P.A Hepburn

Safety evaluation of phytosterol esters. Part 1. Assessment of oestrogenicity using a combination of in vivo and in vitro assays.

Phytosterols are natural constituents of the human diet, and as part of an extensive programme of safety evaluation studies investigating their use as a novel food ingredient, the possible oestrogenic effects of phytosterols have been investigated using a combination of in vitro and in vivo assays. Competitive binding with the immature rat uterine oestrogen receptor (ER) has been used to measure the ability of phytosterols to bind to ERs while the transcriptional activation of oestrogen-responsive genes has been examined in an oestrogen-inducible yeast screen. Phytosterols did not display any activity in these in vitro assays. Uterotrophic assays have been conducted to investigate the potential for phytosterols to elicit an oestrogenic response when administered orally to immature female rats (n = 10) at doses of 0, 5, 50 or 500 mg/kg/day for 3 consecutive days. Phytosterols (a well characterized mixture of beta-sitosterol, campesterol and stigmasterol) and phytosterol esters (the previous phytosterol mixture esterified with fatty acids from sunflower oil) did not exhibit oestrogenic activity in the immature female rat using uterine wet weight as the endpoint. Beta-oestradiol (0.4 mg/kg/day) consistently produced a significant increase in uterus weights. Coumestrol (a known phytoestrogen) was also tested as a weak positive control and produced a dose response at doses of 20, 40 and 80 mg/kg/day in the uterotrophic assay. In conclusion, we have shown that phytosterols do not bind to the ER and do not stimulate transcriptional activity of the human ER in a recombinant yeast strain. In addition, there was no indication of oestrogenicity from the uterotrophic assay when the material was administered by oral gavage to immature female rats.

Safety evaluation of phytosterol esters. Part 5. Faecal short-chain fatty acid and microflora content, faecal bacterial enzyme activity and serum female sex hormones in healthy normolipidaemic volunteers consuming a controlled diet either with or without a phytosterol ester-enriched margarine

A study was conducted in 12 healthy males and 12 healthy females (mean age 36 years, mean body mass index 24 kg/m2), to determine the effect of a margarine enriched with phytosterol esters on faecal short-chain fatty acids (SCFAs) and faecal bacterial enzyme activities, viable faecal microflora count, female sex hormones and serum cholesterol concentrations. The study design was a two-period, parallel dosing, randomized, placebo-controlled dietary study. Under controlled dietary conditions, participants consumed 40 g of the control margarine for 21 and 28 consecutive days for males and females, respectively. This was followed immediately by the second part of the study where subjects were equally and randomly allocated to consume daily 40 g of either the control or the test margarine, containing 8.6 g vegetable oil phytosterols (a mixture of beta-sitosterol, campesterol and stigmasterol), also for 21 or 28 days. All females were shown to have a regular menstrual cycle and were on an established method of contraception not involving oral contraceptives. When compared with the control group values, the test group showed a significant reduction in serum total and LDL cholesterol concentrations of 18 and 23% (P < 0.001; P < 0.001) respectively, in faecal lactic acid concentration (P = 0.039) and in serum progesterone levels (P = 0.021). There were no other significant treatment effects. Within each group a number of significant changes occurred compared to baseline. In the test group, faecal lactic acid concentration and the ratio of acetic acid:total SCFA; and the ratio of butyric acid:total SCFA, in the control group were both significantly reduced (P = 0.016). Compared to baseline, azo-reductase activity was significantly reduced in the control group (P = 0.047). Total faecal aerobes (P = 0.028), lactobacilli (P = 0.003) and staphylococci (P = 0.025) content was also significantly reduced in the control group, while in the test group only lactobacilli content was reduced (P = 0.019). Of the significant findings reported in this study, none was considered to be of biological importance except the beneficial reduction in serum total and LDL-cholesterol concentrations. The daily consumption of a margarine enriched with 8.6 g vegetable oil phytosterols did not affect the bacterial profile or the metabolic activities of the gut microflora, nor did it result in biologically relevant effects on serum female sex hormone levels. The margarine enriched with the vegetable oil phytosterols was well tolerated by both male and female volunteers.

Safety evaluation of phytosterol esters. Part 2. Subchronic 90-day oral toxicity study on phytosterol esters--a novel functional food.

