Linda A. Malley

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.

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.

Subchronic toxicity of cyclohexane in rats and mice by inhalation exposure

Inhalation studies were conducted to determine the potential toxicity and/or potential neurotoxicity of cyclohexane. Groups of rats and mice were exposed to 0, 500, 2000, or 7000 ppm concentrations of cyclohexane vapor 6 hr/day, 5 days/week for 14 weeks. Subgroups of rats and mice were further observed during a 1-month recovery period. Functional observational battery (FOB) and motor activity (MA) behavioral tests were conducted on rats. These tests were conducted prior to the exposure series and during weeks 4, 8, and 13 on non-exposure days. Clinical pathology evaluations were conducted after approximately 7, 13, and 18 weeks. Approximately 14 and 18 weeks after study initiation, tissues from rats and mice were histologically processed and evaluated by light microscopy. During exposure to 2000 or 7000 ppm, rats and mice had a diminished response or an absent response to delivery of a punctate auditory alerting stimulus. Immediately following removal of rats from the inhalation chambers, 7000 ppm males and females and 2000 ppm females displayed a compound-related increase in the incidence of wet and/or stained fur (which occurred in the areas of the mouth, chin, and/or perineum). These signs were transient, were not observed during exposure or prior to exposure the following day, and were not associated with any behavioral or morphological changes. During exposure sessions, mice exposed to 7000 ppm exhibited clinical signs of toxicity which included hyperactivity, circling, jumping/hopping, excessive grooming, kicking of rear legs, standing on front legs, and occasional flipping behavior. Clinical signs of toxicity observed in 7000 ppm mice immediately after exposure included hyperactivity, hyperreactivity, ruffled fur (females only), gait abnormalities, spasms in both rear legs, and excessive grooming (males only). The clinical signs observed in mice during and immediately after exposure were transient, and were not present prior to the subsequent exposure. A few mice exposed to 2000 ppm appeared hyperactive during exposure in the latter portion of the study. There were no compound-related changes in mean body weights, body weight gains, food consumption, food efficiency, or mortality; and there were no ophthalmological abnormalities in rats or mice. In addition, there were no compound-related effects on 37 different behavioral parameters assessed during the FOB or during motor activity tests in rats. Male and female mice exposed to 7000 ppm had slight increases in measures of circulating erythrocyte mass (red blood cells, hemoglobin, hematocrit) and plasma protein concentration (males only). Male rats and male and female mice exposed to 7000 ppm had significantly increased relative liver weights, and 7000 ppm male mice also had significantly increased absolute liver weights at the end of the exposure period. At the end of the 1-month recovery period, absolute and relative liver weights of male and female mice were similar to control. However, relative liver weights of 7000 ppm male rats continued to be significantly higher at the end of the recovery period. Male and female rats exposed to 7000 ppm had a significantly increased incidence of hepatic centrilobular hypertrophy at the end of the exposure period, which was not observed at the conclusion of the 1-month recovery period. No microscopic changes were observed in mice. In rats, the no-observed-effect level (NOEL) for acute, transient effects was 500 ppm based on a diminished/absent response to an auditory alerting stimulus at 2000 ppm and above. The NOEL for subchronic toxicity in rats was 7000 ppm based on the lack of adverse effects on body weight, clinical chemistry, tissue morphology, and neurobehavioral parameters. In mice, the NOEL for acute, transient effects was 500 ppm based on behavioral changes during exposure at 2000 ppm and above. The NOEL for subchronic toxicity in mice is 2000 ppm based on hematological changes at 7000 ppm.

Repeated Dose Toxicity Study (28 Days) in Rats and Mice with N-Methylpyrrolidone (NMP)

Twenty-eight day feeding studies were conducted to evaluate the repeated dose toxicity of NMP, a widely used industrial solvent, in Crl:CD BR rats and B6C3F1 mice. Groups of 5 male and 5 female rats each were fed either 0, 2,000, 6,000, 18,000, or 30,000 ppm NMP; similar groups of mice were fed either 0, 500, 2,500, 7,500, or 10,000 ppm. In vivo parameters, hematology and clinical chemistry parameters, and complete pathology evaluations were conducted after approximately 28 days. Decrements in mean body weight gains, reflecting decreases in food consumption and efficiency, were seen in male rats fed 18,000 ppm and in both sexes fed 30,000 ppm. In rats, clinical chemical changes, indicating possible compound-related alterations in lipid, protein, and carbohydrate metabolism, occurred at 18,000 ppm in males and 30,000 ppm in both sexes. No histopathological changes in rats were judged to be directly related to NMP exposure. Hematological (mild to moderate leukopenia) and histopathological alterations (hypocellular bone marrow, testicular degeneration and atrophy, and thymic atrophy) were judged to be secondary to nutritional and body weight effects in male and/or female rats at 30,000 ppm. In mice, cloudy swelling of the epithelia of the distal parts of the renal tubuli was observed in 4 males and 3 females at 10,000 ppm and in 2 male mice at 7,500 ppm. For both rats and mice, abnormal urine coloration was observed (in mice at 2,500 ppm and above, and in rats at 18,000 ppm and above). The discoloration was interpreted as a sign of systemic availability of the test substance, but not as an adverse effect. The NOAEL was 6,000 ppm for male rats and 18,000 ppm for female rats. In mice, the NOAEL was 2,500 ppm based on the kidney histopathology.

