Abstract
Rats were fed diets containing 0%, 1 %, 3%, or 5% mixed tocopheryl phosphates for 90 days. No abnormal clinical signs related to treatment appeared. Some statistically significant changes in hematology and clinical chemistry parameters appeared, but the majority were not dose dependent, occurred in only one sex or group, and/or remained within the historical control range for this strain of rat. A statistically significant apparent reduction in blood protein was observed in animals treated with the tocopheryl phosphates, but further investigation showed that the test substance interfered with the protein assay. Repeat analysis using a method unaffected by plasma test substance levels showed no difference in plasma proteins among all groups. Gross necropsy revealed no abnormalities; reduced relative heart and epididymal weights were observed, but were not dose dependent and were considered incidental. Histopathological changes occurred only in the mesenteric lymph node and small intestine. Foreign material in a crystal-like form appeared in macrophages in both organs, and increased in a dose-related fashion. In the lymph node, sinus histiocytosis increased with dose, but the severity was similar between the control and low-dose groups. Foreign-body granulomatous inflammation, associated with Maltese cross birefringence of the crystals was seen in the mid- and high-dose animals, but not the low-dose group. Similarly, the small intestine showed increasing amounts of foreign material and inflammation in the mid- and high-dose but not in the 1 % diet. The 1 % diet (equivalent to 587 and 643 mg mixed tocopheryl phosphates/kg body weight/day for male and female rats, respectively) was considered the no observed adverse effect level.
Mixed tocopheryl phosphate (MTP) is a mixture of three active components, d-α-tocopheryl phosphate (TP), d-α-di-tocopheryl phosphate (T2P), and α-tocopherol and is intended to be formulated into oil and powder products for use as a nutrient in fortified foods. Of these three active components in MTP, only α-tocopherol and tocopheryl phosphate are found naturally in tissues and food (Tomassi and Silano 1986; Tsallas et al. 1986; Vanderveen and Vanderveen 1990; Ogru et al. 2003; Gianello et al. 2005). Both of these compounds have been identified in various seeds and nuts, dairy products, green vegetables, fruits, and cereals (Ogru et al. 2003).
α-Tocopherol, also known as vitamin E, has been extensively studied and widely considered to be one of the safest vitamins. In a recent review, Hathcock et al. (2005) determined the tolerable upper intake level (UL) of
Specific administration, distribution, metabolism and excretion (ADME) studies on MTP are very limited. In vitro and in vivo studies showed that the major metabolite of tocopheryl phosphate is α-tocopherol (Topi and Alessandrini 1953; Nakayama et al. 2003; Rezk et al. 2004). A study conducted in our laboratory (unpublished) has indicated that metabolism of T2P in vivo can result in increased levels of TP and tocopherol. In the study, C57BL6J ob/ob mice were gavaged with 7 daily doses of T2P prepared in medium chain triglyceride (MCT) as the vehicle, or with MCT alone. Livers and adipose tissues were analyzed for tocopherol and TP content. Tocopherol and TP content were increased more in adipose tissue than in liver. A second study (unpublished) indicated that rat liver homogenate incubated with TP results in increased amounts of tocopherol compared to control. The primary excretory route of tocopherol is via the urine and feces. Tocopheryl phosphate-arginine administered to rats through their drinking water indicated conversion of tocopheryl phosphate compounds to α-tocopherol and an increase of tocopheryl phosphate in liver and adipose tissue (Ogru et al. 2003).
α-Tocopherol has properties other than those of an antioxidant. At a cellular level it can also regulate signal transduction, cell proliferation, and gene expression (Ricciarelli et al. 2001). Two major signal transduction pathways that it modulates are those that involve protein kinase C and phosphatidylinositol 3-kinase, and the downstream effects of such modulation are changes to cell proliferation (in particular, of aortic smooth muscle cells), platelet aggregation, and NADPH-oxidase activation (Azzi et al. 2004). In animal models it helps protect against the progression of atherosclerosis caused by a high cholesterol diet (Terasawa et al. 2000).
