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.

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.

Pathology Working Group review and evaluation of proliferative lesions of mammary gland tissues in female rats fed ammonium perfluorooctanoate (APFO) in the diet for 2 years.

Perfluorooctanoate (PFO) is a perfluorinated carboxylate that is widely distributed in the environment. A 2-year chronic study was conducted in rats fed either 30 or 300 ppm of ammonium perfluorooctanoate (APFO). To investigate the possible relationship of APFO exposure to proliferative mammary lesions, a Pathology Working Group (PWG) review of the original slides was performed. The consensus reached by the PWG was that the incidence of mammary-gland neoplasms was not affected by chronic dietary administration of APFO. Therefore, feeding female rats up to 300 ppm of APFO resulted in no increase in proliferative lesions of the mammary tissue.

Immunotoxicity of perfluorooctanoic acid and perfluorooctane sulfonate and the role of peroxisome proliferator-activated receptor alpha.

Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) are environmentally widespread and persistent chemicals with multiple toxicities reported in experimental animals and humans. These compounds can trigger biological activity by activating the alpha isotype of peroxisome proliferator-activated receptors (PPARs), ligand-activated transcription factors that regulate gene expression; however, some biological effects may occur independently of the receptor. Activation of the peroxisome proliferator-activated receptor alpha (PPARalpha) modulates lipid and glucose homeostasis, cell proliferation and differentiation, and inflammation. Reported immunomodulation in experimental animals exposed to PFOA and PFOS has included altered inflammatory responses, production of cytokines and other proteins, reduced lymphoid organ weights, and altered antibody synthesis. Mounting experimental animal evidence suggests PPARalpha independence of some immune effects. This evidence originates primarily from studies with PPARalpha knockout models exposed to PFOA that demonstrate hepatic peroxisome proliferation, reduced lymphoid organ weights, and altered antibody synthesis. As human PPARalpha expression is significantly less than that of rodents, potential PPARalpha independence indicates that future research must explore mechanisms of action of these compounds, including PPARalpha-dependent and -independent pathways. This multiauthored review contains brief descriptions of current and recently published work exploring immunomodulation by PFOA and PFOS, as well as a short overview of other PPARalpha ligands of therapeutic and environmental interest.

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 F(1) 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.

Differential activation of nuclear receptors by perfluorinated fatty acid analogs and natural fatty acids: a comparison of human, mouse, and rat peroxisome proliferator-activated receptor-alpha, -beta, and -gamma, liver X receptor-beta, and retinoid X receptor-alpha.

Administration of ammonium salts of perfluorooctanoate (PFOA) to rats results in peroxisome proliferation and benign liver tumors, events associated with activation of the nuclear receptor (NR) peroxisome proliferator-activated receptor-alpha (PPARalpha). Due to its fatty acid structure, PFOA may activate other NRs, such as PPARbeta, PPARgamma, liver X receptor (LXR), or retinoid X receptor (RXR). In this study, the activation of human, mouse, and rat PPARalpha, PPARbeta, PPARgamma, LXRbeta, and RXRalpha by PFOA (including its linear and branched isomers) and perfluorooctane sulfonate (PFOS) was investigated and compared to several structural classes of natural fatty acids and appropriate positive control ligands. An NR ligand-binding domain/Gal4 DNA-binding domain chimeric reporter system was used. Human, mouse, and rat PPARalpha were activated by PFOA isomers and PFOS. PPARbeta was less sensitive to the agents tested, with only PFOA affecting the mouse receptor. PFOA and PFOS also activated human, mouse, and rat PPARgamma, although the maximum induction of PPARgamma was much less than that seen with rosiglitazone, suggesting that PFOA and PFOS are partial agonists of this receptor. Neither LXRbeta nor the common heterodimerization partner RXRalpha was activated by PFOA in any species examined. Taken together, these data show that of the NRs studied, PPARalpha is the most likely target of PFOA and PFOS, although PPARgamma is also activated to some extent. Compared to naturally occurring long-chain fatty acids, e.g. linoleic and alpha-linolenic acids, these perfluorinated fatty acid analogs were more selective and less potent in their activation of the NRs.

