Thirteen week feeding study with transgenic maize grain containing event DAS-Ø15Ø7-1 in Sprague–Dawley rats

www.elsevier.com/locate/foodchemtox

Food and Chemical Toxicology 45 (2007) 551–562

Thirteen week feeding study with transgenic maize graincontaining event DAS-Ø15Ø7-1 in Sprague–Dawley rats

Susan A. MacKenzie a, Ian Lamb b, Jean Schmidt b, Lora Deege b, Michael J. Morrisey b,Marc Harper b, Raymond J. Layton b, Lee M. Prochaska b, Craig Sanders b, Mary Locke c,

Joel L. Mattsson d, Angel Fuentes e, Bryan Delaney b,*

a DuPont Haskell Laboratory, Newark, DE, USAb Pioneer Hi-Bred International, Inc., 7250 NW 62nd Avenue, P.O. Box 552 Johnston, IA 50131-0552, USA

c DuPont Experimental Station, Wilmington, DE, USAd Dow AgroSciences, Indianapolis, IN, USA

e Pioneer Overseas Corporation, Brussels, Belgium

Received 14 June 2006; accepted 27 September 2006

Abstract

Maize line 1507, containing event DAS-Ø15Ø7-1 (1507), is a genetically modified (GM) maize plant that expresses the cry1F genefrom Bacillus thuringiensis (Bt) sbsp. aizawai and the phosphinothricin-N-acetyltransferase (pat) gene from Streptomyces viridochromo-

genes throughout the plant including in the grain expression of the Cry1F protein confers in planta resistance to the European corn borer(ECB; Ostrinia nubilalis Hubner: Crambidae) and other lepidopteran pests. Expression of the PAT protein confers tolerance to theherbicidal active ingredient glufosinate–ammonium. The current study evaluated the nutritional performance of rats fed diets containing1507 maize grain in a subchronic rodent feeding study. The grains in this study, 1507, its near-isogenic control (33P66), and a non-GMcommercial hybrid (33J56) contained similar amounts of proximates, amino acids, minerals, anti-nutrients, and secondary metabolites.The subchronic feeding study compared standard toxicology response variables in rats fed diets containing 1507 maize grain with thosein rats fed diets containing non-GM maize grains. All diets were prepared according to the specifications of PMI� Nutrition Interna-tional, LLC Certified Rodent LabDiet� 5002 (PMI� 5002). Diets were fed ad libitum to Sprague–Dawley rats for approximately 90 days.In-life response variables included indicators of dietary performance and weekly evaluations for clinical signs of toxicity. Notoxicologically significant differences were observed in the nutritional performance variables, clinical and neurobehavioral signs, ophthal-mology, clinical pathology (hematology, clinical chemistry, coagulation, and urinalysis), organ weights, and gross and microscopicpathology between any pair of treatment groups. These results demonstrate that 1507 maize grain is as safe and as nutritious as non-GM maize grain.� 2006 Elsevier Ltd. All rights reserved.

Keywords: European corn borer; GM food; Subchronic feeding study

1. Introduction

The European corn borer (ECB; Ostrinia nubilalis

Hubner: Crambidae) is responsible for more than $1 bil-lion USD in annual damage to crops including maize

0278-6915/$ - see front matter � 2006 Elsevier Ltd. All rights reserved.doi:10.1016/j.fct.2006.09.016

* Corresponding author. Tel.: +1 515 334 7086; fax: +1 515 334 4478.E-mail address: [email protected] (B. Delaney).

(Zea mays L.) in the United States (Mason et al., 1996).The larvae of ECB damage maize plants by feeding onthe leaves and midribs, tunneling within the stalks, feedingon the shanks resulting in increased ear droppage, andincreasing susceptibility to fungal infection (Fusarium mon-

iliforme and Gibberrela zaea) leading to stalk rot (Masonet al., 1996). Techniques to control the impact of ECB oncrop yield have included in-field application of organo-phosphate insecticides. While these substances are toxic

Primer set Genomic DNA targetsite

Unique identifierfor 1507

Expected sizeof PCRproduct (bp)

obc1081/1082

Insert internaljunction

Yes 194

o147/o144

30 insert-plantgenome border

Yes 252

o149/o144

30 insert-plantgenome border

Yes 342

1068/1069

50 insert-plantgenome border

Yes 426

1068/o49 50 insert-plantgenome border

Yes 734

552 S.A. MacKenzie et al. / Food and Chemical Toxicology 45 (2007) 551–562

to ECB larvae, they also impact beneficial organismsincluding those that serve as predators of ECB larvae. Fur-ther, because the larvae move inside the plant shortly afterhatching, sprayed insecticides may not provide adequateplant protection even after multiple applications (Floodet al., 1995; Rinkleff et al., 1995).

Maize line 1507, containing event DAS-Ø15Ø7-1 (1507),was produced by insertion of the cry1F gene from Bacillus

thuringiensis (Bt) sbsp. aizawai and the phosphinothricin-N-acetyltransferase (pat) gene from Streptomyces virido-

chromogenes. Expression of the Cry1F protein confers in

planta resistance to ECB and other lepidopteran pests.Toxicity to insects observed following ingestion of Cry1Fand other selective Bt proteins is attributable to interactionbetween Bt proteins and high affinity receptors in gutepithelial cells in target Lepidopteran species (but not innon-target species including mammals) leading to pore for-mation and lysis of gut cells which causes the insects to stopfeeding and ultimately die (Schnepf et al., 1998; McClin-tock et al., 1995; IPCS, 1999; Betz et al., 2000). Expressionof the PAT protein confers tolerance to the herbicidalactive ingredient glufosinate-ammonium.

Safety testing of GM crops includes an evaluation oftransgenic proteins for indicators of allergenicity and acutetoxicity. Guidelines to assess possible allergenicity of trans-genic proteins include a comparison of the amino acidsequence to that of known allergenic proteins (FAO/WHO., 2001; ILSI, 1997; FAO, 1996; OECD, 1993;OECD, 1996; OECD., 1997; and WHO., 1991; WHO.,1995) and a test of the digestibility of the protein in simu-lated gastric fluid (SGF) (Astwood et al., 1996). In the caseof 1507 maize grain, neither the Cry1F nor PAT protein issimilar in sequence to allergenic proteins and both readilydigest in SGF (Meyer, 1999 and Schafer, 2002). Addition-ally, neither protein is acutely toxic in mice (unpublishedresults Herouet et al., 2005 and Kuhn, 1998).

In addition to the safety studies conducted with trans-genic proteins, crops containing the transgenic proteinsare evaluated for unintended changes. Because it cannotbe determined if any crop, whether GM or not, is abso-lutely safe, the goal of these studies is to determine if theGM crop is ‘‘substantially equivalent’’ to non-GM coun-terparts (FAO, 1996; Codex, 2003). This assessment typi-cally includes a combination of analytical studies of keynutritional components in the GM crop and animal-feed-ing studies. In the case of maize, the concentration rangeof key nutritional components has been published (Ridleyet al., 2004 and OECD., 2002). The nutritional equivalenceof GM and non-GM maize grains is typically determinedin feeding studies with broiler chickens (OECD., 2003;Sidhu et al., 2000; Taylor et al., 2003a,b,c). Poultry studiesare particularly useful because broilers are extremely sensi-tive to changes in the nutritional value of their diet. Safetystudies of GM crops have been conducted with rats wherestandard toxicology response variables (per OECD 408;OECD., 1998) were compared among groups fed subch-ronically with diets containing GM crops (grains) or non-

GM crops (Hammond et al., 2004 and Hammond et al.,2006). Nutritional equivalence studies conducted with1507 maize grain in broiler chickens demonstrated that itsupports growth performance as well as non-GM grain(Zeph et al., in press).

The current investigation was conducted in accordanceto OECD 408 guidelines to evaluate the nutritional equiv-alence of 1507 maize grain by comparison of the nutritionalperformance of this GM grain with non-GM controls fol-lowing subchronic consumption by rats.

2. Materials and methods

2.1. Maize grains

Maize grains evaluated in the current study included GM maize grain1507, its near isogenic non-GM hybrid (33P66), and a commerciallyavailable non-GM hybrid (33J56). All were grown in isolated field plots inJefferson County, Iowa in 2001.

