Risk assessment of farmers handling pelleted seeds containing crystalline silica and attapulgite.

OBJECTIVES: This study aimed to assess the risk to farmers from handling pelleted seeds that include crystalline silica and attapulgite. METHODS: We measured personal exposure levels to respirable crystalline silica and attapulgite in the experimenter representing a farmer in a simulated workplace. From these values, the annual occupational exposure levels were estimated and compared with the established occupational exposure limits. To assess the toxicity of respirable crystalline silica and attapulgite, digital chest images of workers in a factory producing pelleted seeds were examined. RESULTS: The personal exposure measurement results showed that the concentrations of total dust, respirable dust, and respirable crystalline silica generated during work handling of pelleted seeds were 0.27, 0.06, and 0.00043 mg/m3 , respectively. The estimated annual occupational exposure level to total dust, respirable dust, and respirable crystalline silica in farmers was 103 to 104 times lower than established occupational exposure limits. Attapulgite was not detected by analysis of the pelleted seeds themselves or dust collected during the personal exposure measurements. No pulmonary parenchymal or pleural lesions were detected in the digital chest images of the factory workers. CONCLUSION: We found that farmers handling pelleted seeds would not be exposed to levels of total dust, respirable dust, respirable crystalline silica, and attapulgite derived from pelleted seeds exceeding occupational exposure limits. These results suggest that the risk to farmers of handling pelleted seeds is negligible.

Boosted sono-oxidative catalytic degradation of Brilliant green dye by magnetic MgFe2O4 catalyst: Degradation mechanism, assessment of bio-toxicity and cost analysis.

The magnetic MgFe2O4 nanoparticles (NPs) were fabricated via a facile co-precipitation technique and was comprehensively characterized by XRD, FTIR, SEM, EDX and VSM. The prepared NPs were used as catalyst in presence of ultrasound (US) irradiation to activate persulfate (PS) for generation of sulfate radicals (SO4·-) for boosted degradation of toxic Brilliant Green (BG) dye. Preliminary experiments revealed that highest BG dye degradation efficiency of 91.63% was achieved at MgFe2O4 catalyst dose of 1.0 g/L, PS dose of 300 mg/L, and initial dye concentration of 70 ppm within 15 min of US irradiation. However, only US, US in presence of PS oxidation and US in presence of MgFe2O4 catalyst have shown 20.2%, 83.6% and 45.0% of BG dye removal, respectively. Furthermore, response surface methodology (RSM) based central composite design (CCD) was executed to investigate the effect of interaction between independent variables such as MgFe2O4 catalyst dose (0.5-1.5 g/L), PS dose (150-350 mg/L), initial BG dye concentration (50-150 ppm) and US irradiation time (4-12 min). The RSM based quadratic model was used to predict the experimental data, and the prediction accuracy was confirmed by analysis of variance (R2 = 0.98). The established RSM model has predicted the optimum experimental conditions as MgFe2O4 catalyst dose of 0.75 g/L, PS dose of 300 mg/L, initial dye concentration of 75 ppm and sonication time of 10 min. Subsequently, the treatment cost analysis was performed for all thirty experimental runs of CCD, and the RSM predicted response was found to be evidently optimum as this has delivered best economic condition (140 $/kg of BG removed) with respect to relative dye removal (%). COD removal and residual sulfate analysis have demonstrated satisfactory reduction of COD (90.31%) as well as sulfate ions (42.87 ppm) in the dye solution after treatment. Results of degradation pathway analysis portrayed the transformation of BG molecule (M/Z ratio 385) into simpler fractions with M/Z ratio of 193, 161, 73, and 61. Moreover, the toxicity analysis revealed that sono-catalytically activated PS system has efficiently reduced the toxicity level of BG dye from 93.9% to 5.13%.

Improvement of in vitro behavior of an Mg alloy using a nanostructured composite bioceramic coating.

