In the multiyear combined-site analysis, comparisons of proximates, fiber, and minerals in grain (paddy), straw, and bran samples derived from GR2E and control PSBRc82 rice resulted in no statistically significant differences in any of the measured parameters ( Table 2 ).

Fiber is the predominant form of carbohydrate present in rice straw, with measured fractions comprising neutral detergent fiber (NDF; insoluble lignin, cellulose, and hemicellulose), acid detergent fiber (ADF; cellulose and lignin), and total dietary fiber (TDF) consisting of insoluble and soluble fiber (pectin). The macro minerals include calcium, phosphorus, and potassium. The micronutrient minerals, iron, copper, sodium, and zinc are incorporated in plant tissues in only trace amounts.

A comparison of the amino acid composition of event GR2E and control PSBRc82 rice grain is shown in Table 3 . Across locations and growing seasons, there were no statistically significant differences in the concentrations of any amino acids between samples of GR2E and control PSBRc82 rice.

The complete fatty acid profile of grain from GR2E and control rice was determined, and the results are summarized in Table 4 . The concentrations of a number of fatty acids occurring in trace amounts in both GR2E and PSBRc82 control rice grain samples were below the limit of quantification and are not reported (these are itemized in the footnotes to Table 4 ). In the combined-site analysis over both growing seasons, the only statistically significant difference observed between GR2E and control PSBRc82 rice samples was in the concentration of stearic (C18:0) acid, which was approximately 6.5% higher for GR2E rice but was still within the combined literature range for this parameter.

Grain samples from GR2E and control PSBRc82 rice were analyzed for phytic acid and trypsin inhibitor activity. There were no statistically significant differences in the concentrations of phytic acid or in the levels of trypsin inhibitor between samples of GR2E and PSBRc82 control rice ( Table 5 ).

Trypsin inhibitors are proteins known to interfere with digestion and ultimately absorption of food material and are typical antinutritional components in soybeans, cereals, and potatoes. (16) In rice, both phytic acid and trypsin inhibitors are mainly concentrated in the bran, which is a component of rice grain (paddy).

Phytic acid (inositol hexakisphosphate; phytate when in salt form) is the main storage form of phosphorus in plant tissues, which is not in a bioavailable form for monogastric animals that lack the digestive enzyme phytase. Phytic acid also has strong binding affinity to important minerals such as calcium, magnesium, iron, and zinc, thus reducing the absorption of these minerals.

Rice contains few compounds that are not favorable for human or animal nutrition, and these are generally not present at concentrations that would raise any safety concerns. (16) Both phytic acid and trypsin inhibitors are considered antinutritional factors present in a range of food crops, including rice.

Samples of event GR2E and control PSBRc82 rice grain were analyzed for concentrations of the water-soluble B vitamins (thiamine, riboflavin, niacin, pantothenic acid, pyridoxine, and folic acid) and α-tocopherol (vitamin E). With the exception of riboflavin, where concentrations were below the limit of quantification (LOQ) for all samples, there were no statistically significant differences noted in concentrations of B vitamins and vitamin E between GR2E and control PSBRc82 rice ( Table 5 ). Samples of milled rice prepared from grain (paddy) samples were also analyzed at IRRI for β-carotene and other provitamin A carotenoids. In these analyses, the concentrations of-β-carotene ranged between 1.96 and 7.31 μg/g across locations and years ( Table 5 ) and on average comprised ca. 59% of the total carotenoids as determined by HPLC. Concentrations of provitamin A carotenoids in samples derived from control PSBRc82 rice were below the LOQ in each case.

3.5. Nutritional Effect of Elevated β-Carotene in GR2E Rice

β-Carotene is the major carotenoid present in the human diet and is an effective source of vitamin A in both conventional foods and vitamin supplements. It is generally considered virtually nontoxic because humans tolerate high dietary dosages without apparent harm (46) and there are no reports of adverse effects arising from the consumption of naturally occurring β-carotene in food. There is similarly no evidence that conversion of β-carotene to vitamin A contributes to vitamin A toxicity, even when β-carotene is ingested in large amounts. Standard toxicological tests, including teratogenic, mutagenic, and carcinogenic assays, have been performed on β-carotene without any evidence of harmful effects, as reviewed in ref (47)

On the basis of a pooled analysis of 10 prospective cohort studies in North America and Europe that included more than 500000 women, the mean daily intake of β-carotene is in the range of 2.7–6.4 mg. (48) Other data from the National Health and Nutrition Examination Survey (NHANES) published by the US Department of Agriculture indicate that average daily intake of β-carotene from food sources by adult men and women living in the United States is ca. 2.5 mg.

