Considerations for determining safety of probiotics: A USP perspective.

A history of safe use is a backbone of safety assessments for many current probiotic species, however, there is no global harmonization regarding requirements for establishing probiotic safety for use in foods and supplements. As probiotic manufacturers are increasingly seeking to use new strains, novel species, and next-generation probiotics, justification based on a significant history of use may be challenged. There are efforts underway by a variety of stakeholders, including the United States Pharmacopeia (USP), to develop best practices guidelines for assessing the quality and safety of probiotics. A current initiative of the USP seeks to provide expert advice specific to safety considerations for probiotics. Toward this goal, this review provides a helpful summary guide to global regulatory guidelines. We question the suitability of traditional animal toxicology studies designed for testing chemicals for relevance in assessing probiotic safety. This includes discussion of the use of excessive dose levels, the length of repeated dose toxicity studies needed, and the most suitable animal species used in toxicology studies. In addition, the importance of proper manufacturing practices with regard to final product safety are also included. Thus, an outline of essential parameters of a comprehensive safety assessment for a probiotic are provided.

Subchronic and genetic safety evaluation of a calcium fructoborate in rats.

A branded calcium fructoborate product, a nature-identical calcium salt of bis (fructose) ester of boric acid found in plants and a natural source of boron in the human diet and sold under the trade name FruiteX-B(®) Brand Calcium Fructoborate ("FrxB"), was evaluated in a 90-day dietary toxicity study and two genotoxicity studies. In the 90-day study, four groups of 10 male and 10 female Crl:SD CD(®) IGS rats were fed diets with FrxB admixtures of 0.56, 1.12, and 1.68% dietary concentration, providing mean overall daily intakes of FrxB in male rats of 385.8, 774.9, and 1161.3 mg/kg bw/day, and 392.1, 784.4, and 1171.1 mg/kg bw/day in female rats. There were no mortalities, no clinical or ophthalmologic signs, body weight, body weight gain, food consumption, food efficiency, Functional Observational Battery (FOB), or Motor Activity (MA) findings associated with the administration of FrxB. There were no adverse changes in hematology, coagulation, clinical chemistry, or urinalysis parameters in male or female rats considered the result of test substance administration. At necropsy, there were no macroscopic, histopathological findings, or organ weight changes deemed related to administration of the test substance. Under the conditions of this study, based on the toxicological endpoints evaluated, the no-observed-adverse-effect level (NOAEL) for FrxB in the diet was 1161.3 and 1171.1 mg/kg bw/day in male and female rats, respectively. Bacterial mutagenicity studies and a micronucleus test using Chinese hamster V79 cells demonstrated no mutagenic or genotoxic potential of the tested brand of calcium fructoborate.

Effects of genetic, processing, or product formulation changes on efficacy and safety of probiotics.

Commercial probiotic strains for food or supplement use can be altered in different ways for a variety of purposes. Production conditions for the strain or final product may be changed to address probiotic yield, functionality, or stability. Final food products may be modified to improve flavor and other sensory properties, provide new product formats, or respond to market opportunities. Such changes can alter the expression of physiological traits owing to the live nature of probiotics. In addition, genetic approaches may be used to improve strain attributes. This review explores whether genetic or phenotypic changes, by accident or design, might affect the efficacy or safety of commercial probiotics. We highlight key issues important to determining the need to re-confirm efficacy or safety after strain improvement, process optimization, or product formulation changes. Research pinpointing the mechanisms of action for probiotic function and the development of assays to measure them are greatly needed to better understand if such changes have a substantive impact on probiotic efficacy.

Tamarind seed polysaccharide: a 28-day dietary study in Sprague-Dawley rats.

