognized as Safe self-determination for coagulated potatoprotein in hydrolyzed and un-hydrolyzed form (‘‘potato proteinpreparations’’) for addition to a variety of food products as a waterbinder, foaming aid, or emulsifier at use-levels in the range of 0.1–3.0% resulting in dietary exposures of 1.9 g/day (GRN 000086) (U.S.FDA, 2002). The primary difference between coagulated potato pro-tein (un-hydrolyzed) and HMW/LMW potato proteins is the methodused to obtain the protein from the potato. As the name implies,while heat coagulated potato protein is obtained as a result of coag-ulation of the protein following heat-treatment at temperatures>80–105 �C, HMW and LMW potato proteins are isolated by chro-matographic separation techniques; however, neither process dis-rupts the primary chemical structure of the proteins containedwithin each fraction, and in both cases, the final product is compara-ble to the protein in a boiled potato. Additionally, a potato proteinextract, containing potato proteinase inhibitor II, a protein identifiedin the LMW ingredient, is currently being marketed in the US as adietary supplement under the Dietary Supplements Health and Edu-cation Act of 1994 (DSHEA, 1994). For this product, Slendesta�, therecommended daily dose is 600 mg, standardized to 5% proteinaseinhibitor content (i.e., 30 mg/day).

No traditional safety toxicology studies on potato proteins wereidentified in the scientific literature. Thus, as part of the evaluationof the safety of the potato protein isolates manufactured by SolanicBV, a 90-day oral toxicity study in rats was conducted with each ofthe HMW, LMW, and TP fractions. The results of this study are re-ported herein.

1 The study was conducted at NOTOX B.V. (‘s-Hertogenbosch, The Netherlands) incompliance with standards as described under: OECD, 1998a. OECD Principles ofGood Laboratory Practice (as Revised in 1997). Organisation for Economic Co-Operation & Development (OECD), Environment Directorate, Chemicals Group andManagement Committee, OECD Environmental Health and Safety Publications, Paris,France. OECD Series on Principles of Good Laboratory Practice and ComplianceMonitoring, No. 1, ENV/MC/CHEM(98)17. Available from: http://www.oecd.org/officialdocuments/displaydocumentpdf/?cote=nv/mc/chem(98)17&doclanguage=en;U.S. FDA, 2010. Part 58—Good laboratory practice for nonclinical laboratory studies.In: U.S. Code of Federal Regulations (CFR). Title 21—Food and Drugs (Food and DrugAdministration). U.S. Government Printing Office (GPO), Washington, DC, pp. 526–533. Available at: http://www.access.gpo.gov/nara/cfr/waisidx_10/21cfr58_10.html;EC, 2001. Commission Directive 2001/59/EC of 6 August 2001 adapting to technicalprogress for the 28th time Council Directive 67/548/EEC on the approximation of thelaws, regulations and administrative provisions relating to the classification, pack-aging and labelling of dangerous substances (Text with EEA relevance): Annex V ofthe EEC Directive 67/548/EEC, Part B: Methods for determination of toxicology. B.26.Subchronic oral toxicity test: repeated dose 90-day toxicity study in rodents [L225].Off. J. Eur. Communities 44, 1–333. Available at: http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=CELEX:32001L0059:EN:HTML. OECD, 1998b. Repeated dose 90-day oral toxicity study in rodents. In: OECD Guidelines for the Testing of Chemicals.Organisation for Economic Co-operation and Development (OECD), Paris, France.OECD Guideline No. 409 [Adopted 21st September 1998].; Japanese ChemicalSubstances Control Law 1987 according to the notification of November 21, 2003by Ministry of Health, Labor and Welfare (No. 1121002), Ministry of Economy, Tradeand Industry (No. 2) and Ministry of Environment (No. 031121002).

2. Materials and methods1,2

2.1. Test articles and dose formulation

The test articles included three protein extracts derived from the juice of po-tato tubers (Solanum tuberosum). The three isolates included a HMW proteinfraction (NPP-CMA-07-04-16-02, batch No. 444 7322), a LMW protein fraction(NPP-CMA-07-04-16-01, batch No. 442 7322), and a TP isolate containing boththe HMW and LMW fractions (NPP-CMA-07-04-16-03, batch No. 443 7322). Allof the potato protein extracts were in the form of a freeze-dried powder thatwas either brown (HMW and TP isolates) or beige (LMW protein isolate) inappearance.

The compositions of the HMW, LMW, and TP isolates are presented in Table 1.Like most agricultural commodities, potatoes contain small quantities of naturallyoccurring anti-nutrients (trypsin inhibitors) and toxins (a-chaconine and a-sola-nine glycoalkaloids), and each test article was assayed for glycoalkaloid contentand trypsin inhibitory activity. The concentration of total glycoalkaloids (sum ofa-chaconine and a-solanine) were determined using validated methodology as de-scribed in AOAC official method 997.13, and are presented in Table 1 (AOAC, 2001)3.The trypsin inhibitory activities of the potato protein test articles were evaluatedusing the well described, and validated, L-BAPA (L-Na-benzoyl-L-arginine 4-nitroani-lide hydrochloride) method (ISO, 2001)4. This method measures the quantity of tryp-sin inhibited per gram of protein as determined using the following formula: TIA = i/100% �mi/m0. Where TIA is trypsin inhibitor activity in mg/g; i represents inhibitionpercentage in percent; m0 represents mass of the test sample in grams, and mi repre-sents mass of trypsin in milligrams. Trypsin inhibitor activity was used for this exper-iment rather than total quantity of trypsin inhibitor present since the intended fooduses of Solanine’s potato proteins are typically subject to thermal processing condi-tions that results in significant inactivation of the proteases. Thus, to approximatefood processing conditions, each test article was subject to pasteurization at 80 �Cfor 30 min. Trypsin inhibitory activity was determined for each test article followingthis thermal processing step.

Each of the test articles were blended with a control pelleted diet (SSNIFF�

Spezialdiäten GmbH, Germany) which contained 20% casein as the proteinsource. The LMW potato protein isolate (freeze-dried powder) was blended intothe control diet at a concentration of 7.5% while the HMW and TP isolates wereincorporated at 15%. The potato protein isolates replaced the correspondingpercentage of casein. The diets were certified to be homogenous prior to receiptat the testing laboratory. The compositions of each of the test diets (control,HMW potato protein, LMW potato protein, and TP protein) are presented inTable 2.

The dietary concentrations of potato extract to be administered were deter-mined on the basis of the results of a non-Good Laboratory Practice 14-day oraldose-range finding study and on considerations of the maximum amounts of testarticle that could be incorporated into the diet without causing nutritionalimbalance.

The formulated diets were stable at room temperature, in storage bags, for theduration of the study.

2.2. Animals

A total of 40 (body weights not specified), 5-week-old Wistar Crl:(WI) BR (out-bred-SPF) rats of each sex were obtained from Charles River Deutschland, Sulzfeld,Germany. The rats were quarantined and acclimatized for at least 5 days prior tothe scheduled start of treatment. During this period, the general appearance ofthe animals was monitored daily. Animals were distributed randomly, by com-puter-generated algorithm, to 4 groups of 10 male and 10 female rats per groupaccording to body weight prior to the start of dosing. At the initiation of dosing,the rats were approximately 6 weeks old. At this time, body weights ranged from191 to 218 g in males and from 153 to 175 g in females.

2 All animal welfare aspects were conducted in accordance with all applicable lawsand guidelines pertaining the humane treatment of animals as referenced below:Ministry of Health, Welfare and Sport. The Dutch ‘‘Experiments on Animals Act’’Which Entered Into Force on 5 February, 1997 [Dutch Act on Animal Experimentation(February, 1997)]. The Hague, The Netherlands: Ministry of Health, Welfare and Sport.Available at: http://www.vet.uu.nl/nca/userfiles/other/The_Dutch_Experi-ments_on_Animals_Act.pdf. ‘‘Animal Welfare Officer and the Ethical Committee ofNOTOX’’.