2.2. Characterization of maize grain nutrients, anti-nutrients,

secondary metabolites, mycotoxins, and pesticide residues

The nutrient composition of all maize grains was evaluated analyticallyto determine the percent composition of proximates (crude protein, crudefat, and ash), moisture, dry matter, carbohydrates, crude fiber, acid andneutral detergent fiber, individual amino acids, minerals (calcium andphosphorus) at Woodson–Tenent Laboratories, Inc. (a division of Euro-fins Scientific Inc., Memphis, TN). Gross energy content (kcal/100 g) wasdetermined using bomb calorimetry at Woodson-Tenent. Percent com-position of anti-nutrients (trypsin inhibitor and phytic acid) and secondarymetabolites (furfural, ferulic acid, p-coumaric acid, inositol, and raffinose)were also analyzed at Woodson-Tenent. Mycotoxin concentrations (afla-toxin B1 [AFB1], B2, G1, G2, and M2, zearalenone, oosporein, ergosine,ergotamine, ergocornine, ergocryptine, ergocristine, deoxynivalenol, 13-acetyl-deoxynivalenol, 15-acetyl-deoxynivalenol, cyclopiazonic acid,fumonisin B1 [FB1], B2, and B3, moniliformin, and T-2 toxin) weremeasured at Romer Laboratories, Inc. (Union, MO). All maize grainswere tested for a battery of more than 150 possible pesticide residues(Exygen Research, State College, PA).

2.3. Characterization of transgenic insert in maize grains

All maize grains were analyzed using event specific (event DAS-Ø15Ø7-1) and gene specific (cry1F and pat) qualitative polymerase chainreaction PCR methods (DuPont Experimental Station, Wilmington, DE).Genomic DNA was extracted from maize grains and subjected in dupli-cate to a series of eight PCR reactions with the following primer sets:

Table (continued)

Primerset

Genomic DNAtarget site

Unique identifier for1507

Expected sizeof PCR product(bp)

1070/1071

cry1F gene No 366

1022/1023

pat gene No 482

o197/o198

invertase gene1 No 226

1 Primers for the endogenous maize invertase gene were used to deter-mine whether extracted DNA was of suitable quality for PCR analysis(Hurst et al., 1999).

S.A. MacKenzie et al. / Food and Chemical Toxicology 45 (2007) 551–562 553

Template DNA (50 ng) was combined with each of the indicated pri-mer sets (10 lM each), 5x GC 2 PCR buffer, GC-Melt (5.0 M), Advan-tage�-GC 2 polymerase mix (Advantage�-GC 2 PCR Kit, #K1913-1, BDClontech, Mountain View, CA ), 50· dNTP mix (10 lM of each dNTP),and ddH2O. The reaction mixture was subjected to an initial denaturationcycle of 95 �C for 5 min, 35 cycles each of denaturation (95 �C for 1 min),annealing (60 �C for 2 min), and extension (72 �C for 3 min), and a finalelongation cycle of 72 �C for 7 min. Following completion of the PCR,10 ll of each sample was evaluated individually on a 1% agarose gelstained with ethidium bromide.

2.4. Characterization of transgenic protein concentration

in maize grains

The concentrations of Cry1F and PAT proteins were evaluated in allmaize grain samples at Pioneer Hi-Bred International, Inc. (Johnston, IA)using antibody specific enzyme linked immunosorbent assays (ELISAs).

2.5. Diet formulation

Diets were formulated and prepared in accordance with a commonlyused commercially available rodent diet specification (PMI� NutritionInternational, LLC Certified Rodent LabDiet� 5002 [PMI 5002]) using1507, 33P66, or 33J56 maize grains at a total concentration that did notexceed 33% (wt/wt). Two experimental diets contained 1507 maize grain:one with 33% 1507 maize grain (Group 50207) and the other with 11%1507 maize grain and 22% 33J56 maize grain (Group 50210). Three con-trol diets were formulated to contain either 33% 33P66 maize grain(Group 50208), 33% 33J56 maize grain (Group 50209), or a combinationof both control maize grains (11% 33P66 and 22% 33J56; Group 50211).All diets were formulated in consultation with Purina Mills Labdiet�

(Richmond, IN) to meet the nutritional standards of PMI� 5002, and wereprepared and packaged in 20-kg allotments prior to study start. All dietswere guaranteed to be stable for 6 months. Diets were refrigerated uponarrival at DuPont Haskell Laboratory for Health and EnvironmentalSciences (Newark, DE).

2.6. Characterization of diets

Nutrient, mycotoxin, and pesticide residue analyses were conducted onall diets prior to administration to the rats. In addition to the analytes listedabove for maize grains, additional minerals (potassium, magnesium,sodium, chloride, iron, zinc, manganese, copper, cobalt, iodine, chromium,fluoride), heavy metals (arsenic, cadmium, lead, mercury, and selenium),and vitamins (biotin, choline, folic acid, niacin, pantothenic acid, vitaminsA, B6, B12, and D3, and a-, b-, d-, and c-tocopherol) were quantified.These analyses were conducted at Woodson-Tenent. Mycotoxin screeningwas conducted at Romer Laboratories, Inc (Union, MO) and pesticidescreening was conducted at Exygen Research (State College, PA).

2.7. Analysis of homogeneity and stability of Cry1F in

rodent diets

Samples of each diet produced at Purina Test Diet were collected at thebeginning, middle, and near the end of their respective production runs andshipped to Pioneer Hi-Bred International, Inc. (Johnston, Iowa). Prior toinitiating the feeding study, these samples were analyzed for homogeneity ofblending by measuring the concentrations of the Cry1F protein using anti-body specific ELISA. The limit of quantitation for the Cry1F protein in thisassay was 13.30 pg/mg of diet. Concentrations of PAT were not determinedbecause prior studies demonstrated that the concentrations were below theanalytical detection limits (data not shown). The stability of Cry1F trans-genic protein in the experimental diets was evaluated by determination of theconcentrations at the beginning and end of the feeding study.

2.8. Bioactivity against ECB of Cry1F in rodent diets

Experimental ECB diets were prepared by mixing the indicated rodentdiets with ECB diets (Southland Products, Inc., Lake Village, AR) at dilutionsranging from 1 g rodent diet per 2 g ECB diet (1:2) and threefold serial dilu-tions (1/6; 1/18, 1/54; 1/162; 1/486; and 1/1458). The activity of Cry1F in thesediets was evaluated at the beginning and end of the feeding trial by quantifyingthe growth of ECB larvae. One 1st instar was placed into the well of a 96 welltissue culture plate (n = 32 per treatment group) and covered with a perfo-rated lid. The tissue culture plates were incubated at 25 �C in 65% humidity for7 days. At the end of this incubation period, the larvae were removed fromtissue culture plates, weighed, and evaluated for mortality. The percentweight-gain inhibition (GI50) for each diet was determined using the equation:

%Wt gain inhibition ¼ 1� ðMean weight per larva½treatment�=Average maximum larva weight½control�Þ � 100

2.9. Test species

Male and female Crl:CD� (SD)IGS BR rats (n = 12/sex/group) wereobtained from Charles River Laboratories, Inc., (Raleigh, NC). Rats werehoused singly in stainless steel, wire-mesh cages suspended above cageboards. Animal rooms were maintained at a temperature of 22 ± 3 �C anda relative humidity of 50% ± 20%, and were artificially illuminated(fluorescent light) on an approximate 12-h light/dark cycle. All rats wereprovided with tap water ad libitum (United Water Delaware, Wilmington,DE). During the pretest period, animals were fed PMI� Nutrition Inter-national, LLC Certified Rodent LabDiet� 5002 ad libitum (Purina TestDiet, Richmond, IN). During the test period, animals were fed theirrespective diets ad libitum (except when fasted). Body weight and feedconsumption of all rats were determined daily for the first week of thefeeding trial and weekly thereafter. From the food consumption and bodyweight data, mean daily food efficiency was calculated.