Magnesium (Mg) alloys as a new group of biodegradable metal implants are being extensively investigated as a promising selection for biomaterials applications due to their apt mechanical and biological performance. However, as a foremost drawback of Mg alloys, the high degradation in body fluid prevents its clinical applications. In this work, a bioceramic composite coating is developed composed of diopside, bredigite, and fluoridated hydroxyapatite on the AZ91 Mg alloy in order to moderate the degradation rate, while improving its bioactivity, cell compatibility, and mechanical integrity. Microstructural studies were performed using a transmission electron microscope (TEM), scanning electron microscope (SEM), X-ray diffraction (XRD) analysis, and energy dispersive spectroscopy (EDS). The degradation properties of samples were carried out under two steps, including electrochemical corrosion test and immersion test in simulated body fluid (SBF). Additionally, compression test was performed to evaluate the mechanical integrity of the specimens. L-929 fibroblast cells were cultured on the samples to determine the cell compatibility of the samples, including the cell viability and attachment. The degradation results suggest that the composite coating decreases the degradation and improves the bioactivity of AZ91 Mg alloy substrate. No considerable deterioration in the compression strength was observed for the coated samples compared to the uncoated sample after 4 weeks immersion. Cytotoxicity test indicated that the coatings improve the cell compatibility of AZ91 alloy for L-929 cells.

Microwave-assisted magnesium phosphate coating on the AZ31 magnesium alloy.

Due to the combination of many unique properties, magnesium alloys have been widely recognized as suitable metallic materials for fabricating degradable biomedical implants. However, the extremely high degradation kinetics of magnesium alloys in the physiological environment have hindered their clinical applications. This paper reports for the first time the use of a novel microwave-assisted coating process to deposit magnesium phosphate (MgP) coatings on the Mg alloy AZ31 and improve its in vitro corrosion resistance. Newberyite and trimagnesium phosphate hydrate (TMP) layers with distinct features were fabricated at various processing times and temperatures. Subsequently, the corrosion resistance, degradation behavior, bioactivity and cytocompatibility of the MgP coated AZ31 samples were investigated. The potentiodynamic polarization tests reveal that the corrosion current density of the AZ31 magnesium alloy in simulated body fluid (SBF) is significantly suppressed by the deposited MgP coatings. Additionally, it is seen that MgP coatings remarkably reduced the mass loss of the AZ31 alloy after immersion in SBF for two weeks and promoted precipitation of apatite particles. The high viability of preosteoblast cells cultured with extracts of coated samples indicates that the MgP coatings can improve the cytocompatibility of the AZ31 alloy. These attractive results suggest that MgP coatings, serving as the protective and bioactive layer, can enhance the corrosion resistance and biological response of magnesium alloys.

Nutrient recovery from airplane wastewater: composition, treatment and ecotoxicological assay.

For the 2014 World Cup and the 2016 Olympic Games, Brazil has expanded its airport infrastructure. This will lead to an increase in wastewater generation from aircrafts. This wastewater is traditionally taken from the aircrafts and disposed in the public sewage collection system. However, this residual water may have a different composition than the usual sanitary sewage. Therefore, it is important to study an alternative to treat this kind of wastewater. Thus, the objective of this study was to characterize and analyze the treatment of wastewater from airplane toilets through chemical precipitation for the removal of ammonia in the form of struvite. The airplanes' effluent showed a composition similar to human urine with pH 8.9, ammonia nitrogen 4,215 mg L-1, phosphorus 430 mg L-1 and a very high acute toxicity (Vibrio fischeri). The best treatment for struvite formation was with pH 9.0 and molar ratio Mg:NH4:PO4 equal to 1.5:1.0:1.0. In this case, the removal of ammonia and phosphorus achieved 97.0% and 95.3%, respectively. After this procedure, the toxicity by Vibrio fischeri decreased.

Investigating palygorskite's role in the development of mesothelioma in southern Nevada: Insights into fiber-induced carcinogenicity.

Similar to asbestos fibers, nonregulated mineral fibers can cause malignant mesothelioma (MM). Recently, increased proportions of women and young individuals with MM were identified in southern Nevada, suggesting that environmental exposure to carcinogenic fibers was causing the development of MM. Palygorskite, a fibrous silicate mineral with a history of possible carcinogenicity, is abundant in southern Nevada. In this study, our aim was to determine whether palygorskite was contributing to the development of MM in southern Nevada. While palygorskite, in vitro, displayed some cytotoxicity toward primary human mesothelial (HM) cells and reduced their viability, the effects were roughly half of those observed when using similar amounts of crocidolite asbestos. No Balb/c (0/19) or MexTAg (0/18) mice injected with palygorskite developed MM, while 3/16 Balb/c and 13/14 MexTAg mice injected with crocidolite did. Lack of MM development was associated with a decreased acute inflammatory response, as injection of palygorskite resulted in lower percentages of macrophages (p = .006) and neutrophils (p = .02) in the peritoneal cavity 3 d after exposure compared to injection of crocidolite. Additionally, compared to mice injected with crocidolite, palygorskite-injected mice had lower percentages of M2 (tumor-promoting) macrophages (p = .008) in their peritoneal cavities when exposed to fiber for several weeks. Our study indicates that palygorskite found in the environment in southern Nevada does not cause MM in mice, seemingly because palygorskite, in vivo, fails to elicit inflammation that is associated with MM development. Therefore, palygorskite is not a likely contributor to the MM cases observed in southern Nevada.