The biochemistry of the phosphate form of tocopherol has only recently begun to be investigated. α-Tocopheryl phosphate shares some of the nonantioxidant properties displayed by α-tocopherol, but is more effective at lower concentrations, and the possibility exists that it may be the active form of tocopherol (Negis et al. 2005). The effects of MTP on cells have been investigated in vitro in aortic smooth muscle cells and THP-1 monocytes (Ogru et al. 2004). Increased smooth muscle cell proliferation is a pivotal factor implicated in the pathogenesis of atherosclerotic vessel disease, and in these studies, MTP reduced their proliferation at concentrations below those seen with tocopherol at which tocopherol was equally inhibitory. The MTP not only also reduced the proliferation of human THP-1 monocytes more effectively than did tocopherol, but also reduced the uptake of oxidized low-density lipoprotein (oxLDL) by the monocytes. The reduced uptake was the result of decreased expression of CD36 mRNA and total protein, the scavenger receptor specific for oxLDL, and less expression of the receptor on the surface. The reduced uptake of oxLDL suggests that treatment with MTP could reduce the amount of foam cell formation. In a rabbit model of atherosclerosis it has been shown that phosphorylated tocopherol can slow the progression of plaque formation by 62% compared to 17% for tocopherol acetate (Negis et al. 2006). Furthermore, ApoE lipoprotein knockout mice that are fed a normal diet supplemented with tocopheryl phosphate have reduced lipid levels (cholesterol, triglycerides, LDL), and when placed on an atherogenic diet high in fat and cholesterol, a significant reduction in plaque formation is also observed, to levels similar to those seen in the rabbit model (unpublished; manuscript in preparation).
The safety of MTP has been previously studied. In several animal studies (i.e., acute toxicity, irritation, sensitization, mutagenicity/genotoxicity, and 28-day studies) using a related product (Vital ET, a personal-care product made by mixing MTP with a surfactant), no consistent or dose-dependent adverse effects were reported at doses up to 565 mg MTP/kg body weight [bw]/day (Libinaki et al. 2006). Two 28-day gavage studies were conducted with MTP at doses up to 984 mg MTP/kg bw/day and it was concluded that there were no significant changes that could be related to the test substance (Libinaki et al. 2006).
To further examine the safety of MTP, a 13-week dietary study was conducted with rats.
MATERIALS AND METHODS
This study was conducted according to the Organization of Economic Cooperation and Development (OECD) Guidelines for the Testing of Chemicals, No. 408 (Repeated Dose 90-day Oral Toxicity Study in Rodents), and the operation of the testing facility complied with the OECD Principles of Good Laboratory Practice (NATA 14320) and AS/NZS ISO 9001:2000 (NCSI 8116). All work undertaken by the testing laboratory was in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, 2004 (National Health and Medical Research Council).
Test Substance
The substance tested was MTP, which is a mixture of
Vehicle and Diet
MTP was administered via pelleted low-vitamin E modification of AIN93G Rodent Diet (Table 2). Referring to Table 2, the pellets were prepared by adding in order, and thoroughly mixing for 5 to 10 min each time, the following ingredients—ingredients 1 and 4 were mixed, followed by ingredients 5, which was added with mixing, followed by ingredients 6, which was added with mixing, followed by ingredients 2, 3, 7 to 17 with mixing. The MTP is a light brown solid, which was ground into a fine powder using a mechanical grinder for addition to the mixtures. Pellets were prepared with concentrations of MTP of 0%, 1%, 3%, or 5% w/w, which were the four rat treatment groups. The pellets were packed in vacuum-sealed polythene-lined paper bags and stored in the dark, at 4°C. Stability and homogeneity was assessed by randomly removing from the bags approximately 100 g of pellets, which were ground to a powder using a mortar and pestle. The MTP was extracted into solvent and assayed by high-performance liquid chromatography (HPLC) for the concentration of the three tocopherol species. The three species were stable at the last time point of assay during stability tests, which was 105 days.