2.10. Clinical observations

Cage-side examinations to detect moribund or dead rats and abnormalbehavior and/or appearance were conducted at least twice daily through-out the study. Detailed clinical observations were conducted weekly in astandardized arena by trained personnel that were blinded to treatmentstatus of the rats. The detailed clinical observations included evaluation offur, skin, eyes, mucous membranes, occurrence of secretions and excre-tions, autonomic nervous system activity (lacrimation, piloerection, andunusual respiratory pattern), changes in gait, posture, response to han-dling, presence of clonic, tonic, stereotypical, or bizarre behavior.

2.11. Ophthalmology

An ophthalmological examination was conducted on all rats prior toinitiation and near the end of the feeding study. Both eyes of each rat wereexamined by focal illumination and indirect ophthalmoscopy in subduedlight after production of mydriasis.

554 S.A. MacKenzie et al. / Food and Chemical Toxicology 45 (2007) 551–562

2.12. Neurobehavioral evaluation

Prior to initiation of diet administration and during week 13, sensoryfunction and motor activity were evaluated on all surviving animals.Sensory motor function was assessed by evaluating grip strength, responseto approach/touch, tail pinch, and sharp auditory stimulus. Fore- andhind-limb grip strengths were measured by a strain gauge device (Chatil-lon� Digital Force gauge; Largo, FL). Pupil size was evaluated byassessment of pupillary response to a beam of light prior to removing therats from the motor activity chambers. Presence of diarrhea and polyuriaon the cageboards below the motor activity cages was evaluated followingeach motor activity session. Motor activity was assessed for 60 minfollowing sensory motor function evaluation on the same day usingautomated activity monitors (Coulbourn�; Allentown, PA).

2.13. Clinical pathology evaluation

A clinical pathology evaluation was conducted on all rats during week7 and all surviving rats during week 13. Rats were placed in metabolismcages the day before collection of blood samples and fasted overnight (atleast 15 h), and urine was collected from each rat. Blood samples forhematology and serum chemistry for the week 7 and 13 evaluations werecollected from the orbital sinus of each animal while it was under carbondioxide anesthesia. Blood samples for coagulation parameters were col-lected at necropsy from the abdominal vena cava while animals were undercarbon dioxide anesthesia.

2.14. Hematology and coagulation

Red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT),mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH),mean corpuscular hemoglobin concentration (MCHC), red cell distribu-tion width (RDW), absolute reticulocyte count (ARET), platelet count(PLT), white blood cell count (WBC), differential white blood cell count(ANEU [neutrophils], ALYM [lymphocytes], AMON [monocytes], AEOS[eosinophils], ABAS [basophils], and ALUC [large unstained cells]) wereevaluated with a Bayer Advia 120 Hematology Analyzer (Bayer Diag-nostics Division, Norwood, MA). Prothrombin time (PT) and activatedpartial thromboplastin time (APTT) were evaluated with a SysmexCA-1000 Coagulation Analyzer (Dade Behring, Deerfield, IL).

2.15. Serum chemistry

Serum aspartate aminotransferase (AST), alanine aminotransferase(ALT), sorbitol dehydrogenase (SDH), alkaline phosphatase (ALKP),total bilirubin (BILI), urea nitrogen (BUN), creatinine (CREA), choles-terol (CHOL), triglycerides (TRIG), glucose (GLUC), total protein (TP),albumin (ALB), globulin (GLOB), calcium (CALC), inorganic phospho-rous (IPHS), sodium (NA), potassium (K), and chloride (CL) were eval-uated using an Olympus AU640 Clinical Chemistry Analyzer (OlympusAmerica, Irving, TX).

2.16. Urinalysis

Urine volume (VOL), specific gravity (SG), pH, bilirubin, and urobilin-ogen (URO) were evaluated using a Bayer Clinitek Atlas Automated UrineChemistry Analyzer (Bayer Diagnostics). Urine protein concentrations(UMTP) were evaluated using an Olympus AU640 Clinical ChemistryAnalyzer (Olympus America). Urine osmolality (UOSM) was evaluated usingan Advanced Osmometer 390 (Advanced Instruments Inc., Norwood, MA).

2.17. Anatomic pathology

At the end of the feeding period, rats were fasted for at least 15 h andthen euthanized by carbon dioxide anesthesia and exsanguination. Fol-lowing sacrifice, the following organs were weighed: liver, kidneys, adrenal

glands, thymus, brain, spleen, heart, ovaries and uterus (female) or testesand epididymides (male). Absolute and relative organ weights (percent offinal body weight; ratio to brain weight) were calculated. Tissues from thefollowing organ systems were processed to slides, stained with hematoxylinand eosin, and examined microscopically: digestive system (liver, esopha-gus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, salivaryglands, and pancreas), urinary system (kidneys and urinary bladder),respiratory system (lungs, trachea, nose, larynx, and pharynx), cardiovas-cular system (heart and aorta), hematopoietic system (spleen, thymus,mandibular lymph node, mesenteric lymph node, and bone marrow),endocrine system (pituitary gland, thyroid gland, parathyroid glands, andadrenal glands), nervous system (brain [including cerebrum, cerebellum,and medulla/pons], spinal cord [cervical, mid-thoracic, and lumbar] andsciatic nerve), musculoskeletal system (skeletal muscle, femur/knee joint,sternum), reproductive system of males (testes, epididymides, prostate, andseminal vesicles) and females (ovaries, uterus, mammary glands, andvagina), skin, and eyes (including retina and optic nerve).

2.18. Statistical analysis

Statistical comparisons were designed to determine whether differenceswere attributable to consumption of diets produced with 1507 graincompared to diets containing non-GM grains. Male and female rats wereanalyzed separately. Preliminary tests were conducted to verify homoge-neity and normality of variances for all continuous data andneurobehavioral response variables (Levene’s test; [Levene, 1960] andShapiro–Wilks test [Shapiro and Wilk, 1965]). If neither preliminaryhomogeneity test was significant, a one-way analysis of variance(ANOVA) was conducted. If one or more of the preliminary tests weresignificant, Dunn’s Type 1 p-adjusted linear contrasts were used (Dunn,1964). Response variables from groups consuming 33% 1507 maize werecompared to those consuming 33% 33P66 maize. When statistically sig-nificant differences were observed, further analyses were conducted tocompare the responses in the 33% 1507 group to the response variables inthe other treatment groups. For all analyses, a p-value of less than 0.05was considered statistically significant.

3. Results

3.1. Analysis of maize grains and diets

The nutrients, anti-nutrient and secondary metaboliteprofiles of all maize grains were similar and within pub-lished ranges for corn (data not shown). Similarly, the nutri-tional composition of all diets was similar regardless of theidentity of the maize grain (data not shown). Use of the con-trol primers to detect the endogenous maize invertase geneyielded the expected 226 bp fragment in all samples andindicated that the extracted DNA in both the control maizegrain and 1507 maize grain samples were of suitable qualityfor PCR (Hurst et al., 1999). PCR products of the expectedsizes for detecting the cry1F and pat genes in 1507 maizegrain were observed only in 1507 maize grain followingamplification of genomic DNA with the indicated primers(Fig. 1). These results indicated that the transgene was pres-ent only in the 1507 maize grain.

Concentrations of Cry1F protein in diets formulatedwith 33% 1507 maize grain were approximately 3· greaterthan in the diets produced with 11% 1507 maize grain butit was not detected in any of the control diets (Table 1).The Cry1F protein was stable over the course of the feed-ing study. Similarly, Cry1F bioactivity was present only in

Fig. 1. PCR analysis of event DAS-Ø15Ø7-1 in DNA from 1507 maize grain and unmodified near-isogenic control and reference maize grain. (a) PCRamplification with primer set OBC1081/OBC1082 targeting a unique internal junction in Event DAS-Ø15Ø7-1 with an expected target sequence size of 194bp and with primer sets o149/o144 and o147/o144 targeting the unique 3 0 border junction of the insertion in Event DAS-Ø15Ø7-1 with maize genomicDNA; (b) PCR amplification with primer sets 1068/1069 and 1068/o49 targeting the unique 5 0 border junction of the insertion in Event DAS-Ø15Ø7-1with maize genomic DNA and primer set 1070/1071 targeting the cry1F gene in Event DAS-Ø15Ø7-1; (c) PCR amplification with primer set 1022/1023targeting the pat gene in Event DAS-Ø15Ø7-1 with primer set o197/o198 targeting the endogenous maize invertase (control) gene with an expected targetsequence size of 226 bp.

