Carolina Dizioli Rodrigues Oliveira1,*, Camila Queiroz Moreira2, Lilian Rose Marques de Sá2, Helenice de Souza Spinosa2, Mauricio Yonamine1
Article first published online: 25 OCT 2010
DOI: 10.1002/bdrb.20271 © 2010 Wiley-Liss, Inc.
To the Editor:
Response to the Letter to the Editor Ref: BDRD-10-0049—Toxicity of chronic ayahuasca administration to the pregnant rat: how relevant it is regarding the human, ritual use of ayahuasca? Author: Rafael Guimarães dos Santos.
First of all, we thank the author for his important comments regarding our recently published article entitled “Maternal and developmental toxicity of ayahuasca in Wistar rats.” This is a great opportunity to discuss some points that were not detailed in the original text. As the author states in his letter, this is the first study that shows toxic effects of chronic exposure to ayahuasca in pregnant rats and their offspring.
A basic tenet of toxicology is that “experimental results in animals, when properly qualified, are applicable to humans,” but it is recognized that differences in responses to toxic substances may occur among different species. Another principle is that exposure of experimental animals to chemicals in high doses is a necessary and valid method to determine the potential hazards posed to humans. This principle is based on the quantal dose–response concept that the incidence of an effect in a population is greater as the dose or exposure increases. Practical considerations in the design of experimental model systems require that the number of animals used in toxicology experiments always be small compared with the size of at risk human populations (Eaton and Gilbert, 2008). This is the reason why high doses are also used in experimental studies and not necessarily to reproduce the conditions to which humans are generally exposed.
The main objective of reproductive and developmental toxicology tests is to identify the effects caused by exposure to all types of pharmaceuticals and a variety of chemical substances on mammalian reproductive functions in all stages within the developmental process (U.S. FDA, 2000). If a drug has been shown to have teratogenic effects in animals, but other preclinical tests show that it is a safe and effective drug, its use may be permitted for humans, but restriction of its use during pregnancy must be recommended until its safety is totally proven through other experimental assays or clinical trials.
The most commonly used protocol for assessing developmental toxicity in laboratory animals involves the administration of a test substance to pregnant animals (usually mice, rats, or rabbits) during the period of major organogenesis followed by evaluation of maternal responses throughout pregnancy and examination of the dam and the uterine contents just prior to term (U.S. EPA, 1998; U.S. FDA, 2000; OECD, 2001).
When teratogenic information about a test substance is already available, it is possible to define a day (or days) for administration of the substance. When prior information on teratogenic toxicity is not available, it is recommended that the test substance be administered during all days of organogenesis. In the context of the ritual use of ayahuasca, it is known that members may consume the tea as often as twice monthly or even more. It is possible that pregnant women, in general, drink less frequently but certainly there appears to be no unique pattern for its use. Moreover, given the differences which exist between the average gestational period of Wistar rats and that of humans (22 days in Wistar rats×259–294 days in humans) it would not be reasonable to infer the same absolute frequency of ayahuasca consumption observed in humans to the experimental design conducted in rats. Even with the use of ayahuasca twice a month, a woman could have exposed her fetus 18 times during her gestation.
The definition of the period and the doses for administration of ayahuasca in animals was based on the international governmental guidelines. The guideline protocols require experiments to have a minimum of three concentration levels plus a current vehicle control (four groups total). The top concentration level should be chosen to result in demonstrable a slight maternal and possibly developmental toxicity but not cause clinical signs of toxicity. The spacing of the doses may be arithmetic or logarithmic. The low dose should be selected, if possible, to allow application of safety factors during risk assessment and still be above any expected human exposure levels (U.S. EPA, 1998; OECD, 2001; Hood, 2006). Thus, in our study, the lowest dose used for the treatment of animals was a dose arithmetically calculated, and still below of the dose recommended by the guidelines.
