Toxicity Studies of Blockal,a Dietary Supplement Containing Phase 2 Starch Neutralizer (Phase 2),a Standardized Extract of the Common White Kidney Bean ( Phaseolus vulgaris )

Abstract

The number of available dietary supplements containing “starch blockers” intended for weight loss has risen dramatically in recent years. These supplements are believed to reduce carbohydrate-derived calories by interfering with α-amylase, the digestive enzyme responsible for conversion of complex carbohydrates to simple absorbable sugars. The present paper reports the findings of single- and multiple-dose (4-week) oral toxicity studies in rats of the marketed dietary supplement Blockal. Blockal contains as its main ingredient Phase 2 Starch Neutralizer (Phase 2 or Phaseolamin 2250), a standardized extract derived from the common white kidney bean (Phaseolus vulgaris) that has been shown to have α-amylase-inhibiting activity. The Blockal acute oral LD₅₀ exceeded the highest dose tested (3 g/kg body weight [bw]), which provided a single dose of 1668 mg/kg bw of Phase 2 white kidney bean extract. The no-observed-effect level (NOEL) seen in the 4-week study was equivalent to the highest Blockal dose tested (2 g/kg bw/day), which provided 1112 mg/kg/day of Phase 2 white kidney bean extract. The results of these studies support and are consistent with the safety of the marketed dietary supplement Blockal, and indirectly, the safety of its main ingredient, Phase 2 Starch Neutralizer (Phase 2 or Phaseolamin 2250), a standardized extract derived from the common white kidney bean.

The term “low-carb” has become part of our vernacular and the number of available reduced-carbohydrate food products and dietary supplements containing “starch blockers” intended to assist with consumer weight loss has risen dramatically in recent years. Starch blockers are thought to promote weight loss by interfering with the digestion of complex carbohydrates through inhibition of α-amylase, the digestive enzyme responsible for the breakdown of complex carbohydrates (i.e., starches) into simple sugars that can then be absorbed in the small intestine. The end result is a potential reduction in carbohydrate-derived calories.

α-Amylase inhibitors have been identified in many plant species. In 1943, Kneen and Sandstedt described the amylase-inhibiting activity of substances derived from wheat and rye, and certain sorghums. Bowman (1945) later found similar activity in simple aqueous extracts of ground navy beans. Since then, α-amylase inhibitors have been isolated from various other plant species, including many varieties of the common (i.e., white, red, and black kidney) bean, Phaseolus vulgaris (Marshall and Lauda 1975; Powers and Whitaker 1977a, 1977b; Wilcox and Whitaker 1984; Lajolo and Finardi Filho 1985).

In addition to α-amylase inhibitors, raw P. vulgaris beans are reported to contain a variety of antinutritional and potentially toxic substances. Reduced feed efficiency, impaired weight gain, histopathological changes, and occasional death in livestock and laboratory animals have been observed following ingestion of raw P. vulgaris beans. In humans, consumption of raw or undercooked kidney beans has been associated with severe but transient gastrointestinal disturbances (Haidvogl, Fritsch, and Grubauer 1979; Rodhouse et al. 1990; Sockett et al. 1993). These effects have been attributed largely to phytohemagglutinins (PHAs). Dependent upon natural variation, the red kidney bean, Phaseolus vulgaris, is known to contain up to five tetrameric isolectins: L₄, L₃E₁, L₂E₂, L₁E₃, and E₄ (Green and Baenziger 1987). Lectins are a class of carbohydrate-binding glycoproteins found in both plants and animals. Varying proportions of leukocyte reactive (L-PHA) and erythrocyte reactive (E-PHA) polypeptide subunits may be present at high levels in raw kidney beans but are reduced by cooking. For example, raw kidney beans may contain 20,000 to 70,000 HAU compared to 200 to 400 HAU in cooked beans. Moreover, there is some variation among bean types in PHA concentrations. Colored beans, particularly large-seeded kidney beans, have high levels of PHAs, whereas small white navy beans are reported to have negligible levels of these lectins (Uebersax, personal communication). Red kidney beans have about three times the amount present in white kidney beans (U.S. FDA/CFSAN 1992).

