Journal of the Science of Food and Agriculture

J Sci Food Agric 88:382388 (2008)

Toxicological evaluation of transgenic rice our with an Escherichia coli phytase gene appA by sub-chronic feeding study in Wistar rats
Cheng-Chih Tsai,1 Chieh-Hsien Lai,1 Chi-Sheng Yang,2 Chien-Ku Lin1 and Hau-Yang Tsen1
1 Department 2 Department

of Food Science and Nutrition, Hungkuang University, Shalu, Taiwan, ROC of Nursing, Hungkuang University, Shalu, Taiwan, ROC

Abstract
BACKGROUND: Phytase is increasingly used as an ingredient in swine and poultry feeds to improve the bioavailability of phytate-bound phosphorus and reduce the supplement of elemental phosphorus in feeds. Recently, a transgenic rise (TGR) with phytase appA gene from E. coli has been developed in Taiwan. To assure the food safety of this TGR, we performed a sub-chronic whole-food feeding study with Wistar rats. RESULTS: Weaned feeding male and female rats were divided into TGR and non-TGR (NTGR) groups (20 rats per group), and fed diets containing 76.8% rice our of TGR and NTGR, respectively. After 93 days feeding, although some hematological ndings and blood chemistry values differed signicantly between the TGR and NTGR groups, all values were still within the normal range for rats of this age and sex. No adverse effects of TGR were observed in terms of animal behavior, weight gain, or feed utilization rate. Necropsy at the end of the experiment indicated that neither pathological lesions nor histopathological abnormalities were present in organs such as liver, kidney, intestines and testes of rats in TGR and NTGR groups. In addition, no signicant differences (P > 0.05) were observed in relative organ weights, hemograms and blood indices of rats between the two groups. CONCLUSION: These results demonstrated that in a 93-day feeding study with Wistar rats a diet containing 76.8% of TGR our having a phytase activity of about 1500 U kg1 body weight per day has no adverse effects. 2007 Society of Chemical Industry

Keywords: phytase; transgenic rice; food safety; sub-chronic feeding study

INTRODUCTION Phosphorous is an essential nutrient for growing animals. It is important for bone formation and mineralization, cell metabolism, protein synthesis, and is a constituent of cell membranes and intracellular buffers for acidalkaline balance. Since monogastric animals lack intestinal phytase and the bioavailability of plant phosphorous in feedstuffs is limited, phytase is increasingly used as an ingredient in swine and poultry feeds to improve the bioavailability of phytate-bound phosphorus and reduce the supplement of elemental phosphorus in feeds.1 Plant phytases are normally not produced in sufcient quantities; various phytase-transgenic plants have therefore been developed with the aim to reduce production cost compared to that microbial fermentation.2 Phytase genes have been cloned from Aspergillus species and expressed in transgenic tobacco

seeds and leaves,3,4 in transformed soybean cell suspension cultures,5 in transgenic soybean and alfalfa,6,7 and in transgenic wheat, rice and canola seeds.8 11 The transgenic plant-produced phytase has a lower molecular mass than the fungal enzyme due to differences in glycosylation.3,5 Nevertheless, the plant-produced phytase efciently substitutes for the microbial enzyme in poultry-feeding studies.3,6,12 Genetically modied (GM) crops are being increasingly employed in both agricultural and livestock industries.13 The safety of these GM crops, however, needs to be assured according to the ofcial process. The approaches used in the safety assessment of crops and foods derived from genetically modied organisms have been developed in collaborative work with international agencies such as the Organization for Economic Coordination and Development14,15 and the United Nations World Health Organization/Food

Correspondence to: Hau-Yang Tsen, Department of Food Science and Nutrition, HungKuang University, 34 Chung-Chi Rd, Taichung County 433, Taiwan, ROC E-mail: hytsen@sunrise.hk.edu.tw Contract/grant sponsor: Department of Health, Taipei, Taiwan; contract/grant number: DOH94-FS033 (Received 6 March 2007; revised version received 4 July 2007; accepted 23 July 2007) Published online 26 November 2007; DOI: 10.1002/jsfa.3096

