Follow-up of the re-evaluation of quillaia extract (E 999) as a food additive and safety of the proposed extension of uses.

Quillaia extract (E 999) was re-evaluated in 2019 by the EFSA Panel on Food Additives and Flavourings (FAF). EFSA derived an acceptable daily intake (ADI) of 3 mg saponins/kg bw per day for E 999. Following a European Commission call for data to submit data to fill the data gaps, the present follow-up opinion assesses data provided by interested business operators (IBOs) to support an amendment of the EU specifications for E 999. Additionally, this opinion deals with the assessment of the proposed extension of use for E 999 in food supplements supplied in a solid and liquid form, excluding food supplements for infants and young children and, as a carrier in botanical nutrients. The Panel concluded that the proposed extension of use, if authorised, could result in an exceedance of the ADI at the maximum of the ranges of the mean for children, adolescents and the elderly, and for all populations at the 95th percentile. An additional proposed extension of use for E 999 to be used as a carrier for glazing agents on entire fresh fruits and vegetables has been received. Since no information on the proposed use levels of E 999 on a saponins content basis has been provided by this applicant, the Panel was not able to evaluate the safety of this extension of use. Considering the technical data submitted, the Panel recommended some modifications of the existing EU specifications for E 999, mainly to lower the limits for lead, mercury and arsenic and to include a maximum limit for cadmium and for calcium oxalate. The Panel also recommended that the limits would be expressed on a saponins basis. The Panel proposed to revise the definition of E 999 to better describe the composition in a qualitative way.

The rat prepubertal uterine myometrium and not the luminal epithelium is predominantly affected by a chronic dietary genistein exposure.

Current knowledge about dietary soy isoflavone-induced hormonal effects and potential priming effects for the responsiveness of the organism to other estrogens is insufficient. The present study examined the effects of pre- and postnatal soy isoflavone exposure on estrogen responsiveness by estrogen receptor agonists in the uteri of prepubertal Wistar rats. To this end, offspring were generated from dams already maintained on three dietary groups, (1) a phytoestrogen-free diet, (2) a soy isoflavone-rich diet with 232 ppm daidzein and 240 ppm genistein or (3) a custom-made diet supplemented with 700 ppm genistein (GEN). Then, F1 females continuously exposed to isoflavones from GD1 to PND21 and non-exposed controls were subjected to an immature uterotrophic assay to compare physiological parameters and the response to subcutaneous treatment with 17ß-estradiol, GEN or an estrogen receptor subtype (ERα and ERß)-specific agonist. Uterine wet weight (UWW), luminal epithelial height (LEH) and myometrial thickness (MMT) were determined. In addition, isoflavone plasma levels and mRNA expression profiles of relevant steroid receptors and of molecular markers for proliferation and estrogenicity were assessed for all groups. The influence of dietary isoflavones on the sensitivity to various estrogenic stimuli in these prepubertal animals was minor. Yet, the uterus of immature rats with high chronic GEN exposure alone showed already an increase in UWW, LEH and MMT. The myometrial response to GEN was more pronounced than that of the luminal epithelium, which may be due to a non-uniform distribution of steroid receptors, in particular the progesterone receptor. In conclusion, although the impact of a continuous, prenatally initiated exposure to dietary isoflavones on the uterine physiology of juvenile rats is modest, the possible priming effects of this exposure for beneficial or adverse late-onset consequences in adults should not be neglected.

Urinary isoflavone phytoestrogens in German children and adolescents--a longitudinal examination in the DONALD cohort.