Comparative responses of rats and mice exposed to linear/branched, linear, or branched ammonium perfluorooctanoate (APFO).

The purpose of this study was to compare the toxicity of linear/branched ammonium perfluorooctanoate (APFO) with that of linear and branched APFO. Linear/branched APFO (approximately 80% linear and 20% branched isomers) was formerly used in the production of commercial products. The extensive toxicologic database for APFO has been developed essentially using this mixture of isomers. The trend now is to use APFO containing only the linear isomer. The current study was performed to determine if the toxicological database developed for the linear/branched isomer is applicable to the linear isomer. To determine the contribution of branched APFO to the toxicity of linear/branched APFO, a form of APFO that was 100% branched was synthesized. Rats and mice were given doses by oral gavage ranging from 0.3 to 30 mg/kg of either the linear/branched, linear, or branched APFO for 14 days. Clinical signs, body weights, food consumption, selected hematology and serum lipid parameters, liver and kidney weights, hepatic peroxisomal beta-oxidation, and serum PFOA concentrations were evaluated. Mean body weights were about 20% lower in rats and mice dosed with 30 mg/kg of linear/branched or linear APFO compared to controls, and 3-5% lower in animals dosed with 30 mg/kg of branched APFO. In rats, all three forms reduced lipids. In mice, all three forms reduced total and HDL cholesterol similarly but triglycerides were increased at lower doses. Increased peroxisomal beta-oxidation activity and serum PFOA concentrations were seen in both species but these effects were least pronounced in rats dosed with the branched material. In rats, serum PFOA levels were 20-51 ppm at Lowest Observed Effect Levels (LOEL) of 0.3-1 mg/kg, based primarily upon lipid parameters. In mice, serum PFOA levels were 10-14 ppm at the LOEL of 0.3 mg/kg, based primarily upon relative liver weight. In both rats and mice, the overall responses to the linear/branched and the linear forms of PFOA were similar, but the branched form appears to be less potent. Based on these results, and for the endpoints evaluated in this study, the toxicological database developed primarily from testing linear/branched APFO is applicable to linear APFO.

Characterization and reclassification of titanium dioxide-related pulmonary lesions.

OBJECTIVE: Using current diagnostic criteria, this work summarizes the microscopic review of 16 proliferative squamous lesions, previously diagnosed as cystic keratinizing squamous cell carcinoma, in the lungs of rats from a 2-year inhalation study with pigment-grade titanium dioxide particles. METHODS: In the aftermath of two international pathology workshops designed, in part, to establish histological criteria for classifying pulmonary keratin lesions, these lesions were evaluated by four pathologists using current diagnostic criteria. RESULTS: Unanimous agreement was reached as to the diagnosis of each of the lesions. Two of the lesions were diagnosed as squamous metaplasia and one as a poorly keratinizing squamous cell carcinoma. The remaining 13 lesions were diagnosed as non-neoplastic pulmonary keratin cysts. CONCLUSIONS: These keratin cysts are a species-specific lesion that is unique to the rat lung under conditions of particle overload exposure.

Absorption, distribution, metabolism, excretion, and kinetics of 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)propanoic acid ammonium salt following a single dose in rat, mouse, and cynomolgus monkey.

Ammonium, 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)-propanoate has been developed as a processing aid used in the manufacture of fluoropolymers. The absorption, distribution, elimination, and distribution (ADME) and kinetic behavior of this substance has been evaluated in rats, mice, and cynomolgus monkeys by oral and intravenous routes of exposure and studied in both plasma and urine. The test substance is rapidly and completely absorbed in both rats and mice and both in vivo and in vitro experiments indicate that it is not metabolized. The test substance is rapidly eliminated exclusively in the urine in both rats and mice, with rats eliminating it more quickly than mice (approximately 5h elimination half-life in rats, 20 h half-life in mice). Pharmacokinetic analysis in monkeys, rats, and mice indicate rapid, biphasic elimination characterized by a very fast alpha phase and a slower beta phase. The beta phase does not contribute to potential accumulation after multiple dosing in rats or monkeys. Comparative pharmacokinetics in rats, mice, and monkeys indicates that the rat is more similar to the monkey and is therefore a more appropriate rodent model for pharmacokinetics in primates.