Animals and Environment
A total of 80 specific pathogen-free Sprague-Dawley rats (40 males and 40 females), weighing between 190 and 220 g and aged 6 to 8 weeks upon receipt, were used for the study. Rats were acclimatized a minimum of 5 days prior to study commencement and after acclimatization they were randomly allocated into four treatment groups (consisting of 10 rats per sex per treatment group). Rats were individually housed in wire bottom cages in rooms maintained at 22°C ± 3°C with a relative humidity of 30% to 70% and an automated light/dark cycle of 12-h light/12-h dark. Test diet and tap water were provided to the rats ad libitum.
Observations
Body Weight
Animal weights were recorded immediately prior to study commencement, weekly thereafter, at death or one day prior to study termination.
Feed Intake
Feed was added twice a week and its consumption (ad libitum) was calculated twice a week by subtracting the mass of the feed remaining (including feed spilled) from the known mass provided to the rats.
Water Intake
Water was provided to the rats in bottles with sipper tubes ad libitum. Ingestion of water was calculated weekly by subtracting the volume of the water remaining from the initial volume of water given to the rats.
Clinical Signs
Animals were observed twice daily with at least 5 h between observations for signs of toxicity (morbidity and mortality) and abnormal behavior. Detailed clinical examinations were conducted prior to study commencement and weekly thereafter to study termination. All observations were recorded.
Ophthalmological Examinations
Eyes were examined prior to study commencement and during the week prior to study termination using an indirect ophthalmoscope after the application of the mydriatic agent, tropicamide.
Hematology and Clinical Chemistry
Blood samples were taken from all surviving rats on the day of study termination (day 91) following an overnight fast. Animals were anesthetized by intraperitoneal injection of sodium pentobarbitone and approximately 4 ml of blood was removed by cardiac puncture. The blood samples were divided into two aliquots: one into a tube containing EDTA and one into a Li-heparin tube.
The following hematology parameters were analyzed: red blood cell count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, white cell count, neutrophils, lymphocytes, monocytes, eosinophils, and basophils. In addition, blood smears were evaluated for cell morphology and parasites. Only abnormal findings on smear evaluation were reported.
The following clinical chemistry parameters were analyzed: sodium (Na), potassium (K), chloride (Cl), bicarbonate, urea, creatinine, glucose, calcium (Ca), phosphate, total protein, albumin, total bilirubin, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, creatine kinase, γ-glutamyltranspeptidase, cholesterol, and triglyceride. In addition, the following parameters were calculated: Na:K ratio, anion gap, Ca:P ratio, globulin, and A:G ratio.
Urinalysis
Urine samples were collected from rats during the final week of the study. Rats were placed in individual metabolic cages for 16 h following a 24-h fast. Using Bayer Multistix, the urine was analyzed for glucose, bilirubin, ketone, specific gravity, blood, pH, protein, urobilinogen, nitrite, and leucocytes.
Gross Necropsy and Organ Weights
Following lethal injection of sodium pentobarbitone on day 91, all animals were necropsied under the supervision of a veterinary pathologist and gross observations recorded. Wet organ weights were recorded for liver, kidneys, adrenals, brain, gonads, uterus, heart, thyroid/parathyroid, epididymis, and spleen. Eyes were placed in Bouin’s fluid for 24 h and then transferred to ethanol. All other tissues and carcasses were preserved in 10% formalin. Tissues were retained in formalin for at least 48 h prior to processing and embedding in paraffin. The paraffin blocks were cut at 4 to 6 μm; the sections were placed on glass slides and stained with hematoxylin and eosin.
Histopathological Examination
The following tissues were examined by light microscopy from all animals of the control and high-dose groups: mesenteric and inguinal lymph nodes, mammary glands, salivary glands, skeletal muscle, femur and bone marrow, pituitary gland, thymus, trachea, lung, heart, aorta, thyroid/parathyroid glands, esophagus, stomach, small intestines including Peyer’s patches, large intestine, liver, pancreas, spleen, kidneys, adrenals, urinary bladder, prostate, epididymides, gonads, seminal vesicles, uterus, fallopian tubes, vagina, brain (cerebrum, cerebellum, brain stem), eye, nerve, spinal cord (cervical, mid-thoracic and lumbar), skin, harderian glands, and nasal turbinates. The mesenteric lymph nodes and small intestine also were examined in all animals from the low- and mid-dose groups.