The present paper reports the findings of rat studies evaluating the acute and subacute toxicity of the dietary supplement Blockal, containing Phase 2 Starch Neutralizer, also known as Phaseolamin 2250 and Phase 2 (Pharmachem Laboratories, Inc.). Phase 2 is a standardized aqueous extract derived from the common white kidney bean (P. vulgaris) that has been shown to inhibit the activity of α-amylase in vitro. Phase 2 is routinely assayed to assure compliance with the established product specifications for the presence of antinutritional or potentially toxic substances such as PHAs (<3400 HAU/g1) and trypsin inhibitors (<40 TIU/mg²). The amount of PHA in Phase 2 typically averages approximately 1500 HAU/g (dry weight). This compares favorably with 32,000 HAU/g and 70 TIU/mg (dry weight) for soybean lectin and defatted soybean flour, respectively.

Previous toxicological studies in rats showed no mortality or significant toxicity following oral (gavage) administration of single doses up to 5 g/kg body weight (bw) or multiple doses (90 days) up to 1 g/kg bw/day of the Phase 2 extract alone (Harikumar et al. 2005). In a double-blind clinical investigation, 1500 mg of Phase 2 administered over the course of 8 weeks has shown potential usefulness in the treatment of obesity and hypertriglyceridemia (Udani, Hardy, and Madsen 2004) in a study population of 27 obese adults.

MATERIALS AND METHODS

Test Material

The test material used in these toxicity studies was standardized dietary supplement marketed as Blockal, containing 55.6% of Phase 2 Starch Neutralizer (Phase 2). Phase 2, a dried aqueous extract of the common white kidney bean (Phaseolus vulgaris) manufactured by Pharmachem Laboratories, Inc., New Jersey, has been standardized to a minimum of 3000 AAIU (α-amylase–inhibiting units) and validated by a modified United States Pharmacopeia (USP) method (SOP 110, Rev. 5). The standard composition of Blockal (Batch D106B) includes 55.6% Phase 2, 20.3% calcium phosphate, 10.1% microcrystalline cellulose, 6.8% vitamin B₃, and small quantities of other ingredients, including polyvinyl pyrrholidone (2.5%), silicone dioxide (2.0%), magnesium stearate (2.0%), vitamin B₆ (0.8%), and chromium picolinate (0.1%). The complete description of the chemical and physical properties of the Blockal test substance, including stability, was conducted by the study sponsor, Roeder 1956 Farmaceutici S.p.A., Italy.

In order to prepare the test article for administration, in both the acute and subacute studies, tablets of Blockal were first ground to a powder and subsequently dissolved in a distilled water vehicle. Prior to commencement of treatment, the proposed formulation procedure was checked by chemical analysis to confirm that the method was acceptable and that the stability of the formulation was satisfactory. Samples of the formulations prepared in weeks 1 and 4 of the study were also analyzed to check the concentration and homogeneity of the formulations. Chemical analyses were carried out by the Analytical Chemistry Department at RTC. All samples were within the established acceptance criteria of concentration (90% to 110%) and coefficients of homogeneity variation (<7.37%).

Acute Oral Toxicity Study

The acute oral toxicity study was conducted in May–June 2001 at Centre de Recherches Biologiques (CERB Study no. 20010265 ST), Baugy, France. The study was carried out in accordance with the following Good Laboratory Practice (GLP) standards: Organization for Economic Cooperation and Development (OECD); Council Directive 87/18/EEC; U.S. FDA GLP, 21 CFR Part 58; The French Ministry of Social Affairs and National Solidarity, State Secretariat for Health GLP; and “Notification No. 424 of the Pharmaceutical Affairs Bureau of the Japanese Ministry of Health and Welfare.” Complete description of the chemical and physical properties of the test substance, including stability, was the responsibility of the sponsor.