Influence of ethanol content in the precipitation medium on the composition, structure and reactivity of magnesium-calcium phosphate.

Biocompatible amorphous magnesium calcium phosphate (AMCP) particles were synthesized using ethanol in precipitation medium from moderately supersaturated solution at pH10. Some synthesis parameters such as, (Mg+Ca):P, Mg:Ca ratio and different drying methods on the structure and stability of as-produced powder was studied and characterized using SEM, XRD and cell cytocompatibility. The results showed that depending on the Mg(2+) concentration, nano crystalline Struvite (MgNH4PO4·6H2O) can also be alternatively formed. However, the as-formed AMCP preserved its amorphous structure after 7 days of incubation in SBF for tested phosphate concentration, and equally ionic concentration of magnesium and calcium.

Chronic trimethyltin chloride exposure and the development of kidney stones in rats.

We recently reported that occupational exposure to trimethyltin (TMT) is a risk factor for developing kidney stones. To further examine the association between TMT exposure and the formation of kidney stones, we conducted a 180-day animal study and exposed the randomly grouped Sprague-Dawley (SD) rats to TMT in the drinking water at doses of 0, 8.2, 32.8 and 131.3 µg kg(-1) day(-1). Transient behavioral changes were observed in the high-dose group during the first 2 weeks of exposure. TMT exposure led to a significant dose-dependent inhibition of renal H(+)/K(+)-ATPase and an increase in urinary pH. In comparison to no kidney stones being identified in the control and the lowest dose group, 1 rat in the 32.8 µg kg(-1) day(-1) dose group and 3 out of 9 rats in the 131.3 µg kg(-1) day(-1) dose group were found to have stones in the kidney/urinary tract. Pathological analysis showed that more wide spread calcium disposition was observed in kidneys of rats with TMT exposure compared with the rats in the control group. However, X-ray diffraction (XRD) analysis found that the kidney stones were mainly composed of struvite with the formula: NH4MgPO4 6H2O, while calcium-containing components were also detected. Together, this study further demonstrates through animal studies that chronic exposure to a relatively low level of TMT induces nephrotoxicity and increases the risk for developing kidney stones.

Fabrication and cytocompatibility of spherical magnesium ammonium phosphate granules.

Magnesium phosphate compounds, as for example struvite (MgNH4PO4·6H2O), have comparable characteristics to calcium phosphate bone substitutes, but degrade faster under physiological conditions. In the present work, we used a struvite forming calcium doped magnesium phosphate cement with the formulation Ca0.75Mg2.25(PO4)2 and an ammonium phosphate containing aqueous solution to produce round-shaped granules. For the fabrication of spherical granules, the cement paste was dispersed in a lipophilic liquid and stabilized by surfactants. The granules were characterized with respect to morphology, size distribution, phase composition, compressive strength, biocompatibility and solubility. In general, it was seen that small granules can hardly be produced by means of emulsification, when the raw material is a hydraulic paste, because long setting times promote coalescence of initially small unhardened cement droplets. Here, this problem was solved by using an aqueous solution containing both the secondary (NH4)2HPO4 and primary ammonium phosphates NH4H2PO4 to accelerate the setting reaction. This resulted in granules with 97 wt.% having a size in the range between 200 and 1,000 µm. The novel solution composition doubled the compressive strength of the cement to 37 ± 5 MPa without affecting either the conversion to struvite or the cytocompatibility using human fetal osteoblasts.

In vitro degradation behavior and biocompatibility of Mg-Nd-Zn-Zr alloy by hydrofluoric acid treatment.