SCOPE: In light of concerns about hormonally active agents, it is important to assess human exposure to such compounds, especially in children as a susceptible subgroup. Estrogenic plant constituents are present in the human diet in varying levels, in particular the isoflavones daidzein (DAI) and genistein (GEN). We aimed to examine age-dependent and secular trends in phytoestrogen exposures and to investigate equol (EQ) excretion of German children using biomarker analysis in 24-h urine samples from a longitudinally designed study. METHODS AND RESULTS: The concentrations of DAI, its metabolite EQ and GEN were determined by GC-MS analysis in 24-h urines (510 samples) collected between 1985 and 2000 in 90 (47 boys) German children (6-18 years old), who are participants in the Dortmund Nutritional and Anthropometric Longitudinally Designed study. The results from the urinary biomarker analysis indicate isoflavone exposures at quite variable levels in German children: Analyte concentrations in over 500 urine samples cover the range reported previously in adults on typical German diet and with soy intake. EQ, the DAI metabolite produced by the gastrointestinal microflora, was detected in a high fraction of all samples, with 28/90 children (31%) excreting EQ in all their urines, and 62/90 children (68%) in at least one sample. Interestingly, when multiple urines obtained from individuals at different ages (6-18 years) were analyzed, EQ formation did not appear to be a constant trait over time. When stratified by sex, DAI, EQ and GEN concentrations (ng/mL) in urines and excretion rates (µg/day) were similar in boys and girls. Total isoflavone excretion rates (µg/day) increased during childhood (6-12 years) (p=0.02) and were constant during adolescence (13-18 years) (p=0.6). No clear trend for changes in dietary isoflavone exposure over the total study period was seen (p=0.7). CONCLUSIONS: In conclusion, biomarkers in urine of German children and adolescents indicate a frequent, but widely variable dietary isoflavone intake and suggest no secular increase (1985-2000) in the exposure to isoflavone phytoestrogens among German children and adolescents.

Relation of isoflavones and fiber intake in childhood to the timing of puberty.

BACKGROUND: It has been suggested that phytoestrogens and dietary fiber can affect puberty timing. OBJECTIVE: We examined whether intake of isoflavone and fiber in healthy white children before their pubertal growth spurt [age at take-off (ATO)] was associated with puberty timing. DESIGN: Multivariate regression analyses were performed in 227 DONALD (DOrtmund Nutritional and Anthropometric Longitudinally Designed) Study participants with 3-d weighed dietary records and information on potential confounders at baseline (1 and 2 y before ATO). In a subsample (n = 111), urinary isoflavones were determined in 24-h urine samples by gas chromatography-mass spectrometry analysis. Puberty timing was examined by using ATO and chronologic ages at pubertal stage 2 for breast development (B2) or gonadal development, peak height velocity (PHV), and menarche or voice break. RESULTS: Girls whose diet was in the highest dietary isoflavone tertile experienced Tanner stage 2 for breast development ap 0.7 y later and reached PHV ap 0.6 y later than did girls whose diet was in the lowest isoflavone tertile [age (95% CI) at B2: 10.7 y (10.4, 10.9 y) compared with 10.0 y ( 9.7, 10.3 y), respectively; P for trend = 0.04; age at PHV: 11.9 y (11.6, 12.2 y) compared with 11.3 y (11.0, 11.6 y), respectively; P for trend = 0.04; adjusted for body mass index z score and fiber intake]. In boys, dietary isoflavones were not associated with pubertal markers. Urinary isoflavone and dietary fiber intakes were not associated with pubertal markers. CONCLUSIONS: Girls, but not boys, with higher prepubertal isoflavone intakes appear to enter puberty at a later age. Fiber intake in this sample of healthy white girls and boys was not relevant for puberty timing.

In utero and postnatal exposure to a phytoestrogen-enriched diet increases parameters of acute inflammation in a rat model of TNBS-induced colitis.