2007 Society of Chemical Industry. J Sci Food Agric 00225142/2007/$30.00

Toxicological evaluation of transgenic rice our with E. coli phytase gene

and Agricultural Organization.16 18 Although substantial equivalence based on the comparison of the compositions, including those of the macro- and micronutrients, antinutrients, amino acids and fatty acids between a GM organism and its parent crop and sequence alignment between the cloned gene and the donor gene, are the priority chosen for safety assessment, to assure the safety for whole food, animal feeding studies are in some cases performed. The use of animal feeding for sub-chronic toxicity study has recently been reviewed in detail by Concerted Action Food Safety in Europe (FOSIE).19 Since high-level expression of the desired proteins has the possibility to provoke not only nutritional change but also metabolic disturbance in the host crops, the safety assessment standard substantial equivalence hitherto used is not always sufcient for application to the safety assessment of GM crops thus created.20 In recent years, the Academic Senica in Taiwan has developed a GM rice cloned with phytase appA gene from E. coli strain B (ATCC 11 303).12 The phytase activity is about 19 386 U kg1 rice our. The composition, including macro- and micro-nutrients, total amino acid and fatty acid composition, between GMO rice and its parent rice (Tainung 67) are equivalent to each other; however, the molecular mass of the phytase product from this transgenic rice is about 54 kDa, while that from the gene donor E. coli is 45 kDa. Based on the above described facts and the review by FOSIE,19 and because rice is a staple diet for many people throughout the world, toxicity associated with GM rice should be carefully evaluated. In this study, we therefore performed a sub-chronic (93 days) feeding evaluation experiment using Wistar rats. The 90-day animal study is now the core study for the safety assessment of GM foods in the SAFOTEST project, an EU project entitled New methods for the safety testing of transgenic food.21

from the InGene Co., Taichung, Taiwan, and the National Agriculture Research Institute (NART), Taichung, Taiwan. The phytase activity, proximate composition, amino acid composition, fatty acid composition, minerals such as Ca, P, Na, Fe, micronutrients, including vitamin B1 , B2 , niacin etc. of the dehusked rice our have been assayed by the InGene Biotechnology Co., Taichung, Taiwan and the Food Industry Research and Development Institute, Hsin-Chu, Taiwan. No signicant differences in these nutritional compositions were observed. Phytase activity of the TGR and NTGR rice our was 19 386 U kg1 and 0 U kg1 , respectively. Such activity was measured at 37 C and pH 5.5 using 1.5 mmol L1 sodium phytate as substrate according to the method of Shimizu.22 Denition of 1 enzyme unit is 1 mol of Pi released per minute. Ingredients of the TGR and NTGR diets are shown in Table 1. These diets were prepared based on the nutritional composition of commercial diet AIN93G and the method used by Zhuo et al .23 and Wang et al .24 The animal room was ventilated and maintained at a temperature of 22 2 C with a relative humidity of 65 5%. Articial lighting was sequenced at 12-h light/dark cycles. The treatments lasted for 93 days, during which activity, behavior, and hair luster of each rat were observed and recorded daily, while water intake (WI), feed intake (FI), and body weight were measured twice weekly. The specic growth rate (SGR) was expressed as the average weight gain (g) twice every week. On day 94, all animals were sacriced humanely and their blood and tissue samples were collected for further laboratory analysis. Hematology, blood biochemistry, and pathology Blood samples were collected into ethylenediaminetetraacetic acid (EDTA)-treated tubes. Leukocytes, erythrocytes and platelet counts, hemoglobin concentration (HB), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and hematocrit (HT) were determined with an auto-ow blood cell counter (Sysmex KX-21, Kobe, Japan).
Table 1. Ingredients of diets for TGR and NTGR groups of rats

MATERIALS AND METHODS Animals and experimental design Forty Wistar rats aged 4 weeks and weighing 8085 g were obtained from BioLASCO Taiwan Co., Ltd (Taipei, Taiwan) and acclimatized to laboratory conditions for 1 week. At day zero, the rats were randomly allocated to two groups of 10 rats per sex. Every two rats were conned in each grid. Animals were identied on the tail. Rats were offered basal diet (AIN 93G) and water ad libitum. After 1 week, male and female rats were divided into two groups each with 20 rats; the two groups were transgenic rice (TGR) and non-transgenic rice (NTGR) groups. Rats were fed diets containing 76.8% rice our from the transgenic rice (Nat-ANN) with phytase appA gene from E. coli strain B (ATCC 11 303);12 and non-transgenic parent rice (Oryza sativa L. cv. Tainung 67), respectively. Rice our was obtained by grinding dehusked rice grains of TGR and NTGR (Tainung 67), respectively. These TGR and NTGR rice samples were obtained
J Sci Food Agric 88:382388 (2008) DOI: 10.1002/jsfa

Ingredient (%) Cornstarch Casein (>85% protein) Sucrose Soybean oil (no additives) Fiber (alphacel, non-nutritive bulk) Mineral mix (AIN-93G-MX) Vitamin mix (AIN-93-VX) L-Cystine Choline bitartrate (41.1% choline) TGR rice our NTGR rice our Totala

TGR 12.28 4.64 2.32 1.62 1.16 0.81 0.23 0.07 0.06 76.8 0 100

NTGR 12.28 4.64 2.32 1.62 1.16 0.81 0.23 0.07 0.06 0 76.8 100

a For each diet preparation, 0.0003% of tert-butylhydroquinone (TBHQ) was mixed.

