Brain Cortical and Hippocampal Dopamine: A New Mechanistic Approach for Eurycoma longifolia Well-Known Aphrodisiac Activity and Its Chemical Characterization

Eurycoma longifolia Jack (Fam.: Simaroubaceae), known as Tongkat Ali (TA), has been known as a symbol of virility and sexual power for men. Metabolic profiling of the aqueous extract of E. longifolia (AEEL) using UPLC-MS/MS in both positive and negative modes allowed the identification of seventeen metabolites. The identified compounds were classified into four groups: quassinoids, alkaloids, triterpenes, and biphenylneolignans. AEEL is considered safe with oral LD50 cut-off >5000 mg/kg. Oral administration of 50, 100, 200, 400, or 800 mg/kg of AEEL for 10 consecutive days to Sprague-Dawley male rats caused significant reductions in mounting, intromission, and ejaculation latencies and increased penile erection index. AEEL increased total body weight and relative weights of seminal vesicles and prostate. Total and free serum testosterone and brain cortical and hippocampal dopamine content was significantly elevated in treated groups with no significant effects on serotonin or noradrenaline content.


1.
OECD Guidelines for the Testing of Chemicals are periodically reviewed in the light of scientific progress or changing assessment practices. The original Guideline 423 was adopted in March 1996 as the second alternative to the conventional acute toxicity test, described in Test Guideline 401. Based on the recommendations of several expert meetings, revision was considered timely because: i) international agreement has been reached on harmonised LD50 cut-off values for the classification of chemical substances, which differ from the cut-offs recommended in the 1996 version of the Guideline, and ii) testing in one sex (usually females) is now considered sufficient.

2.
The acute toxic class method (1) set out in this Guideline is a stepwise procedure with the use of 3 animals of a single sex per step. Depending on the mortality and/or the moribund status of the animals, on average 2-4 steps may be necessary to allow judgement on the acute toxicity of the test substance. This procedure is reproducible, uses very few animals and is able to rank substances in a similar manner to the other acute toxicity testing methods (Test Guidelines 420 and 425). The acute toxic class method is based on biometric evaluations (2)(3)(4)(5) with fixed doses, adequately separated to enable a substance to be ranked for classification purposes and hazard assessment. The method as adopted in 1996 was extensively validated in vivo against LD50 data obtained from the literature, both nationally (6) and internationally (7).

3.
Guidance on the selection of the most appropriate test method for a given purpose can be found in the Guidance Document on Acute Oral Toxicity Testing (8). This Guidance Document also contains additional information on the conduct and interpretation of Test Guideline 423.

4.
Definitions used in the context of this Guideline are set out in Annex 1.

5.
Test substances, at doses that are known to cause marked pain and distress due to corrosive or severely irritant actions, need not be administered. Moribund animals, or animals obviously in pain or showing signs of severe and enduring distress shall be humanely killed, and are considered in the interpretation of the test results in the same way as animals that died on test. Criteria for making the decision to kill moribund or severely suffering animals, and guidance on the recognition of predictable or impending death, are the subject of a separate Guidance Document (9).

6.
The method uses pre-defined doses and the results allow a substance to be ranked and classified according to the Globally Harmonised System for the classification of chemicals which cause acute toxicity (10).
In principle, the method is not intended to allow the calculation of a precise LD 50 , but does allow for the determination of defined exposure ranges where lethality is expected since death of a proportion of the animals is still the major endpoint of this test. The method allows for the determination of an LD50 value only when at least two doses result in mortality higher than 0% and lower than 100%. The use of a selection of pre-defined doses, regardless of test substance, with classification explicitly tied to number of animals observed in different states improves the opportunity for laboratory to laboratory reporting consistency and repeatability.