Inflammatory bowel disease (IBD) is very common in Europe and USA. Its incidence in East Asia has been traditionally low, albeit the risk of IBD increases in Asian immigrants adopting western lifestyles, suggesting a strong role of environmental/dietary factors in IBD. A lifelong exposure to phytoestrogen-rich diets has been associated with a decreased risk of developing breast cancer and might also be protective against IBD. We studied the influence of in utero and postnatal exposure to a phytoestrogen (PE)-rich diet on acute inflammation in an animal model of TNBS-induced colitis. Wistar rats were exposed in utero and postnatally to high (genistein: 240 microg/g feed; daidzein: 232 microg/g feed) or very low levels (genistein and daidzein <10 microg/g feed) of phytoestrogen isoflavones fed to pregnant dams with the diet and throughout nursing. After weaning, the offspring had free access to these diets. At the age of 11 weeks, colitis was induced with an enema of TNBS. After 3 days, animals were sacrificed and tissues were collected for histological evaluation and analysis of molecular markers of inflammation. Animals kept on a PE-rich diet (PRD) had higher colon weights than animals on low PE-levels (PDD), suggesting enhanced acute inflammation by phytoestrogens. This result was supported by histological findings and by analysis of myeloperoxidase activity. Interestingly, relative mRNA and protein expression of cyclooxygenase-2 (COX-2) were modulated in rats on PRD, providing evidence that COX-2, the inducible isoform of the enzyme, is involved in the management of colonic inflammation. Our results suggest that early-in-life exposure to PE might not protect against the development of IBD but enhances the extent of acute inflammation.

Simultaneous determination of daidzein, equol, genistein and bisphenol A in human urine by a fast and simple method using SPE and GC-MS.

Human diet contains weakly estrogenic compounds such as daidzein (DAI) and genistein (GEN), phytoestrogens present in soy and many vegetables as well as bisphenol A (BPA), a contaminant from packing materials and plastic containers for foods and beverages. In light of concerns about hormonally active agents, biomonitoring methods are needed to assess human exposure to such compounds. A method for simultaneous determination of DAI, its metabolite equol (EQ), GEN, and BPA by GC-MS analysis was established, validated and applied to measure concentrations in human urine. Sample preparation involves enzymatic conjugate cleavage, SPE and derivatization by silylation. For GC/MS analysis, deuterated DAI and GEN and( 13)C-BPA are used as internal standards. LOD are 4, 4, 5 and 3 ng/mL urine for DAI, EQ, GEN and BPA, respectively. Interassay variations were 9% for DAI, 15% for EQ, 18% for GEN and 10% for BPA. Simple workup and accuracy of the method are suited for biomonitoring. An analysis of urine samples from 15 adults consuming typical German food revealed dietary exposure to phytoestrogens in all samples: GEN concentrations ranged between 13 and 238 ng/mL, those for DAI ranged from 12 to 356 ng/mL. More than half of the individuals excreted also the more estrogenic metabolite EQ, at levels of 8-128 ng/mL. Higher concentrations (GEN: 820, DAI: 960 and EQ: 1740 ng/mL) were measured in a 24 h urine sample upon ingestion of soy protein (50 g with 12.9 mg DAI and 25.2 mg GEN). Only urine collected after some days on strict phytoestrogen-free diet had undetectable isoflavone levels. BPA was detected in 9 of 15 urine samples ranging from 3 to 11 ng/mL, and at 55 ng/mL in one sample. In conclusion, a reliable method to determine BPA and isoflavones in urine was established and applied in a pilot study: Biomonitoring results show much higher dietary exposure to phytoestrogens than to BPA in German adults.

Combined effects of physical activity, dietary isoflavones and 17beta-estradiol on movement drive, body weight and bone mineral density in ovariectomized female rats.