Table 1Cry1F concentrations and stability in rodent diets evaluated by ELISA

Maize line Percentage composition

1507 33 – – 11 –33P66 – 33 – – 1133J56 – – 33 22 22

Study start (pg/mg) 166 NDa ND 53 NDStudy end (pg/mg) 193 ND ND 59 NDStability (%) 116 – – 111 –

a Limit of quantitation for the Cry1F protein was 13.30 pg/mg of diet.

S.A. MacKenzie et al. / Food and Chemical Toxicology 45 (2007) 551–562 555

those rodent diets containing 1507 maize grain (Table 2).Although the calculated GI50-values of the diets contain-ing 11% and 33% 1507 maize grain were similar, the larvabody weights in the 33% 1507 were considerably lowerthan those in the 11% 1507 group in the first two dilutions(Table 2). Further, the GI50-value of ECB diets containingeither dilution of 1507 were more than 4· lower thanGI50-values observed for diets containing control maize(Table 2). The Cry1F bioactivity at the beginning andend of the feeding trial was approximately the same sup-porting the results from the ELISA analysis that this pro-tein was stable over the course of the feeding trial (datanot shown).

3.2. Body weight and feed consumption

There were no toxicologically significant differences inmean body weight between any of the treatment groups(Figs. 2 and 3). Mean daily feed consumption of malesconsuming 33% 1507 diet (27.5 ± 2.6 g/day) was 7% higherthan that of males consuming the near isogenic control diet(25.7 ± 1.7 g/day of 33% 33P66 diets; p < 0.05) at nearly allweekly intervals following week 3; however, no statisticallysignificant differences were observed between 33% 1507 andother male groups (Fig. 4). This observation was attributedto the higher mean body weight of males in the 33% 1507maize grain group compared to the 33% 33P66 maize graingroup (p > 0.05). The difference was not considered nutri-tionally significant because the overall mean feed efficiencyvalues between the 33% 1507 and 33% 33P66 groups(0.111 ± 0.009 and 0.115 ± 0.010 g body weight gain/g feedconsumed) were not significantly different. Further, therewere no statistically significant differences in mean overallfeed efficiency values between the 33% 1507 group andany other group of male rats (33% 33J56 [0.111 ± 0.009 gbody weight gain/g feed consumed], 11% 1507/22% 33J56[0.108 ± 0.012 g body weight gain/g feed consumed], or11% 33P66/22% 33J56 [0.113 ± 0.012 g body weight gain/g feed consumed]). There were no statistically significant

Table 2Bioactivity of Cry1F in rodent diets

Maize line Percentagecomposition

1507 33 – – 11 –33P66 – 33 – – 1133J56 – – 33 22 22Ratio1 Weight2

1:2 0.07 5.95 4.20 1.22 5.361:6 0.39 11.71 12.49 3.02 11.101:18 10.06 16.72 10.99 7.93 12.791:54 12.95 14.35 11.51 12.69 14.601:162 15.74 12.93 12.96 13.42 13.971:486 15.18 12.86 11.27 12.76 14.451:1458 14.33 14.95 10.99 16.13 13.01GI50

3 0.062 0.284 0.262 0.063 0.251CI (0.048, 0.077) (0.198, 0.345) (0.202, 0.305) (0.037, 0.089) (0.200, 0.295)

1 Ratio of rat diet/total diet.2 Mean larval weight (mg).3 Estimated for 50% growth inhibition with 95% confidence intervals.

0

100

200

300

400

500

600

0 7 14 21 28 35 42 49 56 63 70 77 84 91

1507 (33%)

33P66 (33%)

33J56 (33%)

1507/33J56(11%/22%)33P66/33J56(11%/22%)

Bo

dy

Wei

gh

t (g

)

Study Day

Fig. 2. Mean male body weights.

0

50

100

150

200

250

300

350

0 7 14 21 28 35 42 49 56 63 70 77 84 91

1507 (33%)

33P66 (33%)

33J56 (33%)

1507/33J56(11%/22%)33P66/33J56(11%/22%)

Bo

dy

Wei

gh

t (g

)

Study Day

Fig. 3. Mean female body weights.

0

5

10

15

20

25

30

35

0 7 14 21 28 35 42 49 56 63 70 77 84 91

1507 (33%)

33P66 (33%)

33J56 (33%)

1507/33J56(11%/22%)33P66/33J56(11%/22%)

Fee

d C

on

sum

pti

on

(g

/day

)

Study Day

Fig. 4. Mean male feed consumption.

0

5

10

15

20

25

0 7 14 21 28 35 42 49 56 63 70 77 84 91

1507 (33%)

33P66 (33%)

33J56 (33%)

1507/33J56(11%/22%)33P66/33J56(11%/22%)

Fee

d C

on

sum

pti

on

(g

/day

)

Study Day

Fig. 5. Mean female body weights.

556 S.A. MacKenzie et al. / Food and Chemical Toxicology 45 (2007) 551–562

differences observed in nutritional performance variablesbetween any of the female treatment groups (Fig. 5).

3.3. Survival, clinical observations, ophthalmological

evaluations, and neurobehavioral evaluations

All rats survived for the duration of the feeding trial,and no clinical signs of toxicity or ophthalmalogical lesions

were observed in any male or female treatment group (datanot shown). Additionally, no diet-related changes in gripstrength (forelimb or hindlimb), sensory motor function,or motor activity were observed in any treatment group(data not shown).

S.A. MacKenzie et al. / Food and Chemical Toxicology 45 (2007) 551–562 557

3.4. Hematology and coagulation

There were no differences in hematology response vari-ables among any of the male treatment groups, and nodifferences in coagulation parameters of any group fromeither sex (Table 3). Mean RBC of females in the 33%1507 group were approximately 3.4% lower (p < 0.05)lower than in the 33% near-isogenic maize grain (33P66)group. This change was not considered biologically signif-icant because mean HGB values, the most biologically rel-evant indicator of RBC mass and function between thesetreatment groups (and all other groups) were not signifi-cantly different. Other indicators of RBC function thattypically correlate with decreased RBC function (MCV,

Table 3Hematology and coagulation values ± SD

Maize line Percentage composition

1507 33 –33P66 – 3333J56 – –

Males

RBC (·106/lL) 8.56 ± 0.43 8.40 ± 0.45HGB (g/dL) 15.9 ± 0.5 15.5 ± 0.7HCT (%) 46.1 ± 1.6 45.1 ± 2.0MCV (fL) 54.0 ± 1.9 53.7 ± 1.9MCH (pg) 18.6 ± 0.7 18.4 ± 0.9MCHC (G/DL) 34.5 ± 0.6 34.3 ± 0.7RDW (%) 12.6 ± 0.7 12.6 ± 0.6ARET (·103/lL) 186.0 ± 31.0 172.6 ± 34.0PLT (·103/lL) 1116 ± 173c 1199 ± 224f

WBC (·103/lL) 10.50 ± 2.08 12.14 ± 3.07ANEU (·103/lL) 1.70 ± 0.53 1.73 ± 0.46ALYM (·103/lL) 8.29 ± 1.71 9.86 ± 2.90AMON (·103/lL) 0.20 ± 0.07 0.24 ± 0.09AEOS (·103/lL) 0.17 ± 0.09 0.14 ± 0.05ABAS (·103/lL) 0.08 ± 0.04 0.09 ± 0.03ALUC (·103/lL) 0.06 ± 0.03 0.08 ± 0.04PT (s) 16.1 ± 1.0 15.8 ± 0.7APTT (s) 22.2 ± 2.7 22.6 ± 2.1

