Rodent and broiler feeding studies with maize containing genetically modified event DP-915635-4 show no adverse effects on health or performance.

Several regulatory agencies continue to require animal feeding studies to approve new genetically modified crops despite such studies providing little value in the safety assessment. Feeding studies with maize grain containing event DP-915635-4 (DP915635), a new corn rootworm management trait, were conducted to fulfill that requirement. Diets fed to Crl:CD®(SD) rats for 90 days contained up to 50% ground maize grain from DP915635, non-transgenic control, or non-transgenic reference hybrids (P1197, 6158, and 6365). Ross 708 broilers received phase diets containing up to 67% maize grain from each source for 42 days. Growth performance was compared between animals fed DP915635 and control diets; rats were further evaluated for clinical and neurobehavioral measures, ophthalmology, clinical pathology, organ weights, and gross and microscopic pathology, whereas carcass parts and select organ yields were determined for broilers. Reference group inclusion assisted in determining natural variation influence on observed significant differences between DP915635 and control groups. DP915635 maize grain diet consumption did not affect any measure evaluated in either feeding study. Results demonstrated DP-915635-4 maize grain safety and nutritional equivalency when fed in nutritionally adequate diets, adding to the existing literature confirming the lack of significant effects of feeding grain from genetically modified plants.

Evaluation of the safety and nutritional equivalency of maize grain with genetically modified event DP-Ø23211-2.

Feeding studies were conducted with rats and broiler chickens to assess the safety and nutrition of maize grain containing event DP-Ø23211-2 (DP23211), a newly developed trait-pyramid product for corn rootworm management. Diets containing 50% ground maize grain from DP23211, non-transgenic control, or non-transgenic reference hybrids (P0928, P0993, and P1105) were fed to Crl:CD®(SD) rats for 90 days. Ross 708 broilers were fed phase diets containing up to 67% maize grain from each source for 42 days. Body weight, gain, and feed conversion were determined for comparisons between animals fed DP23211 and control diets in each study. Additional measures included clinical and neurobehavioral evaluations, ophthalmology, clinical pathology, organ weights, and gross and microscopic pathology for rats, and carcass parts and select organ yields for broilers. Reference groups were included to determine if any observed significant differences between DP23211 and control groups were likely due to natural variation. No diet-related effects on mortality or evaluation measures were observed between animal fed diets produced with DP23211 maize grain and animal fed diets produced with control maize grain. These studies show that maize grain containing event DP-Ø23211-2 is as safe and nutritious as non-transgenic maize grains when fed in nutritionally adequate diets. The results are consistent with previously published studies, providing further demonstration of the absence of hazards from edible-fraction consumption of genetically modified plants.

DP-2Ø2216-6 maize does not adversely affect rats in a 90-day feeding study.

Maize plants containing event DP-2Ø2216-6 (DP202216), which confers herbicide tolerance through expression of phosphinothricin acetyltransferase and enhanced grain yield potential via temporal modulation of the native ZMM28 protein, were developed for commercialization. To address current regulatory expectations, a mandatory 90-day rodent feeding study was conducted to support the safety assessment. Diets containing 50% by weight of ground maize grain from DP202216, non-transgenic control, and 3 non-transgenic reference varieties, were fully characterized, along with the grain, and diets were fed to Crl:CD®(SD) rats for at least 90 days. As anticipated, no biologically-relevant effects or toxicologically-significant differences were observed on survival, body weight/gain, food consumption/efficiency, clinical and neurobehavioral evaluations, ophthalmology, clinical pathology (hematology, coagulation, clinical chemistry, urinalysis), organ weights, or gross and microscopic pathology parameters in rats fed a diet containing up to 50% DP202216 maize grain when compared with rats fed diets containing control or reference maize grains. The results of this study support the conclusion that maize grain from plants containing event DP-2Ø2216-6 is as safe and nutritious as maize grain not containing the event and add to the significant existing database of rodent subchronic studies demonstrating the absence of hazards from consumption of edible fractions of genetically modified plants.

Safety evaluation of E12, W8, X17, and Y9 potatoes: Nutritional evaluation and 90-day subchronic feeding study in rats.

