Greg P. Sykes

Subchronic feeding study of herbicide-tolerant soybean DP-356Ø43-5 in Sprague-Dawley rats.

Optimum GAT1 soybean is a genetically modified (GM) soybean containing event DP-356Ø43-5 (356043) that was produced by integration of the coding sequences of the GAT4601 and GM-HRA proteins. In planta expression of these proteins confers tolerance to glyphosate and sulfonylurea/imidazolinone herbicides, respectively. This paper reports the results from a subchronic rat feeding study conducted with 356043 soybeans. Dehulled/defatted toasted meal and toasted ground hulls were prepared from soybeans from untreated plants (356043), herbicide-treated plants (356043+Gly/SU), non-transgenic isoline control (091), and three commercial non-transgenic reference varieties (93B86, 93B15, and 93M40). Individual diets conforming to standard certified rodent chow formulation (Purina Rodent LabDiet) 5002) were prepared with 20% meal (w/w) and 1.5% hulls (w/w). Diets were fed to young adult Sprague-Dawley rats (12/sex/group) for at least 93 days. Compared with rats fed the isoline control or conventional reference diets, no biologically-relevant, adverse effects were observed in rats fed diets containing 356043 or 356043+Gly/SU soybean with respect to body weight/gain, food consumption/efficiency, clinical signs, mortality, ophthalmology, neurobehavioral assessments (sensory response, grip strength, motor activity), clinical pathology (hematology, coagulation, serum chemistry, urinalysis), organ weights, and gross and microscopic pathology. The results from this study indicate that 356043 soybeans are as safe and nutritious as conventional non-GM soybeans.

Subchronic feeding study of high oleic acid soybeans (Event DP-3Ø5423-1) in Sprague-Dawley rats.

DP-3Ø5423-1 (305423) is a genetically-modified (GM) soybean that was produced by biolistic insertion of a gm-fad2-1 gene fragment and the gm-hra gene into the germline of soybean seeds. The gm-fad2-1 gene fragment cosuppresses expression of the endogenous FAD2-1 gene encoding the seed-specific omega-6 fatty acid desaturase resulting in higher concentrations of oleic acid (18:1) relative to linoleic acid (18:2). The gm-hra gene encoding a modified acetolactate synthase (ALS) enzyme was used as a selectable marker. In the current study, processed fractions (meal, hulls, and oil) from 305423 soybeans, non-GM soybeans with a similar genetic background (near isoline control) and three commercially-available non-GM varieties were used to formulate diets that were nutritionally comparable to PMI Certified Rodent LabDiet 5002. Diets were fed to young adult Crl:CD(SD) rats (12/sex/group) for approximately 90 days. Compared with rats fed the non-GM control diet, no biologically relevant differences were observed in rats fed the 305423 diet with respect to body weight/gain, food consumption/efficiency, mortality, clinical signs of toxicity, or ophthalmological observations. No test diet-related effects were observed on neurobehavioral assessments, organ weights, or clinical or anatomic pathology. These results demonstrated that 305423 soybeans are as safe and wholesome as non-GM soybeans.

Evaluation of the Immune System in Rats and Mice Administered Linear Ammonium Perfluorooctanoate

Repeated high doses of ammonium perfluorooctanoate (APFO) have been reported to affect immune system function in mice. To examine dose-response characteristics in both rats and mice, male CD rats and CD-1 mice were dosed by oral gavage with 0.3-30 mg/kg/day of linear APFO for 29 days. Anti-sheep red blood cell (SRBC) IgM levels, clinical signs, body weights, selected hematology, and lipid parameters, liver weights, spleen, and thymus weights and cell number, selected histopathology, and serum corticosterone concentrations were evaluated. In rats, linear APFO had no effect on production of anti-SRBC antibodies. Ten and 30 mg/kg/day resulted in systemic toxicity as evidenced by decreases in body weight gain to 74 and 37%, and increases in serum corticosterone levels to 135 and 196% of control, respectively. In mice dosed with 10 and 30 mg/kg/day, marked systemic toxicity and stress were observed, as evidenced by a loss in body weight of 3.8 and 6.6 g, respectively (despite a tripling of liver weight), approximately 230% increase in serum corticosterone, and increases in absolute numbers of peripheral blood neutrophils and monocytes with an accompanying decrease in absolute lymphocyte numbers. Immune-related findings at 10 and 30 mg/kg/day that likely represent secondary responses to the systemic toxicity and stress observed at these doses include: decreased IgM antibody production at 10 (20% suppression) and 30 mg/kg/day (28% suppression); decreased spleen and thymus weights and cell numbers; microscopic depletion/atrophy of lymphoid tissue at 10 (thymus) and 30 mg/kg/day (spleen). In summary, no immune-related changes occurred in rats, even at doses causing systemic toxicity. In mice, immune-related changes occurred only at doses causing significant and profound systemic toxicity and stress.