3 AOAC, 2001. AOAC Official Method 997.13. [Glycoalkaloids (a-solanine and a-chaconine) in potato tubers? Liquid chromatographic method first action 1997]. In:Official Methods of Analysis of the Association of Official Analytical Chemists, 17th ed.Association of Official Analytical Chemists (AOAC), Arlington, VA.

4 ISO, 2001. Animal Feeding Stuffs: determination of Trypsin Inhibitor Activity ofSoya Products. International Organization for Standardization (ISO), Geneva, Switz.International Standard, No. 14092:2001. Information available at: http://www.i-so.org/iso/iso_catalogue/catalogue_tc/catalogue_detail.htm?csnumber=25890&commid=47858.

Table 1Compositions of the HMW, LMW, and TP Protein Isolates.

Parameter Potato protein test articlesa

HMW LMW TP

ProximatesDry matter (%) 96.12 96.22 96.34Nitrogen (%) 14.111 14.596 14.975Crude protein, N � 6.25 (%) 88.19 91.23 93.59Crude ash (%) 2.1 2.45 1.00

MineralsCalcium (g/kg) 0.67 0.31 0.33Phosphorus (g/kg) 0.20 0.08 0.13Sodium (g/kg) 17.00 14.00 6.10Chloride (g/kg) 2.10 <1.50 <1.50Potassium (g/kg) 0.36 <0.05 <0.05

Amino acidsLysine (%) 6.69 7.32 7.06Methionine (%) 2.37 1.33 1.84Cysteine (%) 0.96 2.18 1.90Threonine (%) 5.93 4.55 5.08Tryptophan (%) 1.03 1.26 1.28Lysine:methionine and cysteine (ratio) 0.498 0.480 0.530Lysine:threonine (ratio) 0.886 0.622 0.720Lysine:tryptophan (ratio) 0.154 0.172 0.181Arginine (%) 3.74 4.52 4.50Histidine (%) 2.15 2.43 2.22Valine (%) 5.05 7.39 6.67Isoleucine (%) 4.70 5.20 5.18Leucine (%) 9.34 8.85 9.17Phenyalanine (%) 5.60 6.18 6.05Tyrosine (%) 5.41 5.36 5.44Glycine (%) 3.92 5.35 4.80Glutamic acid (%) 10.11 7.69 8.67Aspartic acid (%) 10.95 12.24 11.96Proline (%) 4.32 4.61 5.18Alanine (%) 4.78 2.86 3.37Serine (%) 4.92 5.50 5.54

Anti-nutrients and toxinsTrypsin inhibitor activity (TIA)b (mg trypsin inhibition/g product) 8.3 59.8 17.8Glycoalkaloids (a-cachonine + a-solanine) (mg/kg product)c 96 157 172

HMW, high molecular weight potato protein fraction; LMW, low molecular weight potato protein fraction; TP, total proteinfraction (HMW + LMW).

a To simulate partial inactivation of trypsin inhibitor occurring during food processing, each potato protein test article waspasteurized at 80 �C for 30 min, and all analyses reported above were conducted on the pasteurized materials.

b Trypsin inhibitory activity was assessed using the L-BAPA (L-Na-benzoyl-L-arginine 4-nitroanilide hydrochloride) methodas described under ISO14902:2001E.

c Glycoalkaloid concentrations were determined using the AOAC 997.13 method.

B. Lynch et al. / Food and Chemical Toxicology 50 (2012) 373–384 375

2.3. Animal husbandry

During the acclimation and treatment periods, the rats were group housed, 5per sex per in Macrolon (MIV type) cages with sterilized sawdust (Litalabo, SPPS,Argenteuil, France) provided as bedding material and paper as cage-enrichment.During overnight monitoring associated with the conduct of the motor activity testportion of the functional observations (during study weeks 12 or 13), the rats wereindividually housed in Macrolon (MIII type) cages. During this period, cage-enrich-ment materials were not provided. Rats were provided free access to the pellet diet(SM R/M-Z from SSNIFF� Spezialdiäten GmbH, Soest, Germany) and tap water [citywater of ‘s-Hertogenbosch (The Netherlands)] supplied by water bottles.

The environmental conditions of the room in which the rats were housed werecontrolled to ensure: approximately 15 air changes per hour, temperature range of19.5–23.8 �C (actual), relative humidity of 42–86% (actual), and 12 h of artificial fluo-rescent light and 12 h of darkness per day. Minor variations (<1 h differential) in thelight and dark cycle were not considered to have affected the outcome of the study.

Analyses of the bedding, diet, and water found no contaminants that could beexpected to have affected the outcome of the study.

2.4. Test article administration

The test article was administered orally by inclusion in the diet. Any remainingfood in the food hopper was replaced with new food from the storage bags on aweekly basis. The amount of test article incorporated into the diet was held at aconstant concentration (7.5% for the LMW potato protein and 15% for the HMWand TP protein) for the duration of the study. Actual intake of the test substancewas estimated based on body weight and food consumption data. Food consump-

tion was measured in grams/animal/day due to group housing, and these valueswere converted to g/kg body weight/day based on the individual weights of the ratsin each cage.

The dose levels for the 90-day study were based on results of a non-GLP 14-day range finding study in the rat in which 7.5% and 15% of each test substance(HMW, LMW and TP) was tested. From the 14-day results, dose levels selectedfor the 90-day dietary toxicity study were 15% HMW and TP, and 7.5% LMW(based on the increase in pancreas weights at 15% LMW). Increased pancreasweights are likely the result of the trypsin inhibiting substances in the LMWprotein isolate fraction.

2.5. Observation, measurement, and examination

2.5.1. Clinical observationsAll animals were observed twice daily for general condition, including at least

once daily detailed clinical examination. These clinical examinations included,but were not limited to, changes in skin, fur, eyes, mucous membranes, occurrenceof secretions, autonomic activity (e.g., lacrimation, piloerection, pupil size, unusualrespiratory pattern), changes in gait, posture and response to handling, clonic or to-nic movements, stereotypies (e.g., excessive grooming, repetitive circling) and bi-zarre behavior (e.g., self mutilation, walking backwards). Any abnormal findingswere recorded with times of onset and disappearance.

2.5.2. Body weight and food consumptionBody weight and food consumption were measured weekly.

Table 2Compositions of the HMW, LMW, and TP Protein Diets.

Parameter Control diet Dose group

15% HMW 7.5% LMW 15% TP

Proximates and macronutrientsDry matter (%) 96.8 97.6 97.2 97.7Crude protein, N � 6.25 (%) 18.0 18.0 18.0 18.0Crude fat (%) 7.1 7.1 7.1 7.1Crude fiber (%) 6.0 6.0 6.0 6.0Crude ash (%) 5.6 5.1 5.4 5.3Casein (%) 20.2 5.0 12.3 4.0Starch (%) 37.0 37.1 37.2 37.1Sucrose and maltodextrin (%) 21.1 22.3 21.9 22.8N-free extracts 60.1 61.4 60.7 61.3

MineralsCalcium (%) 0.89 0.90 0.89 0.89Phosphorus (%) 0.62 0.61 0.61 0.61Sodium (%) 0.25 0.26 0.25 0.25Magnesium (%) 0.21 0.21 0.21 0.21

Amino acidsLysine (%) 1.500 1.500 1.500 1.500Methionine (%) 0.628 0.625 0.625 0.628Cysteine (%) 0.406 0.406 0.410 0.409Threonine (%) 1.074 1.083 1.071 1.073Tryptophan (%) 0.269 0.264 0.265 0.266Lysine: methionine and cysteine (ratio) 0.690 0.687 0.690 0.691Lysine:threonine (ratio) 0.716 0.721 0.714 0.715Lysine:tryptophan (ratio) 0.179 0.176 0.176 0.177

Anti-nutrients and toxinsTrypsin inhibitor activity (TIA) (mg trypsin inhibition/g diet)a – 1.2 4.5 2.7Glycoalkaloids (a-cachonine + a-solanine) (mg/kg diet)a ND 14 12 26

EnergyGross energy (MJ/kg) 18.9 19.1 19.0 19.1Metabolizable energy (pig) (MJ/kg) 14.8 14.9 14.9 14.9

Metabolizable energy (Atwater) (MJ/kg)Protein (%) 15.8 15.9 15.9 15.9Fat (%) 19 19 19 19Carbohydrates (%) 17 17 17 17

64 64 64 64

ND, not determined.a Theoretical concentration based on content reported in Table 1.