Mesenteric Lymph Node Analyses
During the histopathology examination of the mesenteric lymph node, foreign material was observed in the dosed animals, which was in the form of spherulites in the mid and high-dose rats. Lymph nodes from three male control rats (animals 1, 2, and 3) and from five high-dose male rats (animals 66 to 70) were blotted to remove excess moisture, weighed (typically 30 to 35 mg), extracted with 10% acetic acid in isopropanol, and analyzed by high-performance liquid chromatography with mass spectrometry (LC-MS) for tocopheryl phosphate and di-tocopheryl phosphate. The equipment was a Waters Acquity UPLC with a Quattro-micro MS with a Waters BEH C18 column (1.7 μm, 2.1 × 50 mm). The chromatography used a 0.3 ml/min flow rate, with 0.1% (v/v) aqueous formic acid as solvent A and 0.1% (v/v) formic acid in isopropanol as solvent B, with the following gradient parameters. Initial conditions were 50% B, then to 95% B at t = 3 min using curve 6 on the programmer, then to 100% B at 4.5 min using curve 6, then to 0% B at 5 min using curve 11. The column was maintained at 30°C and the samples at 20° C. Analyses were performed with both 2-μl and 20-μl injections in positive ion mode with single ion recording at 511.4 and 923.9, respectively, for TP and T2P. A standard curve for each species, TP and T2P, was prepared in 10% acetic acid in isopropanol over the range 10 to 10,000 ng/ml. Confirmation analyses were done in negative ion mode with single ion recording at 509.4 and 921.9, respectively, for TP and T2P.
Statistical Analyses
Urinalysis and clinical observation data were summarized by descriptive analysis, means, and standard deviations. Significance level was set at p < .05. Statistical analyses were performed using the two-tailed Student’s t test for blood data.
Weekly feed intake, weekly water intake and bodyweight data were analyzed using a mixed linear model for repeated measures. The model included fixed effects of sex, treatment, day of study and the interactions of those effects. Random effects included block (within sex), animal, and error.
Percentage organ weight was analyzed using a mixed linear model. The arcsine square root transformation was applied to the data prior to analysis. The model included fixed effects of sex, treatment and the interactions of those effects. Random effects included block (within sex) and error.
Least squares and back-transformed least squares means were used for estimates of treatment means. Standard errors of least squares means were estimated and 95% confidence intervals were constructed. Priori contrasts were used to assess treatment differences where the treatment main effect or the treatment by day of study interaction effects was significant. Treatment differences were assessed at the 5% level of significance (p < .05).
RESULTS
Observations
Survival
One control male died on day 85 but the death was considered accidental due to a fall after jumping from its cage.
Clinical Signs
Ingestion of MTP was well tolerated by rats. No abnormal clinical signs or behavior were observed during the study in any of the treatment groups.
Body Weight
There were no consistent, statistically significant, dose-dependent, treatment-related adverse effects on body weight gain (Figures 1 and 2).
Feed Intake
The dietary concentrations tested in this study (1%, 3%, and 5%) provided mean daily doses of 587, 1866, and 3064 mg MTP/kg bw for males and 643, 1956, and 3334 mg MTP/kg bw for females, respectively, over the course of the study. Feed consumption was statistically significantly increased in males (days 36 to 43, 50 to 57, 57 to 64, and 78 to 85) and females (days 1 to 8, 8 to 15, and 15 to 22) fed the lowest concentration of MTP compared to corresponding controls. At the mid dose, feed intake was statistically significantly increased throughout the study in males and on days 1 to 8 in females when compared with controls. High-dose males initially had a statistically significant decrease in feed intake (days 1 to 8). However, there was a statistically significant increase in feed intake by days 57 to 64. No other statistically significant differences from controls were noted in high-dose animals.
