13-Week Oral Toxicity Study with Stanol Esters in Rats

Regulatory Toxicology and Pharmacology 29, 216 –226 (1999) Article ID rtph.1999.1291, available online at http://www.idealibrary.com on

13-Week Oral Toxicity Study with Stanol Esters in Rats Duncan Turnbull,* Margaret H. Whittaker,* Vasilios H. Frankos,* ,1 and Diana Jonker† *ENVIRON Corporation, 4350 North Fairfax Drive, Arlington, Virginia 22203; and †TNO Nutrition and Food Research Institute, P.O. Box 360, 3700 AJ Zeist, The Netherlands Received February 11, 1999

Plant sterols and their saturated derivatives, known as stanols, reduce serum cholesterol when consumed in amounts of approximately 2 g per day. Stanol fatty acid esters have been developed as a highly fat-soluble form that may lower cholesterol more effectively than stanols. Stanol esters occur naturally in human diets, but at levels far below those known to lower cholesterol. The present study was conducted to assess the safety of stanol esters upon subchronic ingestion at levels comparable to or exceeding those recommended for lowering cholesterol. Two stanol fatty acid ester preparations, wood-derived stanol esters and vegetable oil-derived stanol esters, were fed to groups of 20 male and 20 female Wistar rats for 13 weeks, at dietary concentrations of 0, 0.2, 1, and 5% total stanols (equivalent to 0, 0.34, 1.68, and 8.39% wood-derived stanol esters and 0, 0.36, 1.78, and 8.91% vegetable oil-derived stanol esters). Both preparations were well tolerated as evidenced by the absence of clinical changes or major abnormalities in growth, food and water consumption, ophthalmoscopic findings, routine hematological and clinical chemistry values, renal concentrating ability, composition of the urine, appearance of the feces, estrus cycle length, organ weights, gross necropsy findings, and histopathological findings. Plasma cholesterol and phospholipids were slightly decreased in males fed the stanol esters. In both sexes, plasma levels of plant sterols were decreased whereas those of stanols tended to increase. Fecal excretion of sterols, including cholesterol, and stanols was markedly increased in the stanol ester groups. Compared to controls, male rats fed stanol esters showed somewhat lower liver weights and more pronounced glycogen depletion. These hepatic changes were considered to reflect an altered nutritional condition and not a pathological condition. Plasma levels of vitamin E, vitamin K 1 , and, to a lesser extent, vitamin D were decreased in males and females fed the high-dose diets. Hepatic levels of vitamins E and D showed similar changes (vitamin K 1 in the liver was not determined). For both preparations, the mid-dose level (1% total 1

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stanols in the diet) was a no-observed-adverse-effect level. This dietary level provided approximately 0.5 g total stanols/kg body wt/day. © 1999 Academic Press

INTRODUCTION

Plant sterols are a group of steroid-based alcohols having hydrocarbon side chains of 8 –10 carbon atoms. Plant sterols are derived from various plant sources including pine trees and vegetables. One unique property of plant sterols and stanols (the saturated derivatives of the sterols) is their ability to reduce cholesterol levels when consumed at certain levels. The cholesterol-lowering effect of sitostanol may be increased when ingested in a soluble, esterified form (Vanhanen et al., 1993). Sitostanol ester has been incorporated into a margarine product and has been shown to reduce cholesterol in clinical studies (Vanhanen et al. 1993; Gylling and Miettinen, 1994; Vanhanen et al., 1994). To examine the health effects of stanol esters, a subchronic toxicity test in rats using two stanol ester preparations was performed utilizing dietary concentrations of 0, 0.2, 1, and 5% total stanols. MATERIALS AND METHODS

Animals Young, male and female Wistar rats [Crl:(WI)WU BR] were obtained from a colony maintained under SPF conditions at Charles River Wiga GMBH, Sulzfeld, Germany. Animals were quarantined upon arrival and checked for overt signs of ill health and anomalies. During the quarantine period, their microbiological status was checked by the conduct of serological controls in random samples. At initiation of the study, the rats were about 7 weeks old. They were housed conventionally, in groups of five, in suspended stainless steel cages with wire-mesh floors and fronts, in a controlled environment (temperature 19623°C, humidity 30 –55%, and 12-h light/dark cycle).

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TABLE 1 Compositional Analysis of TNO Rodent Diet Nutrient

Found upon analysis

Moisture Crude fat Crude protein Crude fiber Ash Calcium Phosphorus Sodium Chloride Potassium Magnesium Iron Copper Manganese Zinc Vitamin A Vitamin E

9.3% 5.8% 21.0% 2.8% 5.2% 0.91% 0.60% 0.23% 0.41% 0.65% 0.15% 242 mg/kg 17 mg/kg 63 mg/kg 67 mg/kg 7.6 iu/g 89 mg/kg

Diets and Test Materials Feed and water were provided ad libitum from the arrival of the rats until study termination, unless precluded by test observations or measurements. The feed was provided as a powder, in stainless steel cans, covered by a perforated stainless steel plate to prevent spillage. Until initiation of treatment, the rats were fed the institute’s cereal-based rodent diet (TNO rodent diet, see Table 1 for a mean compositional analysis of this diet). From the start of treatment, they were kept on the experimental diets. Drinking water (tap water) was given in polypropylene bottles that were cleaned approximately weekly and filled when necessary. Two stanol esters were tested in this study. The first was a wood-derived stanol ester while the second was a vegetable oil-derived stanol ester. Both substances had a purity of approximately 99%. Rapeseed oil was used

to balance the level of added energy in the various experimental diets, assuming that both the test substance-derived fatty acids and the rapeseed oil contained 9 kcal/g. The rapeseed oil had a purity of approximately 100%. The test substances were administered in the diet, at constant concentrations, for 13 consecutive weeks. The TNO rodent diet was used as the carrier. The test substances (melted in a water bath at 65–70°C prior to use) and the rapeseed oil were incorporated into the diet by mixing in a mechanical blender. Control rats received the TNO diet supplemented with rapeseed oil. Fresh batches of experimental diets were prepared monthly and stored in a refrigerator (2–10°C) until use. The stability of the test substances under simulated experimental conditions was checked before the initiation of the 13-week study. Samples of prepared diets were analyzed to examine the stability during storage for up to 7 days in the animal room (open container) and during storage for up to 5 weeks in the refrigerator (2–10°C) (closed container). The test diets administered during the study were determined to be acceptable with respect to concentration, homogeneity, and stability. Experimental Design As indicated in Table 2, the study comprised seven groups of 20 rats per sex per group, including one control group, three test groups fed different levels of wood-derived stanol esters, and three test groups fed different levels of vegetable oil-derived stanol esters. For each test substance, the lowest dose level was in the range of the recommended daily intake for lowering plasma cholesterol in humans (i.e., 2 g of total stanols per person per day) and was intended to be a noobserved-adverse-effect level (“NOAEL”). The highest dose level was 25 times higher than the lowest level. This high-dose level, corresponding to dietary levels of

TABLE 2 Experimental Design Dietary percentage of stanols (% stanol esters)