376 B. Lynch et al. / Food and Chemical Toxicology 50 (2012) 373–384

2.5.3. Water consumptionBy subjective appraisal during the course of the study. No quantitative mea-

surements taken.

2.5.4. OphthalmologyOphthalmologic examinations were conducted on all animals prior to dose

administration and during week 13 of administration.

2.5.5. Functional observationsDuring week 12 to 13 of treatment, the following tests were conducted on all

animals: hearing ability, papillary reflex, static righting reflex, and grip strength.At this time, a motor activity test, using a computerized monitoring system (Pear-son Technical Services, Debenham, Stowmarket, England) was conducted on all ani-mals (individually housed) during a 12-h overnight period.

2.5.6. Hematology and blood chemistryBlood samples were collected from all surviving animals for hematology and

blood chemistry immediately prior to scheduled necropsy at the end of the studyperiod. All animals were fasted for a maximum of 20 h prior to blood collection be-tween 7:00 and 10:30 am. Blood was collected into tubes (Greiner Bio-One, BadHaller, Austria) from the retro-orbital sinus under isoflurane (Abbott LaboratoriesLtd., Zwolle, The Netherlands) anesthesia.

For hematology analysis, clotting tests, and clinical biochemistry evaluations,the collected blood was treated with ethylenediamine tetraacetic acid (0.5 mL), cit-rate (0.9 mL), and lithium–heparin (0.5 mL), respectively.

Hematological analyses were conducted by ADVIA 120 (Bayer Diagnostics) andassessed: red blood cell count, red blood cell distribution width, hematocrit, hemo-globin concentration, mean corpuscular volume, mean corpuscular hemoglobin,mean corpuscular hemoglobin concentration, reticulocyte count, platelet count,white blood cell count (WBC), and differential count of WBC.

Blood clotting parameters, prothrombin time and activated partial thrombo-plastin time, were evaluated using an STA Compact from Roche Diagnostics.

Blood chemistry parameters were determined using an Olympus AU 400 fromGoffin Meyvis. Parameters evaluated included: aspartate aminotransferase, alanineaminotransferase, alkaline phosphatase, total protein, albumin, total bilirubin, urea,creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, and inorganicphosphate.

2.5.7. NecropsyAt the end of the study, all animals were deeply anesthetized under isoflurane

vapor and subsequently exsanguinated. All animals were subject to a macroscopicevaluation and any abnormalities recorded. The following organs and tissues wereexamined macroscopically and fixed and preserved in 10% neutral buffered forma-lin solution (Klinipath, Duiven, The Netherlands): adrenal glands, aorta, brain (cer-ebellum, mid-brain, cortex), cecum, cervix, colon, duodenum, epididymides, heart,ileum, jejunum, kidneys, liver, lung, lymph nodes (mandibular and mesenteric),esophagus, ovaries, pancreas, Peyer’s patches (jejunum and ileum), pituitary gland,prostate gland, rectum, sciatic nerve, seminal vesicles, spinal cord (cervical, midtho-racic, lumbar), spleen, sternum with bone marrow, stomach, testes, thymus, thyroidand parathyroid, trachea, urinary bladder, uterus, vagina, and all grossly abnormaltissues/lesions.

2.5.8. Organ weightsAbsolute and relative organ weights (based on terminal body weights) were

determined for the adrenal glands, brain, epididymides, heart, kidneys, liver, ova-ries, spleen, testes, thymus, uterus, and pancreas. Bilateral organs were measuredtogether.

2.5.9. HistopathologyAll organs and tissues fixed and preserved at necropsy from all animals at the

scheduled sacrificed and from those that died spontaneously or sacrificed in extre-mis were examined histopathologically. Tissues identified as grossly abnormal atmacroscopic evaluation were also examined histopathologically. The samples wereembedded in paraffin wax, sectioned (2–4 lm), and stained with hematoxylin and

Table 3Body weights and body weight gains of male and female rats administered the HMW, LMW, and TP protein diets for 90 days.

Studyday

Treatment group (body weight in % of day 1 value in parentheses)

Males Females

Control 15% HMWprotein

7.5% LMWprotein

15% TP protein Control 15% HMWprotein

7.5% LMWprotein

15% TP protein

1 202 ± 8.2 202 ± 8.8 203 ± 7.5 201 ± 4.3 162 ± 5.2 162 ± 6.1 164 ± 4.0 162 ± 5.38 264 ± 14.4 (31) 260 ± 11.8 (29) 264 ± 9.9 (30) 258 ± 6.6 (29) 191 ± 7.64 (18) 189 ± 11.0 (17) 195 ± 7.7 (18) 191 ± 8.9 (18)

15 326 ± 18.4 (61) 321 ± 17.3 (59) 325 ± 15.8 (60) 313 ± 11.0 (56*) 220 ± 11.5 (35) 215 ± 13.2 (33) 219 ± 11.3 (33) 216 ± 13.5 (33)22 367 ± 23.1 (83) 365 ± 21.9 (80) 368 ± 20.8 (81) 352 ± 17.9 (75) 235 ± 11.2 (45) 230 ± 13.0 (42) 237 ± 12.7 (44) 233 ± 17.7 (44)29 406 ± 29.5 (102) 403 ± 25.8 (99) 407 ± 24.9 (100) 385 ± 20.8 (92) 247 ± 15.3 (52) 241 ± 17.0 (49) 251 ± 12.8 (53) 245 ± 15.5 (51)36 433 ± 31.4 (115) 432 ± 28.2 (114) 431 ± 30.4 (112) 410 ± 20.2 (104) 260 ± 14.1 (61) 258 ± 19.0 (59) 264 ± 14.9 (60) 258 ± 16.4 (59)43 466 ± 34.7 (132) 465 ± 30.8 (130) 462 ± 36.4 (127) 439 ± 22.7 (119) 275 ± 18.2 (70) 271 ± 19.1 (67) 276 ± 17.6 (68) 273 ± 16.3 (68)50 492 ± 41.2 (145) 491 ± 33.0 (143) 486 ± 40.3 (139) 462 ± 24.7 (130) 280 ± 17.7 (73) 275 ± 20.3 (70) 281 ± 14.3 (71) 279 ± 15.2 (72)57 510 ± 43.7 (154) 511 ± 35.7 (153) 507 ± 43.1 (149) 484 ± 28.3 (141) 286 ± 17.2 (77) 278 ± 25.0 (72) 299 ± 21.7 (82) 284 ± 14.0 (75)64 528 ± 44.6 (163) 534 ± 39.0 (165) 527 ± 45.5 (160) 496 ± 28.7 (147) 291 ± 17.0 (79) 286 ± 25.6 (77) 295 ± 18.3 (79) 291 ± 17.9 (80)71 542 ± 45.3 (170) 550 ± 40.5 (172) 541 ± 48.6 (166) 510 ± 31.4 (154) 301 ± 19.9 (85) 297 ± 25.9 (83) 302 ± 21.7 (83) 296 ± 16.7 (83)78 556 ± 47.0 (177) 563 ± 42.3 (179) 554 ± 50.8 (173) 521 ± 33.8 (159) 304 ± 20.7 (88) 299 ± 24.9 (85) 311 ± 20.5 (89) 305 ± 17.3 (89)85 566 ± 49.7 (181) 576 ± 43.5 (185) 559 ± 52.6 (175) 528 ± 38.5 (163) 302 ± 21.2 (86) 299 ± 26.0 (85) 314 ± 22.1 (91) 308 ± 17.8 (90)91 573 ± 48.7 (185) 578 ± 45.1 (186) 559 ± 57.3 (175) 533 ± 36.8 (165) 302 ± 20.0 (86) 303 ± 28.0 (87) 311 ± 23.6 (89) 304 ± 16.4 (88)

All values represent the mean ± S.D.n = 10 per treatment group.* p < 0.05.