CHRONIC TOXICITY AND ONCOGENICITY OF N-METHYLPYRROLIDONE (NMP) IN RATS AND MICE BY DIETARY ADMINISTRATION

A two-year feeding study in rats and an 18-month feeding study in mice were conducted to evaluate the potential chronic toxicity and oncogenicity of NMP in Crl : CD® (SD)BR rats and B6C3F1/CrlBR mice. Groups of 62 male and female rats were administered diets containing 0, 1600, 5000, or 15 000 ppm of NMP for approximately 2 years. Groups of 50 male and female mice were administered diets containing 0, 600, 1200, or 7200 ppm NMP for approximately 18 months. In vivo parameters were evaluated weekly during the first 3 months of the study, and every other week or monthly during the remainder of the study. For rats, an ophthalmoscopic examination was conducted prior to study start and near the end of the study. Periodically, blood samples were collected from rats and mice for determination of leukocyte differential counts, and from mice for red blood cell morphology. After approximately 2 years of dietary administration in rats and 18 months in mice, all surviving animals were sacrificed. Selected tissues were processed for morphological evaluation. Over the course of the two-year study in rats, test substance-related decrements in body weight and weight gain occurred in 15 000 ppm males and females, which correlated with decreased food consumption and food efficiency. A toxicologically significant, test substance-related increase in the incidence of severe chronic progressive nephropathy occurred in 15 000 ppm males. Several morphological changes noted grossly and/or microscopically were secondary to the increased severity of chronic progressive nephropathy. NMP was not oncogenic in male or female rats at dietary concen trations of 15000 ppm and below. A test substance-related decrease in the percentage of 15 000 ppm males surviving to the end of the two-year study compared to the control group resulted from the higher incidence of severe chronic progressive nephropathy. However, a sufficient population of 15000 ppm rats were at risk for potential oncogenicity, so the lower survival did not impair the ability to detect an oncogenic response in this study. There were no adverse, test substance-related effects on the incidences of clinical observations, ophthalmic observations, or differential leukocyte counts in males or females, or on survival of females at any dietary concentration. Male and female mice administered dietary concentrations of 7200 ppm had significantly increased liver weight, significantly increased incidence of hepatocellular adenoma, and significantly increased foci of cellular alteration in the liver. At 7200 ppm, male mice also had an increased incidence of hepatocellular carcinoma while the increased incidence of hepatocellular carcinoma in female mice fell within the historical control range. In addition, the incidence of hepatocellular hypertrophy was increased in 7200 ppm males. Liver weight and hepatocellular hypertrophy were also increased in 1200 ppm males. There were no adverse, test substance-related effects on the incidences of clinical observations, food consumption, body weight, differential leukocyte counts, red blood cell morphology, or survival in either males or females at any dietary concentration. Under the conditions of the study, the no-observed-effect level for NMP was 5000 ppm for male and female rats, 600 ppm for male mice, and 1200 ppm for female mice.

90-day oral gavage toxicity study of 8-2 fluorotelomer alcohol in rats.

8-2 fluorotelomer alcohol is a fluorinated chemical intermediate used to manufacture specialty polymers and surfactants. The potential subchronic toxicity and the reversibility of the effects of this chemical were evaluated following approximately 90 days of oral gavage dosing to Crl:CD®(SD)IGS BR rats. A complete toxicological profile, including neurobehavioral assessments and hepatic β-oxidation, were conducted at selected intervals and a group of rats was included for a 90-day postdosing recovery period. Dose levels tested were 0 (control), 1, 5, 25, and 125 mg/kg. No test-substance-related mortality occurred at any dose level. Rats at 125 mg/kg developed striated teeth, such that these animals were switched to ground chow at 77 days. No treatment-related alterations in body weight, food consumption, neurobehavioral parameters, or hematology/clinical chemistry were found. Hepatic β-oxidation was increased in males at 125 mg/kg and in females at 25 and 125 mg/kg. In both males and females, plasma fluorine levels were increased at 125 mg/kg and urinary fluorine was elevated at ≥5 mg/kg. Degeneration/disorganization of enamel organ ameloblast cells was observed at 125 mg/kg in males, but not females. Liver weight increases accompanied by focal hepatic necrosis were observed at both 25 and 125 mg/kg, and chronic progressive nephrotoxicity occurred in female rats at 125 mg/kg. With the exception of hepatocellular necrosis in males at 125 mg/kg and the increased incidence and severity of chronic progressive nephropathy in females at 125 mg/kg, all other changes showed evidence of reversibility. The no-observed-adverse-effect level was 5 mg/kg.