Number of males

Number of females

Daily test substance intake (mg stanol esters/kg body wt/day)

Control 0.2% Wood-derived stanols (0.34% wood-derived stanol esters) 1% Wood-derived stanols (1.68% wood-derived stanol esters) 5% Wood-derived stanols (8.39% wood-derived stanol esters) 0.2% Vegetable oil-derived stanols (0.36% vegetable oil-derived stanol esters) 1% Vegetable oil-derived stanols (1.78% vegetable oil-derived stanol esters) 5% Vegetable oil-derived stanols (8.91% vegetable oil-derived stanol esters)

20 20

20 20

20

20

20

20

20

20

20

20

20

20

0 174 (males) 193 (females) 872 (males) 979 (females) 4579 (males) 5093 (females) 187 (males) 206 (females) 938 (males) 1049 (females) 4934 (males) 5509 (females)

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the test substances of 8 –9% (as esterified stanols), was selected as the maximum dose feasible without having to fortify the diet with vitamins and minerals. Each animal was observed daily for clinical signs of toxicity, moribundity, and mortality. On working days, all cages were again checked in the afternoon for dead or moribund animals. During weekends and holidays, only one check per day was performed. The body weight of each rat was recorded at the initiation of treatment (day 0), once every week thereafter, and on the day of scheduled autopsy. Food consumption was measured per cage, over successive 7-day periods, by weighing the feeders and expressed in grams per rat per day. In addition, food consumption was measured per rat (3 rats/sex/group) over 1-day periods during the 3-day feces collection period. The intake of the test substances per kilogram of body weight was calculated from the nominal dietary level of the test substances, the food intake, and the body weight. Water consumption was measured per cage on 5 consecutive days in weeks 1, 6, and 12. Ophthalmoscopic observations were made at the start and in week 13 of the treatment period. At autopsy, blood was sampled from the abdominal aorta of 10 rats/sex/group while under ether anesthesia. The following hematological determinations were made: hemoglobin (HB), packed cell volume (PCV), red blood cell count (RBC), total white blood cell count (WBC) and differential white blood cell count, prothrombin time (PTT), and thrombocyte count (Thromboc). The following hematological parameters were calculated: mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC). Clinical chemistry determinations were made on 10 rats/sex/ group (the same as those used for hematology). At autopsy, blood was collected in heparinized tubes from the abdominal aorta, and the following measurements were made in the plasma obtained after centrifugations: alkaline phosphatase (ALP), aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), gamma glutamyl-transferase (GGT), total protein (TP), albumin (Album), ratio of albumin to globulin (A/G ratio), urea, creatinine (Creatin), bilirubin-total (BiliTot), total cholesterol (Chol), HDL-cholesterol (HDLChol), non-HDL cholesterol (non-HDL), triglycerides (Trigly), phospholipids (Phospho-Lip), calcium (Ca), sodium (Na), potassium (K), chloride (Cl), and inorganic phosphate (Inorg-P). Fasting glucose was determined in blood collected from the tip of the tail at the end of the urine collection period. On days 86 – 87, 10 rats/sex/group (the same as those used for hematology and clinical chemistry) were deprived of water for 24 h and of food during the last 16 h of this period as part of a renal concentration test. During the last 16 h of deprivation, the rats were kept in stainless steel metabolism cages (1 rat per cage) and urine was collected. The following determinations were

carried out on individual samples: volume, density, appearance, semiquantitative observations (pH, glucose, occult blood, ketones, protein, bilirubin, urobilinogen), and microscopy of the sediment (erythrocytes, leukocytes, epithelial cells, amorphous material, crystals, casts, bacteria, sperm cells, and worm eggs). On days 85– 88, feces were collected from 3 rats/sex/ group (not the same rats as those used for hematology, clinical chemistry, urinalysis, and plasma sterols). Feces samples were collected from each animal individually, while housed in open, plexiglass metabolism cages for 3 consecutive days. The fecal concentrations of neutral steroids (including sitosterol, campesterol, cholesterol, and their 5a/b-saturated derivatives) and bile acids were measured in samples from the control group and from the two top-dose groups. In addition, one pooled sample of each group was analyzed for free and total (free plus esterified) sterols and stanols. For feces, the analytical methods included alkaline hydrolysis (omitted when free sterols were determined), silylation of sterols and stanols, butylation of bile acids, and gas chromatography/flame ionization detection (GC/FID). At the end of treatment, plasma concentrations of sterols and stanols (including sitosterol, campesterol, cholesterol, their 5a-saturated derivatives, and cholesterol precursors) were measured in 10 rats/sex/group (the same as those used for hematology, clinical chemistry, and urinalysis). For plasma, the analytical method comprised alkaline hydrolysis, silylation, and GC/FID. The plasma concentrations of b-carotene, vitamin A, vitamin E (a-tocopherol), vitamin D (25-hydroxy-cholecalciferol), and vitamin K 1 (phylloquinone) were measured in 10 rats/sex/group (not the same as those used for hematology, clinical chemistry, and plasma sterols). In addition, the levels of vitamins A, E, and D were measured in the liver of females of the control group and the vegetable oil-derived stanol 0.2, 1, and 5% dose groups (the same animals as used for the analysis of plasma vitamins). Levels of these vitamins were measured in the liver to determine whether vitamin depletion occurred in the liver before the plasma. On days 72– 81, vaginal smears were made for all females. The smears from the control females and the top-dose females were stained (Cyto-Tek slide stainer) and examined microscopically to determine the estrus cycle length. At the end of the treatment period, on 4 successive days, the rats were killed by exsanguination from the abdominal aorta under light ether anesthesia, and a thorough necropsy was performed. The adrenals, brain, epididymides, heart, kidneys, liver, ovaries, pituitary, prostate, seminal vesicles with coagulating glands, spleen, testes, thymus, thyroid with parathyroids, and uterus of all rats were weighed and relative organ weights were calculated on the basis of the terminal body weights. Samples of the weighed organs