In this paper, Mg-Nd-Zn-Zr alloy (denoted as JDBM) coated with hydrofluoric acid (HF) chemical conversion film (MgF2) was researched as a potential biodegradable cardiovascular stent material. The microstructures, in vitro degradation and biocompatibility were investigated. The field emission scanning electron microscopy (FE-SEM) and X-ray photoelectron spectroscopy (XPS) showed that a compact MgF2 film was formed on the surface of JDBM. The corrosion rate decreased in artificial plasma from 0.337 to 0.253 mm·y(-1) and the electrochemical measurement demonstrated that the corrosion resistance of JDBM alloy could be obviously improved due to the protective MgF2 film on the surface of the substrate. Meanwhile, the hemolysis ratio of JDBM decreased from 52.0% to 10.1% and the cytotoxicity met the requirement of cellular application after HF treatment. In addition, JDBM and MgF2 film showed good anti-platelet adhesion, which is a very favorable property for implant material in contact with blood directly.

Total allowable concentrations of monomeric inorganic aluminum and hydrated aluminum silicates in drinking water.

Maximum contaminant levels are used to control potential health hazards posed by chemicals in drinking water, but no primary national or international limits for aluminum (Al) have been adopted. Given the differences in toxicological profiles, the present evaluation derives total allowable concentrations for certain water-soluble inorganic Al compounds (including chloride, hydroxide, oxide, phosphate and sulfate) and for the hydrated Al silicates (including attapulgite, bentonite/montmorillonite, illite, kaolinite) in drinking water. The chemistry, toxicology and clinical experience with Al materials are extensive and depend upon the particular physical and chemical form. In general, the water solubility of the monomeric Al materials depends on pH and their water solubility and gastrointestinal bioavailability are much greater than that of the hydrated Al silicates. Other than Al-containing antacids and buffered aspirin, food is the primary source of Al exposure for most healthy people. Systemic uptake of Al after ingestion of the monomeric salts is somewhat greater from drinking water (0.28%) than from food (0.1%). Once absorbed, Al accumulates in bone, brain, liver and kidney, with bone as the major site for Al deposition in humans. Oral Al hydroxide is used routinely to bind phosphate salts in the gut to control hyperphosphatemia in people with compromised renal function. Signs of chronic Al toxicity in the musculoskeletal system include a vitamin D-resistant osteomalacia (deranged membranous bone formation characterized by accumulation of the osteoid matrix and reduced mineralization, reduced numbers of osteoblasts and osteoclasts, decreased lamellar and osteoid bands with elevated Al concentrations) presenting as bone pain and proximal myopathy. Aluminum-induced bone disease can progress to stress fractures of the ribs, femur, vertebrae, humerus and metatarsals. Serum Al ≥100 µg/L has a 75-88% positive predictive value for Al bone disease. Chronic Al toxicity is also manifest in the hematopoietic system as an erythropoietin-resistant microcytic hypochromic anemia. Signs of Al toxicity in the central nervous system (speech difficulty to total mutism to facial grimacing to multifacial seizures and dyspraxia) are related to Al accumulation in the brain and these symptoms can progress to frank encephalopathy. There are four groups of people at elevated risk of systemic Al intoxication after repeated ingestion of monomeric Al salts: the preterm infant, the infant with congenital uremia and children and adults with kidney disease. There is a dose-dependent increase in serum and urinary Al in people with compromised renal function, and restoration of renal function permits normal handling of systemically absorbed Al and resolution of Al bone disease. Clinical experience with 960 mg/day of Al(OH)(3) (~5 mg Al/kg-day) given by mouth over 3 months to men and women with compromised renal function found subclinical reductions in hemoglobin, hematocrit and serum ferritin. Following adult males and females with reduced kidney function found that ingestion of Al(OH)(3) at 2.85 g/day (~40 mg/kg-day Al) over 7 years increased bone Al, but failed to elicit significant bone toxicity. There was one report of DNA damage in cultured lymphocytes after high AlCl(3) exposure, but there is no evidence that ingestion of common inorganic Al compounds presents an increased carcinogenic risk or increases the risk for adverse reproductive or developmental outcomes. A number of studies of Al exposure in relation to memory in rodents have been published, but the results are inconsistent. At present, there is no evidence to substantiate the hypothesis that the pathogenesis of Alzheimer's Disease is caused by Al found in food and drinking water