Females

RBC (·106/lL) 8.17 ± 0.26 8.46 ± 0.35*

HGB (g/dL) 15.9 ± 0.4 16.3 ± 0.4HCT (%) 45.4 ± 1.0 46.8 ± 1.5*

MCV (fL) 55.6 ± 1.3 55.3 ± 1.9MCH (pg) 19.5 ± 0.5 19.3 ± 0.8MCHC (G/DL) 35.0 ± 0.6 34.8 ± 0.7RDW (%) 11.3 ± 0.4 11.3 ± 0.3ARET (·103/lL) 163.1 ± 31.0 172.8 ± 34.9PLT (·103/lL) 1103 ± 158b 1170 ± 83f

WBC (·103/lL) 9.34 ± 1.54 10.09 ± 2.02ANEU (·103/lL) 1.22 ± 0.32 1.36 ± 0.36ALYM (·103/lL) 7.72 ± 1.31 8.26 ± 1.67AMON (·103/lL) 0.18 ± 0.12 0.18 ± 0.06AEOS (·103/lL) 0.11 ± 0.03 0.16 ± 0.07@

ABAS (·103/lL) 0.06 ± 0.03 0.08 ± 0.03ALUC (·103/lL) 0.06 ± 0.01 0.06 ± 0.02PT (s) 15.0 ± 0.5 15.0 ± 0.9APTT (s) 17.1 ± 2.1 17.0 ± 1.5

a–g Except where indicated n = 12 (an = 11; bn = 10; cn = 9; dn = 8; en = 7; fn =animal to conduct the full battery of hematologic analyses. Data from clumpe

* p < 0.05 compared to 33% 1507 group by linear contrasts test.@ p < 0.05 compared to 33% 1507 group by Dunn’s type I test.

MCH, MCHC, and RDW) were not significantly differentbetween these groups and the values observed between the33% 1507 maize-grain group and all other treatmentgroups were not significantly different. An approximate3% decrease (p < 0.05) in the HCT concentration wasobserved between the females consuming the 33% 1507and those consuming the 33% 33P66 maize grain. Thiseffect was not considered to be related to consumption ofthe 1507 maize grain as the HCT values in females consum-ing the 1507 maize grain were not significantly differentfrom those of all other treatment groups. Finally, a statis-tically significant (p < 0.05) decrease in AEOS concentra-tion was observed in the 33% 1507 maize grain femalescompared to the 33% 33P66 and 33% 33J56 maize grain

– 11 –– – 1133 22 22

8.43 ± 0.47 8.31 ± 0.44 8.24 ± 0.9315.8 ± 0.5a 15.4 ± 0.9 15.5 ± 1.345.5 ± 1.5a 44.6 ± 2.2 44.9 ± 3.154.6 ± 1.5a 53.7 ± 1.4 54.9 ± 4.218.9 ± 0.6a 18.6 ± 0.6 18.9 ± 1.034.6 ± 0.5a 34.5 ± 0.7 34.4 ± 1.012.7 ± 0.4a 12.6 ± 0.5 13.4 ± 2.6

190.1 ± 20.7 154.7 ± 14.5 235.4 ± 192.91018 ± 371e 1019 ± 152d 1175 ± 264e

11.53 ± 3.00a 10.41 ± 2.81 11.66 ± 1.701.89 ± 0.56a 1.97 ± 0.63 1.91 ± 0.519.03 ± 2.58a 7.94 ± 2.40 9.20 ± 1.430.26 ± 0.27a 0.21 ± 0.09 0.28 ± 0.080.16 ± 0.06a 0.15 ± 0.06 0.14 ± 0.070.10 ± 0.08a 0.07 ± 0.04 0.07 ± 0.030.08 ± 0.04a 0.06 ± 0.03 0.06 ± 0.0315.6 ± 0.6 15.6 ± 0.7 15.8 ± 0.921.2 ± 1.5 21.9 ± 3.3 22.2 ± 1.9

8.40 ± 0.41 8.17 ± 0.34 8.14 ± 0.3316.2 ± 0.6 15.8 ± 0.6 16.0 ± 0.645.8 ± 1.5 45.4 ± 1.6 45.5 ± 1.554.6 ± 1.6 55.6 ± 1.4 55.9 ± 1.619.3 ± 0.5 19.4 ± 0.7 19.7 ± 0.435.3 ± 0.7 34.8 ± 0.8 35.2 ± 0.611.3 ± 0.4 11.7 ± 0.4 11.3 ± 0.4

157.1 ± 26.3 175.3 ± 32.9 160.2 ± 28.61185 ± 90g 1112 ± 232d 1002 ± 95f

10.28 ± 2.61 10.04 ± 1.78 9.57 ± 2.811.68 ± 0.50 1.38 ± 0.55 1.70 ± 0.888.08 ± 2.19 8.22 ± 1.81 7.43 ± 1.980.23 ± 0.11 0.19 ± 0.09 0.18 ± 0.090.16 ± 0.07@ 0.13 ± 0.07 0.13 ± 0.060.05 ± 0.02 0.06 ± 0.03 0.07 ± 0.030.07 ± 0.04 0.07 ± 0.03 0.06 ± 0.0315.1 ± 0.7 14.7 ± 0.5a 14.9 ± 0.417.4 ± 1.6 17.1 ± 1.1a 16.7 ± 1.0

6; and gn = 4). An insufficient amount of blood was collected from eachd PLT were excluded from analysis.

558 S.A. MacKenzie et al. / Food and Chemical Toxicology 45 (2007) 551–562

groups (Table 3). This was also not considered to be a bio-logically significant change because it was attributable to asmaller SD within the 33% 1507 maize grain group com-pared to the control groups. Furthermore, there are noknown adverse effects associated with decreased concentra-tions of eosinophils. It was concluded that there were nodiet-related hematologic or coagulation changes associatedwith consumption of 1507 diets.

3.5. Serum chemistry

Compared to males consuming 33% 33P66 maize grain,mean serum ALKP activity was decreased (p < 0.05) in ratsconsuming 33% 1507 maize grain diets (Table 4). This dif-

Table 4Serum chemistry mean values ± SD

Maize line Percentage composition

1507 33 –33P66 – 3333J56 – –

Males

AST (U/L) 89 ± 21 89 ± 17ALT (U/L) 37 ± 6 45 ± 16SDH (U/L) 16.5 ± 5.3 19.4 ± 2.4a

ALKP (U/L) 91 ± 19 112 ± 25b

BILI (mg/dL) 0.08 ± 0.03 0.09 ± 0.03BUN (MG/DL) 17 ± 2 17 ± 2CREA (mg/dL) 0.45 ± 0.05 0.43 ± 0.05CHOL (mg/dL) 67 ± 16 69 ± 15TRIG (mg/dL) 77 ± 32 63 ± 20GLUC (mg/dL) 101 ± 8 97 ± 4TP (g/dL) 7.0 ± 0.3 7.0 ± 0.2ALB (g/dL) 4.3 ± 0.2 4.2 ± 0.2GLOB (g/dL) 2.7 ± 0.2 2.8 ± 0.2CALC (mg/dL) 10.6 ± 0.4 10.7 ± 0.3IPHS (mg/dL) 7.4 ± 0.5 7.6 ± 0.9NA (mmol/L) 149.6 ± 1.8 149.2 ± 1.6K (mmol/L) 5.98 ± 0.42 5.95 ± 0.41CL (mmol/L) 102.6 ± 1.6 102.7 ± 2.6

Females

AST (U/L) 80 ± 9 81 ± 10ALT (U/L) 37 ± 6 38 ± 7SDH (U/L) 14.4 ± 3.2 13.9 ± 3.0ALKP (U/L) 59 ± 21 61 ± 13BILI (mg/dL) 0.15 ± 0.02 0.14 ± 0.03BUN (MG/DL) 17 ± 2 17 ± 2CREA (mg/dL) 0.54 ± 0.04 0.53 ± 0.07CHOL (mg/dL) 83 ± 15 87 ± 16TRIG (mg/dL) 60 ± 26 42 ± 9GLUC (mg/dL) 98 ± 6 91 ± 7TP (g/dL) 8.0 ± 0.4 7.9 ± 0.5ALB (g/dL) 5.3 ± 0.4 5.1 ± 0.4GLOB (g/dL) 2.7 ± 0.2 2.8 ± 0.2CALC (mg/dL) 11.3 ± 0.6 11.2 ± 0.4IPHS (mg/dL) 6.6 ± 0.6 6.5 ± 0.6NA (mmol/L) 151.5 ± 2.2 151.4 ± 2.2K (mmol/L) 5.63 ± 0.31 5.86 ± 0.39CL (mmol/L) 104.2 ± 2.4 105.4 ± 2.1

Except where indicated n = 12 (an = 10). An insufficient amount of blood waanalyses.

b p < 0.05 compared to 33% 1507 group by Linear Contrasts Test.

ference was not considered biologically significant becauseserum ALKP activities in males consuming 33% 1507maize grain were not significantly different from othertreatment groups. Further, all values in the 33% 1507 maizegrain group were well within the historical age-matchedcontrol mean serum ALKP activity recorded within thislaboratory (66–109 U/L) whereas the values in the 33%33P66 near-isogenic control group approached the upperlimits. There were no other statistically significant differ-ences observed between the serum chemistry response vari-ables of the 1507 diet groups compared with any othermale treatment group. There were no statistically signifi-cant differences in any serum chemistry response variablesin females.