Molecular characterization of thyroid toxicity: anchoring gene expression profiles to biochemical and pathologic end points.

Organic iodides have been shown to induce thyroid hypertrophy and increase alterations in colloid in rats, although the mechanism involved in this toxicity is unclear. To evaluate the effect that free iodide has on thyroid toxicity, we exposed rats for 2 weeks by daily gavage to sodium iodide (NaI). To compare the effects of compounds with alternative mechanisms (increased thyroid hormone metabolism and decreased thyroid hormone synthesis, respectively), we also examined phenobarbital (PB) and propylthiouracil (PTU) as model thyroid toxicants. Follicular cell hypertrophy and pale-staining colloid were present in thyroid glands from PB-treated rats, and more severe hypertrophy/colloid changes along with diffuse hyperplasia were present in thyroid glands from PTU-treated rats. In PB- and PTU-treated rats, thyroid-stimulating hormone (TSH) levels were significantly elevated, and both thyroxine and triiodothyronine hormone levels were significantly decreased. PB induced hepatic uridine diphosphate-glucuronyltransferase (UDPGT) activity almost 2-fold, whereas PTU reduced hepatic 5 -deiodinase I (5 -DI) activity to < 10% of control in support of previous reports regarding the mechanism of action of each chemical. NaI also significantly altered liver weights and UDPGT activity but did not affect thyroid hormone levels or thyroid pathology. Thyroid gene expression analyses using Affymetrix U34A GeneChips, a regularized t-test, and Gene Map Annotator and Pathway Profiler demonstrated significant changes in rhodopsin-like G-protein-coupled receptor transcripts from all chemicals tested. NaI demonstrated dose-dependent changes in multiple oxidative stress-related genes, as also determined by principal component and linear regression analyses. Differential transcript profiles, possibly relevant to rodent follicular cell tumor outcomes, were observed in rats exposed to PB and PTU, including genes involved in Wnt signaling and ribosomal protein expression.

Evaluation of chronic toxicity and carcinogenicity of ammonium 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)-propanoate in Sprague-Dawley rats.

Ammonium 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)-propanoate, developed for use as a polymerization processing aid in the manufacture of fluoropolymers, was tested for its potential chronic toxicity and carcinogenicity in a 2-year oral dosing study in Sprague-Dawley rats. Male rats were given daily doses of either 0, 0.1, 1 or 50 mg/kg; females were given either 0, 1, 50 or 500 mg/kg. Body weights, food consumption and clinical signs were monitored daily; clinical pathology was conducted at designated intervals and animals were given a complete pathological evaluation after 12 months and 24 months of dosing. Normal survival was seen in all groups, no abnormal clinical signs were seen, and body weight gain was reduced only in female rats at 500 mg/kg. Both sexes at the high dose had mild decreases in red cell mass which were somewhat more pronounced in females. Clinical pathology indicative of liver injury was present in males that received 50 mg/kg and correlated with histomorphological liver changes that included both hypertrophic and degenerative/necrotic lesions. Similar histomorphological lesions were seen in the livers of females at 500 mg/kg. Previous shorter term toxicity studies have identified this chemical as a PPARα agonist and the finding of benign tumors of the liver, pancreas and/or testes in males at 50 mg/kg and females at 500 mg/kg is consistent with the rat response to peroxisome proliferators and is of questionable human relevance. Changes in the kidney, tongue, and stomach were observed only at the highest dose of 500 mg/kg in females. The no-observed-adverse-effect-level in this study lies between 1 and 50 mg/kg for males and between 50 and 500 mg/kg for females.

Evaluation of a 15-day screening assay using intact male rats for identifying steroid biosynthesis inhibitors and thyroid modulators.