The nutritional and health effects of four biotech potato events, E12, W8, X17, and Y9, were evaluated in a subchronic rodent feeding study. E12 contains pSIM1278 insert DNA derived from potato and designed to down regulate potato genes through RNAi. These changes result in reduced black spot and reduced acrylamide. W8, X17, and Y9 contain the DNA inserts from pSIM1278 and pSIM1678 to further reduce acrylamide and express a gene from wild potato that protects against late blight. Rats were fed diets containing 20% cooked, dried potatoes from these four events and three conventional potato varieties. Compositional analyses of the processed potatoes and the rodent diets demonstrated comparability between the four events and their respective conventional varieties. Rats consumed the diets for 90 days and were evaluated for body weight, dietary intake, clinical signs, ophthalmology, neurobehavioral parameters, clinical pathology, organ weights, gross pathology, and histopathology. No adverse effects were observed as a result of test diet consumption. These results support the conclusion that foods containing E12, W8, X17, or Y9 potatoes are as safe, wholesome and nutritious as foods from conventional potato varieties.

An Integrated Multi-Omics Analysis Defines Key Pathway Alterations in a Diet-Induced Obesity Mouse Model.

Obesity is a multifactorial disease with many complications and related diseases and has become a global epidemic. To thoroughly understand the impact of obesity on whole organism homeostasis, it is helpful to utilize a systems biological approach combining gene expression and metabolomics across tissues and biofluids together with metagenomics of gut microbial diversity. Here, we present a multi-omics study on liver, muscle, adipose tissue, urine, plasma, and feces on mice fed a high-fat diet (HFD). Gene expression analyses showed alterations in genes related to lipid and energy metabolism and inflammation in liver and adipose tissue. The integration of metabolomics data across tissues and biofluids identified major differences in liver TCA cycle, where malate, succinate and oxaloacetate were found to be increased in HFD mice. This finding was supported by gene expression analysis of TCA-related enzymes in liver, where expression of malate dehydrogenase was found to be decreased. Investigations of the microbiome showed enrichment of Lachnospiraceae, Ruminococcaceae, Streptococcaceae and Lactobacillaceae in the HFD group. Our findings help elucidate how the whole organism metabolome and transcriptome are integrated and regulated during obesity.

Safety assessment of coleopteran active IPD072Aa protein from Psuedomonas chlororaphis.

The ipd072Aa gene from Pseudomonas chlororaphis encodes the IPD072Aa protein which confers protection against certain coleopteran pests when expressed in genetically modified (GM) plants. A weight of evidence approach was used to assess the safety of the IPD072Aa protein. This approach considered the history of safe use of the source organism and bioinformatic comparison of the protein sequence with known allergenic and toxic proteins. The IPD072Aa protein was assessed for resistance to degradation in the presence of simulated gastric fluid containing pepsin as well as heat stability. There was no hazard identified with the IPD072Aa protein. Furthermore, an acute oral toxicity study found no evidence of adverse effects. Collectively, these studies support the human health safety assessment of the IPD072Aa protein.

Safety evaluation of HOWARU® Restore (Lactobacillus acidophilus NCFM, Lactobacillus paracasei Lpc-37, Bifidobacterium animalis subsp. lactis Bl-04 and B. lactis Bi-07) for antibiotic resistance, genomic risk factors, and acute toxicity.

Although probiotic lactobacilli and bifidobacteria are generally considered safe by various regulatory agencies, safety properties, such as absence of transferable antibiotic resistance, must still be determined for each strain prior to market introduction as a probiotic. Safety requirements for probiotics vary regionally and evaluation methods are not standardized, therefore methodologies are often adopted from food ingredients or chemicals to assess microbial safety. Four individual probiotic strains, Lactobacillus acidophilus NCFM®, Lactobacillus paracasei Lpc-37®, Bifidobacterium animalis subsp. lactis strains Bl-04®, and Bi-07®, and their combination (HOWARU® Restore) were examined for antibiotic resistance by broth microdilution culture, toxin genes by PCR and genome mining, and acute oral toxicity in rats. Only B. lactis Bl-04 exhibited antibiotic resistance above a regulated threshold due to a tetW gene previously demonstrated to be non-transferable. Genomic mining did not reveal any bacterial toxin genes known to harm mammalian hosts in any of the strains. The rodent studies did not indicate any evidence of acute toxicity following a dose of 1.7-4.1 × 1012 CFU/kg body weight. Considering a 100-fold safety margin, this corresponds to 1.2-2.8 × 1012 CFU for a 70 kg human. Our findings demonstrate a comprehensive approach of in vitro, in silico, and in vivo safety testing for probiotics.