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Following completion of the hematology assays, plasma was separated from blood samples by centrifugation. Glucose concentrations, blood urea nitrogen (BUN), glutamic oxaloacetic transaminase (GOT), glutamic pyruvate transaminase (GPT), lowdensity lipoproteins (LDL), high-density lipoproteins (HDL), total protein, albumin, urate, creatinine, total bilirubin, cholesterol (CHO), cholesterol esters (CHE), alkaline phosphatase, Ca2+ , Na+ , K+ , Cl and inorganic phosphate in plasma were determined using a COBAS Mira Plus analyzer and Roche diagnostics biochemicals (Roche, Nutley, NJ, USA). Following sacrice, a thorough necropsy was performed on all animals. The following organs were weighed (paired organs together) after dissection: adrenals, testicles, heart, kidneys, liver, ovaries and spleen. The organto-body weight ratios (relative organ weights) were calculated from the absolute organ weights and the terminal body weight of the rats. Samples of the weighed organs were preserved in neutral 4% formaldehyde in PBS. Histopathological analyses were conducted on 5 m sections of appropriate parafnembedded tissues, stained with hematoxylin and eosin, from all animals. Additionally, histopathological analyses were conducted on the adrenals, testicles, heart, kidneys, liver, ovaries and spleen from all experimental groups. Statistical analysis Statistical analysis was that used by Schrder et al .25 Data were expressed as mean SD and the statistical signicance of the difference of means between male rats or female rats fed non-transgenic and transgenic rice diets was conducted by one-way analysis of variance (ANOVA) followed by Duncans tests. Statistical signicance is indicated as a or b for P -values <0.05.

uctuated with time, mean feed and water intake for TGR and NTGR rat groups was in general the same. There was no signicant difference in daily feed intake among animals fed with 76.8% TGR or NTGR (Table 2). Although mean feed intake per rat for females was lower than that for the males, their relative feed intake (g feed per kg body weight per day) was generally the same. Although data on SGR uctuated with time in each group, there were no signicant differences in SGR between the TGR and NTGR groups for male rats during the 93-day experimental period (P > 0.05) (Table 3). For female rats, however, it seems that after 51 days feeding TGR may enhance the growth of female rats (Table 3). Hematology The effects of feeding TGR diet and NTGR diet on hematological parameters are shown in Table 4. There were no signicant differences in hematocrit, MCHC, or platelets among the rats in TGR and NTGR groups and different sex groups. However, signicant increases were noted in leukocytes, MCH, and MPV in TGR groups compared with NTGR groups for different sex groups. There were signicant differences in erythrocytes, hemoglobin and MCV in the TGR and NTGR for female groups and in leukocytes for males. Increases were noted in hemoglobin and MCV in TGR females compared with NTGR females. Although a few parameters were signicantly different for TGR and NTGR male and/or female groups, these values appeared to be within the normal reference intervals for rats of the same age and sex as compared with the data from control groups of Wistar rats fed 90 days with diets containing 60% of non-transgenic rice, as reported by Poulsen et al .21 Therefore, these changes had no toxicological signicance. Clinical chemistry Forty rats in each group for each sex were assayed for clinical chemistry. Clinical chemistry values at terminal sampling time for TGR and NTGR groups showed that statistically there were no signicant differences in the plasma concentrations of cholesterol, cholesterol esters, HDL, LDL, GOT, GPT, albumin, alkaline phosphatase, BUN, creatinine, Na+ , and Cl (Table 5). The creatinine levels for our TGR and NTGR groups, although higher than those of the literature data, are similar to each other. Signicant

RESULTS Feed, water intake, body weight and specic growth rate (SGR) Throughout the experimental period, there was no noticeable change in activity, behavior, or hair luster in any of the groups of rats. No dead case, diarrhea or other treatment-related sickness was recorded. At the end of the experimental period all animals were healthy. Although the daily intake of feed and water

Table 2. Absolute and relative feed consumption (means SD) for TGR and NTGR group rats during the rst and second periods of the feeding study

