When Bad Study Design Comes Back to Bite You (Part 2): Critical Importance of the Selection of Dose

The importance of proper study design was introduced in Part One of this series, which appeared in the March 2013 issue of the Burdock Advisor. A critical issue not discussed at that time is the selection of the top dose in subchronic or chronic toxicity studies. The need for subchronic or chronic studies in rodents to detect toxicities that could occur in humans over a lifetime of exposure has led to the hypothesis that the maximum tolerated dose (MTD) should be used as the highest dose in long-term rodent toxicity studies. The FDA advocates use of the MTD in subchronic toxicity studies for new dietary ingredients.1 As defined by the National Toxicology Program, the MTD is a dose that causes no more than a 10% decrement in weight gain compared to the control group, and no deaths, clinical signs of toxicology, or pathological lesions that would be predicted to shorten the animal’s lifespan.2 Typically, the MTD is determined from either an acute toxicity or short-term (e.g. 14 day) repeated dose-range finding study.

The rationale for use of the MTD as the highest dose in subchronic or chronic studies stems from the assumptions that the dose response curves for toxicokinetics and toxicodynamics are linear.3 However it is altogether possible that the dose response curve is not linear and toxicity only occurs when a certain threshold is exceeded. In order to do things “by the book”, investigators may not hesitate to use the MTD as the upper dose in long-term rodent studies. However, this approach may overestimate the potential of a food ingredient to produce toxicity at doses consumed by humans, as demonstrated for the low calorie sweeteners saccharin and aspartame. Both of these substances are rat carcinogens, but neither has been shown to be a human carcinogen.4 Bruce Ames has estimated that more than half of the chemicals (including “natural” chemicals) tested in both rats and mice are carcinogens in chronic rodent bioassays conducted with the MTD.5 Ames has hypothesized that the increases in tumor incidence at the MTD are due to secondary effects of administering high doses, such as excessive cell death and mitogenesis, such that any chemical, whether synthetic or natural, or genotoxic or nongenotoxic, is a likely rodent carcinogen at the MTD. Others have shown that high doses of “macroingredients” such as carbohydrates or fiber may produce toxicological effects due to secondary effects on nutrition.6

Clearly, use of the MTD as the upper bound for toxicity studies is not appropriate for all substances. One may be tempted to use the MTD, particularly if large doses of a substance need to be provided to rats to derive a suitable safety factor for the estimated human dose. The million dollar question is: “When should I use a dose lower than the MTD for the maximum dose?” If any of the following conditions applies, use of a dose lower than the MTD should be considered:

The substance is unpalatable at the MTD: The MTD should not be used if it affects the palatability of the food. In general, effects on palatability can be uncovered in a range-finding dietary study (e.g. 7-14 day). Food consumption will be decreased if the rats or mice have an aversion to the taste of the food. A potential effect on palatability of food will not be uncovered if the substance is administered by gavage.  So, in order to be certain that palatability of feed is not altered by high doses of your substance, use a dietary method of administration for range-finding studies.

The MTD could cause nutritional imbalance or nutrient displacement: Typically, at levels >5% of the diet, ingredients designed to replace a “macronutrient” such as fat, carbohydrate or protein will cause nutritional imbalances, influencing the toxicity6; that is, an animal has a maximum volume it can consume per day and if the diet is diluted by the test ingredient to the extent that even at maximum intake, the animal cannot get adequate nutrition (i.e. “nutrient displacement”), the effects of inadequate nutrition may compound or even mask toxic effects of the test substance. In some cases however, depending on the “macroingredient”, it may be possible to provide greater than 5% in the diet. The 13-week dietary no observable adverse effect level of the oligosaccharide fibermalt in rats is 15%, without alteration of the diet7, and the fat replacer olestra has been administered to rats and mice at approximately 9-10% in feed for up to two years, when test diets are supplemented with vitamins A, D, E, and K.8 Whether or not a “macroingredient” can be administered to rodents at a dietary concentration higher than 5% should be examined on a case-by-case basis and should not be attempted unless a suitable safety factor will not be achieved from administration of the test substance at 5%.

Toxicokinetics is altered at the MTD: The MTD should not be used if the dose could saturate enzymes involved in metabolism or block detoxification pathways, or cause the substance to metabolized in a manner in which it is not normally metabolized. Good examples of substances that behave in this manner are trans-anethole and fructose trans-anethole has been determined generally recognized as sage (GRAS) for use as a flavor ingredient by the Flavor Extract Manufacturer’s association (FEMA) despite the fact that high doses cause liver tumors in female rats.9 At low levels of exposure, trans-anethole is efficiently detoxified in rodents and humans primarily by O-demethylation and omega-oxidation, respectively, while epoxidation is only a minor pathway. At high dose levels in rats (particularly females), a metabolic shift occurs resulting in increased epoxidation and formation of toxic epoxides. This overwhelms the capacity for detoxification of the epoxides by glutathione S-transferase, resulting in increased potential for the epoxides to react with DNA or tissue. Because trans-anethole undergoes efficient metabolic detoxification in humans at low levels of exposure, the neoplastic effects in rats associated with high doses were not considered by FEMA to be indicative of any significant risk to human health from the use of trans-anethole as a flavoring substance.

In rats, administration of 10% fructose in water (approximately 10 g/kg bw/day) for two weeks promotes synthesis of triglycerides.10  However, plasma concentrations of triglycerides are not increased in humans after long term ingestion of up to 95th percentile intake levels of fructose (approximately 140 g/day or 2 g/kg bw/day for a 70 kg person).11 Thus, the shift in metabolism of fructose to triglycerides that occurs in rats exposed to high levels of fructose does not occur in humans ingesting fructose in a normal dietary manner.

