Development and Validation of Triticum Phytobiological Method as an Alternative Procedure for Investigating In Vivo Acute Toxicity on Mice

Abstract

The goal of this study was to validate an alternative method for determining in vivo acute toxicity using vegetal material instead of laboratory animals, starting from the phytobiological method known also as the Triticum technique. We set out to demonstrate that vegetal cells have similar sensitivity to some toxic agents as animal cells, in which case a statistical correlation could be established. A series of new compounds synthesized by the Romanian National Institute for Chemical Pharmaceutical Research and Development as potential β3 adrenergic receptors agonists were tested for their acute toxicity using classic animal exposure models, before investigating possible anti-diabetic and anti-obesity effects. We then determined whether similar conclusions might be reached exposing vegetal material to the same agents. We successfully demonstrated that plants are affected in a very similar way as animals when exposed to some potentially toxic agents, providing new possibilities for ending unethical animal experiments.

Introduction

Toxicity studies are an essential part of any research meant to develop new drugs. At present no study on toxicity can be undertaken without animal testing, and in order to achieve statistical significance, it may be necessary to use a relatively high number of individuals. Bioethical considerations, however, emphasize the need to use as few animals as possible in any given test. Thus emerges a conflict between the need for statistics validation and the bioethics constraints of any experimental study. Solving this ongoing dilemma will require new methods for determining toxicity without the use of lab animals. A series of in silico/ in vitro/ in vivo correlations have already been established regarding toxicity tests by our research team [1,2].

Generally, the humane use of animals in research is governed by three principles: replace, reduce, refine. Replacement refers to the use of non-sentient animals or materials instead of conscious live animals. Reduction involves decreasing the number of animals used in a specific procedure or experiment. Refinement implies the use of advanced techniques, able to decrease the magnitude and incidence of animal pain and distress. These three principles were established in 1959 based on the writings of Russell and Burch, with the intention of advancing a more ethical and humane perspective regarding the use of animals in experimental studies [3].

We developed the Triticum phytobiological method as an alternative method to determine in vivo acute toxicity by introducing a new and original quantitative parameter: the inhibitory concentration 50% (IC50) calculated by the graphic method of the regression curves. This in vitro parameter was intended as an alternative to the in vivo parameter – lethal dose 50% (LD50) [4].

Materials and methods

The Triticum phytobiological method consists of exposing wheat seeds to 6 molar dilutions of a water soluble compound and measuring radicular elongation of the germinating seeds for five consecutive days. Microscopic cellular changes are also observed. IC50 is meant to show the correlation between the radicular growth and the concentration of the substance in contact with the seeds.

The in vivo acute toxicity in mice was determined for 2 new series of compounds, potentially β3 adrenergic receptor agonists by using the regression curve method. Taking into account the observed LD50, compounds were classified using the 1956 Hodge – Sterner toxicity scale.

The new method was developed by observing the correlation between the phytobiologic toxicity (IC50) and in vivo acute toxicity in mice (LD50) using compounds C1, C2, C3. The validation of the Triticum method was accomplished using compounds belonging to series A (A1-A8) [5,6,7].

Statistical evaluation of the results was performed using special software, GraphPad Prism version 5.01. This software analyzes two group populations, either with normal distributions using the Student t test, or with skewed distributions using the Mann-Whitney test. More than 2 groups are analyzed using ANOVA. D`Agostino – Pearson test was used to determine whether the population is distributed normally.

All experimental procedures were carried out in accordance with the European Directive 2010/63/UE of 22nd September 2010, and The Romanian Government Ordinance 37/30.01.2002 regarding the protection of animals used for experimental and other scientific purposes. All experimental procedures were approved by the Ethical Committee of the Faculty of Pharmacy Bucharest.

Results

The LD50 values were determined using regression curves. The classification of the compounds belonging to series C, in accordance to the Hodge – Sterner toxicity scale, is shown in

Table 1. Correlation between the in vivo acute toxicity (LD50%) and the phytobiologic toxicity (IC50%). Hodge-Sterner classification of the compounds from series C.

. In the case of compounds belonging to series A, LD1 could not be determined because it was higher than the maximum dose that can be administrated as a suspension in mice (1000 mg/kg).

Regression curves for the IC50 determination were linear for a domain of 3-4 concentrations and the regression factors generally have values higher than 0.8. The regression curve for compound C2 is shown in the following Figure 1.

The regression curve for compound C3 is shown in Figure 2.

