Thinking about placing a toxicological test on your novel ingredient? Go no further until you read this article.

As consultants in the food and dietary supplement ingredient industry, we are involved daily with new and exciting ingredients that have emerged after decades of thought and effort. It is fulfilling to help usher these new ingredients safely and successfully to the larger market. We know all too well early choices for ingredients need to be made with careful consideration in order to be set up for success.

The Three Assays to US REgulatory status

When an ingredient is so novel, and so unlike anything available on the market, it is not uncommon for companies to know that some level of safety testing is required. There are many articles and webinars available on the internet that share general outlines for getting a novel ingredient to some type of regulatory status. Options such as new dietary ingredients notifications (NDINs), generally recognized as safe (GRAS) opinions, or food additive petitions are pathways that are commonly discussed. All of these pathways require evidence of safety and often, for novel ingredients, this means safety studies. In doing their due diligence by learning the requirements to get a novel ingredient to market, companies often hear about three assays the FDA generally wants performed to get regulatory status in the U.S. They include:

• Bacterial reverse mutation assay.
• In vitro chromosomal aberration (or micronucleus) assay.
• 90-day repeated dose rodent oral toxicity study.

THE COMPLEXITIES OF SAFETY STUDIES

Armed with this knowledge, we have witnessed companies that want to get a “jump start” on their regulatory journey go out into the contract research organization (CRO) marketplace, find the best priced services, get their ingredient to the lab, and then patiently wait for the results. When the next step in the regulatory journey leads the company to the door of consultants such as GRAS Associates, a SGS Nutrasource company, to initiate the completion of an NDIN or GRAS dossier, the results of the safety studies are handed over along with other background information on the novel ingredient (all confidentially, of course) and it is off to the races. Unfortunately, as with most things, it can turn out to not be that simple.

Not only are there many ways to perform those three studies, but there are also more options in the world of safety studies than just these studies and they may be applicable to your ingredient. Safety study recommendations and the specific protocols for conducting the studies are dependent on a number of factors, including the ingredient, the intended uses, and existing background data, just to name a few. Let’s take a deeper look at these points to see how different factors can influence how safety studies are performed.

1. THE INGREDIENT

Ingredients that are sent to CROs for safety studies need thorough and robust specifications. Creating a specification is no small undertaking. The ingredient’s manufacturing process and specifications should be finalized before testing is initiated to ensure that the ingredient utilized for the safety studies will not be substantially different from the ingredient of commerce. Other characteristics of the ingredient, such as whether it is water soluble, fat soluble, or insoluble, inform study protocols regarding the most appropriate administration vehicle or administration method. There should be a reason why test animals are administered the ingredient in their food (dietary), gavage (tube fed into rats), or drinking water. The ingredient, in whatever form it is being administered to the test animals, will also need to be measured for concentration, homogeneity, and stability to confirm that what the testing protocol says the animals received is indeed what they received. A 90-day repeated dose rodent oral toxicity study shouldn’t be started until you have evidence of stability of the test material under recommended conditions of storage for at least 6 months. This will allow for some wiggle room if a study has to be delayed for some reason.

2. EXISTING DATA

Toxicological study recommendations are also dependent on information about the substance that is in the existing scientific literature. A thorough comb through of the literature can inform the discerning reader if there are organs that may be the target of toxicological effects. For example, finding published data that shows the ingredient may influence estrogen levels means that additional tests should be added onto the 90-day rodent toxicity study, such as sex hormone levels or sperm count, which is affordable and easy to add onto studies that are already being performed. However, if the additional tests were not included in the 90-day study, there is the possibility that another study would need to be performed in order to thoroughly understand the ingredient’s estrogenic potential. Clearly time and money can be saved by examining existing data on an ingredient.

3. INTENDED CONSUMPTION LEVELS

Choosing the best dose groups the test animals will be administered in the repeated-dose rodent toxicity studies is a big decision and is nothing short of a balancing act. In designing a 90-day study, you want to strive for the level at which no effects were seen (called the no adverse effect level or NOAEL) to have a sufficient uncertainty factor for its intended use level with consumers. Uncertainty factors are buffers applied to accommodate for the differences between humans and rats, and between one individual and another. Usually, a 100-fold safety factor is considered adequate in the U.S. If dose groups are too high, there may be no NOAEL. In this case, another 90-day toxicity study would need to be performed to determine a NOAEL. If dose groups are too low, the study’s findings may not allow you to achieve your intended use level. Performing a 14-day dose range finding study can be helpful to prevent these situations but having assistance in choosing the dose groups can be the difference between study results that help you achieve your goal and those that do not.

CONSIDERATIONS FOR MACRONUTRIENTS

As always, there are times when what is best for everyone else may not be best for you. Consider macronutrients. With novel macronutrient ingredients, such as novel protein or fat sources, there are other necessary considerations. Macronutrients are consumed by humans at large amounts, which can be difficult to accommodate in toxicity testing. Macronutrients are typically added to the diet of rats for testing, and balancing of the diet or creation of special diets may be required to help prevent nutritional deficiencies or imbalances. Also, it is not uncommon that an additional control group would be added to a macronutrient study. There is usually a control group that receives no test item in all studies, but with macronutrients, use of an additional control group that is nutritionally similar to the test substance should be considered. With two controls, an observed effect can be compared to both and answer the question of if the change was due to the ingredient or the nature of the ingredient (e.g. protein or fat). Additionally, though it is rare, there are circumstances when rats are not the most appropriate animal model.

ADDITIONAL ASPECTS

There are other aspects to the testing guidelines and testing facilities that should be thought through. The test should be performed according to the most recent GLP and OECD guidelines. Other countries may have similar but different standards for testing. However, in a thorough comparison between guidelines, we have determined that many guidelines are not similar enough to the GLP and OECD standards to be sufficient for safety assessments to the U.S. standard.

These are just a handful of considerations to keep in mind when planning a toxicological study for a novel ingredient.

THE FINAL STEPS

Once the results of the studies roll in, the findings need to be sufficiently described so that other toxicologists reading the final study report can draw their own decision about the findings. We often see a lack of historical control data in reports, a vital piece of information to be able to consider if the effects seen during the study were considered “normal” or not. Furthermore, if you plan on utilizing the GRAS regulatory pathway for your ingredient, your toxicological studies will need to be published, which will require a certain expertise.

Having consultants on your team with experience performing gap analyses, toxicological safety assessments and putting together study protocols can help ensure the investment (in both time and money) in toxicological studies is not wasted. GRAS Associates has a team of experienced toxicologists that are adept at understanding the above facets of toxicological testing and more. It also provides study placement and monitoring services, which includes planning and review of toxicological protocols and counseling before the study begins, decision-making about animal welfare and unforeseen circumstances, review of the draft study report, and study publication, helping you ensure that the toxicological tests you place are performed correctly and with your best interests front and center.

About Dr. Preece

Kayla Preece has extensive experience assessing scientific and regulatory compliance of ingredients for food and dietary supplements, including botanicals, microorganisms, and more. These assessments have predominantly been for self-determined and FDA GRAS notifications and New Dietary Ingredient Notifications.

Dr. Preece has been involved in producing and publishing toxicological manuscripts for dietary ingredients and received her Diplomate of the American Board of Toxicology. Kayla is also a full member of the Society of Toxicology. She has a background in integrative medicine and practiced medicine before becoming involved with the world of regulatory affairs and safety assessment.