ACUTE AND SUBCHRONIC NEUROTOXICOLOGICAL EVALUATION OF TETRAHYDROFURAN BY INHALATION IN RATS

Groups of adult male and female rats received exposure to tetrahydrofuran (THF) vapor by inhalation in acute or subchronic exposure scenarios. Acute exposure concentrations were 0, 500, 2500, or 5000 ppm for 6 hr. Evaluations conducted immediately after exposure included clinical observations, motor activity assessments (MA), and a battery of functional tests (FOB) designed to reveal nervous system dysfunction. During exposure to 2500 and 5000 ppm, rats had a diminished or absent startle response to a punctate auditory alerting stimulus. Following exposure to 5000 ppm, male and female rats were lethargic, exhibited abnormal gait or mobility, and splayed rear feet. Lethargy and splayed rear feet were also observed in females esposed to 2500 ppm. During the subsequent FOB, males exposed to 5000 ppm had a lower incidence of palpebral closure, higher incidences of slow or absent righting reflex, and a biphasic pattern of reduced motor activity followed by increased motor activity. Females exposed to 5000 ppm had increased incidences of palpebral closure in the open field, increased incidences of slow or absent righting reflex, and decreased motor activity. During the 14-week subchronic exposure series, daily THF exposure concentrations were 0, 500, 1500, or 3000 ppm, and neurobehavioral evaluations occurred on non-exposure days at approximately monthly intervals. Diminished startle responses to an auditory alerting stimulus were observed during exposure to 1500 or 3000 ppm; however, repeated exposures did not cause additional neurobehavioral or pathological effects. This pattern of effects is suggestive of transient sedation. Despite daily reinstatement of acute sedative effects during repeated exposure with up to 3000 ppm, THF did not produce any persistent or cumulative effects on nervous system structure or function. The demonstrated no-observed-effect level of THF for both acute and subchronic exposure was 500 ppm.

90-Day Subchronic Toxicity Study in Rats and Mice Fed N-Methylpyrrolidone (NMP) Including Neurotoxicity Evaluation in Rats

In mice, there were no effects on body weight or food consumption. As observed in rats, mice fed 2,500 or 7,500 ppm exhibited a change in urine coloration which was not associated with morphological changes in cholesterol, triglycerides, calcium, and alkaline phosphatase occurred at 28 days but not 90 days. These changes are thus assessed as being of minor toxicological relevance. Liver weights were elevated in males fed 2,500 or 7,500 ppm and centrilobular hypertrophy was seen in both sexes fed 7,500 ppm. These changes may be regarded as an adaptation process but are clearly related to NMP exposure. Other toxicological endpoints examined were unaffected by NMP. The NOAEL was 3,000 ppm for both sexes of rats based on body weight effects and changes in 3 neurobehavioral parameters (males only) at higher feeding levels. In mice, the NOAEL was 1,000 ppm based on liver responses to higher concentrations.

Subchronic, Reproductive, and Developmental Toxicity of a Fluorotelomer-Based Urethane Polymeric Product

A commercial fluorotelomer-based urethane polymeric dispersion, consisting of polymer, surfactant, and water, was evaluated in subchronic, reproduction, and developmental toxicity studies. The dispersion was administered daily by gavage to rats at dosages of 0, 50, 250, or 1000 mg polymer/kg/day or with 70 mg/kg/day of the sulfonate surfactant. Dose levels of 0, 50, 250, or 1000 mg polymer/kg/day were also used for the reproductive and developmental studies. Nasal olfactory epithelial degeneration and necrosis occurred in all dose groups in the 90-day study. Nasal adhesions were observed only in rats administered surfactant alone. Liver-enzyme alterations at 250 and 1000 mg/kg were considered to be potentially adverse effects. The subchronic no-observed-adverse-effects level (NOAEL) was 50 mg/kg. For the reproduction study, rats were dosed for 10 weeks prior to cohabitation and throughout mating, gestation, and lactation. There were no effects on reproductive function in males or females at any dosage. Thyroid weight was decreased in the 250 and 1000 mg/kg day F1 groups unaccompanied by microscopic effects. In the developmental toxicity study, female rats were dosed from gestation days 6–20; there was no test-substance-related embryolethality, nor was there any dose-related increase in either fetal malformations. Fetal weight was minimally decreased at 1000 mg/kg/day in the presence of slight maternal toxicity; the NOAEL for developmental parameters was 250 mg/kg/day. The polymeric product was not a specific developmental or reproductive toxin.