Absolute (g feed per cage per day) Feeding week Group TGR NTGR Sex M F M F 17 50.7 4.06 39.07 4.32 49.6 5.52 39.41 4.88 813 52.38 12.71 34.89 8.95 47.35 6.98 35.93 9.68

Relative (g feed kg1 rat per day) Feeding week 17 90.39 32.61 95.06 26.11 91.06 30.82 97.03 29.09 813 54.10 13.90 61.34 16.33 50.38 8.53 64.62 17.99

384

J Sci Food Agric 88:382388 (2008) DOI: 10.1002/jsfa

Toxicological evaluation of transgenic rice our with E. coli phytase gene

Specic growth rate of the rat = (W W0 )/W0 100 (%), where W is rat weight at the dened feeding days and W0 is rat weight at 0 day; P < 0.05 (t-test). Statistical analysis was according to that described in methods. Signicant difference of means between male and female rats fed with TGR and NTGR was observed. P > 0.05 (t-test). Signicant difference of means between male and female rats fed with TGR and NTGR was not observed.

124.9 29.0 103.4 25.13

decreases in total protein, urate, and phosphate were observed in TGR males compared with the NTGR male groups. In male rats, an increase in K+ concentration was observed in TGR rats compared with the NTGR groups. Signicant decreases were noted in Ca2+ in TGR groups compared with NTGR groups for both sex groups. A few parameters were signicantly different but in general, as compared with those available in the reference literature,21,25,26 all were within the range for rats of the same sex and age.21,25,26 Organ weights and pathology The relative organ weights ((organ weight/body weight) 100%) of rats are shown in Table 6. There were no signicant differences in relative heart, liver, kidney, adrenal, spleen, or ovary among rats in the different treatment groups and different sex groups. Gross examination at heart, kidney, adrenal, spleen, testicle, or ovary, and microscopic examination revealed no changes attributable to the ingestion of diets containing TGR and NTGR.

93 days

69 days

Female Male SGR 51 days Female

112.5 24.8 93.5 23.7

267.8 30.8 276.0 55.7

300.6 31.3 304.9 56.1

Male

140.8 32.4 118.4 36.2

Female

DISCUSSION Efcacy of phytase preparations derived from E. coli and phytase transgenic yeast on phytase phosphorus utilization by young chicks and pigs has been reported.27,28 Under the levels suggested for feed supplement use, phytase effectively releases phytate P from the cornsoy diet of chicks and pigs. No toxicity has been reported. In our study, rats were fed with TGR and NTGR rice our composed of up to 76.8% of the dietary ingredients. Such a ratio of TGR or NTGR in diets was the highest ratio of rice ingredient in the diet when a similar protein level based on commercial diet AIN-93G was considered. Previously, Zhuo et al .23 and Wang et al .24 reported that diets with high ratios (up to 76%) of non-transgenic rice our would not cause signicant adverse effects on the growth rates and behavior of rats compared to those fed with the commercial diet. Concerning the growth behavior of Wistar rats, the phytase-TGR diet seems able to enhance the SGR of females after 51 feeding days. Phytase in the TGR may improve the bioavailability of phytate-bound phosphorus and enhance the SGR for rats of specic sex and age. In this regard, it should be mentioned that the phosphorus level in the blood of TGR group rats was no higher than that in the NTGR group rats (data not shown). Thus phytase in TGR did not cause an increase in blood phosphorus level. The safety of TGR was evaluated by 93-day feeding studies on rats and monitoring the food intake and body weight, as well as the detailed blood composition, organ weights and histopathological ndings. Similar approaches have been used for the safety evaluation of the Cry1Ab gene and cowpea trypsin inhibitor gene transformed rices23 25 and, more recently, for the evaluation of transgenic rice with
385

Table 3. Specic growth rate (SGR) (mean SD) of rats orally administrated TGR and NTGR for 93 days

34 days

16 days

Rat weight on 0 day

Group

J Sci Food Agric 88:382388 (2008) DOI: 10.1002/jsfa

TGR NTGR

133.8 11.3 129.3 7.1

Male

127.0 8.6 138.4 14.4

Female

87.2 14.5 83.7 11.7

Male

53.1 11.4 53.8 12.9

Female

166.0 26.5 165.0 21.58

Male

90.4 18.8 92.2 18.0

Female

224.4 27.4 226.7 42.3

Male

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Table 4. Hematological ndings in male (M) and female (F) rats orally fed with TGR (phytase-rice) and NTGR (TN67) diets for 93 days