– 11 –– – 1133 22 22

86 ± 12 101 ± 27 92 ± 2743 ± 7 60 ± 32 44 ± 9

17.9 ± 3.6 24.1 ± 14.3 18.4 ± 5.291 ± 14 110 ± 21 98 ± 15

0.10 ± 0.04 0.10 ± 0.02 0.10 ± 0.0316 ± 2 17 ± 2 17 ± 2

0.42 ± 0.06 0.46 ± 0.03 0.42 ± 0.0569 ± 7 58 ± 8 66 ± 1070 ± 24 85 ± 32 75 ± 2494 ± 8 99 ± 9 105 ± 9

7.1 ± 0.3 7.0 ± 0.3 7.0 ± 0.34.3 ± 0.2 4.2 ± 0.3 4.3 ± 0.22.8 ± 0.2 2.8 ± 0.3 2.7 ± 0.2

10.7 ± 0.5 10.7 ± 0.3 10.8 ± 0.37.2 ± 0.4 7.8 ± 0.8 7.8 ± 0.7

149.3 ± 2.2 149.7 ± 1.5 149.0 ± 2.55.88 ± 0.43 5.98 ± 0.51 6.15 ± 0.54

102.8 ± 1.8 102.6 ± 2.3 103.3 ± 1.9

83 ± 23 89 ± 14 88 ± 942 ± 19 38 ± 8 39 ± 8

13.7 ± 5.2 13.1 ± 2.3 16.5 ± 2.565 ± 26 62 ± 13 57 ± 19

0.15 ± 0.02 0.13 ± 0.03 0.14 ± 0.0318 ± 3 17 ± 3 16 ± 1

0.54 ± 0.06 0.52 ± 0.05 0.54 ± 0.0591 ± 23 96 ± 17 85 ± 2160 ± 30 54 ± 11 51 ± 22

104 ± 16 101 ± 8 106 ± 158.0 ± 0.6 7.9 ± 0.3 7.9 ± 0.45.2 ± 0.5 5.1 ± 0.3 5.2 ± 0.32.8 ± 0.3 2.8 ± 0.2 2.7 ± 0.2

11.3 ± 0.5 11.3 ± 0.4 11.3 ± 0.46.7 ± 0.8 6.6 ± 0.9 6.3 ± 1.4

150.7 ± 1.7 150.8 ± 1.9 152.0 ± 3.25.78 ± 0.32 5.72 ± 0.31 5.79 ± 0.42

104.8 ± 1.8 105.6 ± 1.7 106.6 ± 2.2

s collected from each animal to conduct the full battery of hematologic

S.A. MacKenzie et al. / Food and Chemical Toxicology 45 (2007) 551–562 559

3.6. Urinalysis

There were no statistically significant differencesbetween any urinalysis response variables of males andfemales from any of the treatment groups (Table 5).

3.7. Organ weights and pathology

There were no differences in the relative organ weightsamong any of the female treatment groups (Table 6). The

Table 5Urinalysis mean values ± SD

Maize line Percentage composition

1507 33 –33P66 – 3333J56 – –

Males

VOL (ml) 7.8 ± 6.3 6.6 ± 4.8UOSM (mOsm/kg) 1650 ± 1052 1583 ± 760pH 6.7 ± 0.3 7.0 ± 0.6URO (EU/dL) 0.2 ± 0.0 0.2 ± 0.0UMTP (MG/DL) 112 ± 63 135 ± 78

Females

VOL (ml) 2.9 ± 3.0 4.7 ± 5.6a

UOSM (mOsm/kg) 2003 ± 1045a 1670 ± 1109a

pH 6.3 ± 0.3a 6.4 ± 0.4a

URO (EU/dL) 0.2 ± 0.0a 0.2 ± 0.0a

UMTP (MG/DL) 64 ± 40a 57 ± 59a

Except where indicated n = 12 (an = 11; bn = 10). An insufficient amount of urianalytes.

Table 6Organ/body weight mean values ± SD

Maize line Percentage composition

1507 33 –33P66 – 3333J56 – –

Males

Adrenals 0.012 ± 0.002 0.011 ± 0.002Brain 0.408 ± 0.048 0.428 ± 0.031Epididymides 0.292 ± 0.041 0.301 ± 0.038Heart 0.325 ± 0.038 0.347 ± 0.043Kidneys 0.685 ± 0.073 0.753 ± 0.076b

Liver 2.776 ± 0.158 2.759 ± 0.238Spleen 0.151 ± 0.023 0.160 ± 0.023Testes 0.641 ± 0.053 0.674 ± 0.117Thymus 0.085 ± 0.023 0.078 ± 0.023

Females

Adrenals 0.028 ± 0.005 0.026 ± 0.005Brain 0.696 ± 0.062 0.709 ± 0.077Heart 0.371 ± 0.019 0.390 ± 0.028Kidneys 0.771 ± 0.063 0.780 ± 0.101Liver 2.846 ± 0.173 2.786 ± 0.208Ovaries 0.049 ± 0.008 0.052 ± 0.010Spleen 0.202 ± 0.028 0.209 ± 0.038Thymus 0.106 ± 0.027 0.099 ± 0.020Uterus 0.259 ± 0.090 0.252 ± 0.083

Except where indicated n = 12 (an = 11). An insufficient amount of tissue wasb p < 0.05 compared to 33% 1507 group by one-way ANOVA.

only statistically significant difference observed in maleswas in the relative kidney weights. Specifically, the relativekidney weights from male rats in the 33% 1507 maize graingroup were lower (p < 0.05) than those in the 33% 33P66and 33% 33J56 groups. However, there were no statisticallysignificant differences between absolute kidney weights andkidney weight relative to brain weight among all maletreatment groups (data not shown). The difference wasattributed to the slightly higher final body weight in the33% 1507 maize grain group compared to the control

– 11 –– – 1133 22 22

5.6 ± 4.6 6.2 ± 6.4 8.9 ± 7.01841 ± 1042 1939 ± 1422 1390 ± 894

6.8 ± 0.3 6.7 ± 0.2 6.8 ± 0.40.2 ± 0.0 0.2 ± 0.0 0.2 ± 0.0182 ± 162 177 ± 139 85 ± 49

3.4 ± 3.4 2.3 ± 2.0 4.4 ± 5.31842 ± 1267a 1669 ± 712a 2031 ± 1292

6.6 ± 0.4a 6.5 ± 0.4a 6.5 ± 0.4a

0.2 ± 0.0a 0.2 ± 0.0a 0.2 ± 0.0a

51 ± 50b 49 ± 27a 51 ± 42b

ne was collected from each animal to conduct the full battery of urinalysis

– 11 –– – 1133 22 22

0.012 ± 0.002 0.013 ± 0.002 0.011 ± 0.0020.421 ± 0.020 0.419 ± 0.032 0.418 ± 0.0340.303 ± 0.046 0.298 ± 0.045 0.278 ± 0.0580.328 ± 0.024 0.332 ± 0.024 0.322 ± 0.0280.750 ± 0.052b 0.734 ± 0.040a 0.689 ± 0.0432.769 ± 0.136 2.922 ± 0.232 2.782 ± 0.1620.163 ± 0.025 0.145 ± 0.020 0.161 ± 0.0380.661 ± 0.112 0.646 ± 0.116 0.641 ± 0.1690.075 ± 0.021 0.073 ± 0.015 0.069 ± 0.019

0.025 ± 0.004 0.027 ± 0.006 0.025 ± 0.0050.689 ± 0.047 0.683 ± 0.075 0.679 ± 0.0800.383 ± 0.031 0.407 ± 0.025 0.377 ± 0.0290.727 ± 0.055 0.786 ± 0.049 0.772 ± 0.0802.764 ± 0.241 2.883 ± 0.132 2.762 ± 0.1670.048 ± 0.011 0.056 ± 0.018 0.047 ± 0.0100.199 ± 0.036 0.191 ± 0.024 0.190 ± 0.0260.111 ± 0.015 0.109 ± 0.025 0.108 ± 0.0260.240 ± 0.072 0.231 ± 0.068 0.286 ± 0.102

collected from all animals.