Water Intake
The only statistical significant change in water intake was decreased water intake in low-dose females on days 1 to 8, 8–15, 15 to 22, and 22 to 29 compared to controls.
Ophthalmological Examinations
There were no differences between treated and control animals.
Hematology and Clinical Chemistry
The hematology results for male and female rats fed MTP are presented in Tables 3 and 4. There were no consistent, statistically significant, dose-dependent, adverse effects on any of the hematological parameters evaluated although a few changes were noted. Red blood cell counts were statistically significantly decreased in low-dose females and in high-dose rats of both sexes, but these changes were not dose dependent. Mean corpuscular volume was statistically significantly increased in high-dose males and females of all dose groups but not in a dose-dependent manner. Mean corpuscular hemoglobin was statistically significantly increased in both sexes at the highest dose but remained within the historical control range for this strain of rat (Giknis and Clifford 2006). Platelets were statistically significantly reduced only in low-dose females and not in males at any dose level. Neutrophils were statistically significantly increased in males at the highest dose, but not in a dose-dependent manner and values remained within the historical control range for this strain of rat (Giknis and Clifford 2006). Because of the lack of dose dependence, all these change were considered incidental findings.
The clinical chemistry findings for male and female rats fed MTP are presented in Tables 5 and 6. There were no consistent, statistically significant, dose-dependent, adverse effects on any of the clinical chemistry parameters evaluated although a few changes were noted. There was a statistically significant reduction in total protein and albumin values of male and female rats at all dose levels compared to controls. Globulin levels also were statistically significantly reduced compared to controls at all dose levels in males and at the two highest doses in females. These findings were not supported by any change in the histopathology of the spleen or liver, the major source of production of most of the proteins. All proteins had been assayed with a Biuret procedure. Subsequent studies (unpublished) indicated that the Biuret procedure was interfered with by the MTP in blood plasma. Repeat assay of the samples with an alternative assay (Coomassie) that was not affected by the MTP revealed no effect of the compound MTP on protein values in any of the groups compared to controls. A further test on the interference was performed by serially diluting a stock solution of tocopherol and TP into fresh rat plasma to five concentrations, and then assaying with the Biuret assay. A decrease in apparent protein concentration was found to occur at the mid-point of the dilution set (i.e., at 500 ng/ml TP and 125 ng/ml tocopherol). In mid- and high-dose animals of both sexes, cholesterol values were statistically significantly lower than control values. Low-dose females had a statistically significant increase in total bilirubin compared to controls. Creatine kinase values were statistically significantly increased compared to control values in low- (males only), mid- (males only), and high-dose rats (both sexes). Although there was a reduction in heart weights in low- and mid-dose males, and all dosed females, no correlative changes were observed in heart or skeletal muscle to account for the slight increases observed; other enzymes that correlate with myocardial degeneration (aspartate aminotransferase) were not elevated. Other statistically significant differences from control values included: increased chloride in mid-dose males and high-dose females; increased potassium in high-dose females; increased bicarbonate in mid-dose females; decreased Na:K ratio in high-dose females; decreased anion gap in low-dose males and mid- and high-dose females; decreased urea in high-dose females; decreased creatinine in high-dose males and females and mid-dose females; decreased calcium in high-dose males and females; increased phosphate in mid-dose males; decreased Ca:P ratio in mid- and high-dose males; and increased alanine aminotransferase in high-dose females. These changes were not considered biologically significant because the differences were very small, occurred only in one sex, were not dose-dependent, or fell within historical control ranges for this strain and age of rat (Giknis and Clifford 2006).
Urinalysis
There were no statistically significant differences between animals fed MTP and corresponding controls.
Gross Necropsy
No gross abnormalities were noted in any treated or control animals other than an enlarged spleen in one low-dose male and a mottled appearance of the liver of one low-dose female.