Reduced estrogen levels occurring during menopause in woman are accompanied by a variety of disorders, e. g., hot flushes, depressions, osteoporosis, increase of body weight, and reduced movement drive. In this study we investigated the combined effects of physical activity, estradiol substitution, and a phytoestrogen-rich diet on bone mineral density, increase of body weight, and movement drive in an animal model. Ovariectomized (OVX) female Wistar rats were either fed an isoflavone-rich diet (IRD) or substituted with 17beta-estradiol (E2) for 3 months. Sham-operated rats (Sham) and vehicle-treated OVX animals served as controls. One half of the animals had the opportunity of voluntary wheel running. OVX rats displayed an eight times lower movement activity than Sham animals. E2 treatment, but not IRD, significantly increased the movement activity of OVX rats. During 3 months the lowest increase of body weight was observed in Sham animals, the highest rate in OVX animals. Along with running activity E2 treatment, but not IRD, also lowered the increase of body weight significantly compared to OVX animals. Bone mineral density (BMD) in the trabecular area of the tibia was strongly reduced in OVX rats compared to Sham animals. In contrast to IRD, E2 substitution resulted in a protection of BMD in this area compared to OVX animals. Our data demonstrate that body weight, movement drive, and BMD are positively influenced by E2. The steroid estrogen acts in the trabecular area of the tibia in a bone-protective manner, increases movement drive and antagonizes the increase of body weight. All these effects could not be observed in animals fed an isoflavone-rich diet.

Hormonal activity of combinations of genistein, bisphenol A and 17beta-estradiol in the female Wistar rat.

Phytoestrogens have been described as weak estrogens, selective estrogen receptor mediators (SERMs) or to exhibit antiestrogenic properties. However, information about their activity in combination with xenoestrogens and 17beta-estradiol in vivo, is limited. Therefore, the combinatory activity of the phytoestrogen genistein (Gen), the industrial chemical bisphenol A (BPA), and ethinylestradiol (EE) in ovariectomized Wistar rats was analyzed in this study. All compounds were administered orally on three consecutive days (EE at 30 microg, Gen at 100 mg and BPA at 200 mg per kg body weight per day). The pure antiestrogen fulvestrant (3 mg/kg) served as estrogen receptor (ER) antagonist control. Effects on uterine wet weight, height of the uterine epithelium, uterine clusterin (Clu) and complement C3 expression, and the height of the vaginal epithelium were examined. Treatment with Gen alone resulted in a moderate stimulation of uterine weight; in the vagina the height of the epithelium was strongly stimulated. BPA did not stimulate any of the above-mentioned parameters significantly. In combination with EE, Gen acted on most of the analyzed parameters in an additive manner, whereas BPA significantly antagonized the effects of EE on the uterine epithelium and uterine Clu expression. Given in combination with Gen, BPA was also able to antagonize the stimulatory effect of Gen on the uterine epithelium. In summary, our results demonstrate that Gen, in contrast to BPA, does not exhibit any antiestrogenic properties, even if given at high concentrations. The results of this study characterize BPA as a functional antiestrogen, very likely the result of a lack of ability to activate ER-mediated transactivation after binding to the receptor. This is not the case for Gen. Our results point to the involvement of complex molecular mechanisms in the action of Gen. These mechanisms, especially the role of ERbeta have to be characterized in further investigations.

Effects of genistein on the mammary gland proliferation of adult ovariectomised Wistar rats.

The effects of phytoestrogens on the female breast are discussed controversially. On the one hand, epidemiological and experimental data provide evidence that dietary phytoestrogens may prevent the development of breast cancer. On the other hand, in breast cancer cell lines and tumour models isoflavone phytoestrogens have been demonstrated to stimulate the growth of estrogen-dependent breast cancer cells. To further investigate the molecular effects of genistein (Gen) on the mammary gland, we treated non-tumour bearing, ovariectomised female Wistar rats with this phytoestrogen either subcutaneously (10 mg/kg body weight) or orally (100 and 200 mg/kg body weight) for 3 days. Estradiol (E(2), 0.004 mg/kg s. c.) and ethynylestradiol (EE, 0.1 mg/kg per os) served as reference compounds. In the breast tissue, mRNA and protein expression of the progesterone receptor (marker for estrogenicity) and PCNA (marker gene for proliferation) were examined by quantitative real-time PCR, Western blotting and immunohistochemistry; the uterotrophic response was assessed also. Treatment with Gen per os or s. c. results in a small but significant stimulation of the uterine wet weight. In the mammary gland, Gen stimulates the expression of progesterone receptor (PR) but, in contrast to E(2), the isoflavone does not stimulate the expression of PCNA. These findings resemble recent data demonstrating a differential ability of Gen to induce uterine gene expression and uterine proliferation. Our data indicate that in non-malignant breast tissue short-term administration of Gen, in contrast to more potent estrogens like E(2), does not induce proliferation. Chronic stimulation of proliferation is believed to be a key mechanism during the development of breast cancer. The limited ability of Gen to stimulate proliferation in this tissue could be an indication for a limited carcinogenic potency of Gen in the breast. In further investigations it is important to identify molecular differences between healthy and malignant breast tissue which may explain the different sensitivity towards Gen treatment.