Young adult (>6 weeks old) male and (nulliparous) female Sprague-Dawley rats (Depre Breeding Centre, Saint Doulchard, France) were acclimatized for 6 days before receiving a single oral dose (10 ml/kg bw in sterile water) of the Blockal test material (Batch D106B), via gavage. Average body weight values at day 1 were approximately 172 g for males and 148 g for females. Blockal was given at 3 g/kg bw, which provided approximately 1668 mg/kg bw of Phase 2 white kidney bean extract. Animals (n = 5/sex) received food (UAR A04C-10 quality controlled/radiation-sterilized Food Product, Lot 10110, Usine d’Alimentation Rationnelle, Epinay-sur-Orge, France) and drinking (tap) water ad libitum, and were kept under standard animal room conditions (19°C to 23°C room temperature, 45% to 65% relative humidity, and artificial 12-h light/12-h dark cycle). Body weights, mortality, morbidity, and clinical signs were monitored at regular intervals for 14 days. Animals were sacrificed on day 15 by subtotal exsanguination after intraperitoneal sodium pentobarbital anesthesia. All animals were necropsied and the principal organs (liver, spleen, kidneys, stomach, intestines, gonads/reproductive tract, lungs, and heart) were examined macroscopically. All organs showing macroscopic signs of pathology were fixed in an appropriate fixative for possible histopathological examination.

Subacute Oral Toxicity Study

The subacute (4-week) oral toxicity study was conducted in October–November 2001 at Research Toxicology Centre S.p.A. (RTC Study No. 9269), Rome, Italy. The study was carried out in accordance with the following Good Laboratory Practice (GLP) standards: U.S. FDA GLP, 21 CFR Part 58; Commission Directive 1999/11/EC of March 8, 1999, adapting to technical progress the principles of good laboratory practice as specified in Council Directive 87/18/EEC; and Decreto Legislativo 27 Gennaio 1992 n. 120 published in the Gazzetta Ufficiale della Repubblica Italiana, 18 Febbraio 1992 (adoption of the Commission Directive of December 18, 1989 adapting to technical progress the Annex to Council Directive 88/320/EEC on the inspection and verification of GLP [90/18/EEC] and subsequent revisions).

A total of 70 Hsd: Sprague-Dawley rats (Harlan Italy s.r.l., San Pietro al Natisone [UD], Italy), 27 to 29-days old and with average body weights of 126 g for males and 93 g for females were acclimatized for 14 days. Animal room controls were set to maintain room temperature at approximately 22°C, relative humidity at 55%, and an artificial 12-h light/12-h dark cycle. On two occasions, relative humidity was out of range and remedial actions were taken. Animals received a commercially available laboratory rodent diet (Altromin MT pelleted diet, D-32770 Lage, Postfack 1120, Germany) and drinking water ad libitum. Seven days prior to dosing, animals were allocated to groups by computerized stratified randomization to give approximately equal initial group mean body weights.

After acclimatization, animals (10/sex/group) received the test material, Blockal (Batch D242B), via gavage for 4 weeks (once per day, 7 days per week). Blockal was administered at 0.7 or 2 g/kg bw/day (10 mL/kg bw in distilled water), which provided approximately 389 and 1112 mg/kg bw/day of Phase 2, respectively. A similarly constituted control group received the distilled water vehicle only, via gavage. Animals were monitored during the study for mortality, clinical signs, body weights, food consumption, hematology, clinical chemistry, and urinalysis. At necropsy, terminal body weights were obtained; organ to body weight ratios were calculated for brain, heart, kidney, liver, pancreas, spleen, thyroid and parathyroid glands, and empty gut. Samples of several tissues were fixed and preserved; sections of brain, cecum, colon, duodenum, heart, ileum, jejunum, kidneys, liver, pancreas, rectum, skeletal muscle, spleen, and stomach from control and high-dose animals were prepared for histopathological examination by Precision Histology International Ltd., London, UK, in compliance with United Kingdom Statutory Instrument No. 1999/3106, the GLP Regulations 1999, which incorporates the revised (1997) OECD GLP Principles as adopted by the European Union via Directives 1999/11/EC and 1999/12 EC.