Postnatal Evaluation of Cervical Ribs in Control Rats.

BACKGROUND: Cervical ribs in rat fetuses have been widely reported to occur in controls as well as in response to various maternal chemical exposures. However, few evaluations of cervical ribs have been reported in rats postnatally. The available literature has indicated that the postnatal incidences of cervical ribs in control rats are no higher than in perinatal fetuses. METHODS: In a developmental toxicity study in rats conducted by the inhalation route, a control group of 44 time-mated female rats was exposed to filtered air only from gestation day (GD) 6 to 20, and divided into two cohorts. For one cohort, fetuses were removed from dams by laparohysterectomy for skeletal evaluation on GD 21. The other cohort of dams was permitted to deliver, and adult offspring were euthanized on postnatal day 65 for a subsequent postnatal skeletal evaluation of cervical ribs. RESULTS: The incidence of cervical ribs (mean percentage of affected fetuses or adults per litter) was observed to increase during postnatal development, from 1.0% on GD 21 to 12.7% on postnatal day 65. Further evaluation is ongoing to determine whether these observations were attributable to the inhalation exposure conditions used in this study. CONCLUSION: These results, while limited to the evaluation of one skeletal alteration in control rats, support the need for additional research into the area of postnatal development of skeletal abnormalities observed in developmental toxicity studies and the relevance of these skeletal observations to human risk assessment. Birth Defects Research 109:1301-1304, 2017. © 2017 Wiley Periodicals, Inc.

Safety Assessment of Food and Feed from GM Crops in Europe: Evaluating EFSA's Alternative Framework for the Rat 90-day Feeding Study.

Regulatory-compliant rodent subchronic feeding studies are compulsory regardless of a hypothesis to test, according to recent EU legislation for the safety assessment of whole food/feed produced from genetically modified (GM) crops containing a single genetic transformation event (European Union Commission Implementing Regulation No. 503/2013). The Implementing Regulation refers to guidelines set forth by the European Food Safety Authority (EFSA) for the design, conduct, and analysis of rodent subchronic feeding studies. The set of EFSA recommendations was rigorously applied to a 90-day feeding study in Sprague-Dawley rats. After study completion, the appropriateness and applicability of these recommendations were assessed using a battery of statistical analysis approaches including both retrospective and prospective statistical power analyses as well as variance-covariance decomposition. In the interest of animal welfare considerations, alternative experimental designs were investigated and evaluated in the context of informing the health risk assessment of food/feed from GM crops.

Development of dietary soluble fibres by enzymatic synthesis and assessment of their digestibility in in vitro, animal and randomised clinical trial models.

The aim was to develop novel fibres by enzymatic synthesis, to determine their total dietary fibre by AOAC method 2009.01 and to estimate their potential digestibility and assess their digestibility in vivo using glycaemic and insulinaemic responses as markers in mice and randomised clinical trial models. We found that fibre candidates to which α-(1,2) branching was added were resistant to digestion in the mouse model, depending on the amount of branching. These results show that in vivo models are needed to reliably assess the digestibility of α-glycosidic-linked oligomeric dietary fibre candidates, possibly due to absence of brush border α-glucosidase activity in the current in vitro assessment. α-(1,3)-linked and α-(1,6)-linked glucose oligomers were completely digested in humans and mice. In conclusion, it is possible to develop dietary soluble fibres by enzymatic synthesis. Adding α-(1,2) branching increases their resistance to digestion in vivo and can thus improve their suitability as potential fibre candidates. Clinical Trial Registry: ClinicalTrials.gov, NCT02701270.

Safety evaluation of AB-LIFE(®) (Lactobacillus plantarum CECT 7527, 7528 and 7529): Antibiotic resistance and 90-day repeated-dose study in rats.