13-WEEK ORAL TOXICITY STUDY WITH STANOL ESTERS IN RATS

and of the aorta, cecum, colon, eyes, lungs, female mammary gland, mesenteric lymph nodes, muscle (thigh), esophagus, pancreas, rectum, salivary glands, sciatic nerve, small intestine (duodenum, ileum, jejunum), spinal cord (three levels), sternum (with bone marrow), stomach, trachea, urinary bladder, vagina, and gross lesions were fixed in Bouin’s fixative (testes and epididymides) or 10% neutral buffered formalin. In addition, part of the liver of 10 females/group was frozen in liquid nitrogen and stored at 270°C (used for analysis of fat-soluble vitamins). Tissue samples of the preserved organs from all rats of the control group and the high-dose groups were processed, embedded in paraffin, sectioned at 5 mm, stained with hematoxylin and eosin (testes and epididymides were also stained with periodic acid–Schiff), and examined by light microscopy. The kidneys, liver, lungs, and gross lesions were also examined in all rats of the intermediate dose groups. Furthermore, the uterus, ovaries, vagina, and mammary gland of all females were subjected to a detailed light-microscopic evaluation with histomorphological classification of the stage of estrous in individual animals. This evaluation was conducted by Robert F. McConnell, D.V.M. (Consulting Pathology Services, Flemington, NJ), an external consulting pathologist with a broad expertise in reproductive organ pathology. Statistical Analysis All pairwise comparisons were two tailed. Body weight data were analyzed by one-way analysis of covariance using preexposure (day 0) weights as the covariate. When group means were significantly different (P , 0.05), individual pairwise comparisons were made using Dunnett’s multiple-comparison method. Food and water consumption, food conversion efficiency, red blood cell and clotting potential variables, total and differential white blood cell counts (absolute numbers), urinary volume and density, clinical chemistry values, plasma and liver vitamins, and organ weights were evaluated by analysis of variance (ANOVA). When group means were significantly different (P , 0.05), individual pairwise comparisons were made using Dunnett’s multiple-comparison method. Independent from the results of ANOVA, homogeneity of variances was tested by means of Bartlett’s test. When the variances differed significantly (P , 0.01), ANOVA was performed on transformed data, or Kruskal–Wallis nonparametric ANOVA was used. Percentages of differential white blood cell counts and urinary parameters except for volume and density were analyzed by Kruskal–Wallis nonparametric one-way ANOVA. When this analysis yielded a significant difference, pairwise comparisons between the control and treatment groups were made by means of Mann–Whitney U tests. Plasma levels of sterols and

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stanols were evaluated by ANOVA or, in cases of nonhomogeneity of variances (Levene’s test; P , 0.05), by Welch ANOVA, followed by pairwise comparisons using t tests. Fecal excretions of neutral steroids and bile acids were evaluated by two-sample t tests or by separate variance t tests when variances were nonhomogeneous (Levene’s test, P , 0.05). The incidence of histopathological changes was evaluated by Fisher’s exact probability test. In addition, the incidence of uterine luminal dilation was evaluated by the Cochran–Armitrage trend test. RESULTS

The appearance, general condition, and behavior of the rats were not adversely affected by the test substances at any dose level. The feces of rats fed the test substances was not visibly changed. The few clinical signs observed included areas of sparsely haired skin in control and treated rats and occasional dermal encrustations and malocclusion of the incisors. These changes were not considered treatment related. One control group female was found dead on day 48 of the study and upon histopathological examination was found to have markedly enlarged lymphoid organs. Death of this rat was attributed to leukemia. All other rats survived until their scheduled autopsy. There were no significant differences in mean body weight between the treated groups and the controls. Mean food intake and food conversion efficiency showed no treatment-related intergroup differences in male rats. In females, food consumption tended to be increased in the top-dose groups. The differences with the control group occasionally reached the level of statistical significance, especially in the vegetable oil-derived stanol ester group. Food conversion efficiency in females showed no significant intergroup differences. Water consumption was not affected by treatment. Ophthalmoscopic examination of high-dose rats and controls toward the end of the treatment period revealed no treatment-related ocular changes. As shown in Tables 3 and 4, no significant hematological changes were noted in male rats fed either the wood-derived or vegetable oil-derived stanols at any dose level relative to controls. Females of the woodderived stanol 5% dose group showed a statistically significant increase in thrombocyte count, and females of the vegetable oil-derived stanol 5% dose group had an increased percentage of neutrophils and a decreased percentage of lymphocytes. Although statistically significant, this change in percentage of neutrophils and lymphocytes was not ascribed to treatment because there was no clear dose–response relationship and there were no significant changes in the absolute numbers of these cell types. As shown in Tables 5 and 6, there were several

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TABLE 3 Hematology Values (Red Blood Cell and Coagulation Parameters) in Rats Fed Wood- or Vegetable Oil-Derived Stanol Esters for 13 Weeks

Control 0.2% Wood-derived stanols 1% Wood-derived stanols 5% Wood-derived stanols 0.2% Vegetable oil-derived stanols 1% Vegetable oil-derived stanols 5% Vegetable oil-derived stanols

RBC (310 12 /liter)

HB (mmol/liter)

PCV (liters/liter)

MCV (fl)

MCH (fmol)

MCHC (mmol/liter)

Thrombocy (310 9 /liter)

PTT (s)

Male Female Male Female Male Female Male Female Male Female

8.38 6 0.11 7.55 6 0.13 8.38 6 0.07 7.65 6 0.07 8.38 6 0.08 7.65 6 0.09 8.36 6 0.07 7.55 6 0.06 8.41 6 0.11 7.58 6 0.10

9.4 6 0.1 9.1 6 0.1 9.6 6 0.1 9.2 6 0.1 9.6 6 0.0 9.3 6 0.1 9.6 6 0.1 9.1 6 0.1 9.6 6 0.0 9.2 6 0.1

0.432 6 0.004 0.401 6 0.003 0.430 6 0.004 0.404 6 0.004 0.432 6 0.003 0.413 6 0.005 0.434 6 0.003 0.402 6 0.005 0.431 6 0.002 0.407 6 0.003

51.6 6 0.8 53.2 6 0.8 51.3 6 0.5 52.8 6 0.5 51.6 6 0.7 54.1 6 0.6 52.0 6 0.6 53.2 6 0.6 51.3 6 0.8 53.7 6 0.7

1.13 6 0.02 1.20 6 0.02 1.14 6 0.01 1.20 6 0.01 1.14 6 0.01 1.22 6 0.01 1.15 6 0.01 1.20 6 0.01 1.14 6 0.01 1.21 6 0.01

21.8 6 0.2 22.6 6 0.1 22.3 6 0.1 22.8 6 0.1 22.1 6 0.1 22.6 6 0.2 22.2 6 0.1 22.6 6 0.1 22.2 6 0.1 22.6 6 0.1

945 6 30 850 6 19 888 6 21 838 6 24 959 6 16 857 6 33 986 6 12 950 6 21* 989 6 24 846 6 24

41.1 6 0.6 35.5 6 0.6 42.9 6 1.1 36.4 6 0.7 42.7 6 0.9 36.0 6 0.8 41.8 6 0.5 37.0 6 0.6 42.8 6 0.7 36.3 6 0.6

Male Female

8.39 6 0.11 7.56 6 0.08

9.5 6 0.1 9.2 6 0.1

0.429 6 0.005 0.407 6 0.004

51.1 6 0.7 53.9 6 0.7

1.14 6 0.01 1.21 6 0.01

22.2 6 0.1 22.6 6 0.2

1010 6 20 829 6 25

41.1 6 0.8 35.9 6 0.5

Male Female

8.57 6 0.09 7.51 6 0.05

9.7 6 0.1 9.2 6 0.1

0.435 6 0.003 0.411 6 0.004

50.8 6 0.7 54.7 6 0.5

1.13 6 0.01 1.22 6 0.01

22.2 6 0.1 22.4 6 0.1

1030 6 24 867 6 22

41.7 6 0.3 36.8 6 0.7

Note. Values are means 6 SEM (n 5 10 animals of each sex per dose group). Hematology abbreviations are defined under Materials and Methods. * P , 0.05.

statistically significant changes in clinical chemistry values. In male rats fed wood-derived stanols, statistically significant decreases in total protein (in the 1 and 5% dose groups), total and HDL-cholesterol (in the 0.2 and 1% dose groups), phospholipids (in the 0.2 and 1% dose groups), and calcium (in the 1% dose group) were noted. Females fed wood-derived stanols showed decreases in total protein and albumin (in the 5% group). Male rats fed vegetable oil-derived stanols showed an

increase in the A/G ratio (in the 5% dose group) and decreases in total cholesterol (in the 0.2 and 5% dose groups), HDL-cholesterol (all treated groups), and phospholipids (all treated groups). In females fed vegetable oil-derived stanols, a decrease in albumin (in the 0.2 and 1% dose groups) and an increase in alkaline phosphatase (in the 5% dose group) were observed. As shown in Table 7, plasma levels of sterols and stanols showed multiple treatment-related changes.