B. Lynch et al. / Food and Chemical Toxicology 50 (2012) 373–384 377

eosin. Pathology data were recorded using TOXDATA and histopathology data wererecorded using Pathdata version 6.2B (Pathology Data Systems, Basel, Switzerland).The histopathology data were subject to peer-review.

2.6. Statistical analyses

The following statistical methods were used to analyze the data:

� If the variables could be assumed to follow a normal distribution, Dunnett’s test,based on pooled variance estimates, was applied.� Steel’s test was applied when the data could not be assumed to follow a normal

distribution.� The Fisher exact test was applied to frequency data.

All tests were two-sided where p < 0.05 was accepted as the lowest level of sig-nificance. Group means were calculated for continuous variables and medians werecalculated for discrete (scored) data. Test statistics were calculated on the basis ofexact values for means and pooled variances.

3. Results

3.1. Mortality and clinical signs

No deaths occurred in the HMW, LMW and TP groups adminis-tered potato protein isolates. One control male was found dead on

Table 4Relative food consumption (g/kg body weight/day) of male and female rats administered

Study days Treatment group

Males

Control 15% HMW protein 7.5% LMW protein 15% TP prot

1–8 94 ± 5.2 88 ± 1.4 90 ± 1.2 90 ± 0.48–15 80 ± 4.6 80 ± 0.1 79 ± 0.0 76 ± 0.2

15–22 66 ± 2.0 67 ± 0.5 65 ± 0.4 65 ± 1.222–29 63 ± 2.5 62 ± 0.7 62 ± 1.6 60 ± 0.429–36 59 ± 1.2 58 ± 0.1 57 ± 1.4 57 ± 0.036–43 57 ± 1.5 57 ± 0.3 55 ± 1.2 55 ± 0.343–50 53 ± 1.3 52 ± 0.4 52 ± 0.7 52 ± 1.150–57 51 ± 1.0 50 ± 0.9 51 ± 0.5 49 ± 1.957–64 49 ± 0.1 49 ± 0.8 49 ± 0.4 48 ± 1.664–71 46 ± 0.2 46 ± 0.9 45 ± 1.2 45 ± 1.471–78 44 ± 0.1 44 ± 0.1 44 ± 0.3 44 ± 0.378–85 44 ± 0.3 44 ± 0.3 41 ± 2.9 44 ± 0.985–91 45 ± 0.5 44 ± 0.9 42 ± 2.7 43 ± 1.7

All values represent the mean ± S.D.No statistically significant differences.n = 2 cages of 5 animals per treatment group.

study day 19. A cause of death for this animal could not be deter-mined. No clinical signs of toxicity were noted over the course ofthe study. One male treated with 7.5% LMW potato protein wasnoted to have a swelling on the back at the end of the treatmentperiod. This finding correlated to a well differentiated fibrosarcomaseen upon histopathological examination. Given that this was anisolated finding, and noting the improbability of tumor formationin response to 13-weeks of treatment, this was considered a spon-taneous occurrence with no relation to treatment. Other incidentalfindings, including sporadic occurrences of alopecia and scabs onvarious parts of the body are commonly found in this age andstrain of rats and were considered of no toxicological significance.

3.2. Body weight and food consumption

There were no toxicologically significant changes in bodyweight gain or food consumption between the potato protein trea-ted and control groups (Tables 3 and 4; Figs. 1 and 2). Over thecourse of the study, the achieved intakes were calculated to be8571, 4224, and 8414 mg/kg body weight/day for the 15% HMW,7.5% LMW, and 15% TP protein group males, respectively. The cor-responding values in females were 9698, 4912, and 9768 mg/kgbody weight/day, respectively.

the HMW, LMW, and TP potato protein diets for 90 days.

Females

ein Control 15% HMW protein 7.5% LMW protein 15% TP protein

95 ± 3.8 89 ± 4.1 92 ± 0.1 91 ± 2.876 ± 1.5 78 ± 3.3 80 ± 1.2 76 ± 1.168 ± 0.2 69 ± 1.1 71 ± 0.2 70 ± 4.468 ± 0.5 72 ± 2.2 71 ± 1.4 70 ± 1.865 ± 0.5 67 ± 0.4 67 ± 1.8 66 ± 0.864 ± 0.2 65 ± 0.3 66 ± 0.5 66 ± 2.957 ± 0.2 60 ± 0.8 62 ± 0.2 61 ± 2.856 ± 2.2 60 ± 2.1 58 ± 4.3 61 ± 2.257 ± 0.7 60 ± 2.4 59 ± 1.7 59 ± 3.455 ± 0.8 57 ± 0.2 59 ± 0.8 59 ± 2.352 ± 1.6 54 ± 1.2 56 ± 1.0 57 ± 5.052 ± 2.2 55 ± 1.1 54 ± 0.4 54 ± 3.154 ± 0.7 55 ± 1.6 57 ± 2.6 56 ± 2.8

Fig. 1. Body weight growth curves for male Wistar rats administered 15% HMW, 7.5% LMW, or 15% TP protein for 90 days.

Fig. 2. Body weight growth curves for female Wistar rats administered 15% HMW, 7.5% LMW, or 15% TP protein for 90 days.

378 B. Lynch et al. / Food and Chemical Toxicology 50 (2012) 373–384

B. Lynch et al. / Food and Chemical Toxicology 50 (2012) 373–384 379

In males treated with 15% TP protein, both body weight andbody weight gain (see Table 3 and Fig. 1) were slightly reducedcompared to the controls. However, statistical significance for bodyweight gain occurred only during week 3 (day 15) and the effectwas of a very slight nature. Absolute food consumption (Table 4)also appeared slightly reduced in the males treated with 15% TPprotein. No differences in food consumption were observable whencorrected for body weight. Given the above, the slight differencesin body weight gain and food consumption in males treated with15% TP protein were not considered of any biological significance.

3.3. Water consumption

Based on a subjective analysis, there were no obvious treat-ment-related effects on water consumption.

3.4. Ophthalmological and functional examinations

The ophthalmological examinations (data not shown) revealedno effect of potato protein treatment. Similarly, the functional testsshowed no effect of either HMW, LMW, or TP protein treatment onhearing ability, papillary reflex, static righting reflex, or gripstrength.

During the overnight activity test, a statistically significantreduction in motor activity scores was noted in females treatedwith either 7.5% LMW or 15% HMW potato protein relative to con-trols. Relative to females randomized to the control group, perfor-mance measures obtained in the low beam (control 3810 ± 1562,HMW 1803 ± 1329, LMW 1742 ± 768) and high beam (control6508 ± 1962, HMW 3606 ± 697, LMW 3893 ± 593) recording condi-tions were reduced in females administered the LMW and HMWpotato protein in the diet.