Table 7Summary of microscopic incidence findings

Findings Male Female

33% 1507N = 12

33% 33P66N = 12

33% 1507N = 12

33% 33P66N = 12

Liver Foci of chronic inflammation 9 11 8 10Fatty change, median cleft 0 0 3 2

Pancreas Atrophy 2 1 0 0Foci of basophilic alteration 1 0 0 0Foci of chronic inflammation 0 0 2 1

Kidneys Chronic progressive nephropathy 7 7 2 3Focal infarct 0 0 0 1

Lungs Focal histiocytosis 0 1 0 0Foci of chronic inflammation 0 0 1 0

Pharynx/larynx Foci of chronic inflammation 0 0 1 0Nose Odontodysplasia 0 0 0 1

Odontitis/periodontitis 0 0 2 1Heart Cardiomyopathy 2 5 1 4Pituitary gland Cyst 0 0 0 1Testes Degeneration of seminiferous

tubules0 1 – –

Epididymides Oligospermia 0 1 – –Prostate Chronic inflammation 2 0 – –Ovaries Cyst – – 1 0Uterus Dilated lumen – – 1 0Skin Alopecia 0 0 1 0Eyes Optic nerve not present 0 1 1 0

560 S.A. MacKenzie et al. / Food and Chemical Toxicology 45 (2007) 551–562

groups (5.3% and 4.1% respectively [p > 0.05]). Addition-ally, there were no gross lesions or increased incidence ofmicroscopic lesions beyond those known to occur sponta-neously in rats of this strain and age associated withconsumption of the 1507 diet compared to any other treat-ment group (data not shown). There were no statisticallysignificant differences in organ weights of female rats con-suming the 1507 diets compared to any other treatmentgroup. The incidence of microscopic changes in tissue sam-ples from animals in the 33% 1507 maize grain and 33%33P66 maize grain groups is presented in Table 7. Allobservations are known to occur spontaneously in rats ofthis strain and age and were not considered related to con-sumption of diets containing 1507 maize grain. It was con-cluded from these analyses that consumption of the 1507diets was not associated with an increased incidence of tox-icologically significant effects.

4. Discussion

There are multiple guidelines for the safety assessmentprocess to demonstrate that GM crops are ‘‘as safe as’’conventional non-GM crops (Codex, 2003; FAO, 1996;OECD, 1993; OECD., 1997; WHO., 1991; WHO., 1995).Guidelines also require compositional analyses of keynutrients and anti-nutrients in GM crops. Feeding studiesare also conducted to compare the nutritional performanceof GM crops to non-GM comparators.

In the current study, no differences between the compo-sitional analysis of DAS-Ø15Ø7-1 (1507) maize grain,near-isogenic maize grain (33P66), and another commer-

cially available non-GM hybrid maize grain (33J56) wereidentified. The concentrations of all nutrient and anti-nutrients analytes were consistent with published historicalranges (Ridley et al., 2004; OECD., 2002). These results areconsistent with other studies of maize grains modified toexpress transgenic proteins, including Cry1F, that havereported no significant differences in nutrient compositioncompared to non-GM controls or to historical referenceranges (Herman et al., 2004; George et al., 2004; Ridleyet al., 2002; Sidhu et al., 2000).

Feeding studies are another important component ofestablishing nutritional equivalence between GM andnon-GM grains. Historically, these studies have been con-ducted by comparing nutritional performance variables inbroiler chickens consuming diets prepared with eitherGM grains to those of broiler chickens consuming dietscontaining non-GM maize grain. Response variables typi-cally include body weight, weight gain, and feed efficiencyover the course of 42 days. Numerous GM maize grainshave been shown to be nutritionally equivalent to non-GM maize grains in broiler chickens (Sidhu et al., 2000;Taylor et al., 2003a,b,c). Similarly, our results have demon-strated that 1507 maize grain is nutritionally equivalent tonear-isogenic (33P66) maize grain in broiler chickens (man-uscript submitted).

Subchronic rodent feeding studies have also been con-ducted to compare the performance of GM maize grainwith non-GM maize grains using toxicology responsevariables. Two recent studies reported no significant differ-ences following subchronic consumption of up to 33% her-bicide tolerant (Hammond et al., 2004) or corn rootworm

S.A. MacKenzie et al. / Food and Chemical Toxicology 45 (2007) 551–562 561

protected maize grains (Hammond et al., 2006) comparedto non-GM maize grains. Results from these studies weresimilar to the outcome of feeding trials conducted in broilerchickens in that they demonstrated equivalent nutritionalperformance of GM and non-GM maize grains.

In the current study, 1507, near-isogenic control (33P66)and reference (33J56) maize grains were each used to pro-duce separate batches of rodent feed according to the spec-ifications of Purina Mills Certified Rodent LabDiet� 5002.These diets were fed to separate groups of rats for approx-imately 13 weeks. Over the duration of the feeding study,no biologically significant differences were observed in thein-life nutritional performance response variables betweenrats fed diets formulated with the 1507 maize grain andthose fed the non-GM control diets. Additionally, therewere no toxicologically significant differences in neuro-behavioral, hematological, serum chemistry, urinalysis,organ weights, or pathology identified between rats con-suming diets formulated with 1507 maize grain comparedto rats consuming diets produced with non-GM maizegrains.

The results from this study demonstrate that consump-tion of diets prepared with 1507 maize grain do not causeadverse effects in a subchronic feeding study in rats. Eval-uations included a substantial battery of nutritional perfor-mance, neurobehavioral, and pathological responsevariables. From these results, it was concluded that 1507maize grain is as safe and nutritious as non-GM maizegrain.

References

Astwood, J.D., Leach, J.N., Fuchs, R.L., 1996. Stability of food allergensto digestion in vitro. Nat. Biotechnol. 14, 1269–1273.

Betz, F.S., Hammond, B.G., Fuchs, R.L., 2000. Safety and advantages ofBacillus thuringiensis protected plants to control insect pests. Regul.Pharmacol. Toxicol. 32, 156–173.

Codex, 2003. Joint FAO/WHO Food Standards Programme. CodexAlimentarius Commission. Report of the Fourth Session of the CodexAd Hoc Intergovernmental Task Force on Foods Derived fromBiotechnology.

Dunn, O.J., 1964. Multiple contrasts using rank sums. Technometrics 6,241–252.

FAO/WHO, 2001. Evaluation of the allergenicity of genetically modifiedfoods. Report of a Joint FAO/WHO Expert Consultation. Food andAgriculture Organization of the United Nations and World HealthOrganization. FAO, Rome, Italy.

FAO, 1996. Biotechnology and food safety. Report of a joint FAO/WHOconsultation. Food and Nutrition Paper 61. FAO, Rome, Italy.

Flood, B., Foster, R., Hutchison, B., 1995. Sweet corn. In: Foster, R.,Flood, B. (Eds.), Vegetable Insect Management: With Emphasis on theMidwest. Meister, Willoughby, OH, pp. 19–40.

George, C., Ridley, W.P., Obert, J.C., Nemeth, M.A., Breeze, M.L.,Astwood, J.D., 2004. Composition of grain and forage from cornrootworm-protected corn event MON863 is equivalent to that ofconventional corn (Zea mays L.). J. Argic. Food Chem. 52, 4149–4158.