Statistical Analyses

Statistical analysis of the acute oral toxicity study was performed using RS/1 software (release 6.0.1; BBN SOFTWARE).

For continuous variables in the subacute toxicity study, the significance of the differences among group means was assessed by analysis of variance (ANOVA). Differences between each treated group and the control group were assessed by Dunnett’s test using a pooled error variance. The homogeneity of the data was verified by Bartlett’s test. If data were found to be heterogeneous, a modified t test (Cochran and Cox) was applied. The mean, standard deviation, and statistical values were calculated from the actual values in the computer, without rounding off. Statistical analysis of histopathological findings was carried out by means of the nonparametric Kolmogorov-Smirnov test.

RESULTS

Acute Toxicity Study

Animals were monitored daily for 14 days following administration of a single 3 g/kg bw oral gavage dose of Blockal the supplement. No mortality or clinical signs indicative of toxicity were observed in male or female rats receiving the Blockal supplement, which provided 1668 mg/kg bw of Phase 2 white kidney bean extract. On days 1, 7, and 14, the mean male (n = 5) weight was 172.1 g, 228.9 g, and 278.2 g, respectively. On days 1, 7, and 14, the mean female (n = 5) weight was 147.7 g, 184.9 g, and 201.8 g, respectively. Group mean weight gains were normal when compared with data for the strain (Figure 1). Necropsy examination revealed black spots on the thymus and lungs of one male. No other gross organ or tissue findings were seen at necropsy of other animals and no histopathological variations were noted.

The median lethal dose (LD₅₀) was considered to exceed the highest dose of the Blockal supplement tested (3 g/kg bw), which provided 1668 mg/kg bw of Phase 2 white kidney bean extract. This dose of Phase 2 would be equivalent to more than 120 times the average manufacturer recommended intake of 13.9 mg/kg bw Phase 2 white kidney bean extract. Human intake is premised upon consumption of 1500 mg/day of Blockal or 500 mg taken with each of three daily meals (834 mg/day of Phase 2) in a 60-kg individual.

Subacute Toxicity Study

No mortality or clinical signs indicative of toxicity were observed in any group during the study. Mean body weight gains (Figures 2 and 3) and food consumption were unaffected by treatment. Terminal body weights were likewise unaffected. As illustrated in Tables 1 and 2, compared to control animals, male rats receiving the Blockal supplement in amounts providing 389 or 1112 mg/kg bw/day of Phase 2 white kidney bean extract demonstrated significantly higher (p < 0.01) serum total bilirubin (high- and low-dose) and creatinine (high- and low-dose), and lower total cholesterol and calcium levels (both low-dose). Mean hematological values of female rats receiving the Blockal supplement in amounts providing 389 or 1112 mg/kg bw/day of Phase 2 white kidney bean extract demonstrated significantly higher (p < .01) red blood cell counts (high- and low-dose) and hematocrit (low-dose only). Mean clinical chemistry values of the female rats demonstrated significantly higher (p < .01) total protein (high- and low-dose) and significantly lower (p < .01) total cholesterol, (p < .05), alkaline phosphatase, and potassium (low doses only).

High-dose males also showed a statistically significant increase in urine specific gravity when compared to controls (Table 3). Compared to the study control group, females receiving the Phase 2–containing supplement exhibited significantly higher (p<0.01) red blood cell counts, hematocrit (high-dose), and total serum protein levels, along with lower total serum cholesterol levels (low-dose).

The mean relative organ weights of male and female rats are listed in Tables 4 and 5, respectively. The most significant observation was significantly higher (p<0.01) relative empty gut weights in both high-dose males and females compared to their respective controls. Other differences in organ weights that reached statistical significance at the p<0.05 level included lower absolute and relative liver weights, higher absolute and relative pancreas weights, and higher relative spleen weights, among high-dose males.