Phytosterol esters (PE) are intended for use as a novel food ingredient, primarily in margarines and spreads as a functional component with plasma cholesterol lowering activity. Phytosterols and their esters are present naturally in vegetable oils and on average people consume 200 mg/day, but their consumption at this level is not sufficient to lower plasma cholesterol levels. Therefore, through the incorporation of PE into margarines/spreads, the intake can be increased by approximately 10-fold by consuming the PE-containing margarine/spread at normal intake levels. As part of an extensive programme of safety evaluation studies a subchronic rat toxicity study has been conducted in which groups of Alpk:AP(f)SD (Wistar derived) rats (20 males and 20 females/group) were fed diets containing PE at levels of 0, 0.16, 1.6, 3.2 and 8.1% (w/w) in the diet for 90 days. Throughout the study, clinical observations, body weights, and food and water consumption were measured. At the end of the study the rats were subjected to a full post-mortem examination, cardiac blood samples were taken for clinical pathology, selected organs were weighed, and a full tissue list was taken for subsequent histological examination. There were no treatment-related changes that were considered to be of toxicological significance. Therefore, a nominal PE concentration of 8.1% was considered to be the no-observed-adverse- effect level (NOAEL) following daily oral administration to rats for 90 days. This was equivalent to a dose of 6.6 g/kg body weight/day PE or 4.1 g/kg/day phytosterol.

A 13-Week Feeding Study in the Rat with Shea Oleine and Hardened Shea Oleine

Shea oleine is an oil fraction derived from the nut of the tree, Butyrospermum parkii, which grows in central and western Africa. There are several uses of shea oleine, including its use as a cooking oil and, after hardening, in margarine and toffee fat. As part of a series of studies, a 13-week rat feeding study was conducted in which groups of Colworth-Wistar rats (15 male and 15 female) were fed 27.5% total fat semipurified diets containing 20% (w/w) shea oleine (SU) or hardened shea oleine (SH). Equivalent groups of rats were fed either 20% (w/w) palm oil (PU), soyabean oil (BU), or the hardened (hydrogenated) equivalents (PH and BH, respectively). Assessments of body weight, food and water intake, clinical pathology, organ weights, and macroscopic histopathology were carried out. Results showed that shea oleine diets produced similar biological effects to palm oil and and soyabean oil diets. A slightly reduced body weight gain was noted in rats fed either of the shea oleine diets in comparison with palm oil and soyabean oil. The process of hardening had no significant impact on the normal growth in rats fed shea oleine, although minor differences compared with un-hardened diets comprised some small changes in clinical chemistry parameters, raised fiver weight, and less liver lipid. In addition, raised alkaline phosphatase and an increase in food intake were noted in rats fed SH. All diets were well tolerated in the growing rat and none of the findings in the study were considered to be adverse.

Reproduction studies in the rat with shea oleine and hardened shea oleine.

Shea oleine is an oil fraction derived from the nut of the tree Butyrospermum parkii, which grows in central and western Africa. There are several uses of shea oleine including its use as a frying oil and, after hardening, in margarine and toffee fat. This investigation was performed to examine the toxicity of 7 or 15% hardened shea oleine in comparison with 7 or 15% unhardened shea oleine and various commercially available materials, sheanut and palm oils, cocoa butter and toffee powder following dietary administration to rats during pre-mating, mating, pregnancy and offspring weaning in two separate investigations. Reproduction was assessed using number of litters and pups born plus survival and body weights at birth and at weaning on day 21. Skeletal evaluation using X-ray, clinical pathology and a macroscopic examination were also performed for F1 rats. Study measures for parent animals comprised evaluation of body weight, food consumption, clinical pathology, organ weights and macroscopic examination. Fatty acids and hydrocarbon levels were measured and an evaluation for lipogranulomata was made for various tissues. Results showed that shea oleine, whether unhardened or hardened, produced no evidence of reproduction toxicity and gave a similar profile to the other commercially available materials used in this study in the rat. Minor findings with shea oleine were not related to reproduction performance but comprised slightly reduced body weight gain and reduced cholesterol and raised alkaline phosphatase levels. None of the findings in this study were considered to be of toxicological significance. Thus, no evidence of reproduction toxicity was seen for both unhardened and hardened shea oleine in this investigation in the rat at levels equating to greater than 7.5 g/kg/day.

Studies to Investigate the Absorption and Excretion of Shea Oleine Sterols in Rat and Man

Shea oleine (SU), an oil fraction derived from the nut of the African tree, Butyrospermum parkii, is used as a frying oil and, after hardening (SH), in margarine and toffee fat. Both SU and SH contain a high level (approximately 8%) of 4,4-dimethylsterols (4,4-DMS), mostly as esters of cinnamic acid. As part of a series of studies evaluating SU, investigations to study rat and human dietary utilization were carried out. These comprised fecal fat analysis of groups of Wistar rats and a small number (four subjects) of human volunteers. Groups of rats were administered SU in a semisynthetic diet over 3 weeks at up to 20% in the diet (approximately 10 g/kg/day). In the human study, four male volunteers consumed a single 25-g portion of SU (approximately 0.4 g/kg) and ate no other vegetable fat during the course of the study. No preferential absorption of any of the 4,4-DMS occurred in the rat or man. Apparent absorption of the most prominent sterol fraction in the unsaponifiable material, 4,4-DMS, as estimated by its disappearance from feces, was similar in both species (27% to 52% in the rat compared with 13% to 49% in human subjects). Both rats and humans showed a similar profile of dietary and fecal 4,4-DMS fraction sterol components.