Organ Weights
There were no consistent, statistically significant, dose-dependent adverse effects on any of the organ weights evaluated; statistically significant decreases in relative (to body weight) epididymis weights of low- and mid-dose males and in relative heart weights of low- and mid-dose males and in all treated females were noted (Tables 7 and 8). No histopathological changes were seen in the hearts to account for the differences among the groups.
Histopathological Examination
Histopathological examination of tissues from the control and high-dose animals revealed no treatment-related differences except for the presence of foreign material and inflammation in the mesenteric lymph nodes and small intestine of MTP-treated animals. Because some differences from controls were noted in the high-dose animals, these tissues also were examined in low- and mid-dose animals.
Foreign material was found in macrophages of the mesenteric lymph nodes of dosed animals of both sexes in a dose-related manner but not in controls (Table 9). In the 1/10 low-dose males and 3/10 low-dose females containing foreign material in the node macrophages (sinus histiocytes), the material was amorphous and eosinophilic in most macrophages but had poorly perceived radial striations and an amphophilic appearance in some of the macrophages. No birefringence of the foreign material was observed in the low dose group animals. Moreover, there was no host response against this material in the low dose animals, except for a slightly increased incidence of sinus histiocytosis compared to controls; mean severity of the histiocytosis was similar to that of the controls (Table 9). These changes were not considered adverse.
However, in the mid- and high- dose group animals, a dose-related increased percentage of the foreign material (in macrophages) often had a crystalline appearance with radial striations, was irregularly round and approximately 10 to 30 μm in diameter, had an amphophilic to slightly basophilic color, and demonstrated Maltese cross birefringence under polarized light characteristic of spherulites (Table 9; Figures 3 and 4). There was minimal to moderate granulomatous response associated with the foreign material in the nodes from mid- and high- dose animals, and a dose-related increase in incidence and mean severity of sinus histiocytosis compared to the controls and low dose animals (stated above). The granulomatous response consisted of an increase in size and number of macrophages, many with the appearance of epithelioid cells, and the formation of multinucleated foreign body giant cells; lymphocytic infiltrates were absent in the inflammatory response. Macrophages containing foreign material were observed in the subcapsular sinus where materials enter into the node via the afferent lymphatics from the intestinal tract, and also in the sinusoids of the node where materials had moved from the subcapsular sinus. The foreign material polarized more frequently in the sinusoids than in the subcapsular sinus, suggesting that it matured to become birefringent crystalloid structures. The foreign material could not be identified microscopically: it stained negative for amyloid using Congo red.
To fully investigate the nature of these findings, an examination was conducted of the hematoxylin- and eosin-stained mesenteric lymph node, liver, and spleen sections by an independent pathologist (Hall 2006, unpublished data). Findings in the mesenteric node were similar to those of the study pathologist. Descriptive changes of the mesenteric lymph node from the review are included herein (see above) and data of the percentage of foreign material that polarized is found in Table 9. Liver and spleen sections were included in the independent pathologist’s review to determine if there were any histopathological changes that would support the statistically significant changes in blood protein and cholesterol levels reported in treated rats. There were no microscopic findings that could account for the protein or cholesterol level changes.
The small intestine was examined histologically for the presence of foreign material and host response against it (Table 10). No foreign material or inflammation was seen in control and low-dose animals. Foreign material was seen in macrophages of the lamina propria of the small intestine of all males and high dose females, and in 5/10 mid-dose females. Mild chronic inflammation was associated with the foreign material in 4/10 mid-dose males, 2/10 mid-dose females, 3/10 high-dose males, and 1/10 high-dose females. Some of the affected villi were thickened.
Mesenteric Lymph Node Analyses
The foreign material noted in the mesenteric lymph node specimens taken from MTP-treated rats. Analysis by LC-MS of extracts of mesenteric lymph nodes taken from three control males and five high-dose males identified tocopheryl phosphate at high levels in mesenteric lymph nodes taken from treated rats but not controls. Values from the control nodes were below the limit of quantification (0.1 μg/ml). Nodes from high-dose rats had values, expressed as μg tocopheryl phosphate/mg node wet weight, of 1.3, 1.6, 1.9, <0.1, 2.1. Di-tocopheryl phosphate levels were below the limits of detection.