Although some variations were noted in the macroscopic (gross) and/or microscopic appearance of several organs and tissues, no statistically significant differences were noted between control animals and animals receiving the Phase 2–containing dietary supplement. Table 6 lists the occurrence of variations in the gross appearance of liver, spleen, lungs, thymus, and uterus (filled with clear fluid). Of these, only the liver had corresponding microscopic changes, and the differences between control and treated animals were not statistically significant (p<0.05) (Table 7). Microscopic examination also revealed changes in the heart, kidneys, and skeletal muscle that were not significantly different between groups. No changes were observed in the gross or microscopic appearance of the brain, cecum, colon, duodenum, ileum, jejunum, pancreas, rectum, spleen, or stomach of males or females.

In the absence of any relationship to dose and/or sex, or any corresponding histopathological changes, the variations observed in this study, which were also within the laboratory’s historical values, were not considered of toxicological significance. The no-observed-effect level (NOEL) was equal to or greater than the highest dose of Blockal supplement tested (2 g/kg bw/day), which provided 1112 mg/kg bw/day of Phase 2 white kidney bean extract. The high dose of Phase 2 administered in this subacute study would be equivalent to approximately 80 times the average manufacturer recommended intake of 13.9 mg/kg bw Phase 2 extract. The low dose in this study, wherein rats were exposed to 389 mg/kg bw of Phase 2, would result in exposure of approximately 28 times the average manufacturer recommended intake. Human intake is premised upon consumption of 1500 mg/day of Blockal or 500 mg taken with each of three daily meals (834 mg/day of Phase 2) in a 60-kg individual.

DISCUSSION

Administration of 3 g/kg of Blockal, a dietary supplement providing 1668 mg/kg bw of Phase 2 Starch Neutralizer (Phase 2 white kidney bean extract) to rats as a single oral dose did not result in mortality or signs of toxicity. Likewise, no toxicity was evident following oral gavage administration of 2 g/kg Blockal to rats at levels providing up to 1112 mg/kg bw/day of Phase 2 continuously for 4 weeks. Variations in some hematology, clinical chemistry, and urinalysis parameters were noted in the 4-week study, along with some differences in the weight and gross or microscopic appearance of some organs/tissues. However, these findings were not considered of toxicological significance because they (1) were within the range of the laboratory’s historical data; (2) were not associated with any histopathological changes; and/or (3) did not appear to be related to dose or gender. Moreover, hematologic and pathological changes were observed in rats exposed to test substance were not similar to those reported for PHA exposure, which underscores the possibility that whatever changes which did occur were unrelated to low levels of PHA (<3400 HAU/g) specified in Phase 2 white kidney bean extract.

Ingestion of raw Phaseolus vulgaris beans has been associated with toxicity in various animal species. For example, Carmalt et al. (2003) reported that horses and cattle exhibited signs of toxicity within hours of ingesting feed containing 10% pelleted but uncooked white kidney beans. Horses exhibited signs of abdominal discomfort, anorexia, colic, and hyperthermia, and cattle exhibited anorexia, diarrhea, and decreased milk production. One horse (18-year-old mare) developed tremors, blindness, and seizures within 10 h and was euthanized at 16 h after exposure. There are numerous other reports in the literature of reduced feed efficiency, impaired weight gain, histopathological changes in the gastrointestinal tract, and occasional death in livestock and laboratory animals fed raw P. vulgaris beans (Myer, Froseth, and Coon 1982; Pusztai, King, and Clarke 1982; Hara, Tsukamoto, and Miyoshi 1983; Banwell et al. 1983; Greer, Brewer, and Pusztai 1985; de Oliveira, Vidal Bde, and Sgarbieri 1989; Huisman et al. 1990; Kik et al. 1990; Grant et al. 1995; Cavalle de Moya et al. 2003).