An in vivo screening assay using intact adult male rats has been evaluated for its ability to detect four endocrine-active compounds (EACs) via oral (gavage) administration. The test compounds included the aromatase inhibitor fadrozole (FAD), the testosterone biosynthesis inhibitor ketoconazole (KETO), and the thyroid modulators phenobarbital (PB) and propylthiouracil (PTU). Three of the test compounds (KETO, PB, and PTU) have been previously evaluated in the 15-day intact male assay with compound administration via intraperitoneal injection (ip). For the current studies, male rats were dosed for 15 days via oral gavage and euthanized on the morning of test day 15. The endpoints evaluated included final body and organ weights (liver, thyroid gland, testes, epididymides, prostate, seminal vesicles with fluid, accessory sex gland unit [ASG]), serum hormone concentrations (testosterone [T], estradiol [E2], dihydrotestosterone [DHT], luteinizing hormone [LH,] follicle stimulating hormone [FSH], prolactin [PRL], T(3), T(4), thyroid stimulating hormone [TSH]), and histopathology of the testis, epididymis, and thyroid gland; positive results for each endpoint are described below. In addition, an evaluation of immune system endpoints (humoral immune function, spleen and thymus weights, and spleen cell number) was conducted on a subset of animals dosed with either KETO or PB. FAD and KETO decreased the weights for the androgen-dependent tissues and caused similar patterns of hormonal alterations (decreased serum T and DHT; increased serum FSH and/or LH). In addition, KETO caused spermatid retention. For FAD and KETO, effects on thyroid parameters were not indicative of thyroid toxicity. PB and PTU caused thyroid effects consistent with thyroid modulators (increased thyroid weight, decreased serum T(3) and T(4), increased serum TSH, thyroid follicular cell hypertrophy/hyperplasia, and colloid depletion). In addition, PB increased relative liver weight and altered reproductive hormone concentrations (decreased serum DHT, PRL, LH; increased serum E2). Orally administered KETO and PB did not alter the primary humoral immune response to sheep red blood cells (SRBC), although spleen weights were increased at the highest doses for both compounds. In the current study, all four test substances were identified as endocrine-active. The effects that were observed in the current study via oral (gavage) compound administration were similar to the responses that were observed by the ip route in previous studies for KETO, PB, and PTU. Overall, the sensitivity (i.e., the dose required to elicit similar magnitude responses) between the ip and oral routes of administration were similar for the three EACs that were examined by both routes of administration. This article, in addition to the > 20 compounds that have already been examined using the 15-day intact male assay, supports this assay as a viable screening assay for detecting EACs, and also illustrates that the ability to identify EACs using the intact male assay will be equivalent regardless of the route of compound administration.

Evaluation of a 15-day screening assay using intact male rats for identifying antiandrogens.

An in vivo screening assay using intact adult male rats has been evaluated for its ability to detect six antiandrogenic compounds via oral administration. The test compounds included cyproterone acetate (CPA), flutamide (FLUT), p,p'-DDE (DDE), di-n-butyl phthalate (DBP), linuron (LIN), and vinclozolin (VCZ). Two of the test compounds (DDE and FLUT) have been previously evaluated in the 15-day intact male assay with compound administration via intraperitoneal injection (ip). For the current studies, male rats were dosed for 15 days via oral gavage and euthanized on the morning of test day 15. The endpoints evaluated included final body and organ weights (liver, thyroid gland, testes, epididymides, prostate, seminal vesicles with fluid, accessory sex gland unit [ASG]), serum hormone concentrations (testosterone [T], estradiol [E2], dihydrotestosterone [DHT], luteinizing hormone [LH], follicle stimulating hormone [FSH], prolactin [PRL], T(3), T(4), and thyroid stimulating hormone[TSH]), and histopathology of the testis, epididymis, and thyroid gland; positive results for each endpoint are described below. In addition, an evaluation of immune system endpoints (humoral immune function, spleen and thymus weights, and spleen cell number) was conducted on a subset of animals dosed with either DDE or FLUT. All six endocrine-active compounds (EACs) increased relative liver weight. FLUT and VCZ caused the typical pattern for an androgen receptor (AR) antagonist, although not all endpoints were statistically significant for VCZ: decreased ASG weights, hormonal alterations (increased T, DHT, LH, and FSH), and induced Leydig cell hypertrophy and/or hyperplasia. CPA caused effects consistent with its mixed AR antagonist/progesterone receptor agonist activity: it decreased ASG weights, caused hormonal alterations (increased T and E2; decreased FSH), and caused spermatid retention. DBP, a compound with antiandrogen-like activity via a nonreceptor mediated mechanism, caused hormonal alterations (decreased T, DHT, and E2; increased LH, FSH, and PRL) and induced general testicular degeneration. LIN, a weak AR antagonist, decreased ASG weights, caused hormonal alterations (decreased T, DHT, and LH; increased E2), and caused spermatid retention. Unlike the other AR antagonists evaluated, DDE, a weak AR antagonist, did not alter reproductive parameters. All six antiandrogens caused some effects on thyroid parameters, although only CPA, DDE, and VCZ caused results consistent with a potential thyroid-modulator. FLUT and DDE did not alter the primary humoral immune response to SRBC, spleen or thymus weights, or spleen cell number. In the current study, 5 of the six test substances were identified as endocrine-active substances consistent with their known/proposed mechanism(s) of action. The effects that were observed in the current study via oral (gavage) compound administration were similar to the responses that were observed by the ip route in previous studies for DDE and FLUT. This report, in addition to the > 20 compounds that have already been examined using the 15-day intact male assay, supports this assay as a viable screening assay for detecting EACs, and also illustrates that the ability to identify EACs using the intact male assay will be equivalent regardless of the route of compound administration.