TABLE 4 Hematology Values (White Blood Cell Values) in Rats Fed Wood- or Vegetable Oil-Derived Stanol Esters for 13 Weeks

Control 0.2% Wood-derived stanols 1% Wood-derived stanols 5% Wood-derived stanols 0.2% Vegetable oil-derived stanols 1% Vegetable oil-derived stanols 5% Vegetable oil-derived stanols

Male Female Male Female Male Female Male Female Male Female Male Female Male Female

WBC (310 9/liter)

Neutro (310 9/liter)

Lympho (310 9/liter)

Neutro (%)

Lympho (%)

7.0 6 0.3 6.3 6 0.7 6.5 6 0.6 5.6 6 0.6 7.2 6 0.4 5.7 6 0.3 7.6 6 0.5 5.7 6 0.6 6.4 6 0.5 5.1 6 0.3 7.4 6 0.6 5.6 6 0.8 7.2 6 0.4 5.3 6 0.4

0.5 6 1.0 0.4 6 0.0 0.5 6 0.1 0.6 6 0.1 0.5 6 0.1 0.6 6 0.1 0.6 6 0.1 0.3 6 0.1 0.5 6 0.1 0.5 6 0.1 0.5 6 0.1 0.3 6 0.1 0.5 6 0.1 0.6 6 0.1

6.4 6 0.3 5.8 6 0.7 5.9 6 0.6 5.0 6 0.6 6.6 6 0.4 5.0 6 0.3 6.9 6 0.4 5.2 6 0.5 5.8 6 0.5 4.5 6 0.3 6.8 6 0.6 5.2 6 0.7 6.6 6 0.5 4.6 6 0.3

6.9 6 1 7.0 6 0.8 7.9 6 1.5 10.4 6 1.6 7.1 6 1.1 10.3 6 1.8 7.8 6 0.9 6.3 6 0.9 7.6 6 0.9 9.5 6 1.5 7.0 6 1.5 7.1 6 1.3 7.8 6 1.7 11.7 6 1.0*

91.9 6 1.1 92.4 6 0.8 90.3 6 1.6 88.1 6 1.5 91.8 6 1.2 88.4 6 1.9 91.3 6 0.9 92.1 6 0.8 91.2 6 1.0 89.3 6 1.4 91.7 6 1.5 92.5 6 1.3 91.4 6 1.6 86.7 6 0.9**

Note. Values are means 6 SEM (n 5 10 animals of each sex per dose group). Abbreviations used: WBC, white blood cells; Neutro, neutrophils; Lympho, lymphocytes. * P , 0.02. ** P , 0.002.

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TABLE 5 Clinical Chemistry Values in Rats Fed Wood-Derived Stanols (Stanol Esters) for 13 Weeks Males

Gluc (mmol/liter) ALP (U/liter) ALAT (U/liter) ASAT (U/liter) GGT (U/liter) TP (g/liter) Album (g/liter) A/G ratio Urea (mmol/liter) Creatin (mmol/liter) Bili-Tot (mmol/liter) Cholest (mmol/liter) HDL-Chol (mmol/liter) Non-HDL (mmol/liter) Trigly (mmol/liter) Phos-lip (mmol/liter) Calcium (mmol/liter) Potassium (mmol/liter) Sodium (mmol/liter) Chloride (mmol/liter) Inorg-P (mmol/liter)

Females

Control

0.2% (0.34%)

1% (1.68%)

5% (8.39%)

Control

0.2% (0.34%)

1% (1.68%)

5% (8.39%)

3.73 6 0.12 192 6 11 30 6 1 49 6 2 1.0 6 0.4 70 6 1 35 6 0 1.00 6 0.01 7.4 6 0.4 25 6 1 1.1 6 0.6 2.08 6 0.07 1.49 6 0.06 0.59 6 0.02 1.70 6 0.24 2.20 6 0.06 2.79 6 0.02 3.8 6 0.1 145 6 0 105 6 0 1.63 6 0.14

3.51 6 0.12 200 6 13 31 6 1 47 6 1 0.6 6 0.2 68 6 0 34 6 0 1.04 6 0.02 7.5 6 0.3 26 6 1 0.5 6 0.1 1.81 6 0.09* 1.27 6 0.08* 0.54 6 0.03 1.19 6 0.22 1.88 6 0.09** 2.72 6 0.02 3.8 6 0.1 145 6 0 105 6 0 1.51 6 0.12

3.65 6 0.16 199 6 8 32 6 1 53 6 2 0.4 6 0.2 67 6 1** 34 6 0 1.05 6 0.02 6.5 6 0.3 26 6 1 0.9 6 0.4 1.75 6 0.05* 1.18 6 0.04** 0.56 6 0.04 0.95 6 0.15 1.78 6 0.06** 2.70 6 0.01** 3.8 6 0.1 145 6 0 105 6 0 1.48 6 0.17

3.46 6 0.10 204 6 6 32 6 1 53 6 1 1.4 6 0.4 67 6 0** 34 6 0 1.06 6 0.02 7.1 6 0.4 28 6 2 0.6 6 0.1 1.95 6 0.08 1.35 6 0.07 0.61 6 0.03 1.27 6 0.16 2.01 6 0.07 2.72 6 0.02 4.0 6 0.1 144 6 0* 104 6 1 1.49 6 0.12

4.20 6 0.11 186 6 14 35 6 2 63 6 3 0.0 6 0.0 68 6 1 38 6 0 1.25 6 0.02 8.6 6 0.6 26 6 1 1.0 6 0.1 1.50 6 0.04 1.04 6 0.05 0.45 6 0.03 0.81 6 0.12 1.91 6 0.06 2.54 6 0.02 3.4 6 0.1 143 6 1 105 6 1 1.29 6 0.18

3.95 6 0.08 212 6 15 41 6 3 72 6 4 0.0 6 0.0 67 6 1 37 6 1 1.23 6 0.02 7.6 6 0.5 25 6 1 1.0 6 0.1 1.55 6 0.05 1.09 6 0.06 0.46 6 0.04 0.76 6 0.18 1.88 6 0.07 2.56 6 0.03 3.2 6 0.1 143 6 0 105 6 1 1.32 6 0.21