Table 5Hematology results for male and female rats following dietary exposure to HMW, LMW a

Parameter (units) Treatment group

Male (n = 10, except 9 in controls)

Control 15% HMWprotein

7.5% LMWprotein

15% TPprotein

RBC (�1012/L) 8.34 ± 0.34 8.34 ± 0.29 8.16 ± 0.18 8.08 ±Ht (L/L) 0.431 ± 0.010 0.440 ± 0.018 0.440 ± 0.015 0.428 ±Hb (mmol/L) 9.5 ± 0.3 9.6 ± 0.3 9.6 ± 0.2 9.5 ±MCV (fL) 51.8 ± 2.1 52.8 ± 1.3 53.9 ± 1.3 53.0 ±MCH (fmol) 1.14 ± 0.04 1.16 ± 0.03 1.18 ± 0.03 1.18 ±MCHC (mmol/L) 22.09 ± 0.32 21.87 ± 0.39 21.89 ± 0.39 22.19 ±WBC (�109/L) 10.8 ± 2.1 9.4 ± 2.1 7.8 ± 2.5* 10.6 ±Platelet count (109/

L)945 ± 88 961 ± 117 913 ± 220 974 ±

Retic. count (% RBC) 2.0 ± 0.3 2.0 ± 0.2 2.1 ± 0.5 2.0 ±PT (s) 17.7 ± 0.5 17.7 ± 0.6 17.3 ± 1.1 17.3 ±APTT (s) 20.6 ± 1.3 19.9 ± 2.5 18.3 ± 3.8 18.9 ±

Differential WBC counts (%)Neutrophil (% WBC) 21.6 ± 3.7 17.2 ± 5.1 18.2 ± 5.0 15.4 ±Eosinophil (% WBC) 2.0 ± 0.6 2.4 ± 1.1 2.1 ± 0.6 1.7 ±Basophil (% WBC) 0.6 ± 0.1 0.4 ± 0.2* 0.5 ± 0.3 0.5 ±Monocyte (% WBC) 3.3 ± 1.1 3.5 ± 1.1 3.5 ± 1.1 3.0 ±Lymphocyte (%

WBC)72.5 ± 4.2 76.5 ± 5.8 75.8 ± 4.9 79.5 ±

RDW (%) 12.6 ± 0.3 12.2 ± 0.5 12.4 ± 0.8 12.3 ±

All values represent the mean ± S.D.APTT, activated partial thromboplastin time; Hb, hemoglobin; Ht, hematocrit; MCH, meMCV, mean corpuscular volume; PT, prothrombin time; RBC, red blood cell count; Retic.* p < 0.05.** p < 0.01.

3.5. Hematology and blood chemistry

A few statistically significant differences between control andpotato protein treated groups were noted with respect to bothhematology (Table 5) and clinical chemistry (Table 6) parameters.

Statistically significant changes in hematology parameters in-cluded: reduced relative neutrophil counts and increased relativelymphocyte counts in males treated with 15% TP protein, lower rel-ative basophil counts in males administered 15% HMW protein, re-duced WBC in males administered 7.5% LMW protein and infemales treated with 15% HMW protein, reduced red blood cellsin females treated with 15% HMW protein, increased hematocritin females given 15% TP protein, and increased mean corpuscularvolume in females administered 7.5% LMW protein and 15% TPprotein. All of the changes were minor in nature, limited to onesex, were not internally consistent and did not correlate with anyother changes.

As with the hematology values, several statistically significantdifferences in blood chemistry values between the potato proteintreated groups and the controls were recorded. These are summa-rized in Table 6. Urea was reduced in males given 15% HMW pro-tein, while glucose was increased in males administered 7.5% LMWpotato protein. Calcium concentrations were lower in both malesand females treated with 15% HMW protein, and in males given15% TP protein or 7.5% LMW potato protein. Minor reductions inpotassium and inorganic phosphate were also recorded in severaltreated female groups. While statistically significant, the aboveclinical chemistry changes were all slight in nature and inconsis-tent across sex and dose groups.

3.6. Necropsy

No macroscopic findings related to treatment with either LMW,HMW, or TP potato protein, were observed. A hard nodule noted on

nd TP protein for 90 days.

Female (n = 10)

Control 15% HMWprotein

7.5% LMWprotein

15% TPprotein

0.27 7.76 ± 0.47 7.38 ± 0.27* 7.41 ± 0.34 7.83 ± 0.280.019 0.406 ± 0.203 0.392 ± 0.010 0.402 ± 0.010 0.424 ± 0.017*

0.3 9.2 ± 0.4 8.9 ± 0.3 9.0 ± 0.2 9.5 ± 0.42.8 52.3 ± 1.3 53.2 ± 1.0 54.4 ± 2.6* 54.1 ± 1.2*

0.05 1.19 ± 0.05 1.21 ± 0.03 1.21 ± 0.05 1.22 ± 0.040.47 22.77 ± 0.64 22.67 ± 0.37 22.31 ± 0.44 22.48 ± 0.252.5 6.4 ± 1.3 4.7 ± 0.8⁄ 5.4 ± 1.0 6.0 ± 1.8193 1012 ± 160 942 ± 160 965 ± 101 994 ± 94

0.3 2.1 ± 0.5 2.2 ± 0.4 2.6 ± 1.2 2.2 ± 0.50.5 17.2 ± 0.5 17.6 ± 1.1 17.4 ± 0.5 17.3 ± 0.72.8 17.2 ± 2.4 17.3 ± 2.3 18.2 ± 2.2 17.5 ± 1.9

2.4** 13.5 ± 4.1 16.4 ± 4.8 13.4 ± 5.8 13.5 ± 3.20.6 1.9 ± 0.6 1.9 ± 0.6 2.1 ± 0.5 2.1 ± 0.40.2 0.6 ± 0.2 0.5 ± 0.2 0.5 ± 0.1 0.6 ± 0.20.8 3.0 ± 1.3 2.4 ± 0.6 2.9 ± 0.8 3.3 ± 1.11.8** 81.1 ± 4.4 78.8 ± 4.3 81.1 ± 5.9 80.5 ± 3.3

0.8 11.8 ± 0.5 11.6 ± 0.5 12.1 ± 1.1 11.7 ± 0.6

an corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration;, reticulocyte; RDW, red blood cell distribution width; WBC, white blood cell count.

Table 6Blood chemistry results for male and female rats following dietary exposure to HMW, LMW, and TP protein for 90 days.

Parameter (units) Treatment group

Male (n = 10, except 9 in controls) Female (n = 10)

Control 15% HMW protein 7.5% LMW protein 15% TP protein Control 15% HMW protein 7.5% LMW protein 15% TP protein

Total protein (g/L) 69.3 ± 1.0 66.7 ± 3.2 66.8 ± 2.8 67.2 ± 1.2 72.0 ± 2.7 70.3 ± 2.7 72.3 ± 4.2 70.4 ± 3.8Albumin (g/L) 32.7 ± 0.7 31.9 ± 1.2 31.8 ± 1.3 32.4 ± 0.7 38.2 ± 2.1 37.4 ± 1.8 39.0 ± 2.9 37.5 ± 2.7AST (IU/L) 74.8 ± 8.2 73.8 ± 6.1 87.9 ± 27.2 73.9 ± 14.5 83.8 ± 36.1 71.0 ± 6.7 65.9 ± 6.5 74.8 ± 13.5ALT (IU/L) 29.9 ± 3.7 30.9 ± 5.2 33.4 ± 15.2 31.5 ± 6.3 27.8 ± 4.7 24.6 ± 4.5 24.8 ± 3.6 25.7 ± 5.3ALP (IU/L) 69 ± 14 76 ± 15 61 ± 14 74 ± 15 39 ± 8 42 ± 9 34 ± 4 41 ± 9T-Bil (lmol/L) 3.1 ± 0.3 3.2 ± 0.4 3.3 ± 0.5 3.0 ± 0.3 4.4 ± 0.5 4.3 ± 0.8 3.8 ± 0.5 4.3 ± 0.4Glucose (mmol/L) 9.04 ± 1.78 10.43 ± 1.03 10.70 ± 0.97* 9.87 ± 1.61 8.04 ± 1.26 9.45 ± 1.65 9.12 ± 1.38 8.56 ± 1.26T-Cho (mg/dL) 2.60 ± 0.63 2.17 ± 0.39 2.31 ± 0.41 2.27 ± 0.46 1.97 ± 0.50 1.73 ± 0.24 1.73 ± 0.32 1.71 ± 0.37Urea (mmol/L) 5.5 ± 0.8 4.8 ± 0.6* 5.0 ± 0.6 5.4 ± 0.6 5.5 ± 0.7 5.0 ± 0.8 5.7 ± 0.9 5.8 ± 1.1Creatinine (lmol/L) 42.5 ± 2.0 40.7 ± 1.6 42.3 ± 1.6 41.2 ± 1.5 45.4 ± 2.7 48.7 ± 4.0 47.1 ± 3.0 47.5 ± 3.9Sodium (mmol/L) 143.2 ± 0.7 142.0 ± 1.1 143.0 ± 1.1 143.0 ± 1.5 141.8 ± 1.0 141.3 ± 0.7 142.1 ± 0.5 141.4 ± 1.1Potassium (mmol/L) 4.08 ± 0.17 3.96 ± 0.21 4.17 ± 0.17 4.05 ± 0.18 3.70 ± 0-.27 3.45 ± 0.18* 3.56 ± 0.11 3.47 ± 0.19*