The reproductive toxicology of ammonium perfluorooctanoate (APFO) in the rat.

Ammonium perfluorooctanoate (APFO) is a surfactant used primarily as an aid in processing various fluoropolymers. Many toxicology and epidemiological studies have been conducted with APFO; however, no specific information regarding functional reproduction was previously available. Therefore, the potential reproductive toxicity of APFO across two generations of offspring was studied using current EPA OPPTS 870.3800 guidelines. Male and female Sprague-Dawley rats were dosed orally with 0, 1, 3, 10, or 30 mg/kg APFO. Parental (P) generation rats ( approximately 6 weeks old) were dosed at least 70 days prior to mating and until sacrificed (after mating for male rats; after weaning for female rats). F(1)-generation rats were dosed similarly, beginning at weaning. The F(2)-generation pups were maintained through 22 days of lactation. Reproductive parameters evaluated in P- and F(1)-generation rats included estrous cycling, sperm number and quality, mating, fertility, natural delivery, and litter viability and growth. Age at sexual maturation in F(1), anogenital distance in F(2), and presence of nipples (males) in F(2)-generation pups were also determined. Feed consumption, body-weight gain, selected organ-weights, gross pathology and appropriate histopathology were evaluated. Reproductive endpoints including mating, fertility, and natural delivery were not affected in either generation. P- and F(1)-generation male rats showed decreased body weight, and liver and kidney weight increases at all doses. The 30 mg/kg F(1)-generation pups had decreased birth weight. Viability was reduced in the 30 mg/kg F(1)-generation pups in apparent relationship to reduced body weight at birth and weaning; however, F(2)-generation pups at 30 mg/kg, although somewhat lighter, did not show a loss in viability. Preputial separation and vaginal opening were somewhat delayed at 30 mg/kg, but these rats went on to show normal reproductive performance. No-observed-adverse-effect-levels were >30 mg/kg for reproductive function of P- and F(1)-generation rats, 10 mg/kg for F(1)-generation pup mortality, birth weight, and sexual maturation, and less than 1mg/kg for male body-weight and organ-weight changes.

Pathology review of proliferative lesions of the exocrine pancreas in two chronic feeding studies in rats with ammonium perfluorooctanoate.