Testicular Degeneration and Spermatid Retention in Young Male Rats

The incidence of spontaneous testicular atrophy and its morphological changes in relation to stage-specific spermatogenesis were investigated in young Crl:CDr/BR male rats at 10–12 wk of age used as controls for toxicity screening during 1983–1990. The incidence of testicular degeneration was 2.5% (5/197) in control rats used for oral toxicity studies and 9.4% (31/327) in rats used for inhalation studies. The epididymal tubules of rats with testicular degeneration had exfoliated germ cells and low sperm density. The high incidence of testicular degeneration observed in the control rats used in inhalation studies may be related to the stress associated with immobilization in the restrainer during nose-only exposure conditions. The severity of testicular degeneration in the inhalation studies was mostly minimal. In these minimally affected testes, mature spermatids (step 19) were retained within normal-appearing germinal epithelium at spermatogenic stages IX-XIV. Also, eosinophilic globular bodies (EGBs) were formed with elongated or mature spermatids throughout all spermatogenic stages, but the general architecture of germinal epithelium was normal in appearance. By electron microscopy, EGBs were sequestered necrotic spermatids, and the germ cell degeneration was associated with cytoplasmic vacuolation of Sertoli cells. In moderate testicular degeneration, markedly decreased maturing spermatids (steps 15–19) and a slight depletion of round spermatids were observed in stages I-VIII. In severe testicular degeneration, seminiferous tubules were lined with 1–2 layers of round spermatids and spermatocytes with giant cell formation. The round spermatids served as a marker to identify spermatogenic stages (I-VIII) of the atrophic tubules. Also, in severe testicular degeneration, tubules in spermatogenic stages X-XIV had no elongated spermatids, and spermatocytes were exfoliated with occasional giant cell formation. Many seminiferous tubules were lined with only 1–2 layers of spermatocytes, and specific germ cell markers were not present.

Subchronic feeding study of grain from herbicide-tolerant maize DP-Ø9814Ø-6 in Sprague-Dawley rats.

This 13-week feeding study conducted in Sprague-Dawley rats evaluated the potential health effects from long-term consumption of a rodent diet formulated with grain from genetically modified (GM), herbicide-tolerant maize DP-Ø9814Ø-6 (98140; trade name Optimum GAT (Optimum GAT is a registered trademark of Pioneer Hi-Bred)). Metabolic inactivation of the herbicidal active ingredient glyphosate was conferred by genomic integration and expression of a gene-shuffled acetylase coding sequence, gat4621, from Bacillus licheniformis; tolerance to acetolactate synthase (ALS) inhibiting herbicides was conferred by overexpression of a modified allele (zm-hra) of the endogenous maize ALS enzyme that is resilient to inactivation. Milled maize grain from untreated (98140) and herbicide-treated (98140+Gly/SU) plants, the conventional non-transgenic, near-isogenic control (091), and three commercial non-transgenic reference hybrids (33J56, 33P66, and 33R77) was substituted at concentrations of 35-38% w/w into a common rodent chow formula (PMI) Nutrition International, LLC Certified Rodent LabDiet 5002) and fed to rats (12/sex/group) for at least 91 consecutive days. Compared with rats fed diets containing grain from the conventional near-isogenic control maize, no adverse effects were observed in rats fed diets containing grain from 98140 or 98140+Gly/SU maize with respect to standard nutritional performance metrics and OECD 408-compliant toxicological response variables [OECD, 1998. Section 4 (Part 408), Health Effects: Repeated Dose 90-Day Oral Toxicity Study in Rodents, Guideline for the Testing of Chemicals. Organisation of Economic Co-operation and Development, Paris, France]. These results support the comparative safety and nutritional value of maize grain from genetically modified Optimum GAT and conventional, non-transgenic hybrid field corn.