AB-LIFE(®) is a probiotic product consisting of equal parts of three strains of Lactobacillus plantarum (CECT 7527, 7528, and 7529) blended with inert excipients. Whole genome sequencing was performed on each of the three strains. Antibiotic resistance was evaluated by genomic mining for resistance genes, and assessment for transferability. No risk of transfer potential was identified for any antibiotic resistance genes in the three strains. AB-LIFE(®) was evaluated for potential subchronic oral toxicity in rats, with dosages of 300 and 1000 mg/kg BW/day (equivalent to 5.55 × 10(10) and 1.85 × 10(11) CFU/kg BW/day). Survival of the three test strains through the gastrointestinal tract was supported by fecal analysis. No adverse effects were identified with respect to in-life parameters, clinical or anatomic pathology, translocation, or fecal chemical analyses. The no-observed-adverse-effect level (NOAEL) for AB-LIFE(®) in male and female rats was 1000 mg/kg BW/day (1.85 × 10(11) CFU of AB-LIFE(®)/kg BW/day), the highest dose level evaluated. These results, in conjunction with a previous acute toxicity study in rats, support the conclusion that AB-LIFE(®) is safe for human consumption.

Oral repeated-dose systemic and reproductive toxicity of 6:2 fluorotelomer alcohol in mice.

6:2 fluorotelomer alcohol (6:2 FTOH) was evaluated for potential systemic repeated-dose and reproductive toxicity in mice. 6:2 FTOH was administered by oral gavage to CD-1 mice as a suspension in 0.5% aqueous methylcellulose with 0.1% Tween-80 at dosages of 1, 5, 25, or 100 mg/kg/day. The no-observed-adverse-effect level (NOAEL) for systemic toxicity was 25 mg/kg/day (males) and 5 mg/kg/day (females), based on effects at higher doses on mortality, clinical observations, body weight, nutritional parameters, hematology (red and white blood cell), clinical chemistry (liver-related), liver weights, and histopathology (liver, teeth, reproductive tract, and mammary gland). However, 6:2 FTOH was not a selective reproductive toxicant. The NOAEL for reproductive toxicity was >100 mg/kg/day; no effects on reproductive outcome were observed at any dosage. The NOAEL for viability and growth of the offspring was 25 mg/kg/day, based on clinical signs of delayed maturation in pups, and reductions in pup survival and pup body weight during lactation at 100 mg/kg/day. While the severity of the effects was generally greater in mice than previously reported in CD rats, the overall NOAELs were identical in both species, 5 mg/kg/day for systemic toxicity and 25 mg/kg/day for offspring viability/growth. 6:2 FTOH was not a selective reproductive toxicant in either species; no effects on reproductive outcome occurred at any dose level, and any effects observed in offspring occurred at dose levels that induced mortality and severe toxicity in maternal animals.

Thirteen week rodent feeding study with processed fractions from herbicide tolerant (DP-Ø73496-4) canola.

The potential health effects of meal and oil processed from seed of genetically modified (GM) canola plants (OECD unique identifier: DP-Ø73496-4; hereafter referred to as 73496 canola) containing an insert that expresses the GAT4621 protein conferring tolerance to nonselective herbicidal ingredient glyphosate were evaluated in a subchronic rodent feeding study. Sprague-Dawley rats (12/sex/group) were administered diets containing dehulled, defatted toasted canola meal (DH meal) and refined/bleached/deodorized canola oil (RBD oil) processed from seed of plants that were untreated (73496), sprayed in-field with glyphosate (73496GLY), the non-transgenic near-isogenic (091; control), or one of four commercially available non-GM reference canola varieties (45H72, 45H73, 46A65, 44A89). All diets were formulated as a modification of the standard laboratory chow PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002 (PMI® 5002). DH canola meal and RBD canola oil replaced all commodity soybean fractions typically incorporated in PMI® 5002. No toxicologically significant differences were observed between the test and control groups in this study. The results reported herein support the conclusion that DH meal and RBD oil processed from seed of 73496 canola are as safe and nutritious as DH meal and RBD oil processed from seed of non-GM canola.

Thirteen week rodent feeding study with grain from molecular stacked trait lepidopteran and coleopteran protected (DP-ØØ4114-3) maize.