Acute and subchronic toxicity tests were previously performed to verify clinical and toxic signals of the animals with the aim to select the high dose to be used in our experiments. These tests were based on U.S. EPA guideline (2002), and clinical examinations were made in 30, 60, 120, 240, and 480 min after ayahuasca administration by gavage. The first dose of ayahuasca administered in animals to evaluate the acute toxicity was the TD50 (dose 50 times more concentrated compared to the equivalent typical dose in humans), which causes death of rats (n=2) 480 min after administration. After 120 min, relevant toxic signs were observed, with an increase in the frequency of convulsions and spasms, difficulty in breathing, increased heartbeat, high irritability and vocalization, and death of the animals. The dose was successively decreased and TD35 was estimated as the LD50 for the ayahuasca in rats. The TD10 was the minimum dose found in which no clinical signs were observed and was selected as the high dose for use in our experiments. The high dose was the dose below the estimated LD50, which allowed a safety treatment yet was high enough to fulfill the requirements of the protocols.
In fact, LD50 is a very important parameter for risk assessment. Gable (2007) found that data on the lethality of dimethyltryptamine (DMT) in mice could be successfully extrapolated to humans. As the β-carboline admixtures in ayahuasca appear to be less toxic than DMT, the attention was focused on the DMT component. A traditional rule for scaling unknown differences among species is simply to assume that humans are 10 times more sensitive than rodents. Since there was no previous information on the LD50 of DMT, this author made the assumption that humans are 20 times more sensitive than rodents. This resulted in a LD50 for humans of 1.6 mg/kg body weight when DMT is administered intravenously or 112 mg for a standard 70 kg person. However, the bioavailability of an oral dose is known to be lower. Assuming that the oral bioavailability is 1/5 compared with intravenous administration, Gable (2007) estimated that the oral LD50 of DMT would be 8.0 mg/kg and that the lethal dose of ayahuasca in humans would probably be 20 times higher than the typical ceremonial dose.
Risk assessment is the systematic scientific characterization of potential adverse health effects resulting from human exposures to hazardous agents. It requires qualitative information about the strength of the evidence and the nature of the outcomes as well as quantitative assessment of the exposures, host susceptibility factors, potential magnitude of the risk as well as a description of the uncertainties in the estimates and conclusions (Faustman and Omenn, 2008).
The goal of risk identification is to identify chemicals which, following exposure, may impair biological development in humans. Evaluation of such chemicals is carried out using experimental animals and if developmental toxic effects are observed in the test animals the data is then used to assess the risk to humans. Sometimes, manifestations of developmental toxicity observed in experimental animals are not observed in humans exposed to the same toxicant. Similarly, manifestations of developmental toxicity observed in humans are not always reproduced in experimental animals. The absence of uniformity of response is not surprising; however, differences between human and experimental animal exposures are considered (Mattison, 2002). It has been estimated that more that 4,100 chemicals have been tested for teratogenicity and 9% of them produces equivocal experimental results. An example of this is the famous case of thalidomide that was introduced in 1956 as a sedative/hypnotic and was used throughout the world as a sleep aid and to ameliorate nausea and vomiting during pregnancy. Animal studies revealed differences in species sensitivities; no effects were observed in hamsters and mice but other studies revealed that malformations were induced in rabbits and non-human primates. No apparent toxicity or addictive properties in humans or adult animals were initially observed at therapeutic exposure levels. Further studies of the relationship between periods of drug use and type of malformation established that thalidomide was teratogenic between 20 and 36 days after human fertilization (Rogers and Hoffmann, 2008).
Our study demonstrated that the exposure to ayahuasca can produce dose-dependent maternal and developmental toxicity in Wistar rats. Certainly, the data also suggest the need for additional research addressing the risk of developmental toxicity in humans exposed to ayahuasca. Our study presented the first evaluation of the toxicity of ayahuasca in animals during the gestation period. Confirmation of toxicity and risk associated with its use should be made with additional experiments, particularly with different species. The mechanisms by which these toxic effects occur in animals and whether or not the same process occurs in humans are important issues which need to be clarified.