Chloride (mmol/L) 104 ± 1 103 ± 1 105 ± 1 105 ± 1 104 ± 1 104 ± 2 104 ± 1 103 ± 2Calcium (mmol/L) 2.86 ± 0.04 2.78 ± 0.07** 2.78 ± 0.07* 2.79 ± 0.04* 2.88 ± 0.09 2.74 ± 0.05** 2.87 ± 0.07 2.84 ± 0.09IP (mmol/L) 2.05 ± 0.14 2.06 ± 0.15 2.00 ± 0.08 2.04 ± 0.21 1.87 ± 0.26 1.57 ± 0.25* 1.85 ± 0.17 1.75 ± 0.19

All values represent the mean ± S.D.ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; IP, inorganic phosphorus; T-Bil, total bilirubin; T-Cho, total cholesterol.* p < 0.05.** p < 0.01.

380 B. Lynch et al. / Food and Chemical Toxicology 50 (2012) 373–384

the back of one male treated with 7.5% LMW potato protein corre-lated to a benign neoplasm (fibrosarcoma). This was considered anincidental finding as it was isolated to one animal and could notplausibly be related to treatment over a 13-week period. All othermacroscopic findings were consistent with those occasionallyfound in this age and strain of rat and showed no relationship totreatment.

Table 7Absolute and relative organ weights for male and female rats following dietary exposure

Organ Treatment group

Male (n = 10, except 9 in controls)

Control 15% HMW protein 7.5% LMW protein 15% TP

Absolute organ weights (g, unless stated otherwise)Body weight 554 ± 49 556 ± 45 539 ± 55 513 ±Liver 13.36 ± 2.03 12.75 ± 1.38 12.91 ± 2.08 11.63 ±Kidneys 3.61 ± 0.49 3.70 ± 0.47 3.59 ± 0.35 3.37 ±Spleen 0.93 ± 0.21 1.04 ± 0.23 1.03 ± 0.16 0.89 ±Heart 1.50 ± 0.15 1.60 ± 0.18 1.55 ± 0.13 1.44 ±Brain 2.24 ± 0.11 2.24 ± 0.09 2.24 ± 0.09 2.27 ±Pancreas 1.52 ± 0.31 1.43 ± 0.30 1.78 ± 0.20 1.75 ±Thymus (mg) 360 ± 119 415 ± 92 389 ± 95 390 ±Adrenals (mg) 63 ± 10 66 ± 8 64 ± 10 58 ±Testes 4.00 ± 0.36 4.08 ± 0.41 3.91 ± 0.22 3.64 ±Epididymides 1.43 ± 0.31 1.60 ± 0.31 1.48 ± 0.19 1.31 ±Ovaries (mg) – – – –Uterus – – – –

Relative organ weights (% of bw, unless stated otherwise)Liver 2.40 ± 0.16 2.29 ± 0.11 2.39 ± 0.22 2.27 ±Kidneys 0.65 ± 0.07 0.66 ± 0.05 0.67 ± 0.05 0.66 ±Spleen 0.17 ± 0.03 0.19 ± 0.03 0.19 ± 0.02 0.18 ±Heart 0.27 ± 0.01 0.28 ± 0.02 0.29 ± 0.03 0.28 ±Brain 0.41 ± 0.03 0.40 ± 0.03 0.42 ± 0.03 0.44 ±Pancreas (10�2 %) 28 ± 7 26 ± 5 33 ± 5 34 ±Thymus (10�3 %) 65 ± 21 74 ± 13 72 ± 17 76 ±Adrenals (10-3 %) 12 ± 2 12 ± 1 12 ± 1 11 ±Testes 0.72 ± 0.07 0.74 ± 0.06 0.73 ± 0.07 0.71 ±Epididymides 0.26 ± 0.05 0.29 ± 0.05 0.28 ± 0.04 0.26 ±Ovaries (10�3 %) – – – –Uterus – – – –

All values represent the mean ± S.D.No statistically significant differences.

3.7. Organ weights

The absolute and relative organs weights are summarized in Ta-ble 7. No statistically significant differences between the treatedgroups and the controls were found. Potato protein treatmentwas without effect on both absolute and relative organ weightparameters.

to HMW, LMW, and TP protein for 90 days.

Female (n = 10)

protein Control 15% HMW protein 7.5% LMW protein 15% TP protein

36 290 ± 20 286 ± 28 297 ± 21 293 ± 171.16 7.00 ± 1.13 6.94 ± 0.63 7.44 ± 0.82 6.95 ± 0.520.32 1.92 ± 0.19 2.08 ± 0.27 2.15 ± 0.22 2.07 ± 0.170.15 0.67 ± 0.10 0.61 ± 0.08 0.69 ± 0.08 0.67 ± 0.060.12 0.98 ± 0.12 0.99 ± 0.07 1.01 ± 0.08 0.97 ± 0.050.13 1.99 ± 0.12 2.05 ± 0.07 1.99 ± 0.07 2.00 ± 0.080.40 0.85 ± 0.23 1.03 ± 0.23 1.12 ± 0.43 1.05 ± 0.3287 289 ± 71 263 ± 32 315 ± 54 297 ± 587 79 ± 14 81 ± 12 84 ± 11 79 ± 120.68 – – – –0.30 – – – –

0.15 ± 0.02 0.13 ± 0.02 0.16 ± 0.02 0.16 ± 0.030.67 ± 0.30 0.71 ± 0.24 0.60 ± 0.12 0.66 ± 0.19

0.15 2.41 ± 0.32 2.44 ± 0.21 2.51 ± 0.25 2.38 ± 0.150.05 0.66 ± 0.06 0.73 ± 0.05 0.73 ± 0.08 0.71 ± 0.060.03 0.23 ± 0.03 0.21 ± 0.02 0.23 ± 0.02 0.23 ± 0.020.02 0.34 ± 0.03 0.35 ± 0.03 0.34 ± 0.03 0.33 ± 0.020.03 0.69 ± 0.03 0.72 ± 0.06 0.67 ± 0.05 0.69 ± 0.057 30 ± 9 36 ± 8 38 ± 15 36 ± 1112 99 ± 23 92 ± 10 107 ± 20 102 ± 222 27 ± 5 28 ± 4 28 ± 5 27 ± 30.13 – – – –0.06 – – – –

50 ± 9 47 ± 7 53 ± 8 53 ± 80.23 ± 0.11 0.25 ± 0.07 0.20 ± 0.04 0.22 ± 0.06

B. Lynch et al. / Food and Chemical Toxicology 50 (2012) 373–384 381

3.8. Histopathology

Histopathological analyses revealed a spectrum of lesions nor-mally observed in rats of this age and strain. These are summarizedin Table 8.

Notable findings are presented in Table 9. As shown in Table 9,there was a higher incidence of inflammatory infiltrate in the lungsof males administered 15% TP protein (9/10 versus 0 or 1/10 in theother dose groups and controls). This corresponded to a slightlyhigher incidence of alveolar macrophages in this dose group aswell. These findings were not replicated in females. There was norelationship between any treatment group and the average sever-ity of finding of inflammatory infiltrate/alveolar macrophages.