DISCUSSION
The three diets containing MTP at 1%, 3%, and 5% w/w provided doses of approximately 600, 1900 and 3200 mg MTP/kg bw/day. The actual doses from the 1% diet were (for males and females, respectively) 587 and 643 mg/kg bw/day, from the 3% diet were 1866 and 1956 mg/kg bw/day, and from the 5% diet were 3064 and 3334 mg/kg bw/day. The doses are discussed below in relation to findings in similar 90-day studies.
Ingestion of MTP was well tolerated in the rats used in this study with no abnormal clinical signs or behavior noted. There were some statistically significant fluctuations in body weight gain, feed intake, and water intake throughout the study. The high-dose males had a statistically significant lower weight gain during the experimental period that could not be attributed to decreased food intake. A cause could not be determined for the reduced weight gain. However, by the end of the study there were no differences between treated animals and controls.
A few statistically significant changes in hematology (i.e., decreased red blood cells, increased MCV, increased MCH, reduced platelets, and increased neutrophils) and clinical chemistry parameters (i.e., decreased cholesterol, increased total bilirubin, increased creatine kinase, increased chloride and potassium, increased bicarbonate, decreased Na:K ratio, decreased anion gap, decreased urea, decreased creatinine, decreased calcium, increased phosphate, decreased Ca:P ratio, and increased alanine aminotransferase) were noted. Except for cholesterol, these changes were not considered biologically significant because the differences were very small, occurred only in one sex, were not dose dependent, and/or fell within historical control ranges for this strain of rat. The cholesterol changes were statistically significant in a dose-related reduction, and are consistent with some of the atherosclerotic-preventing effects of MTP reported in the introduction. The effects seen in the rabbit model (Negis et al. 2006) were more pronounced than that of tocopherol (administered to the rabbits as tocopheryl acetate), and suggest that at least in this respect this may be a beneficial anti-inflammatory effect of MTP on cholesterol metabolism. The changes in protein values were not supported by any histopathological changes in the liver and spleen, and could not be substantiated by the Coomassie protein assay. Moreover, it was shown that the MTP itself interfered with the Biuret protein assay and the reduced values observed with that assay were considered an artifact (Ogru et al. 2006). The possibility that the cholesterol assay was interfered with by the MTP is considered unlikely because the enzyme-based reaction (performed on the Olympus AU400 analyzer) has ascorbic acid as the only antioxidant known to interfere with the assay; in this case it is less than 10% at concentrations up to 80 μg/ml.
The esterified form d-α-tocopherol, namely, d-α-tocopheryl acetate is the most common form of vitamin E that is used to fortify foods, d-α-Tocopheryl succinate is another form that is used. Although several feeding studies in rats have reported hemorrhagic diathesis with α-tocopherol (Abdo et al. 1986; Wheldon et al. 1983; Takahashi et al. 1990), no such events were seen with MTP.
The 90-day studies with d-α-tocopheryl acetate and d-α-tocopheryl succinate also indicated other possible abnormalities from the treatments. Enlarged livers in female rats were noted with d-α-tocopheryl acetate at 500 and 2000 mg/kg bw/day, but no histopathological changes were observed. The d-α-tocopheryl succinate study indicated increased alanine and aspartate amino transferases, which was the only significant change that was found, and the NOAEL was 265 mg/kg bw/day. The d-α-tocopheryl acetate study also reported that lesions were found in the lung at doses above 125 mg/kg bw/day.