Using the regression equation, the value of IC50 was determined as illustrated for compound C2 and C3:

The calculated value of this innovative parameter is consistent with a low level of toxicity, confirmed by the vivo tests on mice. The statistical significance of radicular elongation variations for the groups treated with different concentrations of compound C2 in the 3^rd day of measurements is shown in

Table 2. Statistical significance of radicular elongation variation under treatment with C2 in 3rd day of measurements.

.

The statistical significance of radicular elongation variations for the groupstreated with different concentrations of compound C3 in the 3^rd day of measurements is shown in

Table 3. Statistical significance of radicular elongation variation under treatment with C3 in the 3rd day of measurements.

.

The influence of various concentrations of compound C2 on radicular elongation is illustrated in Figure 3:

The influence of various concentrations of compound C3 on radicular elongation is illustrated in Figure 4:

The validation of the Triticum method as a reliable alternative to the classic in vivo toxicity tests was accomplished by applying the technique to the compounds of the series A (A1-A8). The regression curve for compound A2 is shown in Figure 5.

The regression curve for compound A5 is shown in Figure 6.

Using the regression equation, the value of IC50 was determined as illustrated for compound A2:

The calculated value of the inhibitory concentration 50% (IC50) is consistent with a low level of toxicity confirmed by in vivo tests on mice and reflected through a high lethal dose 50%.

Using the regression equation, the value of IC50 was determined as illustrated for compound A5:

The statistical significance of radicular elongation variations for the groups treated with different concentrations of compound A2 on the 3^rd day of measurements is shown in

Table 4. Statistical significance of radicular elongation variation under treatment with A2 in the 3rd day of measurements.

:

The statistical significance of radicular elongation variations for the groups treated with different concentrations of compound A5 in the 3^rd day of measurements is shown in

Table 5. Statistical significance of radicular elongation variation under treatment with A5 in the 3rd day of measurements.

:

The influence of various concentrations of compound A2 on radicular elongation is illustrated in Figure 7:

The influence of various concentrations of compound A5 on radicular elongation is illustrated in Figure 8:

A strong correlation between IC50 and LD50 was observed in all studied compounds.

Discussion

Using the phytobiological method known also as the Triticum technique, this study investigatedan alternative method for determining in vivo acute toxicity using vegetal material instead of laboratory animals. We set out to demonstrate that vegetal cells have similar sensitivity to some toxic agents as animal cells, in which case, a statistical correlation could be established. A series of new compounds synthesized by the Romanian National Institute for Chemical – Pharmaceutical Research and Development as potential β3 adrenergic receptors agonists were tested for their acute toxicity using classical animal exposure models, before investigating possible anti-diabetic and anti-obesity effects [5,6,7].

The validation of the phytobiological method as an alternative for preliminary toxicological evaluations is an original contribution toward identifying bioethical ways to assess the toxicity of new substances without using laboratory animals. In order to perfect this method, we have used 2 new series of compounds with potential β3 adrenergic properties. The method proved to be reliable and reproducible. The improved Triticum technique has confirmed the level of toxicity attributed to most of the studied compounds by the classic in vivo acute toxicity tests performed on mice [8,9].

Only in case of compounds A1, A3, and A8 was the correlation between the Triticum method and the in vivo toxicity test weaker, probably due to a significant difference between the in vivo bioavailability after oral administration and the diffusion process through the membranes of vegetal cells.

The ideal observation day in the Triticum test proved to be the 3^rd day, as it showed an evolution of the IC50 in a 2:1 geometric ratio for compounds belonging to different toxicity classes, smilar to sodium fluoride administration [10].

A good correlation was observed between the radicular elongation and the cellular multiplication, as well as between the toxicity of compounds C2, C3 expressed by LD50 in mice and that expressed by IC50 in the Triticum test [11].

The regression curves for the IC50 determination were linear for a domain of 3-4 concentrations and regression coefficients generally had values higher than 0.8 [12,13].

Conclusions

Experimental studies may involve a potential conflict between two distinct but interrelated specialties, represented by statistical reliability (requiring a large number of cases) and bioethics (suggesting the need to reduce the number of casesto as few as possible). Solving this ongoing dilemma requires developing new methods for determining toxicity without the use of lab animals.

In this study we presented an alternative for determining in vivo acute toxicity using vegetal material instead of laboratory animals, starting from the phytobiological method known also as the Triticum technique. This technique successfully showed that plants are affected in a very similar way as animals when exposed to some potentially toxic agents, thus identifying a new possibility for limiting or ending unethical animal experiments.

Although anatomical and morphological differences between animal and vegetal cells limits, to some extent, the use of the Triticum method for determining the toxicity level of new compounds, it nevertheless offers new directions in the field of toxicological research.