Also in Table 9, it can be seen that while the incidence of vacu-olation of the zona fasciculate of the adrenal gland in males treatedwith 7.5% LMW potato protein, and to a lesser extent 15% HMWpotato protein, was elevated in relation to the controls, there wasno evidence of an increase in average severity grade, nor was thisfinding noted in any of the treated females or in the males treatedwith 15% TP.

4. Discussion

A review of the scientific literature revealed no available tradi-tional toxicology data on other potato protein isolates, although anumber of studies have specifically assessed the potential allergen-icity of potato proteins, from variously sourced cultivars, and havegenerally found no evidence of activity following oral exposure(Dearman et al., 2001, 2002, 2003).

Potatoes or potato proteins have been extensively studied fortheir ability to meet nutritional requirements and in respect to po-tential beneficial properties (e.g., energy metabolism, cholesterollowering activity, antioxidant content/activity, anti-cancer andcardioprotective activity, etc.) (Andre et al., 2007; Camire et al.,2009; Henry et al., 2005; Kudoh et al., 2003; Leeman et al., 2008;Radulescu et al., 2010; Robert et al., 2008; Spielmann et al., 2009).

The current 90-day rat oral toxicity study on the HMW, LMW,and TP protein isolates produced by a proprietary manufacturingmethod showed the treatments to be well tolerated without anysign of overt toxicity. No biologically significant effects were attrib-uted to treatment with the HMW, LMW, or TP protein isolates.

Several differences, some attaining statistical significance, be-tween the treated group(s) and controls were noted. In the func-tional/activity testing, the females of the 7.5% LMW and 15%HMW groups showed lower motor activity during the overnightobservation period relative to controls. This observation was notconsidered to be toxicologically significant given that similar find-ings were not observed in males. Also, the lack of any consistent ef-fects is of particular importance given that the composition of thethree test articles (LMW, HMW, and TP protein) (Table 1), and theirassociated formulated diets (Table 2) are very similar, especially gi-ven that the potato proteins would be expected to be degraded tocomponent amino acids in the stomach and small intestine. Thedietary composition provides little in the way of a plausible biolog-ical mechanism to suggest the likelihood that treatment-relatedchanges in motor activity could be expected.

With respect to hematological and clinical chemistry parame-ters, all of the observed statistically significant findings (Tables 5and 6) were minor in nature, were not internally consistent withina treatment group, were inconsistent across treatment groups andsex, and were not correlated with any histopathological or func-tional effects. As a result, these findings were not considered torepresent an adverse effect of potato protein treatment. Moreover,they likely represent incidental (chance) findings of no relationshipto treatment.

The only notable results of the histopathological examinationwere in the lungs and adrenal gland. The higher incidence ofinflammatory infiltrate and alveolar macrophages in the lungs ofmales treated with 15% TP protein was not considered a treat-ment-related effect. The incidence of these observations in thelow and high MW potato protein treated males was not differentfrom the controls. Also, no increases in these findings were ob-served in females treated with 15% TP protein. In addition, theseverity of the inflammatory infiltrate and alveolar macrophagepresence, even in the 15% TP protein treated males, was not differ-ent from the controls in any sex. The presence of inflammatoryinfiltrate and alveolar macrophages in the lungs of rats of thisage and strain is characteristic of a low grade subclinical respira-tory system infection of unknown etiology (Elwell et al., 1997).This is particularly likely given that the findings were clusteredamongst cage mates.

The increased incidence of vacuolation of the zona fasciculate ofthe adrenal glands in males treated with 7.5% LMW potato proteincould not definitively be excluded as a treatment-related effect.However, given that the lowest incidence of this finding in males(2/10) was recorded in the 15% TP protein group, and noting thatthere were no differences in the severity of this lesion betweentreatment groups and that no such changes were noted in any ofthe female groups, appear to argue against a relationship withpotato protein treatment. In any case, the lesions were graded asminimal to mild and are known to occur spontaneously in rats insubchronic studies (Rosol et al., 2001). This can occur as functionalresponse to alterations in steroid biosynthesis as the zona fascicu-late is the site in the adrenal gland at which glucocorticoids areproduced (Greaves, 2000).

None of the above discussed findings were considered to be oftoxicological significance.

Several classes of protease inhibitor proteins have been identi-fied in potatoes (i.e., inhibitors I and II, with variable chymotrypsinand trypsin inhibitory activity, and carboxy-peptidase inhibitors)(Pouvreau et al., 2001). Although heat treatment typically reducesthe inhibitory activity of these proteins (Pouvreau, 2004; van Kon-ingsveld et al., 2001), the effect of the heat treatment depends onthe class, with some classes showing greater ability to retain inhib-itory activity following heating (inhibitor I and carboxypeptidase)than others (inhibitor II). In addition to heat processing, the prote-ase inhibitor proteins are also inactivated due to initial pepsin deg-radation in the stomach following consumption. The trypsininhibitory activity of the potato protein fractions used for prepara-tion of the rodent diets was 59.8, 8.3, and 17.8 mg trypsin inhibitedper g of protein for the LMW, HMW and TP fractions, respectively.These concentrations would correspond to theoretical feed levelsof 4.5, 1.2, and 2.7 mg trypsin inhibited/g feed. Thus, despite theuse of pasteurization (<80 �C for 30 min) during processing, theingredients retained some trypsin inhibitory activity. It is notewor-thy that the LMW fraction did not show any evidence of toxicolog-ically significant effects in rats following oral dosing at 7.5% in thediet for 90 days given that this fraction is predominantly composedof protease inhibitors that are known to inhibit digestive enzymessecreted by the pancreas, and therefore may influence proteindigestion (Livingstone et al., 1980; Mossor et al., 1984). For exam-ple, the long-term dietary consumption of raw soybeans, or soy de-rived trypsin inhibitor isolates results in cellular pancreatichypertrophy and enlargement of the pancreas in rodents (Grantet al., 1993; Gumbmann et al., 1986, 1989; Smith et al., 1989; Stru-thers et al., 1983).

Why pancreatic effects were not observed during this study isunclear. A comparison of the trypsin inhibitory activities of the testdiets used in this study, with dietary exposures resulting in pancre-atic effects in rodents reported in the literature was unfortunatelynot possible due to the differences in reporting of protease compo-

Table 8Histopathological findings of male and female rats following dietary exposure to HMW, LMW, and TP protein for 90 days.

Organ: finding(s) Male (n = 10) Female (n = 10)

Control 15% HMW 7.5% LMW 15% TPl Control 15% HMW 7.5% LMW 15% TP

Spinal cord (lumbar)Rediculoneuropathy 1 1 0 1 1 1 1 0Sciatic nerve myelin fragmentation 3 1 2 7 3 1 4 2

HeartInflammation, lymphocytic 0 1 0 0 0 0 0 0Myofiber necrosis 0 1 1 0 0 0 1 0Arteropathy 0 1 0 0 0 1 0 0

TracheaInflammation, lymphogranular 0 0 1 0 0 0 0 0

LungMineralization, vascular 0 0 0 1 0 0 0 0Osseous metaplasia 1 0 0 0 0 0 0 0Alveolar inflammation 7 4 1 7 0 4 2 1Alveolar macrophages 3 4 3 7 2 6 0 1Inflammatory infiltrate 1 1 0 9 1 5 5 2Lymphoid hyperplasia 2 0 1 1 0 2 1 0

StomachDilatated gastric pits 2 2 1 1 0 1 0 0Epithelial cyst 0 0 2 0 0 0 0 0Peyer’s patchesMineralization, focal 0 0 1 0 0 0 0 0Cyst(s) 0 1 0 0 0 0 0 0