To determine if the MTD is metabolized differently than lower doses, one could conduct a pharmacokinetic study with the MTD and a lower dose. Alternatively, the metabolic fate of a high dose of the ingredient could be modeled based on results of pharmacokinetic studies for substances with similar structures.

Toxicodynamics is altered at the MTD: The MTD should not be used if the mechanism of action of the substance is different at the MTD than at lower doses. An example of a substance that has a dose-dependent mechanism of action is resveratrol, which acts as a mixed estrogen receptor agonist/antagonist (also known as partial agonist), depending on concentration. A structure activity analysis could screen for the potential of the substance to exhibit altered toxicodynamics at high doses.

The MTD causes spurious effects at high doses: Certain substances are known to produce rodent-specific toxicities at high doses, such as saccharin (produces male rat-specific bladder cancer at high doses due to formation of a urinary precipitate) and limonene (produces male rat-specific kidney cancer at high doses due to binding to alpha 2U-globulin).4, 12 The chemical structure and physical properties of high doses of the material in biological fluids should be analyzed to determine whether high doses could produce rodent-specific toxicities that have no relevance for human risk assessment.

As mentioned in the first article in this series, proper planning of toxicity tests is essential. When contemplating whether or not to use the MTD as the upper dose in a subchronic or chronic toxicity study, make certain to take a good look at the type of substance you are testing, determine whether the MTD is unpalalatable or could cause nutritional imbalances, and perform a structure-activity analysis to determine if substances with similar structures have altered toxicokinetics or toxicodynamics or produce rodent-specific toxicities at high doses. Just because others advocate use of the MTD as highest dose in rodent toxicity studies does not necessarily mean that it should be used as the highest dose for all substances. Some ingredients (e.g. flavors) are only manufactured and used in very small amounts, and dosing at an MTD might require an entire year’s production of ingredient. The MTD, which is typically determined from a short term study, may overshoot the MTD in a longer-term study and cause effects in rodents that are inhumane or irrelevant for risk assessment of lower doses in humans. Clearly, use of the MTD as the upper bound for rodent toxicity studies is not appropriate for some substances and may derail the possibility of approval of an ingredient that is safe for use in humans at lower levels. Use of the MTD as the highest dose will require skill on the part of the protocol designer − the low dose should produce no toxicity and the middle dose should provide either no or minimal toxicity, and a suitable safety factor should be derived from one of the doses for use of the substance in humans. Burdock Group can assist with all elements of toxicity study design, including dosage levels.

References

1. FDA (2013). Draft guidance for industry: Dietary supplements. Available at http://www.fda.gov/Food/GuidanceRegulation/GuidanceDocumentsRegulatoryInformation/DietarySupplements/ucm257563.htm

2. Chhabra, R.S., Huff, J. E., Schwetz, B. S. and Selkirk, J. (1990). An overview of prechronic and chronic toxicity/carcinogenicity experimental study designs and criteria used by the National Toxicology Program. Environmental Health Perspectives 86: 313-321.

3. Dybing, E., Doe, J., Groten, J., Kleiner, J., O’Brien, J., Renwick, A.G., Schlatter, J., Steinberg, P., Tritscher, A., Walker, R. and Younes, M. (2002).  Hazard characterization of chemicals in food and diet: dose response, mechanisms and extrapolation issues. Food and Chemical Toxicology 40:237-282.

4. Nordmann, H. (2012). Low calorie foods, beverages and sweeteners. Can they really contribute to a healthier future? (Part 1). AgroFood Industry Hi-tech. 23:

5. Ames, B. N. and Gold, L. S. (1990). Chemical carcinogenesis: Too many rodent carcinogens. Proceedings National Academy Science, USA, 87: 7772-7776.

6. Borzelleca, J. F. (1996). A proposed model for safety assessment of macronutrient substitutes. Regulatory Toxicology and Pharmacology 23:S15-S18.

7. Dolan, L. C., Gietl, E., LaCognata, U., Landschütze, Marone, P.A., and Matulka, R. A. (2012). Safety evaluation of fibermalt. Food and Chemical Toxicology 50:2515-2523.

8. Wilke, D.L. (2007). GRAS Notification for olestra. Available at http://www.accessdata.fda.gov/scripts/fcn/gras_notices/707728a.pdf.

9. Newberne, P., Smith, R.L., Doull, J., Goodman, J.I., Munro, I.C., Portoghese, P.S., Wagner, B.M., Weil, C.S., Woods, L.S., Adams, T.B., Lucas, C.D., and Ford, R.A. (1999). The FEMA GRAS assessment of trans-anethole used as a flavouring substance. Flavour and Extract Manufacturer’s Association. Food and Chemical Toxicology 37: 789-811.

10. Kazumi, T., Vranic, M. and Steiner, G. (1986). Triglyceride kinetics: effects of dietary glucose, sucrose, or fructose alone or with hyperinsulinemia. American Journal of Physiology 250: E325-30.

11. Dolan, L.C., Potter, S.M., and Burdock, G.A. (2010). Evidence-based review on the effect of normal dietary consumption of fructose on development of hyperlipidemia and obesity in healthy, normal weight individuals. Critical Reviews Food Science and Nutrition 50: 53-84.

12. Hard, G. C. and Whysner, J. (1994). Risk assessment of d-limonene: an example of male-rat specific renal tumorigens. Critical Review Toxicology 24: 231-54.