3.95 6 0.08 212 6 17 36 6 3 65 6 2 0.0 6 0.0 66 6 1 36 6 1 1.21 6 0.02 7.6 6 0.4 26 6 1 1.0 6 0.1 1.50 6 0.06 1.09 6 0.06 0.41 6 0.03 0.72 6 0.11 1.78 6 0.07 2.55 6 0.02 3.3 6 0.0 143 6 0 105 6 0 1.23 6 0.18

3.84 6 0.13 229 6 10 39 6 4 69 6 4 0.1 6 0.0 64 6 1* 35 6 1* 1.21 6 0.02 8.3 6 0.4 27 6 1 0.9 6 0.0 1.53 6 0.05 1.06 6 0.05 0.47 6 0.03 0.72 6 0.11 1.82 6 0.06 2.53 6 0.03 3.4 6 0.1 143 6 1 105 6 1 1.33 6 0.17

Note. Values are means 6 SEM (n 5 10 animals of each sex per dose group). Clinical chemistry abbreviations are defined under Materials and Methods. * P , 0.05. ** P , 0.01.

The individual unsaturated plant sterols sitosterol, campesterol, and stigmasterol, as well as their sum, and the sum of saturated and unsaturated sterols (total sterols) were dose dependently decreased in the stanol ester groups. Plasma sitostanol (1D 5-avenasterol) was increased in males of the 1 and 5% dose groups and in females of all treatment groups. Campestanol was increased in all groups fed vegetable oil-derived stanols. The cholesterol precursors desmosterol and/or lathosterol were increased in females of the 1 and 5% dose groups in response to both test substances. In contrast, males showed decreases in desmosterol (in the 0.2 and 1% dose groups of both test substances) and lathosterol (in the 0.2% wood-derived stanol group). The renal concentration test, semiquantitative urinary observations, and microscopic examination of the urinary sediment did not reveal any treatment-related changes in the wood-derived stanol or vegetable oilderived stanol groups (data not shown). Fat-soluble vitamin levels in both the plasma and liver are presented in Tables 8 and 9. The plasma levels of b-carotene and vitamin A were comparable among treated rats and controls. The plasma levels of vitamin E (a-tocopherol), vitamin D (25-hydroxy-cholecalciferol), and vitamin K 1 (phylloquinone) were decreased in males and females of the 5% dose groups of both test substances. The mean values of vitamin D were decreased by about 15% while those of vitamins E and K 1 were de-

creased by at least 50%. The levels of vitamins A, D, and E in the liver (measured only in the females fed vegetable oil-derived stanols and controls) showed the same pattern as did the plasma levels; the liver level of vitamin A showed no treatment-related changes, whereas the levels of vitamins D and E were decreased in the high-dose group (by about 20 and 55%, respectively). Based on an analysis of feces collected from 3 rats/sex/ group during days 85–88, sterols and stanols in fecal samples were present in free form. Numerous statistically significant changes were noted in the feces from rats of the wood- and vegetable oil-derived stanol ester groups. These changes included increased cholesterol in fecal samples from the two high-dose groups (about 10fold), increased levels of sitosterol and campesterol (about 5-fold), and increased levels of sitostanol (at least 300fold) and campestanol (about 50-fold in response to woodderived stanol esters and about 250-fold in response to vegetable oil-derived stanol esters). These results indicate that the wood- and vegetable oil-derived stanols were not significantly absorbed from the gastrointestinal tract. Table 10 shows the daily consumption and fecal excretion of total plant stanols in male and female rats in the high-dose group. The daily excretion of lithocholic acid, hyodeoxycholic acid, total bile acids, and total secondary bile acids in feces did not change statistically significantly in response to stanol feeding. The absolute weight of the kidneys was slightly (about 9%) but statistically significantly decreased in

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TABLE 6 Clinical Chemistry Values in Rats Fed Vegetable Oil-Derived Stanols (Stanol Esters) for 13 Weeks Males

Gluc (mmol/liter) ALP (U/liter) ALAT (U/liter) ASAT (U/liter) GGT (U/liter) TP (g/liter) Album (g/liter) A/G ratio Urea (mmol/liter) Creatin (mmol/liter) Bili-Tot (mmol/liter) Cholest (mmol/liter) HDL-Chol (mmol/liter) Non-HDL (mmol/liter) Trigly (mmol/liter) Phos-lip (mmol/liter) Calcium (mmol/liter) Potassium (mmol/liter) Sodium (mmol/liter) Chloride (mmol/liter) Inorg-P (mmol/liter)

Females

Control

0.2% (0.36%)

1% (1.78%)

5% (8.91%)

Control

0.2% (0.36%)

1% (1.78%)

5% (8.91%)

3.73 6 0.12 192 6 11 30 6 1 49 6 2 1.0 6 0.4 70 6 1 35 6 0 1.00 6 0.01 7.4 6 0.4 25 6 1 1.1 6 0.6 2.08 6 0.07 1.49 6 0.06 0.59 6 0.02 1.70 6 0.24 2.20 6 0.06 2.79 6 0.02 3.8 6 0.1 145 6 0 105 6 0 1.63 6 0.14

3.60 6 0.08 189 6 6 33 6 1 53 6 1 0.5 6 0.2 69 6 1 35 6 0 1.03 6 0.01 6.9 6 0.4 25 6 1 0.8 6 0.1 1.86 6 0.07* 1.31 6 0.07* 0.55 6 0.03 1.04 6 0.15 1.86 6 0.07** 2.70 6 0.02* 3.9 6 0.1 144 6 0 105 6 1 1.50 6 0.10

3.73 6 0.13 191 6 8 30 6 2 50 6 2 1.2 6 0.3 68 6 0 35 6 0 1.02 6 0.02 6.6 6 0.2 24 6 1 0.5 6 0.2 1.90 6 0.04 1.30 6 0.05* 0.60 6 0.03 1.41 6 0.22 1.97 6 0.05** 2.78 6 0.02 3.9 6 0.1 145 6 0 104 6 0 1.66 6 0.13

3.62 6 0.09 217 6 10 36 6 3 56 6 3 0.8 6 0.4 67 6 0 35 6 0 1.07 6 0.01** 6.8 6 0.1 25 6 1 1.3 6 0.6 1.74 6 0.05** 1.17 6 0.03** 0.57 6 0.03 1.27 6 0.18 1.86 6 0.05** 2.75 6 0.02 3.9 6 0.1 145 6 0 104 6 1 1.67 6 0.11

4.20 6 0.11 186 6 14 35 6 2 63 6 3 0.0 6 0.0 68 6 1 38 6 0 1.25 6 0.02 8.6 6 0.6 26 6 1 1.0 6 0.1 1.50 6 0.04 1.04 6 0.05 0.45 6 0.03 0.81 6 0.12 1.91 6 0.06 2.54 6 0.02 3.4 6 0.1 143 6 1 105 6 1 1.29 6 0.18