Parameter Erythrocytes (104 L1 ) Leukocytes (103 L1 ) Hemoglobin (g dL1 ) Hematocrit (%) MCV (fL) MCH (pg) MCHC (g dL1 ) Platelet (103 L1 )
a

TGR M F M F M F M F M F M F M F M F 726.5 31.65 748.75 68.83a 5.71 2.05a 3.37 2.61a 15.26 1.68 15.23 1.27a 46.7 5.58 42.85 3.99 60.28 2.10 57.23 0.89a 19.73 0.51a 20.31 0.61a 32.73 1.02 35.59 0.71 696.5 304.83 678.12 379.93

NTGR 718.33 16.2 841.3 208.13b 2.62 0.96b 1.36 0.49b 14.26 1.08 13.78 1.01b 42.82 3.90 39.38 4.98 52.5 1.43 52.9 2.92b 17.56 0.89b 18.62 1.55b 33.38 1.61 35.3 3.16 884.8 381.20 918.9 461.89

Literature dataa 864 40 812 40 5.5 0.2 3.0 0.2 15.5 0.5 14.8 0.6 45.8 1.3 43.6 1.6 53 2 54 1 17.9 0.7 18.2 0.6 33.7 0.7 33.8 0.4 640 24 663 11

Data were taken from those of the control groups of Wistar rats fed diets containing 60% of non-transgenic rice as reported by Poulsen et al.21 All values are means SD. Different letters (a, b) within the same treatment indicate that a signicant difference (P < 0.05) was found. M, male; F, female.

Table 5. Blood chemistry in rats treated with TGR and NTGR diets orally for 93 days

Literature dataa Parameter Glucose (mmol L1 ) Blood lipid tests Cholesterol (mg dL1 ) Cholesterol esters (mg dL1 ) HDL (U L1 ) LDL (U L1 ) Liver function tests Total protein (g dL1 ) Total bilirubin (mg dL1 ) GOT (U L1 ) GPT (U L1 ) Albumin (g dL1 ) Alkaline phosphatase (U L1 ) Renal function tests BUN (mg dL1 ) Creatinine (mg dL1 ) Urate (mg dL1 ) Electrolytes P (mg dL1 ) M F M F M F M F M F M F M F M F M F M F M F M F M F M F M F TGR 6.6 3.2 6.1 0.82 50.38 7.53 64.22 12.63 249.35 111.61 1573.87 645.87 24.31 11.34 51.39 12.67 4.75 1.03 5.89 3.11 6.51 0.44b 7.61 0.62 0.21 0.03 0.27 0.07a 227.6 6.41 143.1 3.95 25 3.32 20.89 2.26 3.71 0.19 3.99 0.34 80.85 8.73 51.08 9.91 20.35 3.34 22.59 3.57 0.84 0.16 0.66 0.14 1.75 0.32b 2.21 0.48 8.63 2.8b 7.24 3.02 NTGR 6.1 2.29 5.18 0.29 56.8 10.21 62.78 24.13 204.23 47.65 1517.14 829.93 31.35 8.59 47.79 22.36 7 1.63 8.22 7.19 7.14 0.31a 7.47 0.53 0.14 0.05 0.16 0.07b 228 22.28 121.11 22.78 25.7 3.05 20.78 4.36 3.48 0.14 3.91 0.22 116.38 12.06 76.03 8.82 16.36 2.90 20.04 4.98 0.88 0.12 0.74 0.07 2.18 0.57a 2.1 0.46 12.68 2.04a 11.99 5.85 I 9.72 2.58 8.67 1.64 69.2 14.6 78.8 18.1 II 7.8 0.9 7.8 1.2 III 7.5 0.8 10.5 13.5

6.98 0.34 6.43 0.5 0.5 0.15 0.38 0.09 189 70.6 124 53.3 51.2 4.34 47.9 13.8 4.61 0.24 4.15 0.36 279 51.3 152 43.4 22.55 1.98 25.88 5.39 0.52 0.07 0.57 0.14 3.05 1.49 1.88 0.9 9.11 0.91 6.12 1.1

6.1 0.21 6.55 0.42

6.44 0.22 6.39 0.31

28.6 7.3 20.8 4.6 3.9 0.09 4.56 0.37

35.3 4.7 24.3 8.1 4.08 0.15 4.55 0.32

0.336 0.1 0.58 0.11

0.379 0.084 0.275 0.09

386

J Sci Food Agric 88:382388 (2008) DOI: 10.1002/jsfa

Toxicological evaluation of transgenic rice our with E. coli phytase gene

Table 5. Continued

Literature dataa Parameter Na (mmol L

+ 1

TGR ) M F M F M F M F 147.04 4.45 138.38 5.05 7.88 0.44a 6.13 0.76 103.8 1.40 100.38 3.02 4.25 1.08b 6.5 0.66b