at the levels consumed by people living in North America and Western Europe. Attapulgite (palygorskite) has been used for decades at oral doses (recommended not to exceed two consecutive days) of 2,100 mg/day in children of 3-6 years, 4,200 mg/day in children of 6-12 years, and 9,000 mg/day in adults. Chronic ingestion of insoluble hydrated Al silicates (in kg) can result in disturbances in iron and potassium status, primarily as a result of clay binding to intestinal contents and enhanced fecal iron and zinc elimination. Sufficiently high doses of ingested Al silicates (≥50 g/day) over prolonged periods of time can elicit a deficiency anemia that can be corrected with oral Fe supplements. There is essentially no systemic Al uptake after ingestion of the hydrated Al silicates. Rats fed up to 20,000 ppm Ca montmorillonite (equivalent to 1,860 ppm total Al as the hydrated Al silicate) for 28 weeks failed to develop any adverse signs. The results of dietary Phase I and II clinical trials conducted in healthy adult volunteers over 14 days and 90 days with montmorillonite found no adverse effects after feeding up to 40 mg/kg-day as Al. Since the Al associated with ingestion of hydrated Al silicates is not absorbed into the systemic circulation, the hydrated Al silicates seldom cause medical problems unless the daily doses consumed are substantially greater than those used clinically or as dietary supplements. A no-observable-adverse-effect-level (NOAEL) of 13 mg/kg-day as total Al can be identified based on histologic osteomalacia seen in adult hemodialysis patients given Al hydroxide for up to 7 years as a phosphate binder. Following U.S. EPA methods for calculation of an oral reference dose (RfD), an intraspecies uncertainty factor of 10x was applied to that value results in a chronic oral reference dose (RfD) of 1.3 mg Al/kg-day; assuming a 70-kg adult consumes 2 L of drinking water per day and adjusting for a default 20% relative source contribution that value corresponds to a drinking water maximum concentration of 9 mg/L measured as total Al. A chronic NOAEL for montmorillonite as representative of the hydrated Al silicates was identified from the highest dietary concentration (20,000 ppm) fed in a 28-week bioassay with male and female Sprague-Dawley rats. Since young rats consume standard laboratory chow at ~23 g/day, this concentration corresponds to 56 mg Al/kg-day. Application of 3x interspecies uncertainty factor and a 3x factor to account for study duration results in a chronic oral RfD of 6 mg Al/kg-day. Of note, this RfD is 5-10 fold less than oral doses of Al silicates consumed by people who practice clay geophagy and it corresponds to a maximum drinking water concentration of 40 mg Al/L. To utilize the values derived here, the risk manager must recognize the particular product (e.g., alum) or source (e.g., groundwater, river water, clay or cement pipe) of the Al found in tap water, apply the appropriate analytical methods (atomic absorption, energy dispersive X-ray diffraction, infrared spectral analysis and/or scanning transmission electron microscopy) and compare the results to the most relevant standard. The drinking water concentrations derived here are greater than the U.S. EPA secondary maximum contaminant level (MCL) for total Al of 0.05-0.2 mg/L [40 CFR 143.3]. As such, domestic use of water with these concentrations is likely self-limiting given that its cloudy appearance will be greater than the maximum permitted (0.5-5.0 nephalometric turbidity units; 40 CFR Parts 141 and 142). Therefore, the organoleptic properties of Al materials in water determine public acceptance of potable water as contrast to any potential health hazard at the concentrations ordinarily present in municipal drinking water.

Flocculation of harmful algal blooms by modified attapulgite and its safety evaluation.

Natural attapulgite (N-AT) and modified attapulgite (M-AT) were used in this study to evaluate their flocculation efficiencies and mechanisms in freshwater containing harmful algal blooms through conventional jar test procedure. The experimental results showed that the efficiency of flocculation can be significantly improved by M-AT under appropriate conditions. It was found that the attapulgite modified by hydrochloric acid was similar to polyaluminum ferric silicate chloride (PAFSiC). The high efficiency for M-AT to flocculate Microcystis aeruginosa in freshwater was due to the mechanism of bridging and netting effect. Caenorhabditis elegans was used to detect the toxicity of N-AT and M-AT. The results showed that there was no significant toxicity on this organism. Attapulgite is a natural material, which can be readily available, abundant, and relatively inexpensive. Using modified attapulgite to remove the harmful algal blooms could have the advantages of high effectiveness, low cost, and low impact on the environment.