The conversion efficiency of dietary β-carotene to retinol is in the range of (3.6–28):1 by weight (49) and is greatly influenced by the type of study, the vitamin A status, disease and genetic background of the individuals under study, and the food matrix. Rice has a simple and easily digestible food matrix, which allows for a high bioavailability and bioconversion of β-carotene to vitamin A. The bioconversion efficiency of β-carotene in golden rice was found to be 3.8:1 for healthy adults (50) and 2.3:1 for children in China. (51) A bioconversion efficiency of 4.4:1 was reported for β-carotene biofortified cassava (52) and 3.2:1 for yellow maize with high β-carotene. (53) A vitamin A equivalency for β-carotene of approximately 4:1 is applicable for biofortified cassava, yellow maize, and golden rice. (54)

all-trans-β-carotene (3.57 mg/kg) measured in samples of milled rice, it was estimated that substitution of total daily rice consumption with rice containing event GR2E could potentially contribute 89–113% and 57–99% of the estimated average requirement (EAR) for vitamin A for children in Bangladesh and the Philippines, respectively ( Using mean concentrations of-β-carotene (3.57 mg/kg) measured in samples of milled rice, it was estimated that substitution of total daily rice consumption with rice containing event GR2E could potentially contribute 89–113% and 57–99% of the estimated average requirement (EAR) for vitamin A for children in Bangladesh and the Philippines, respectively ( Table 6 ). Expressed another way, a 100 g (uncooked weight) portion of GR2E rice could supply 30–40% of the recommended daily intake (RDI) of vitamin A for children and 11–13% of the RDI for nonpregnant or pregnant adult women.

Table 6. Potential Contribution of Rice Containing Event GR2E to Meeting Vitamin A Needs in Bangladesh and the Philippines mean daily rice consumption (g, raw)a EARb (μg/day RAEc) RDId (μg/day RAE) β-carotene equive from GR2E Rice (μg/day) % of EARf % of RDI Bangladesh preschool-aged children 157 210 300 561 89 62 school-aged children (6–14 years) 261 275 400 932 113 78 nonpregnant, nonlactating women (15–49 years) 360 500 700 1285 64 46 Philippines preschool-aged children (6 months–5 years) 100 210 300 357 57 40 school-aged children (6–12 years) 229 275 400 818 99 68 adult (19–59 years) 332 500 700 1186 59 42 pregnant women 287 550 800 1025 47 32 lactating women 342 800 1100 1221 38 28

Practically, nutritional effects will be significantly influenced by substitution rates of conventional rice with rice containing event GR2E, varietal (background germplasm) effects on provitamin A expression, actual bioconversion efficiency in target populations, and the known loss of β-carotene over time due to storage, processing, and cooking methods. Assessment of the realistically achievable contribution of rice varieties containing event GR2E to improving vitamin A status in at-risk populations will require additional bioefficacy testing that can only be accomplished following the regulatory authorization of event GR2E for food use in the target geographies.

Over all, among the 62 compositional parameters quantified in samples derived from GR2E rice grain, and components that were measured in samples of straw and derived bran, there were no meaningful differences between GR2E and control rice, except for the intended production of provitamin A carotenoids measured in milled GR2E rice. With the exception of provitamin A carotenoids, the compositional parameters measured in samples of GR2E rice were within or similar to ranges reported in the literature for conventional rice varieties with a history of safe consumption. In those instances where the analyte concentrations measured for GR2E rice were slightly outside the combined literature range for that parameter, there were no significant differences between GR2E rice and the control rice. Overall, no consistent patterns emerged to suggest that biologically meaningful changes in composition or nutritive value of the grain, straw, or bran had occurred as an unexpected, unintended, consequence of the genetic modification. Other than the intended production of β-carotene and related provitamin A carotenoids in the endosperm, GR2E rice was found to be compositionally equivalent to conventional rice.