Forty male and 40 female Crl:SD® CD® IGS rats were fed diets containing 0, 40,000, 80,000, or 120,000 ppm tamarind seed polysaccharide (equivalent to 3450.8, 6738.9, or 10 597.1 mg/kg bw/day and 3602.1, 7190.1, or 10,690.7 mg/kg bw/day for males and females, respectively) for 28 days. Animals were observed for adverse clinical signs, body weight, feed consumption, hematology and clinical chemistry parameters, urinalysis values were recorded, and at the end of the study the rats underwent a full necropsy. Functional Observational Battery (FOB) and Motor Activity (MA) tests were performed on all animals. There were no mortalities, no clinical or ophthalmologic signs, body weight, body weight gain, food consumption and food efficiency, FOB or MA findings associated with the administration of tamarind seed polysaccharide. Initial statistically significant decreases in body weight gain and food consumption resolved after the first week and were considered the result of reduced palatability. There were no adverse changes in hematology, coagulation, clinical chemistry or urinalysis parameters in male or female rats considered the result of test substance administration. At necropsy, there were no macroscopic, histopathological findings, estrus cycle, or organ weight changes deemed related to administration of the test substance. Under the conditions of this study and based on the toxicological endpoints evaluated, the no-observed-adverse-effect level (NOAEL) for tamarind seed polysaccharide in the diet was the highest concentration tested of 120,000 ppm (equivalent to 10,597 mg/kg bw/day and 10,691 mg/kg bw/day for male and female rats, respectively).

Safety assessment and caloric value of partially hydrolyzed guar gum.

Guar gum and partially hydrolyzed guar gum (PHGG) are food ingredients that have been available for many years. PHGG is the partially hydrolyzed product from guar gum obtained from the Indian cluster bean (Cyanopsis tetragonolopus). The gum (CAS Registry No. 9000-30-0) is composed of galactomannan, a gel-forming polysaccharide with a molecular weight ranging from 200 to 300 kDa. The intact and partially hydrolyzed forms have multiple food applications. The intact material can be used to control the viscosity, stability, and texture of foods. PHGG is highly soluble and has little physical impact on foods. Both forms are indigestible but are excellent sources of fermentable dietary fiber. The caloric value of intact guar gum is accepted as 2.0, whereas the caloric value of PHGG has not been firmly established. It is the goal of this paper to review the chemistry, safety, in vivo effects, and caloric value of PHGG.

Health claims substantiation for probiotic and prebiotic products.

The topic of "Health Claims Substantiation for Probiotic and Prebiotic Products" was discussed at the 8 (th) annual International Scientific Association for Probiotics and Prebiotics (ISAPP) meeting. The topic is especially timely considering that the regulatory review process for health benefit claims on probiotic and prebiotic products in Europe has not resulted in a single claim being approved (120 negative opinions on probiotic claims and 19 negative opinions on prebiotic claims through February 2011). This situation in Europe and elsewhere has driven companies to seek clarity on a research path that would stand up to scientific scrutiny as well as satisfy regulatory demands for health claim substantiation. It can be challenging to negotiate rigid regulatory distinctions, such as between health and disease, when these states are more realistically represented by continua. One research approach focused on improved homeostasis is explored as a statistically robust approach to measuring physiological parameters in healthy populations, which are the required target for food and supplement claims. Diverse global regulatory frameworks complicate this issue, and harmonization of different approaches globally would simplify requirements for industry, decrease consumer confusion and improve the scientific framework for the research community to set up appropriate research pathways. This report highlights key points from this discussion. 

Safety assessment of probiotics for human use.

The safety of probiotics is tied to their intended use, which includes consideration of potential vulnerability of the consumer or patient, dose and duration of consumption, and both the manner and frequency of administration. Unique to probiotics is that they are alive when administered, and unlike other food or drug ingredients, possess the potential for infectivity or in situ toxin production. Since numerous types of microbes are used as probiotics, safety is also intricately tied to the nature of the specific microbe being used. The presence of transferable antibiotic resistance genes, which comprises a theoretical risk of transfer to a less innocuous member of the gut microbial community, must also be considered. Genetic stability of the probiotic over time, deleterious metabolic activities, and the potential for pathogenicity or toxicogenicity must be assessed depending on the characteristics of the genus and species of the microbe being used. Immunological effects must be considered, especially in certain vulnerable populations, including infants with undeveloped immune function. A few reports about negative probiotic effects have surfaced, the significance of which would be better understood with more complete understanding of the mechanisms of probiotic interaction with the host and colonizing microbes. Use of readily available and low cost genomic sequencing technologies to assure the absence of genes of concern is advisable for candidate probiotic strains. The field of probiotic safety is characterized by the scarcity of studies specifically designed to assess safety contrasted with the long history of safe use of many of these microbes in foods.