Wear of teeth due to occupational exposure to airborne olivine dust.

OBJECTIVES: To clarify whether high tooth wear of employees in a mining industry that extracts the mineral olivine could be associated with airborne dust exposure in their working environment. METHOD: The cumulative exposure to airborne mineral dust for the workers in the company was calculated on the basis of their period of employment multiplied by the airborne olivine-dust concentrations, which have been monitored continuously during the past 20 years for all divisions of the company. After invitation, 85% of the employees (n = 191) were examined clinically and their dentitions were photographed and duplicated in plaster casts. Four clinicians, working independently, examined the sets of casts/photographs for tooth wear and ranked these from most to least. Two groups of employees were compared with regard to tooth wear, i.e. the 30% with the highest (case) and the lowest (control) estimated dust exposure levels. Tooth wear in the case and control groups was compared using a non-parametric test based on rankings (Mann-Whitney test). RESULTS: Tooth wear differed significantly between the workers in the low and the high mineral dust exposure groups (p < 0.001). The differences were also apparent within three age subsets, although statistical significance was reached only in the 34-44 years subset (p = 0.002). Considerable individual variation was noted within the three exposure groups. CONCLUSION: Workers with high exposure to airborne olivine dust may contract considerable tooth wear.

Effect of coal mine dust and clay extracts on the biological activity of the quartz surface.

Modification of the quartz surface by aluminium salts and metallic iron have been shown to reduce the biological activity of quartz. This study aimed to investigate the ability of water soluble extracts of coal mine dust (CMD), low aluminium clays (hectorite and montmorillonite) and high aluminium clays (attapulgite and kaolin) to inhibit the reactivity of the quartz surface. DQ12 induced significant haemolysis of sheep erythrocytes in vitro and inflammation in vivo as indicated by increases in the total cell numbers, neutrophil cell numbers, MIP-2 protein and albumin content of bronchoalveolar lavage (BAL) fluid. Treatment of DQ12 with CMD extract prevented both haemolysis and inflammation. Extracts of the high aluminium clays (kaolin and attapulgite) prevented inhibition of DQ12 induced haemolysis, and the kaolin extract inhibited quartz driven inflammation. DQ12 induced haemolysis by coal mine dust and kaolin extract could be prevented by pre-treatment of the extracts with a cation chellator. Extracts of the low aluminium clays (montmorillonite and hectorite) did not prevent DQ12 induced haemolysis, although the hectorite extract did prevent inflammation. These results suggest that CMD, and clays both low and rich in aluminium, all contain soluble components (possibly cations) capable of masking the reactivity of the quartz surface.

[Adrenal cortex response to prolonged administration of low doses of magnesium chlorate in rats]. / Reaktsiia kory nadpochechnikov krys na dlitel'noe vvedenie khlorata magniia v malykh dozakh.

Rat adrenals were studied using histological and histochemical methods during prolonged intoxication with pesticide magnesium chlorate which was administered in a dose equal to 1/100 of LD50 (41 mg/kg of body weight). Animals that received distilled water and were kept in similar conditions were used as control. It was demonstrated that intoxication of rats for 3-7 days results in increased secretory activity of all the zones of adrenal cortex. Later (after 14-90 days) the zonal response to pesticide administration was variable. Magnesium chlorate treatment results in the disturbances of hormonal synthesis in adrenocorticocytes. Compensatory-adaptive capacities of zona fasciculata were found to be greater than those in zona glomerulosa and zona reticularis.

[A 90-day repeated dose oral toxicity study of magnesium chloride in F344 rats].

In order to examine the toxicity of magnesium chloride hexahydrate, four groups of 10 male and 10 female F344 rats received the compound by dietary supplementation at 2.5, 0.5, 0.1 or 0% for 90 days. No treatment-related death was observed during the study. Transient soft stool and sustained increase in water consumption were observed both in males and females of the 2.5% group and slight reduction in body weight gain was noted in the high-dose males. There were no toxic changes in food consumption, organ weights, hematology and biochemistry, and histopathological examinations in any treated-groups. Based on these results, the no-observed-adverse-effect-level was estimated to be 0.5%, and 2.5% is considered to be appropriate as highest dose for a 2-year carcinogenicity study.