4.04 6 0.13 228 6 14 37 6 4 71 6 4 0.0 6 0.0 66 6 1 36 6 0* 1.21 6 0.03 7.5 6 0.3 24 6 1 1.0 6 0.1 1.66 6 0.06 1.16 6 0.05 0.49 6 0.03 0.60 6 0.07 1.94 6 0.04 2.52 6 0.03 3.4 6 0.1 143 6 1 105 6 1 1.19 6 0.17

4.11 6 0.10 230 6 14 40 6 3 71 6 5 0.0 6 0.0 65 6 1 35 6 1** 1.19 6 0.02 8.3 6 0.6 27 6 1 1.1 6 0.1 1.56 6 0.04 1.07 6 0.04 0.49 6 0.02 0.61 6 0.09 1.82 6 0.06 2.51 6 0.03 3.4 6 0.1 142 6 1 105 6 1 1.29 6 0.17

4.10 6 0.15 261 6 23** 38 6 3 71 6 6 0.0 6 0.0 65 6 1 36 6 0 1.24 6 0.02 7.7 6 0.5 26 6 1 1.1 6 0.1 1.53 6 0.06 1.04 6 0.05 0.48 6 0.03 0.59 6 0.07 1.87 6 0.06 2.53 6 0.02 3.5 6 0.2 143 6 0 106 6 0 1.23 6 0.18

Note. Values are means 6 SEM (n 5 10 animals of each sex per dose group). Clinical chemistry abbreviations are defined under Materials and Methods. * P , 0.05. ** P , 0.01.

males of both high-dose groups. The absolute and relative weights of the liver were slightly decreased (between 5 and 13%) in males of all groups fed the test substances. In all cases but one (in the 0.2% vegetable oil-derived stanol group), these changes were statistically significant, and in the vegetable oil-derived stanol groups they showed a dose– effect relationship. The relative weight of the spleen was statistically significantly increased in females of the vegetable oil-derived stanol 0.2% dose group only. This finding was not attributed to ingestion of the test substance because it was not seen at the higher dose levels. Gross examination of animals upon autopsy did not reveal any treatment-related changes. Microscopic examination did not reveal adverse effects of the test substances on the gastrointestinal tract or any of the other organs and tissues examined. The only noteworthy findings were glycogen depletion in the liver and luminal dilatation of the uterus. Compared to controls, depletion of liver glycogen was slightly more pronounced in the males of the test groups, most notably in the wood-derived stanol top-dose group. Incidences of slight and moderate glycogen depletion for male rats from the control, low-, mid-, and high-dose wood-derived stanol ester groups, and the low-, mid-, and highdose vegetable oil-derived stanol ester groups were 16, 15, 13, 20, 16, 15, and 20, respectively (of 20 animals in

each group). Uterine luminal dilatation was observed more frequently in females fed vegetable oil-derived stanols than in controls. Incidences of luminal dilatation for females from the control and low-, mid-, and high-dose vegetable oil-derived stanol ester groups were 4, 7, 6, and 10, respectively (of 19 animals examined in the control group and 20 animals examined in each of the vegetable oil-derived groups). Though the differences were not statistically significant, the incidence in the vegetable oil-derived stanol 5% dose group was relatively high in comparison to the background incidence in rats of this strain and age. Apart from luminal dilatation, there were no microscopic observations in the uterus. Examination of the vaginal smears from controls and 5% dose group females revealed no treatment-related changes in the length of the estrus cycle. DISCUSSION

The objective of this 13-week study was to examine the possible subchronic oral toxicity of wood-derived stanol esters and vegetable oil-derived stanol esters in rats. These substances were fed at dietary concentrations of 0, 0.2, 1, and 5% total stanols (equivalent to 0, 0.34, 1.68, and 8.39% wood-derived stanol esters and 0, 0.36, 1.78, and 8.91% vegetable oil-derived stanol es-

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TABLE 7 Concentration of Sterols and Stanols in Plasma (mg/ml) in Rats Fed Wood- or Vegetable Oil-Derived Stanol Esters for 13 Weeks Percent wood-derived stanols (% stanol esters) Control 0%

0.2% (0.34%)

1% (1.68%)

Percent vegetable oil-derived stanols (% stanol esters)

5% (8.39%)

0.2% (0.36%)

1% (1.78%)

5% (8.91%)

4.48** 1.32 1.34 4.87*** 2.12 0.18*** 7.34*** 8.50*** NC 23.0*** 12.4***

4.97** 0.75*** 0.97 28.0*** 4.29*** 0.30*** 21.6*** 4.39 NC 58.6*** 49.9***

4.94** 0.97* 1.10 12.2*** 6.91*** 0.21*** 10.7*** 6.41** 5.76 36.5*** 23.1***

4.36*** 1.35 1.15 5.22*** 6.66*** 0.33** 6.70*** 7.47*** NC 26.4*** 12.3***

2.06** 4.90*** 1.00*** 2.90*** 1.09 0.15** 4.90*** 5.69** NC 14.7*** 7.95***

3.09 2.44 0.66 20.5*** 4.54*** 0.28** 19.2** 5.39** NC 49.9* 40.0***

2.71 3.02** 0.71* 7.98*** 5.75*** 0.09*** 7.81*** 6.11*** 5.63 27.7*** 15.9***

3.14 4.93*** 0.99*** 3.21*** 4.47*** 0.15*** 3.97*** 4.75*** NC 16.5*** 7.32***

Males Cholestanol Desmosterol Lathosterol Campesterol Campestanol Stigmasterol Sitosterol Sitostanol 1 D 5-avenasterol a Sitostanol calculated b Total plant sterols Total unsaturated plant sterols

6.43 1.36 1.14 50.2 1.42 0.83 36.6 4.61 1.48 93.6 87.6

4.34** 0.82*** 0.81* 25.2*** 1.42 0.21*** 19.6*** 4.39 NC 50.9*** 45.1***

3.74*** 0.74*** 0.91 9.55*** 1.68 0.09*** 9.68*** 6.46* 5.93 27.5*** 19.3*** Females

Cholestanol Desmosterol Lathosterol Campesterol Campestanol Stigmasterol Sitosterol Sitostanol 1 D 5-avenasterol a Sitostanol calculated b Total plant sterols Total unsaturated plant sterols

3.12 2.38 0.51 29.2 1.02 0.79 26.3 3.20 0.80 60.5 56.3

2.37* 2.14 0.52 19.2*** 1.41 0.22** 17.7*** 5.61*** NC 44.2*** 37.2***

1.93*** 3.28* 0.61 6.38*** 1.37 0.01*** 7.28*** 6.58** 6.11 21.6*** 13.7***

Note. Values are means of 10 animals. NC, not calculated. a Sitostanol coeluted with D 5-avenasterol. b To obtain calculated sitostanol concentration, the mean concentrations of sitostanol 1 D 5-avenasterol were multiplied by the mean ratios of sitostanol/(sitostanol 1 D 5-avenasterol) [the mean ratios were 32.09% (males) and 24.96% (females) for control group, 91.76% (males) and 92.89% (females) for 1.0% wood stanol group, and 89.82% (males) and 92.20% (females) for 1.0% vegetable oil stanol ester group]. * P , 0.05. ** P , 0.01. *** P , 0.001.