RectumInfiltration, lymphocytic 0 1 0 0 0 0 0 0LiverHepatodiaphragmatic nodule 0 1 0 0 0 1 0 0Vacuolization 2 1 1 0 0 3 1 0Inflammatory cell foci 10 10 10 10 10 9 10 10PancreasPigment, yellow–brown 1 0 2 1 0 0 0 0Vacuolization 0 0 1 0 1 0 0 0Apoptosis, exocrine 0 0 1 0 0 0 0 0Atrophy, exocrine 1 1 0 0 0 0 0 0KidneysPelvic dilation 1 1 0 0 0 0 0 0Lamellar bodies 2 1 2 0 0 0 0 0Cystic tubule(s) 1 0 0 0 0 0 0 0Hyaline cast(s) 2 2 2 3 4 1 1 1Inflammation, interstitial 1 3 5 0 2 0 0 0Basophilia, tubular 5 4 6 4 2 2 2 2Diffuse basophilia 0 0 1 0 0 0 0 0Mineralization, tubular 0 0 0 0 0 0 0 3Urinary bladderInflammation, lymphocytic 0 0 0 0 0 0 2 0

TestesSeminiferous atrophy 0 0 0 1 – – – –

EpididymidesSeminiferous cell debris 0 1 0 1 – – – –Inflammation, lymphocytic 1 0 0 0 – – – –

Prostate glandInflammation, lymphocytic 0 2 2 0 – – – –

OvariesFollicular cyst(s) – – – – 0 0 1 0Cyst – – – – 0 1 0 0

UterusProestrus/estrus epithelium – – – – 2 3 2 1Cystic endometrial glands – – – – 0 0 1 0

Pituitary glandCyst(s) 1 0 1 1 0 1 1 0

Thyroid glandInflammation, lymphocytic 0 0 1 0 0 0 0 0Hyperplasia/hypertrophy 0 0 1 0 0 1 1 0

Adrenal glandsVacuolation, zona fasciculate 3 5 7 2 0 0 0 0SpleenHemopoietic foci 9 7 8 7 6 8 9 7Thymus

382 B. Lynch et al. / Food and Chemical Toxicology 50 (2012) 373–384

Table 8 (continued)

Organ: finding(s) Male (n = 10) Female (n = 10)

Control 15% HMW 7.5% LMW 15% TPl Control 15% HMW 7.5% LMW 15% TP

Congestion 0 0 1 0 1 0 0 0Thymic atrophy 0 0 0 0 0 1 0 0

Mesenteric lymph nodeLymphatic ectasia 0 1 3 0 0 0 0 0Macrophage foci 0 0 0 0 0 0 1 0

Mandibular lymph nodesPlasmacytosis 1 1 0 1 1 0 2 0Skin/subcutisTelogen phase 0 1 0 0 0 0 0 0

Values represent number of animals with findings.

Table 9Selected histopathological findings of male rats following dietary exposure to HMW, LMW, and TP protein for 90 days.

Organ: finding(s) Male (n = 10) Female (n = 10)

Control 15% HMW 7.5% LMW 15% Total Control 15% HMW 7.5% LMW 15% TP

Lung

Alveolar macrophagesGrade 1 2 0 2 6 2 4 0 1Grade 2 1 4 0 1 0 2 0 0Grade 3 0 0 1 0 0 0 0 0Total affected 3 4 3 7 2 6 0 1

1.3 1.0 1.7 1.1 1.0 1.3 – 1.0

Inflammatory infiltrateGrade 1 0 0 0 3 1 3 3 1Grade 2 1 1 0 3 0 1 1 1Grade 3 0 0 0 3 0 1 1 0Total affected 1 1 0 9 1 5 5 2Adrenal gland

Vacuolation, zona fasciculateGrade 1 2 4 5 2 0 0 0 0Grade 2 1 1 2 0 0 0 0 0Total affected 3 5 7 2 0 0 0 0

B. Lynch et al. / Food and Chemical Toxicology 50 (2012) 373–384 383

sition, which, were typically reported as mg trypsin inhibitor/gprotein. Nevertheless, based on the known sensitivity of the ratto dietary trypsin inhibitors, and the fact that pancreatic hypertro-phy has been reported in rats administered potato derived trypsininhibitors, it is likely that the dose of trypsin inhibitor activity pro-vided in the diets was simply insufficient to induce pancreatic ef-fects under the conditions of this study. However, an apparentincrease in pancreatic weights was noted in one animal during pre-liminary findings (non-Good Laboratory Practices studies) in ratsadministered LMW potato protein isolate at dietary concentrationof 15%. Due to the low number of animals used in these experi-ments no dose–response relationship could be determined.

In addition to protease inhibitors, glycoalkaloids are a group ofknown toxins specific to the Solanaceae family of plants, includingpotatoes (Friedman, 2006). a-Solanine and a-chaconine are theprincipal glycoalkaloids identified in potatoes (Camire et al.,2009; Friedman, 2006). In animals, exposure to high oral doses ofpotato glycoalkaloids produces irritant effects on the mucousmembranes of the gastrointestinal tract. Systemic effects include:hemolysis, central nervous system depression, and depression ofrespiratory and motor centers, leading to eventual cardiac arrest(Clarke et al., 1981).

In rabbits consumption of potatoes containing 75 mg of glycoal-kaloids/kg (20–23 mg/kg body weight per day) or greened potatoescontaining 204 mg/kg (49–53 mg/kg body weight) for 20 days, pro-duced impairments in poor protein digestibility, weight loss, anddiarrhea. Decreases in red blood cell and hemoglobin levels also

were observed. The rabbits consuming the lower concentrationdiets (20–23 mg/kg body weight/day) were without evidence oftoxicity. Studies conducted in rhesus monkeys administered potatodiets containing 3.08–4.07 mg of glycoalkaloids/kg of body weight/day for 25 days revealed no adverse effects (Friedman, 2006).

Based on the glycoalkaloid concentrations of the potato proteintest articles reported in Table 1, the glycoalkaloid contents of theHMW, LMW and TP diets were estimated to contain glycoalkaloidsat concentrations of 14, 12, and 26 mg/kg. The absence of glycoal-kaloid induced toxicity is consistent with the low level exposureoccurring in these animals. As glycoalkaloids occur naturally inpotatoes, significant exposure to these toxins via the normal back-ground diet occurs in humans. Based on the per capita potato in-takes reported by the United States Department of Agriculture ofapproximately 147 g/day, exposure to glycoalkaloids from potatoconsumption is estimated to be approximately 12 mg/day. Thesafety of the glycoalkaloids a-solanine and a-chaconine was con-sidered by the Joint FAO/WHO Committee for Food Additives (JEC-FA, 1993). Although the Committee could not establish a safe levelof human consumption at the time of the evaluation, it did con-clude that consumption of potatoes, ‘‘frequently on a daily basis’’,containing glycoalkaloids levels of 20–100 mg/kg, was of no safetyconcern. Based on historical batch analyses of the HMW and LMWpotato protein isolates, the glycoalkaloid content of the ingredientsare expected to be below 300 ppm. Based on typical use levels ofHMW and LMW in food (up to 2%), dietary exposures to potato gly-coalkaloids would be below 10 ppm; given this, and the absence of

384 B. Lynch et al. / Food and Chemical Toxicology 50 (2012) 373–384

glycoalkaloid toxicity in this study, the presence of low concentra-tions of glycoalkaloids in the potato protein isolates is not consid-ered to be of toxicological concern.

In summary, the results of the 90-day study provide no evi-dence of toxicity of HMW, LMW and TP protein isolates at dietaryconcentrations of up to 15%, 7.5%, and 15%, respectively. Thesefindings are consistent with the long history of safe use of the po-tato in the human diet to meet nutritional requirements, includingdietary protein. In conclusion, the results of the 90-day study sup-port the safe use of potato protein isolates at intended use levels infood (up to 2%).

Funding sources statement

Funding for the research and preparation of this publicationwas provided by AVEBE U.A. of Foxhol, The Netherlands.

Conflict of Interest

B. Lynch and R.R. Simon are employees of Cantox Health Sci-ences International, an Intertek Company, and have provided con-sulting services to AVEBE within the last 3 years. F.M.vO. and H.H.E.are employees of NOTOX B.V., and M.L.F.G. and C.K. are employeesof AVEBE U.A.

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