Two chronic dietary studies, conducted years apart, with ammonium perfluorooctanoate (APFO) in Sprague Dawley rats have been previously reported. Although both included male 300 ppm dietary dose groups, only the later study, conducted in 1990-1992 by Biegel et al., reported an increase in proliferative lesions (hyperplasia and adenoma) of the acinar pancreas. An assessment of the significance of the differences between both studies requires careful consideration of: the diagnostic criteria for proliferative acinar cell lesions of the rat pancreas (for example, the diagnosis of pancreatic acinar cell hyperplasia versus adenoma is based on the two-dimensional size of the lesion rather than distinct morphological differences); the basis for those criteria in light of their relevance to biological behavior; and the potential diagnostic variability between individual pathologists for difficult-to-classify lesions. A pathology peer review of male exocrine pancreatic tissues from the earlier study, conducted in 1981-1983 by Butenhoff et al., was undertaken. This review identified an increase in acinar cell hyperplasia but not adenoma or carcinoma in the earlier study. Both studies observed a proliferative response in the acinar pancreas which was more pronounced in the study by Biegel et al. Definitive reasons for the greater incidence of proliferative lesions in the later study were not identified, but some possible explanations are presented herein. The relevance of this finding to human risk assessment, in the face of differences in the biological behavior of human and rat pancreatic proliferative lesions and the proposed mechanism of formation of these lesions, are questionable.

Toxicological evaluation of 6:2 fluorotelomer alcohol.

6:2 fluorotelomer alcohol (6:2 FTOH; CF3[CF2]5[CH2]2OH, CAS# 647-42-7) was evaluated for acute, genetic, and subchronic toxicity using in vitro and in vivo methods. In rats, 6:2 FTOH was considered to be slightly toxic by the oral (LD50=1,750 mg/kg), and dermal (LD50 > 5,000 mg/kg) routes. In rabbits, 6:2 FTOH was not a primary skin or eye irritant, and it did not produce a dermal sensitization response in mice. In a 90-day subchronic study, 6:2 FTOH was administered to rats by oral gavage (0, 5, 25, 125, 250 mg/kg/day). Mortality was observed at 125 and 250 mg/kg/day; deaths occurred after approximately three weeks of dosing and continued sporadically. The NOAEL in the subchronic study was 5mg/kg/day based on hematology and liver effects. 6:2 FTOH was not mutagenic in the bacterial reverse mutation test or in the mouse lymphoma assay and was not clastogenic in a chromosome aberration assay in human lymphocytes. The hazard classification for human health endpoints of 6:2 FTOH according to the United Nations Globally Harmonized System of Classification and Labeling of Chemicals (GHS) is Category 4 for acute oral toxicity based on an LD50 of 1,750 mg/kg. Other acute health endpoints including eye and skin irritation, skin sensitization, as well as genotoxicity, did not meet the criteria for hazard classification. Benchmark Dose Analysis was performed on the most sensitive endpoints from the 90-day oral gavage study and these levels were all above the study NOAEL of 5mg/kg/day. For risk assessment purposes, the recommended point of departure is the more conservative study NOAEL of 5mg/kg/day.

Comparative in vivo and in vitro analysis of possible estrogenic effects of perfluorooctanoic acid.

Previous studies suggested that perfluorooctanoate (PFOA) could activate the estrogen receptor (ER). The present study examined the hypothesis that PFOA can activate ER using an in vivo uterotrophic assay in CD-1 mice and an in vitro reporter assay. Pre-pubertal female CD-1 mice fed an estrogen-free diet from postnatal day (PND)14 through weaning on PND18 were administered 0, 0.005, 0.01, 0.02, 0.05, 0.1, or 1mg/kg PFOA or 17ß-estradiol (E2, 0.5mg/kg) from PND18-20. In contrast to E2, PFOA caused no changes in the relative uterine weight, the expression of ER target genes, or the morphology of the uterus/cervix and/or vagina on PND21. Treatment of a stable human cell line containing an ER-dependent luciferase reporter construct with a broad concentration range of PFOA caused no change in ER-dependent luciferase activity; whereas E2 caused a marked increase of ER-dependent luciferase activity. These data indicate that PFOA does not activate mouse or human ER.

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.

A species difference in the peroxisome proliferator-activated receptor α-dependent response to the developmental effects of perfluorooctanoic acid.