ters) for 13 consecutive weeks. The ingestion of both substances was well tolerated, as evidenced by the normal growth and appearance of the rats and the absence of major abnormalities in hematology and clinical chemistry values, urinalysis results, estrous cycle length, organ weights, gross necropsy findings, and light microscopic findings. The slightly decreased plasma levels of total protein and/or albumin in some of the stanol ester groups were within the range of historical control values and/or showed no clear dose– effect relationship. Therefore, these findings were considered of no toxicological significance. Male rats fed either wood- or vegetable oil-derived stanols showed decreased plasma levels of total cholesterol, HDL-cholesterol, and phospholipids. The differences compared to the controls were statistically significant at the lowand mid-dose levels for wood-derived stanols and at all

dose levels for vegetable oil-derived stanols. There was no clear dose–response relationship, and these changes in plasma lipids are in agreement with the well-known cholesterol-lowering properties of stanols and stanol esters (Heinemann et al., 1986; Miettinen et al., 1995; Sugano et al., 1977; Vanhanen et al., 1993, 1994). The lowering of plasma cholesterol by plant sterols results from inhibition of the intestinal absorption of cholesterol and is reflected in an increased fecal excretion of cholesterol (Jones et al., 1997; Gylling and Miettinen, 1994; Heinemann et al., 1991; Sugano et al., 1977), a change which was also observed in the present study. The reduction of sterol absorption was furthermore reflected in the marked, dose-related decrease in the plasma levels of the unsaturated plant sterols campesterol, sitosterol, and stigmasterol and the concurrent increase in their fecal excretion. The plasma level of sitosta-

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TABLE 8 Fat-Soluble Vitamins in Plasma of Rats Fed Wood-Derived Stanol Esters for 13 Weeks Males

Carotene (mmol/liter) Vit-A (mmol/liter) Vit-E (mmol/liter) Vit-D (nmol/liter) Vit-K (nmol/liter)

Females

Control

0.2%

1%

5%

Control

0.2%

1%

5%

0.01 6 0.00 1.38 6 0.04 22.5 6 1.4 82 6 2 11.3 6 2.9

0.02 6 0.01 1.42 6 0.04 21.3 6 0.6 81 6 3 9.0 6 0.9

0.02 6 0.01 1.34 6 0.06 20.3 6 0.7 77 6 3 7.1 6 1.0

0.01 6 0.00 1.35 6 0.03 7.7 6 0.2** 69 6 3** 2.5 6 0.3**

0.01 6 0.00 0.57 6 0.02 23.2 6 0.9 122 6 4 7.5 6 1.2

0.02 6 0.01 0.64 6 0.04 25.3 6 0.8 123 6 5 12.3 6 2.1

0.01 6 0.01 0.66 6 0.05 24.8 6 0.6 119 6 6 9.9 6 1.8

0.01 6 0.00 0.63 6 0.03 11.4 6 0.3** 102 6 4* 3.1 6 0.6**

Note. Values are means 6 SEM (n 5 9 or 10 animals of each sex per dose group). * P , 0.05. ** P , 0.01.

nol (1D 5-avenasterol), the most abundant stanol in the test substances, was increased in the treated groups. The same was true for campestanol, especially in animals fed vegetable oil-derived stanols, the test substance with the highest campestanol level. Despite the high amounts consumed, the plasma concentration of campestanol and sitostanol remained at very low levels, indicating that only limited amounts of the fed stanols were absorbed. This is in agreement with results from animal and human absorption studies with labeled sitostanol showing that sitostanol is virtually unabsorbed (Czubayko et al., 1991; Hassan and Rampone, 1979; Ikeda and Sugano, 1978; Lu¨tjohann et al., 1993). Both wood-derived stanol and vegetable oil-derived stanol feeding caused dose-dependent reductions in plasma total plant sterols. The fecal excretion of campestanol and sitostanol (1D 5avenasterol) was increased markedly and dose dependently. The daily fecal excretion of these stanols approximated the daily amount consumed, reflecting their poor absorption. The similar ratio of campestanol/sitostanol in feed and feces indicated that there was no preferential absorption of campestanol or sitostanol. Despite the marked increase in cholesterol in the feces of rats fed stanol esters, the excretion of coprostanol (i.e., the prod-

uct of reduction of cholesterol by intestinal bacteria) was only slightly increased, resulting in a decreased proportion of fecal cholesterol converted to coprostanol (from 60% in controls to 20% in top-dose rats). Fecal bile acids, in contrast to neutral sterols, showed no major changes in response to feeding of stanol esters. At the end of treatment, males and females fed the test substances at the 5% dietary level showed markedly decreased plasma levels of vitamins E and K 1 (the decreases were at least 50%) and moderately decreased (about 15%) levels of vitamin D. Despite the markedly lower plasma level of vitamin K 1, there were no indications of compromised blood clotting. Prothrombin time was normal and hemorrhages were not observed. The changes in the plasma levels of vitamins E and D were accompanied by similar changes in the liver levels of these vitamins. A plausible explanation for the effect on the fat-soluble vitamins is solubilization of these vitamins by unhydrolyzed stanol esters in the intestine, leading to decreased absorption of these vitamins. However, this hypothesis could not be verified by data from the present study. The absolute weight of the kidneys was decreased slightly in males of both 5% dose groups. Because there were no accompanying histopathologic le-

TABLE 9 Fat-Soluble Vitamins in Plasma and Liver of Rats Fed Vegetable Oil-Derived Stanol Esters for 13 Weeks Males

Carotene (mmol/liter) Vit-A (mmol/liter) Vit-E (mmol/liter) Vit-D (nmol/liter) Vit-K (nmol/liter) Liver Vit-A (mg/g) Liver Vit-E (mg/g) Liver Vit-D (ng/g)

Females

Control

0.2%

1%

5%

Control

0.2%

1%

5%

0.01 6 0.00 1.38 6 0.04 22.5 6 1.4 82 6 2 11.3 6 2.9 NM NM NM

0.01 6 0.01 1.36 6 0.04 21.1 6 0.8 80 6 3 7.2 6 0.8 NM NM NM

0.02 6 0.01 1.41 6 0.05 19.9 6 0.9 78 6 3 7.1 6 0.8 NM NM NM

0.01 6 0.00 1.28 6 0.03 7.6 6 0.3** 68 6 2** 2.4 6 0.3** NM NM NM

0.01 6 0.00 0.57 6 0.02 23.2 6 0.9 122 6 4 7.5 6 1.2 324 6 7 50.4 6 1.8 4.07 6 0.15

0.02 6 0.01 0.71 6 0.06 25.1 6 1.2 128 6 4 9.5 6 1.6 327 6 6 48.8 6 1.5 4.02 6 0.24

0.01 6 0.01 0.66 6 0.06 24.0 6 1.0 120 6 4 9.3 6 1.3 359 6 12* 51.9 6 1.8 3.56 6 0.22

0.02 6 0.00 0.65 6 0.04 11.5 6 0.3** 100 6 6** 2.7 6 0.3** 338 6 7 21.9 6 1.0** 3.15 6 0.20*

Note. Values are means 6 SEM (n 5 9 or 10 animals of each sex per dose group). NM, not measured. * P , 0.05. ** P , 0.01.