NTGR 115.9 58.62 146.14 6.35 6.81 0.37b 5.98 0.65 101.25 3.42 105.57 5.90 8.2 0.61a 9.48 0.29a

II 146.7 16.2 139.1 23.8 4.8 0.7 4.1 1.0

III 145.1 1.3 143.9 1.3 4.3 0.3 4.1 0.4

K+ (mmol L1 ) Cl (mmol L1 ) Ca2+ (mg dL1 )

a

11.4 0.76 11 0.5

Data were taken from those of the non-transgenic groups fed 90 days with diets containing (I) 0, (II) 60% and (III) 60% of non-transgenic rice to Wistar rats as reported by Liang et al.,26 Schrder et al.,25 and Poulsen et al.21 All values are means SD. Different letters (a, b) within the same treatment indicate that a signicant difference was found (P < 0.05). M, male; F, female. Table 6. Organ relative weights (%)a in rats treated orally with TGR, NTGR, and control for 93 days

Organ (%) Heart Liver Kidney Adrenal Spleen Ovary M F M F M F M F M F F

TGR 0.262 0.009 0.31 0.014 2.287 0.065 2.324 0.261 0.488 0.014 0.568 0.022 0.011 0.001 0.028 0.003 0.164 0.006 0.22 0.015 0.064 0.008

NTGR 0.269 0.005 0.323 0.014 2.428 0.085 2.225 0.088 0.481 0.01 0.502 0.015 0.01 0.001 0.016 0.002 0.175 0.01 0.184 0.01 0.057 0.004

Experimental conditions were as those described in Materials and methods. All values are mean SD of Wistar rats (n = 810 per sex per dose). M, male; F, female. a Relative weight = (organ weight/body weight) 100%.

snowdrop (Galanthus nivalis) lectin (GNA) gene.21 Biochemical assays can be used for detecting moderate to mild deciency of nutrients or imbalance in nutrient metabolism, and these deciencies are usually apparent before any clinical symptoms or changes in body weight.29 To detect adverse subclinical effects of the TGR, a range of hematological and general blood biochemical parameters were assayed. It was noted that for some parameters, although a signicant difference was found for TGR and/or NTGR groups, the uctuating results were within the normal ranges of these parameters as compared to those reported in the literature.21,25,26 Such uctuating results could be considered within the normal ranges of these parameters. Zhuo et al .23 suggested that such differences should not be used to judge that transgenic rice is not safe for food use. Momma et al .20 and Hashimoto et al .,30 in their safety assessments of rice and potatoes genetically modied with soybean glycinin in feeding studies in rats, pointed out that such assessment with laboratory animals is often inuenced by many undened factors other than the property of the transgenic foods, and interpretation
J Sci Food Agric 88:382388 (2008) DOI: 10.1002/jsfa

of the results obtained requires circumspection. In addition, it should be remembered that broad ranges in the blood chemistry data for female and male rats at the same or different growing stages have been reported.21,25,26 The results from this sub-chronic feeding study showed no adverse or toxic effects for our phytase transgenic rice. However, it should be mentioned that the phytase level in the TGR diet used in our feeding study was much lower compared to the level of pure transgenic protein generally used in acute toxicity evaluation studies. Nevertheless, for poultry and porcine feeding, the highest recommended consumption level of phytase activity is 50 units kg1 body weight per day.31 Thus, less than 2.5 g TGR, which offers 50 units of phytase kg1 body per day, would be sufcient for feed phytate digestion and phosphorus ingestion.28 Judged from the daily consumption of feed per kilogram body weight of rats (Table 2), less than 3% of TGR in feed would be sufcient to supply the highest recommended phytase activity. Under such levels, phytase transgenic rice would not cause any adverse or toxic effects.

ACKNOWLEDGEMENTS This study is supported by a project (no. DOH94FS033) from the Department of Health, Taipei, Taiwan. We would like to thank the National Agriculture Research Institute, Taichung, Taiwan, and Dr Pen-Chaur Wu of the InGene Biotechnology Co. for their kind supply of non-transgenic and transgenic rice samples.

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J Sci Food Agric 88:382388 (2008) DOI: 10.1002/jsfa