Hammond, B., Dudek, R., Lemen, J., Nemeth, M., 2004. Results of a13 week safety assurance study with rats fed grain from glyphosatetolerant corn. Food Chem. Toxicol. 42, 1003–1014.

Hammond, B., Lemen, J., Dudek, R., Ward, D., Jiang, C., Nemeth, M.,Burns, J., 2006. Results of a 90 day safety assurance study with rats fed

grain from corn rootworm-protected corn. Food Chem. Toxicol. 44,147–160.

Herman, R.A., Phillips, A.M., Collins, R.A., Tagliani, L.A., Claussen,F.A., Graham, C.D., Bickers, B.L., Harris, T.A., Prochaska, L.M.,2004. Compositional equivalency of Cry1F corn event and TC6275 andconventional corn (Zea mays L.). J. Agric. Food Chem. 52, 2726–2734.

Herouet, C., Esdaile, D.J., Mallyon, B.A., Debruyne, E., Schulz, A.,Currier, T., Hendrickx, K., van der Klis, R-J., Rouan, D., 2005. Safetyevaluation of the phosphoinothricin acetyltransferase proteins encodedby the pat and bar sequences that confer tolerance to glufosinate-ammonium herbicide in transgenic plants. Regul. Toxicol. Pharmacol.41, 134–149.

Hurst, C.D., Knight, A., Bruce, A.J., 1999. PCR detection of geneticallymodified soya and maize in foodstuffs. Mol. Breed. 5, 579–586.

ILSI (International Life Sciences Institute), 1997. ILSI Europe novel foodstask force: The safety assessment of novel foods. Food Chem. Toxicol.34, 931–940.

IPCS (International Programme on Chemical Safety), 1999. Environmen-tal Health Criteria 217: Microbial Pest Control Agent BacillusThuringiensis. World Health Organization, Geneva, Switzerland.

Kuhn, J.O., 1998. Cry1F Bacillus thuringiensis subsp. aizawai Delta-endotoxin. Final report. Acute oral toxicity study in mice. StillmeadowInc., Sugar Land, TX, Study number 4281-98. PHI Document 11171.

Levene, H., 1960. Robust test for equality of variances. In: Olkin, J. (Ed.),Contributions to Probability and Statistics. Stanford University Press,Palo Alto, pp. 278–292.

Mason, C.E., Rice, M.E., Calvin, D.D., Van Duyn, J.W., Showers, W.B.,Hitchison, W.D., Witkowski, J.F., Higgins, R.A., Onstad, D.W.,Dively, G.P., 1996. European Corn Borer Ecology and Management.North Central Regional Extension Publication 327. Iowa StateUniversity, Ames, IA.

McClintock, J.T., Schaffer, C.R., Sjoblad, R.D., 1995. A comparativereview of the mammalian toxicity of Bacillus thuringiensis-basedpesticides. Pestic. Sci. 45, 95–105.

Meyer, T., 1999. Comparison of amino acid sequence similarity of Cry1Fand PAT proteins to known allergenic proteins. Pioneer Hi-BredInternational, Inc., Johnston, IA. Study number PHI-99-013. PHIDocument 11183.

OECD, 1993. Safety evaluation of foods produced by modern biotech-nology: Concepts and principles. Organization of Economic Cooper-ation and Development. Paris, France.

OECD, 1996. OECD documents: food safety and evaluation. Organiza-tion of Economic Cooperation and Development. Paris, France.

OECD, 1997. OECD documents: report of the OECD workshop on thetoxicological and nutritional testing of novel foods. Organization ofEconomic Cooperation and Development. Paris, France.

OECD, 1998. OECD Guidelines for Testing of Chemicals No. 408,Repeated dose 90-day oral toxicity study in rodents. Paris, France.

OECD, 2002. Consensus document on compositional considerations fornew varieties of maize (Zea mays): Key food and feed nutrients, anti-nutrients and secondary plant metabolites. Organization for EconomicCo-operation and Development, ENV/JM/MONO(2002)25.

OECD, 2003. Considerations for the safety assessment of animal feedstuffsderived from genetically modified plants. Organization for Eco-nomic Co-operation and Development. Paris, France. ENV/JM/MONO(2003)10.

Ridley, W.P., Sidhu, R.S., Pyla, P.D., Nemeth, M.A., Breeze, M.L.,Astwood, J.D., 2002. Comparison of the nutritional profile ofglyphosate tolerant corn event NK603 with that of conventional corn(Zea mays L.). J. Agric. Food Chem. 50, 7235–7243.

Ridley, W.P., Shillito, R.D., Coats, I., Steiner, H.Y., Shjawgo, M.,Phillips, A., Dussold, P., Kurtyka, L., 2004. Development of theInternational Life Sciences Institute crop composition database. J.Food Comp. Anal. 17, 423–438.

Rinkleff, J.H., Hutchison, W.D., Campbell, C.D., Bolin, P.C., Bartels,D.W., 1995. Insecticide toxicity in European corn borer (Lepidoptera:Pyralidae): ovicidal activity and residual mortality to neonates. J.Econ. Entomol. 88, 246–253.

562 S.A. MacKenzie et al. / Food and Chemical Toxicology 45 (2007) 551–562

Schafer, B.W. 2002. In vitro simulated gastric fluid digestibility study oftruncated Cry1F Delta-endotoxin derived from Pseudomonas fluores-

cens. Dow AgroSciences LLC, Indianapolis, IN, Study number GH-C5367. PHI Document 12851.

Schnepf, E., Crickmore, N., van Rie, J., Lereclus, D., Baum, J., Feitelson,J., Zeigler, D.R., Dean, D.H., 1998. Bacillus thuringiensis and itspesticidal crystal proteins. Mol. Biol. Rev. 62, 775–806.

Shapiro, S.S., Wilk, M.B., 1965. An analysis of variance test for normality(complete samples). Biometrika 52, 591–611.

Sidhu, R.S., Hammond, B.G., Fuchs, R.L., Mutz, J.N., Holden, L.R.,George, B., Olson, T., 2000. Glyphosate-tolerant corn: The composi-tion and feeding value of grain from glyphosate-tolerant corn isequivalent to that of conventional corn (Zea mays L.). J. Agric. FoodChem. 48, 2305–2312.

Taylor, M.L., Hartnell, G.F., Riordan, S.G., Nemeth, M.A., Kar-unanandaa, K., George, B., Astwood, J.D., 2003a. Comparison ofbroiler performance when fed diets containing grain from YieldGard(MON810), YieldGard · Roundup Ready (GA21), nontransgeniccontrol, or commercial corn. Poultry Sci. 82, 823–830.

Taylor, M.L., Hartnell, G.F., Riordan, S.G., Nemeth, M.A., Kar-unanandaa, K., George, B., Astwood, J.D., 2003b. Comparison of

broiler performance when fed diets containing grain from RoundupReady (NK603), YieldGard · Roundup Ready (MON810 · NK603),non-transgenic control, or commercial corn. Poultry Sci. 82, 443–453.

Taylor, M.L., Hyu, Y., Hartnell, G.F., Riordan, S.G., Nemeth, M.A.,Karunanandaa, K., George, B., Astwood, J.D., 2003c. Comparison ofbroiler performance when fed diets containing grain from YieldGardRootworm (MON863), YieldGard Plus (MON810 · MON863), non-transgenic control, or commercial reference corn hybrids. Poultry Sci.82, 1948–1956.

WHO, 1991. Strategies for assessing the safety of foods produced bybiotechnology. Report of a Joint FAO/WHO Consultation. WorldHealth Organization, Geneva, Switzerland.

WHO, 1995. Application of the principles of substantial equivalence tothe safety evaluation of foods and food components from plantsderived from modern biotechnology. Report of WHO WorkshopWHO/FNU/FOS/95.1. World Health Organization, Geneva,Switzerland.

Zeph. L., Lamb, I., Coates, I., McNaughton, J.L., Hunst, P., Fuentes, A.,and Delaney, B., in press. Feeding performance of broiler chickens feddiets containing Bt Cry1F maize grain. Poultry Sci.