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.

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.

Safety assessment of EPA-rich triglyceride oil produced from yeast: Genotoxicity and 28-day oral toxicity in rats

The 28-day repeat-dose oral and genetic toxicity of eicosapentaenoic acid triglyceride oil (EPA oil) produced from genetically modified Yarrowia lipolytica yeast were assessed. Groups of rats received 0 (olive oil), 940, 1880, or 2820 mg EPA oil/kg/day, or fish oil (sardine/anchovy source) by oral gavage. Lower total serum cholesterol was seen in all EPA and fish oil groups. Liver weights were increased in the medium and high-dose EPA (male only), and fish oil groups but were considered non-adverse physiologically adaptive responses. Increased thyroid follicular cell hypertrophy was observed in male high-dose EPA and fish oil groups, and was considered to be an adaptive response to high levels of polyunsaturated fatty acids. No adverse test substance-related effects were observed on body weight, nutritional, or other clinical or anatomic pathology parameters. The oil was not mutagenic in the in vitro Ames or mouse lymphoma assay, and was not clastogenic in the in vivo mouse micronucleus test. In conclusion, exposure for 28 days to EPA oil derived from yeast did not produce adverse effects at doses up to 2820 mg/kg/day and was not genotoxic. The safety profile of the EPA oil in these tests was comparable to a commercial fish oil.

Subchronic oral toxicity assessment of N-acetyl-L-aspartic acid in rats.

We investigated the systemic effects of subchronic dietary exposure to NAA in Sprague Dawley® rats. NAA was added to the diet at different concentrations to deliver target doses of 100, 250 and 500 mg/kg of body weight/day and was administered for 90 consecutive days. All rats (10/sex/group) survived until scheduled sacrifice. No diet-related differences in body weights, feed consumption and efficiency, clinical signs, or ophthalmologic findings were observed. No biologically significant differences or adverse effects were observed in functional observation battery (FOB) and motor activity evaluations, hematology, coagulation, clinical chemistry, urinalysis, organ weights, or gross pathology evaluations that were attributable to dietary exposure to NAA. Treatment-related increased incidence and degree of acinar cell hypertrophy in salivary glands was observed in both male and female rats in the high dose group. Because there was no evidence of injury or cytotoxicity to the salivary glands, this finding was not considered to be an adverse effect. Based on these results and the actual average doses consumed, the no-observed-adverse-effect-levels (NOAEL) for systemic toxicity from subchronic dietary exposure to NAA were 451.6 and 490.8 mg/kg of body weight/day for male and female Sprague Dawley® rats, respectively.

Two-generation reproductive and developmental toxicity assessment of dietary N-acetyl-L-aspartic acid in rats.

N-acetyl-l-aspartic acid (NAA) is a component of the mammalian central nervous system (CNS) that has also been identified in a number of foods. This paper reports the outcome of a reproductive toxicology study conducted with NAA in Sprague-Dawley rats. NAA was added to diets at target doses of 100, 250 and 500 mg/kg of body weight/day and administered for two consecutive generations. A carrier control group was administered diet with no added NAA and a comparative control group was given aspartate (ASP), the constituent amino acid of NAA, at a target dose of 500 mg/kg of body weight/day. The study evaluated OECD 416 reproductive performance variables and additional segments to assess potential developmental effects, neurobehavioural and ophthalmologic function, and the concentrations of NAA or ASP in brain and plasma. No biologically significant differences were observed in any reproductive response variables, neurobehavioural tests, ophthalmologic examinations, body weights, feed consumption, or organ weights. Further, no test substance related mortalities or adverse clinical, neurohistopathologic or histopathologic findings were observed. Under the conditions of this study, the highest target dose of NAA, 500 mg/kg of body weight/day, represents the no-observed-adverse-effect-level (NOAEL) for reproductive and systemic toxicity, and neurotoxicity for Sprague-Dawley rats.