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.

Approaches and considerations for the assessment of immunotoxicity for environmental chemicals: a workshop summary.

As experience is gained with toxicology testing and as new assays and technologies are developed, it is critical for stakeholders to discuss opportunities to advance our overall testing strategies. To facilitate these discussions, a workshop on practices for assessing immunotoxicity for environmental chemicals was held with the goal of sharing perspectives on immunotoxicity testing strategies and experiences, developmental immunotoxicity (DIT), and integrated and alternative approaches to immunotoxicity testing. Experiences across the chemical and pharmaceutical industries suggested that standard toxicity studies, combined with triggered-based testing approaches, represent an effective and efficient approach to evaluate immunotoxic potential. Additionally, discussions on study design, critical windows, and new guideline approaches and experiences identified important factors to consider before initiating DIT evaluations including assay choice and timing and the impact of existing adult data. Participants agreed that integrating endpoints into standard repeat-dose studies should be considered for fulfilling any immunotoxicity testing requirements, while also maximizing information and reducing animal use. Participants also acknowledged that in vitro evaluation of immunosuppression is complex and may require the use of multiple assays that are still being developed. These workshop discussions should contribute to developing an effective but more resource and animal efficient approach for evaluating chemical immunotoxicity.

Decision trees for evaluating skin and respiratory sensitizing potential of chemicals in accordance with European regulations.

Guidance for determining the sensitizing potential of chemicals is available in EC Regulation No. 1272/2008 Classification, Labeling, and Packaging of Substances; REACH guidance from the European Chemicals Agency; and the United Nations Globally Harmonized System (GHS). We created decision trees for evaluating potential skin and respiratory sensitizers. Our approach (1) brings all the regulatory information into one brief document, providing a step-by-step method to evaluate evidence that individual chemicals or mixtures have sensitizing potential; (2) provides an efficient, uniform approach that promotes consistency when evaluations are done by different reviewers; (3) provides a standard way to convey the rationale and information used to classify chemicals. We applied this approach to more than 50 chemicals distributed among 11 evaluators with varying expertise. Evaluators found the decision trees easy to use and recipients (product stewards) of the analyses found that the resulting documentation was consistent across users and met their regulatory needs. Our approach allows for transparency, process management (e.g., documentation, change management, version control), as well as consistency in chemical hazard assessment for REACH, EC Regulation No. 1272/2008 Classification, Labeling, and Packaging of Substances and the GHS.

Toxicological assessment of tridecafluorohexylethyl methacrylate (6:2 FTMAC).

The toxicity of tridecafluorohexylethyl methacrylate (6:2 FTMAC), an acrylic monomer used in producing polymeric substances, was evaluated. 6:2 FTMAC has low acute oral and dermal toxicity (LD50>5000 mg/kg), was not a skin or eye irritant, and did not demonstrate skin sensitization potential in a local lymph node assay (LLNA). 6:2 FTMAC was not mutagenic in the bacterial reverse mutation (Ames) test or in the mouse lymphoma assay. 6:2 FTMAC induced structural aberrations in human peripheral blood lymphocytes in vitro in the absence of metabolic activation but not in the presence of S9 metabolic activation. No numerical aberrations were detected under any testing condition. Also, no increase occurred in structural or numerical chromosomal aberrations in an in vivo mouse micronucleus assay in 6:2 FTMAC treated animals compared to controls. 6:2 FTMAC was administered at 0, 100, 500 and 1000 mg/kg/day via gavage to male and female SD rats for 14 days. No test substance-related effects on mortality, clinical signs, body weights, nutritional parameters, or clinical pathology were observed at any dose. Test substance-related increases in liver weights in males and females at all dose levels and thyroid and kidney weights in 500 and 1000 mg/kg/day males were noted. While there was no histopathological correlate for thyroid and kidney weight changes, minimal hypertrophy was noted in liver in males and females at 1000 mg/kg/day group. The changes noted in teeth (altered mineralization; retention of basophilic material) and femur (increased mineralization) in all treated groups were not associated with clinical signs or microscopic changes and were likely related to free fluoride formed from 6:2 FTMAC metabolism. Plasma (3-4-fold) and urine (30-50-fold) fluoride was higher in treated groups versus controls. Therefore, the changes noted in organ weights, teeth, femur, plasma or urine were not considered adverse. In the repeated dose toxicity study, the no-observed-adverse-effect-level (NOAEL) was 1000 mg/kg/day. Based on mean measured concentrations, the 96-h LC50 in fathead minnow was >14.5 mg/L and the 72-h EC50 in Pseudokirchneriella subcapitata was >24.6 mg/L, while the 48-h EC50 in Daphnia magna, based on nominal concentrations, was >120 mg/L. Overall, 6:2 FTMAC is considered to have low toxicity potential based on these studies.

Absorption, distribution, metabolism, and excretion of [1-¹4C]-perfluorohexanoate ([¹4C]-PFHx) in rats and mice.

The absorption, tissue distribution, elimination, and metabolism of [1-¹4C]-PFHx in rats and mice dosed orally at 2 or 100 mg/kg was evaluated following a single dose or after 14 consecutive doses. Absorption was rapid in rats as evidenced by a short time to maximum concentration (C(max)) of 30 min in male rats and 15 min in female rats at both the 2 and 100mg/kg dose level. The plasma elimination half-life was somewhat longer in males (1.5-1.7 h) than in females (0.5-0.7 h). Absorption in the mouse was also rapid with the maximum plasma concentration occurring between 15 and 30 min after dosing. The maximum concentration was not appreciably different between male and female mice (8 µg equiv./g at 2 mg/kg; ~350 µg equiv./g at 100 mg/kg). The primary route of elimination was via the urine. PFHx was not metabolized in rat or mouse hepatocytes, nor were any metabolites observed after oral dosing in either rodent species. Essentially 100% of the dose was eliminated in urine within 24 h demonstrating that PFHx is readily absorbed and bioavailability approaches 100%, even at a dose as high as 100 mg/kg. The route and extent of elimination was unchanged after 14 days of daily dosing. Tissues were collected at three time points (rat: 0.5, 2, and 24 h; mice: 0.25, 1, and 24 h) after dosing to investigate the tissue clearance kinetics of PFHx following a single dose at 2 or 100 mg/kg. In all tissues except skin, PFHx was not quantifiable 24 h after dosing in both sexes of the two species.