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13-WEEK ORAL TOXICITY STUDY WITH STANOL ESTERS IN RATS

TABLE 10 Daily Consumption and Fecal Excretion of Total Plant Stanols (Sitostanol plus Campestanol) in the (5%) High-Dose Group a

Dose group

Daily total stanol consumption (mg/rat/day) b

Daily fecal total stanol excretion (mg/rat/day) c

Fecal excretion as percentage of consumption (%)

Wood-derived stanol ester high-dose males Wood-derived stanol ester high-dose females Vegetable oil-derived stanol ester high-dose males Vegetable oil-derived stanol ester high-dose females

730 592 780 597

714 596 842 597

98 101 108 100

Sitostanol measurements included D 5-avenasterol, which coeluted during analysis. Calculated from the mean food consumption during the feces collection period and the nominal concentration of wood-derived or vegetable oil-derived total stanols in the high-dose diets (i.e., 5%). c Calculated from the daily fecal excretion of sitostanol and campestanol. The fecal excretion data used in the calculation were corrected for analytical recovery. Based on a pooled sample from 3 rats/sex/group. a b

sions, and no significant differences in relative kidney weights, or other end points for renal damage, the decreased absolute weight of the kidneys was not considered toxicologically significant. In all groups of males fed the test substances, the weight of the liver (absolute and relative) was slightly (between 5 and 13%) but statistically significantly decreased. Similar changes were observed in a 14-day range-finding study (data not presented), though they reached the level of statistical significance only in females of the wood stanol 5% dose group. The reduced liver weights were not accompanied by any sign of liver damage. Upon histopathological examination of the liver, it was noted that glycogen depletion, observed in most male rats, controls included, was slightly more pronounced in males fed the test substances, especially those of the wood-derived stanol 5% dose group. This change occurred without a dose–response relationship or statistical significance and was considered to reflect an altered nutritional condition and not a pathological condition. It may partly explain the reduced liver weight of the treated males. In addition, sitostanol has been shown to lower hepatic cholesterol (Ikeda et al., 1981; Sugano et al., 1977), which may also have contributed to the lower liver weight. The slightly elevated incidence of uterine luminal dilatation in females fed vegetable oil stanol esters was not accompanied by any histopathological uterine changes indicative of an estrogenic effect nor by treatment-related changes in estrous cycle length or other reproductive organs. Moreover, historical control data on the incidence of luminal dilatation show considerable variation (between 0 and 45%), and the highest incidence in this study (viz. 50% in the vegetable oil-derived stanol 5% dose group) was close to the historical range. Therefore, the uterine luminal dilatation in the vegetable oil stanol ester groups was considered not to represent an estrogenic effect of the test substance. This is further supported by the detailed microscopic evaluation of female reproductive organs with histomorphological classification of the

stage of estrous in individual rats. The pattern of findings in this evaluation was consistent with the variations in estrous cycle length characteristic of the onset of reproductive senescence and provided no evidence of an exogenous estrogenic effect. CONCLUSION

In conclusion, no toxicity was associated with the subchronic ingestion of wood- or vegetable oil-derived stanol esters at dietary concentrations up to 1% (as free stanol; equivalent to about 0.5 g total stanols/kg body wt/day). At dietary levels of 5% (as free stanol), subchronic ingestion of these substances resulted in decreased plasma levels of the fat-soluble vitamins E and K 1 and, to a lesser extent, vitamin D. Hepatic levels of vitamins E and D showed similar changes (vitamin K 1 in the liver was not measured). REFERENCES Czubayko, F., Beumers, B., Lammfuss, S., Lu¨tjohann, D., and von Bergmann, K. (1991). A simplified micro-method for quantification of fecal excretion of neutral and acidic sterols for outpatient studies in humans. J. Lipid Res. 32, 1861–1867. Gylling, H., and Miettinen, T. A. (1994). Serum cholesterol and cholesterol and lipoprotein metabolism in hypercholesterolemic NIDDM patients before and during sitostanol ester-margarine treatment. Diabetology 37, 773–780. Hassan, A. S., and Rampone, A. J. (1979). Intestinal absorption and lymphatic transport of cholesterol and b-sitostanol in the rat. J. Lipid Res. 20, 646 – 653. Heinemann, T., Kullack-Ublick, G. A., Pietruck, B., and von Bergmann, K. (1991). Mechanism of action of plant sterols on inhibition of cholesterol absorption. Eur. J. Clin. Pharmacol. 40(Suppl. 1), S59 –S63. Heinemann, T., Leiss, O., and von Bergmann, K. (1986). Effect of low-dose sitostanol on serum cholesterol in patients with hypercholesterolemia. Atherosclerosis 61, 219 –223. Ikeda, I., Kawasaki, A., Samezima, K., and Sugano, M. (1981). Antihypercholesterolemic activity of b-sitostanol in rabbits. J. Nutr. Sci. Vitamin 27, 243–251.

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Ikeda, I., and Sugano, M. (1978). Comparison of absorption and metabolism of b-sitosterol and b-sitostanol in rats. Atherosclerosis 30, 227–237. Jones, P. J. H., MacDougall, D. E., Ntanios, F., and Vanstone, C. A. (1997). Dietary phytosterols as cholesterol-lowering agents in humans. Can. J. Physiol. Pharmacol. 75, 217–227. Lu¨tjohann, D., Meese, D. C. I., Crouse, J. R., and von Bergmann, K. (1993). Evaluation of deuterated cholesterol and deuterated sitostanol for measurement of cholesterol absorption in human. J. Lipid Res. 34, 1039 –1046. Miettinen, T. A., Puska, P., Gylling, H., Vanhanen, H., and Vartiainen, E. (1995). Reduction of serum cholesterol with sitostanolester margarine in a mildly hypercholesterolemic population. New Engl. J. Med. 333, 1308 –1312.

Sugano, M., Morioka, H., and Ikeda, I. (1977). A comparison of hypocholesterolemic effect of b-sitosterol and b-sitostanol in rats. J. Nutr. 107, 2011–2019. Vanhanen, H. T., Blomqvist, S., Ehnholm, C., Hyo¨nen, M., Jauhiainen, M., Torstila, I., and Miettinen, T. A. (1993). Serum cholesterol, cholesterol precursors, and plant sterols in hypercholesterolemic subjects with different apoE phenotypes during sitostanol ester treatment. J. Lipid Res. 34, 1535–1544. Vanhanen, H. T., Kajander, J., Lehtovirta, H., and Miettinen, T. A. (1994). Serum levels, absorption efficiency, fecal elimination and synthesis of cholesterol during increasing doses of dietary sitostanol esters in hypercholesterolemic subjects. Clin. Sci. 87, 61– 67.