In humans, consumption of raw or undercooked kidney beans has been associated with nausea, vomiting, diarrhea, and abdominal pain within hours. These symptoms can be severe but recovery is typically rapid and spontaneous. The U.S. FDA considers PHAs (kidney bean lectins) to be a natural toxin that induces red kidney bean (Phaseolus vulgaris) poisoning (U.S. FDA/CFSAN 1992). Lectins are glycoproteins with hemagglutinating activity found in many plant varieties. Raw kidney beans contain from 20,000 to 70,000 HAU compared to 200 to 400 HAU in cooked beans, and red kidney beans have about three times the amount present in white kidney beans (U.S. FDA/CFSAN 1992). The amount of hemagglutinin in Phase 2 white kidney bean extract typically averages approximately 1500 HAU/g (dry weight).

In the case of toxicity among horses and cattle described by Carmalt et al. (2003), enzyme-linked immunosorbent assay (ELISA) analysis of the feed revealed the presence of leukoagglutinating phytohemagglutinin (L-PHA) at a concentration of 48 mg/kg in the equine preparation and 12 mg/kg in the ruminant ration, compared to samples of raw white and red kidney beans, which contained 370 mg/kg and 1000 mg/kg of lectin, respectively. Analysis of a different equine feed considered representative of the ration commonly fed to horses revealed lectin levels of 4.8 mg/kg.

In the studies described herein, none of the adverse effects typically seen with raw Phaseolus vulgaris beans were evident following oral administration of the Blockal dietary supplement to rats at levels providing of up to 1668 mg/kg bw of Phase 2 white kidney bean extract. Such effects were, in fact, not expected because Phase 2 is a purified extract that is standardized and routinely tested for the minimized presence of PHAs and other antinutritional or potentially toxic substances. All production lots of Phase 2 are assayed to assure compliance with the established product specifications, <3400 HAU/g for hemagglutinating activity and <40 TIU/mg for trypsin-inhibiting activity, respectively.

The marketed dietary supplement used in these toxicity studies, Blockal (Roeder 1956 Farmaceutici S.p.A., Italy), contains 55.6% Phase 2 white kidney bean extract, along with various other ingredients typically used in multivitamin/mineral preparations, including 0.1% chromium picolinate. In a review of the safety of chromium picolinate, the Institute of Medicine and National Research Council of the National Academies concluded that: “there is neither consistent evidence of reasonable expectation of harm from chromium picolinate nor sufficient evidence to raise concern regarding the safety or toxicity of chromium picolinate when used up to 1.6 mg Cr picolinate/day (200 μg Cr(III)/day) for 3–6 months.” (IOM/NRC 2004). Consumption of the Blockal dietary supplement containing Phase 2 by animals in the studies described herein would have resulted in chromium picolinate exposures of 0.74 mg/day or less, which, as expected, did not produce any toxicity.

Other physiologically active compounds present in the Blockal dietary supplement, addition to Phase 2, include chromium picolinate, vitamin B₃ and vitamin B₆. It is therefore important to recognize that these results support and are consistent with the safety of the Blockal product, including Phase 2 and other components, in amounts consumed under study conditions.

Previous toxicological studies in rats showed no mortality or significant toxicity following oral (gavage) administration of single doses up to 5 g/kg bw or multiple doses (90 days) up to 1 g/kg bw/day of the Phase 2 extract alone (Harikumar et al. 2005). In a double-blind clinical investigation, 1500 mg of Phase 2 administered over the course of 8 weeks has shown potential usefulness in the treatment of obesity and hypertriglyceridemia (Udani, Hardy, and Madsen 2004) in a study population of 27 obese adults. The results of both this acute and subacute study therefore indirectly support and are consistent with the safety in rats of the main ingredient of Blockal, the Phase 2 Starch Neutralizer (Phaseolamin 2250 or Phase 2), a standardized extract derived from the common white kidney bean.