Guide to designing, conducting, publishing and communicating results of clinical studies involving probiotic applications in human participants.

The heterogeneity of human clinical trials to assess the effectiveness of probiotics presents challenges regarding interpretation and comparison. Evidence obtained from clinical trials among a population with a disease or specific risk factors may not be generalizable to healthy individuals. The evaluation of interventions in healthy persons requires careful selection of outcomes due to the absence of health indicators and the low incidence of preventable conditions. Given the tremendous resources invested in such trials, development of consistent approaches to assessing the effectiveness of probiotics would be beneficial. Furthermore, the reporting, presentation and communication of results may also affect the validity of the scientific evidence obtained from a trial. This review outlines the challenges associated with the design, implementation, data analysis and interpretation of clinical trials in humans involving probiotics. Best practices related to their design are offered along with recommendations for enhanced collaboration to advance research in this emerging field.

Health-benefit claims for probiotic products.

Manufacturers wish to communicate the benefits of probiotics in advertising and labeling with lawful and adequately substantiated claims. Regulatory and substantiation requirements differ for products intended to cure, treat, prevent, or mitigate a disease; to reduce a healthy individual's risk of developing a disease; or to affect the structure or function of the body. Food labeling is regulated by the US Food and Drug Administration, and advertising is regulated by the Federal Trade Commission; the standards and methods used by these agencies differ. Food manufacturers must design their claims regarding the benefits of probiotics with the regulatory environment in mind and must develop their research plans to provide evidence that satisfies the agencies' substantiation requirements. This article offers an overview of the applicable laws and regulations, what they mandate regarding legitimate claims, and the issues regarding the design of research to substantiate such claims.

Safety studies regarding a standardized extract of fermented wheat germ.

"Avemar pulvis" is a powder consisting of an aqueous extract of fermented wheat germ, with the drying aids maltodextrin and silicon dioxide, standardized to contain approximately 200 microg/g of the natural constituent 2,6-dimethoxy-p-benzoquinone. The results of toxicological and clinical studies of this product demonstrate its safety for its intended use as a dietary supplement ingredient in the United States. Avemar pulvis has been used in Hungary since 1998 and is approved in that country, as well as in the Czech Republic, Bulgaria, and Romania, as a "medical nutriment for cancer patients." Acute and subacute toxicity studies using rodents orally administered Avemar pulvis showed that dose levels (2000 to 3000 mg/kg body weight [bw]/day) exceeding the normal recommended oral dosage (8.5 g/day or 121 mg/kg bw/day for a 70-kg individual) by up to approximately 25-fold caused no adverse effects. The test substance showed no evidence of mutagenicity or genotoxicity in vitro or in vivo. Clinical studies using Avemar pulvis as a supplement to drug therapy in cancer patients at doses of 8.5 g/day not only showed no evidence of toxicity, but also showed a reduction in the side effects of chemotherapy. Overall, it was concluded that Avemar pulvis would not be expected to cause adverse effects under the conditions of its intended use as an ingredient in dietary supplements.

90-day oral (gavage) study in rats with galactooligosaccharides syrup.

A 90-day oral (gavage) study was conducted in male and female Sprague Dawley rats to investigate the safety of Vivinal galactooligosaccharides (GOS) syrup at 2500 or 5000 mg/kg bw/day. A reference control containing fructooligosaccharides (FOS) was used to match the oligosaccharide and digestible sugars in the test material (approximately 45% and 30%, respectively) and to assess if these had an impact on food consumption. Measurements included clinical observations, body weights, food consumption, hematology, clotting parameters, blood chemistries, urinalysis, ophthalmologic examinations, gross necropsies, organ weights, and histological examinations. There were no effects of feeding GOS syrup at either concentration on any parameter except food consumption. Statistically significant decreases (7-13%) in food consumption were seen in both sexes in the GOS syrup-treated animals at 5000 mg/kg bw/day and animals treated with the FOS control when compared to the reverse osmosis deionized (RODI) water controls. Based on the lack of toxicological effects in the study, the NOAEL for Vivinal GOS syrup is 5000 mg/kg bw/day when administered by gavage for 90 consecutive days.