The results from a subchronic feeding study conducted in Sprague­Dawley rats fed with diets containing grain from 4114 (OECD unique identifier: DP-ØØ4114-3) maize that was untreated (4114) or sprayed in field with glufosinate ammonium (4114GLU) in a design similar to previous studies are reported. The test material, 4114 maize, is a hybrid maize produced by transformation with a DNA construct encoding 4 different transgenic proteins for resistance to lepidopteran pests, coleopteran pests, and tolerance to the herbicidal active ingredient glufosinate ammonium. There were a total of 144 rats divided into 12 groups of 12 rats/sex/group. All experimental diets were formulated by Purina Mills, LLC (St. Louis, MO) in accordance with the standards of Purina Mills Labdiet® Certified Rodent LabDiet® 5002. The incorporation rate of maize grain in all diets was 32% (wt/wt). No biologically significant, treatment related differences in body weight, food consumption, clinical pathology parameters (hematology, blood chemistry, urinalysis, or organ weight) were observed in rats consuming the diets containing 4114 maize grain compared with rats fed conventional maize diets. A number of histologic observations were noted in this study but were background lesions and representative of what would be expected for rats of this age and strain. An independent panel of experts determined certain observations to be spontaneous and not related to the test diet. Accordingly, these results support the conclusion that 4114 maize grain is as safe and nutritious as conventional maize grain.

Safety assessment of EPA-rich oil produced from yeast: Results of a 90-day subchronic toxicity study.

The safety of eicosapentaenoic acid (EPA) oil produced from genetically modified Yarrowia lipolytica yeast was evaluated following 90 days of exposure. Groups of rats received 0 (olive oil), 98, 488, or 976 mg EPA/kg/day, or GRAS fish oil or deionized water by oral gavage. Rats were evaluated for in-life, neurobehavioral, anatomic and clinical pathology parameters. Lower serum cholesterol (total and non-HDL) was observed in Medium and High EPA and fish oil groups. Lower HDL was observed in High EPA and fish oil males, only at early time points. Liver weights were increased in High EPA and Medium EPA (female only) groups with no associated clinical or microscopic pathology findings. Nasal lesions, attributed to oil in the nasal cavity, were observed in High and Medium EPA and fish oil groups. No other effects were attributed to test oil exposure. Exposure to EPA oil for 90 days produced no effects at 98 mg EPA/kg/day and no adverse effects at doses up to 976 mg EPA/kg/day. The safety profile of EPA oil was comparable to that of GRAS fish oil. These results support the use of EPA oil produced from yeast as a safe source for use in dietary supplements.

Toxicological evaluation of sodium perfluorohexanoate.

Sodium perfluorohexanoate [NaPFHx, F(CF(2))(5)CO(2)Na, CAS#2923-26-4] was evaluated in acute, 90-day subchronic, one-generation reproduction, developmental and in vitro genetic toxicity studies. In the subchronic/one-generation reproduction study, four groups of young adult male and female Crl:CD(SD) rats were administered NaPFHx daily for approximately 90 days by gavage at dosages of 0, 20, 100, or 500 mg/kg. Selected groups of rats were evaluated after 1- and 3-month recovery periods. Rats selected for reproductive evaluations were dosed for approximately 70 days prior to cohabitation, through gestation and lactation, for a total of about 4 months. The subchronic toxicity no observed adverse effect level (NOAEL) was 20mg/(kg day), based on nasal lesions observed at 100 and 500 mg/(kg day). No effects were observed for neurobehavioral endpoints. NaPFHx was a moderate inducer of hepatic peroxisomal beta-oxidation with a no observed effect level (NOEL) of 20 (male rats) and 100mg/(kg day) (female rats). Elevated hepatic beta-oxidation levels were observed following 1-month recovery in male and female rats at 500 mg/(kg day). No NaPFHx-related effects were observed on any reproductive parameters. The P(1) adult rat NOAEL was 20mg/(kg day), based on reduced body weight parameters, whereas the NOAEL for reproductive toxicity was 100 mg/(kg day), based on effects limited to reduced F(1) pup weights. In the developmental study, female rats were dosed via gavage on gestation day (GD) 6-20 with the same doses of NaPFHx administered in the subchronic study. The maternal and developmental toxicity NOAEL was 100 mg/(kg day), based on maternal and fetal body weight effects at 500 mg/(kg day). NaPFHx is therefore concluded not to present a reproductive or developmental hazard. NaPFHx genotoxicity studies showed no mutations in the bacterial reverse mutation (Ames) assay or chromosome aberrations in human lymphocytes treated with NaPFHx in vitro. The lowest NOAEL from all of the studies was 20mg/(kg day) in the subchronic study based on nasal lesions. Benchmark doses (BMDL10) for nasal lesions were 13 and 21 mg/(kg day) for male and female rats, respectively. The relevance of the nasal lesions to humans is not known.