This study examined the effect of prenatal perfluorooctanoic acid (PFOA) administration on pre- and postnatal development using peroxisome proliferator-activated receptor α (PPARα)-humanized mice to determine if species differences in receptor activity might influence the developmental effects induced by PFOA. Pregnant mice were treated daily with water or PFOA (3mg/kg) by po gavage from gestation day 1 (GD1) until GD17 and then either euthanized on GD18 or allowed to give birth and then euthanized on postnatal day 20 (PND20). No changes in average fetal weight, crown-to-rump length, or placental weight were observed on GD18. Expression of mRNA encoding the PPARα target genes acyl CoA oxidase (Acox1) and cytochrome P450 4a10 (Cyp4a10) in maternal and fetal liver was increased on GD18 in wild-type and PPARα-humanized mice but not in Pparα-null mice. On PND20, relative liver weight was higher in wild-type mice but not in Pparα-null mice or PPARα-humanized mice. Hepatic expression of Acox1 and Cyp4a10 mRNA was higher in wild-type mice but not in Pparα-null mice or PPARα-humanized mice on PND20. The percentage of mice surviving postnatally was lower in wild-type litters but not in litters from Pparα-null mice or PPARα-humanized mice. No changes in pup weight gain, onset of eye opening, or mammary gland development were found in any genotype. Results from these studies demonstrate that the developmental/postnatal effects resulting from prenatal PFOA exposure in mice are differentially mediated by mouse and human PPARα.

Ninety-day inhalation toxicity study with a vapor grown carbon nanofiber in rats.

A subchronic inhalation toxicity study of inhaled vapor grown carbon nanofibers (CNF) (VGCF-H) was conducted in male and female Sprague Dawley rats. The CNF test sample was composed of > 99.5% carbon with virtually no catalyst metals; Brunauer, Emmett, and Teller (BET) surface area measurements of 13.8 m2/g; and mean lengths and diameters of 5.8 µm and 158 nm, respectively.Four groups of rats per sex were exposed nose-only, 6 h/day, for 5 days/week to target concentrations of 0, 0.50, 2.5, or 25 mg/m3 VGCF-H over a 90-day period and evaluated 1 day later. Assessments included conventional clinical and histopathological methods, bronchoalveolar lavage fluid (BALF) analysis, and cell proliferation (CP) studies of the terminal bronchiole (TB), alveolar duct (AD), and subpleural regions of the respiratory tract. In addition, groups of 0 and 25 mg/m3 exposed rats were evaluated at 3 months postexposure (PE). Aerosol exposures of rats to 0.54 (4.9 f/cc), 2.5 (56 f/cc), and 25 (252 f/cc) mg/m(3) of VGCF-H CNFs produced concentration-related small, detectable accumulation of extrapulmonary fibers with no adverse tissue effects. At the two highest concentrations, inflammation of the TB and AD regions of the respiratory tract was noted wherein fiber-laden alveolar macrophages had accumulated. This finding was characterized by minimal infiltrates of inflammatory cells in rats exposed to 2.5mg/m(3) CNF, inflammation along with some thickening of interstitial walls, and hypertrophy/hyperplasia of type II epithelial cells, graded as slight for the 25mg/m(3) concentration. At 3 months PE, the inflammation in the high dose was reduced. No adverse effects were observed at 0.54mg/m(3). BALF and CP endpoint increases versus controls were noted at 25mg/m(3) VGCF-H but not different from control values at 0.54 or 2.5mg/m(3). After 90 days PE, BALF biomarkers were still increased at 25mg/m(3), indicating that the inflammatory response was not fully resolved. Greater than 90% of CNF-exposed, BALF-recovered alveolar macrophages from the 25 and 2.5mg/m(3) exposure groups contained nanofibers (> 60% for 0.5mg/m(3)). A nonspecific inflammatory response was also noted in the nasal passages. The no-observed-adverse-effect level for VGCF-H nanofibers was considered to be 0.54mg/m(3) (4.9 fibers/cc) for male and female rats, based on the minimal inflammation in the terminal bronchiole and alveolar duct areas of the lungs at 2.5mg/m(3) exposures. It is noteworthy that the histopathology observations at the 2.5mg/m(3) exposure level did not correlate with the CP or BALF data at that exposure concentration. In addition, the results with CNF are compared with published findings of 90-day inhalation studies in rats with carbon nanotubes, and hypotheses are presented for potency differences based on CNT physicochemical characteristics. Finally, the (lack of) relevance of CNF for the high aspect ratio nanomaterials/fiber paradigm is discussed.