Comparative assessment of endocrine modulators with oestrogenic activity. II. Persistent organochlorine pollutants.

Risk assessments of synthetic chemicals with oestrogen-like activity must take into account the high dietary levels of natural endocrine modulators in food. In view of current regulations of the European Union, a hygiene-based margin of safety (HBMOS) for xeno-oestrogens was defined as a quotient of estimated human daily intakes weighted by relative rodent in vivo potencies of the compounds. Such comparisons of intakes and potencies of natural isoflavones, with short half-lives, with those of polychlorinated organic pollutants (POP) displaying significant toxicokinetic accumulation, deserves the special consideration of toxicokinetics. For slowly accumulating compounds such comparison is much more favourable when based on comparative blood and tissue levels, not on scenarios of daily exposures. Observing these principles, the present communication extends the HBMOS concept to POP, using o,p'-DDT, the oestrogenic component of DDT mixtures, as a prototype. An HBMOS of 137 is derived for o,p'-DDT indicative of a sufficient margin of safety to ensure the absence of risk to human health due to its hormonal action, under exposure conditions now prevailing in Western countries.

Estrogenic isoflavones in rodent diets.

Many rodent diets contain components such as soy isoflavones (daidzein and genistein) known to have estrogenic properties. The dietary background of phytoestrogens may modulate some responses to environmental estrogens when these compounds are tested in rodent bioassays. Thus, and since only few data were available on the phytoestrogen content of rodent diets commonly used in European laboratories, it was of interest to analyze the daidzein and genistein contents of our standard animal feeds. Isoflavone contents were determined in seven batches of rodent chow (from two suppliers in Germany, Altromin and Ssniff) by high-performance liquid chromatography, and also analyzed in six rodent diets from the United States. The soy-based rodent diets from Germany contained isoflavone (daidzein plus genistein) concentrations in the range of 0.3-0.55 mg/g feed. These isoflavone contents are similar to those analyzed in the US rodent diets, and similar to values reported by others, including one particular lot of feed (with 0.35 mg isoflavones per g) which produced a large uterotrophic response in immature ovariectomized rats [Environ. Health Perspect., 106 (1998) 369]. Coumestrol was found in a sample of commercial rabbit food at rather high levels (0.27 mg/g), but, this phytoestrogen was not detected (<1 microg/g feed) in any of the other samples we analyzed. The soy components in our rodent diet produce a measurable background of daidzein and genistein in blood of female DA/Han rats, total isoflavones (aglycone plus conjugates) ranging between 90 and 290 ng/ml plasma. The ovariectomized animals kept on this chow, showed no signs of estrogenization of the reproductive tract (uterus, vagina), and responded normally to (xeno-)estrogen administration in a uterotrophic assay [J. Steroid Biochem. Mol. Biol., 73 (2000) 1]. Moreover, ovariectomized Wistar rats on our standard rodent diet (Ssniff R/M H) had lower uterine weights than animals kept on the isoflavone-free (solvent extracted) chow; both groups of rats responded to genistein administration with an increase in uterine weights. These results suggest that--albeit the sensitivity of the rodent uterotrophic assay is not reduced by the use of a diet containing soy isoflavones at commonly encountered levels--attention should be given to a variable dietary phytoestrogen background.