The only significant finding in the present study was the microscopic observation of foreign material in sinusoidal macrophages associated with mild inflammatory changes in the mesenteric lymph node and the presence of similar foreign material in the small intestine of mid- and high-dose animals. The foreign material polarized with Maltese cross birefringence, characteristic of spherulites, and accompanied by minimal to moderate foreign body granulomatous inflammation. In addition, there was an increase in the incidence and mean severity of sinus histiocytosis compared to controls and low-dose animals. In the low-dose group, although less than half the animals (2/10 males and 3/10 females) had nodal macrophages containing foreign material; none showed birefringence and there was no host response against the material except for a slightly increased incidence, but not mean severity, of sinus histiocytosis compared to controls. These changes were not considered adverse. No increase, decrease, or reactive change in lymphoid tissues was observed in the mesenteric lymph nodes. There was an increased incidence of foreign material and inflammation in the small intestine of mid- and high-dose animals, but not the controls or low-dose animals. At the mid and high dose levels, any observed inflammation was very mild and the amount of foreign material present ranged from small to substantial amounts. These changes were not seen in previous animal studies with MTP or the related compound Vital ET in rats at doses up to 984 mg/kg bw/day (Libinaki et al. 2006). It is possible that the semi-purified diet used in this study may have played a role in enhancing any potential effects of MTP since in hamsters fed comparable amounts of dietary α-tocopherol, plasma α-tocopherol concentrations were 233% higher in animals fed semipurified diets than in those fed nonpurified diet (Nicolosi et al. 1998).
The lymph nodes were extracted and examined by LCMS in order to determine if they contained higher than expected amounts of tocopheryl phosphate; if they did then it would most likely be the foreign material. Only the nodes from the treated rats had significant amounts of the compound. The low amount of tissue from the lymph nodes that was available for extraction, namely 30 to 35 mg, is the likely explanation for the low amount of tocopheryl phosphate detected in the controls. The amount found in past projects has been in the order of 0.1 μg tocopheryl phosphate/g tissue in liver and adipose tissue (Gianello et al. 2005).
Spherulites are spherical crystals formed of radial lamellae that give a Maltese cross birefringence under polarized light. They are formed generally of polymers, natural or synthetic, such as carbohydrates (chitin, chitosan, cellulose, amylose, starch granules), proteins and nucleoproteins (bovine insulin, amyloid, lysozyme, carboxypeptidase, hemoglobin S, Na adducts of DNA, uric acid), lipids (cholesterol esters), and other compounds such as polyethylene, polypropylene, polyhydroxybutyrate, polystyrene, and various mineral salts (Krebs et al. 2004). Thus, the list of compounds capable of forming spherulites is large. Under appropriate conditions, the polymer chain folds back on itself in regular radial fashion (self-assembly) forming lamellar crystals of a defined thickness, which when viewed with polarized light, gives a Maltese cross configuration (Krebs et al. 2004). The data (Table 9) suggest that the crystalloids (spherulites) elicit a host response in the lymph nodes, but the non-spherulite form of the foreign material does not. The identity of the material forming the spherulites is not known. It did not stain for amyloid using the Congo red stain. However, the high levels of tocopheryl phosphates in the mesenteric lymph node suggest that the nature of the foreign material is tocopheryl phosphate, likely in a polymerized state. The spherulites have not been isolated for analysis and examination of their physicochemical properties. Foreign material of similar morphology was seen in macrophages of the lamina propria of the small intestine, suggesting that its formation began in that location and was carried to the mesenteric lymph node by regional lymphatics.
Because there was lack of reactive inflammatory change associated with the foreign material in low-dose rats, the severity of histiocytosis was similar among low-dose and control animals, and there were no systemic responses indicated by any clinical pathology parameter, the lowest dietary concentration of 1% (equivalent to approximately 587 and 643 mg MTP/kg bw/day for male and female rats, respectively) was considered to be the no observed adverse effect level (NOAEL).
Footnotes
Figures and Tables
This study was sponsored by Phosphagenics Ltd., Melbourne, Australia.
Conflict of Interest Statement. Phosphagenics Ltd. funded the salaries of R.G. and R.L., and paid W.H. as a pathology consultant and E.K. as a consultant. E.O. is an employee of Phosphagenics Ltd.