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.

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.

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.

Interlaboratory study of the primary antibody response to sheep red blood cells in outbred rodents following exposure to cyclophosphamide or dexamethasone.

EPA guidelines provide a choice in evaluating humoral immune system function in rats and mice immunized with sheep red blood cells (sRBC): an antibody-forming cell (AFC) assay or a sRBC-specific serum IgM enzyme-linked immunosorbent assay (ELISA). Four different laboratories used both methods to detect suppression of the antibody response by cyclophosphamide (CP) or dexamethasone (DEX). Attempts were made to minimize interlaboratory variability through the use of common reagents and vendors; each laboratory used the same source for rodents, immunosuppressive agents, and one sheep for sRBCs, and determined optimal sRBC concentration for immunization and peak day of antibody response in female CD rats and CD1 mice. The CP dose at which statistical significance was first observed in each species was quite similar within each lab using either assay. For DEX, the AFC assay detected significant and greater suppression at lower concentrations compared to the ELISA in both rats and mice. All labs detected DEX suppression using an AFC assay, whereas only one lab detected significant suppression in both species using an ELISA. For both compounds the magnitude of suppression was greater using the AFC assay, and resulted in ID(50) values which were lower in the AFC assay when compared to the ELISA. In addition, cross-species comparisons of ID(50) values suggested rats were more sensitive than mice. These initial experiments with two chemicals indicated that the AFC assay is consistently better at identifying suppression of a T-dependent antibody response across laboratories following xenobiotic exposures in outbred rats and mice. Additional compounds will need to be evaluated before concluding that one method is superior or more sensitive to the other in detecting suppression of the antibody response.

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.

Acute, subchronic, and mutagenicity studies with norbornene fluoroalcohol.

The object of this study was to evaluate the toxicity of norbornene fluoroalcohol (NBFOH), which is used as an intermediate in the production of fluorinated monomers and polymers. NBFOH was evaluated for acute oral, dermal, and inhalation toxicity, dermal sensitization using the Local Lymph Node Assay (LLNA), mutagenesis by the Ames assay, and subchronic toxicity in a 4-week inhalation rat study. NBFOH demonstrated slight acute toxicity in oral, dermal, and inhalation studies. Approximate lethal doses of 3400 and > 5000 mg/kg for the oral and dermal routes, respectively, and an approximate lethal concentration of 4300 mg/m(3) were determined. NBFOH demonstrated moderate skin irritation, was a severe eye irritant, produced dermal sensitization, but did not cause bacterial mutagenicity either in the presence or absence of S9 activation. Male and female rats were exposed nose only to airborne NBFOH at levels of 0, 410, 1400, and 1500 mg/m(3), 6 h/day, 5 days/week for 4 weeks with clinical and histopathology specimens collected 1 day after the final exposure. Due to the vapor pressure of NBFOH, the 1500 mg/m(3) atmosphere was 27% aerosol and 73% vapor; the 1400 mg/m(3) atmosphere was 5% aerosol and 95% vapor, and the 410 mg/m(3) level was only vapor. No test substance-related mortality or clinical signs of toxicity were observed over the course of the study, and male rats demonstrated significant weight loss and decreased food consumption at 1400 mg/m(3). Male rats from the 1500 mg/m(3) group demonstrated an 11% increase in prothrombin time that was significantly higher than the control value. Examination of fluoride in the urine did not demonstrate a concentration-response relationship, with minimal elevations observed in male rats at all exposure levels and sporadic increases in females. Both male and female rats exposed to 1400 mg/m(3) or greater had squamous metaplasia of the laryngeal mucosa and degeneration of the nasal olfactory and respiratory mucosa. Based on the above findings, NBFOH demonstrates the potential to produce allergic contact dermatitis, and subchronic inhalation studies indicate a no-observed-adverse-effect-level (NOAEL) of 410 mg/m(3).

Inhalation toxicity of 1,3-propanediol in the rat.

1,3-Propanediol (504-63-2) was studied to determine the potential effects following repeated inhalation exposures to rats. Rats were exposed 6 hr/day, 5 days/wk for 2 wk (9 exposures) to vapor or vapor/aerosol mixtures of either 0, 41, 650, or 1800 mg 1,3-propanediol/m(3). In vivo responses were observed or measured daily. Clinical pathology and tissue pathology analyses were conducted after the 9th exposure and on half of each group following an 18-day recovery (nonexposure) period. All rats showed normal body weights. No unusual external signs of response were seen, and no deaths were encountered. Clinical pathology (blood counts, serum chemical parameters) and tissue pathology (gross pathology, organ weights, and histopathology) examinations in the 1,3-propanediol exposed rats were similar to those in the unexposed controls. The highest concentration tested, 1800 mg/m(3), which was the highest concentration that could practically be generated, was the no-observed-effect level (NOEL) for this study. 1,3-Propanediol does not appear to pose a significant hazard via inhalation of either the vapor or a vapor/aerosol mixture.