8.
The testing laboratory should consider all available information on the test substance prior to conducting the study. Such information will include the identity and chemical structure of the substance; its physico-chemical properties; the result of any other in vivo or in vitro toxicity tests on the substance; toxicological data on the structurally related substances; and the anticipated use(s) of the substance. This information is necessary to satisfy all concerned that the test is relevant for the protection of human health and will help in the selection of the most appropriate starting dose.

9.
It is the principle of the test that, based on a stepwise procedure with the use of a minimum number of animals per step, sufficient information is obtained on the acute toxicity of the test substance to enable its classification. The substance is administered orally to a group of experimental animals at one of the defined doses. The substance is tested using a stepwise procedure, each step using three animals of a single sex (normally females). Absence or presence of compound-related mortality of the animals dosed at one step will determine the next step, i.e.; − no further testing is needed, − dosing of three additional animals, with the same dose − dosing of three additional animals at the next higher or the next lower dose level.

10.
Details of the test procedure are described in Annex 2. The method will enable a judgement with respect to classifying the test substance to one of a series of toxicity classes defined by fixed LD50 cut-off values.

11.
The preferred rodent species is the rat, although other rodent species may be used. Normally females are used (9). This is because literature surveys of conventional LD50 tests show that, although there is little difference in sensitivity between the sexes, in those cases where differences are observed females are generally slightly more sensitive (11). However if knowledge of the toxicological or toxicokinetic properties of structurally related chemicals indicates that males are likely to be more sensitive, then this sex should be used. When the test is conducted in males adequate justification should be provided.

12.
Healthy young adult animals of commonly used laboratory strains should be employed. Females should be nulliparous and non-pregnant. Each animal, at the commencement of its dosing, should be between 8 and 12 weeks old and its weight should fall in an interval within + 20 % of the mean weight of any previously dosed animals.

3/14
Housing and feeding conditions

13.
The temperature in the experimental animal room should be 22ºC (+ 3ºC). Although the relative humidity should be at least 30% and preferably not exceed 70% other than during room cleaning the aim should be 50-60%. Lighting should be artificial, the sequence being 12 hours light, 12 hours dark. For feeding, conventional laboratory diets may be used with an unlimited supply of drinking water. Animals may be group-caged by dose, but the number of animals per cage must not interfere with clear observations of each animal.

14.
The animals are randomly selected, marked to permit individual identification, and kept in their cages for at least 5 days prior to dosing to allow for acclimatisation to the laboratory conditions.

15.
In general test substances should be administered in a constant volume over the range of doses to be tested by varying the concentration of the dosing preparation. Where a liquid end product or mixture is to be tested however, the use of the undiluted test substance, ie at a constant concentration, may be more relevant to the subsequent risk assessment of that substance, and is a requirement of some regulatory authorities. In either case, the maximum dose volume for administration must not be exceeded. The maximum volume of liquid that can be administered at one time depends on the size of the test animal. In rodents, the volume should not normally exceed 1mL/100g of body weight: however in the case of aqueous solutions 2 mL/100g body weight can be considered. With respect to the formulation of the dosing preparation, the use of an aqueous solution/suspension/emulsion is recommended wherever possible, followed in order of preference by a solution/suspension/emulsion in oil (e.g. corn oil) and then possibly solution in other vehicles. For vehicles other than water the toxicological characteristics of the vehicle should be known. Doses must be prepared shortly prior to administration unless the stability of the preparation over the period during which it will be used is known and shown to be acceptable.

16.
The test substance is administered in a single dose by gavage using a stomach tube or a suitable intubation canula. In the unusual circumstance that a single dose is not possible, the dose may be given in smaller fractions over a period not exceeding 24 hours.

17.
Animals should be fasted prior to dosing (e.g. with the rat, food but not water should be withheld over-night, with the mouse, food but not water should be withheld for 3-4 hours). Following the period of fasting, the animals should be weighed and the test substance administered. After the substance has been administered, food may be withheld for a further 3-4 hours in rats or 1-2 hours in mice. Where a dose is administered in fractions over a period it may be necessary to provide the animals with food and water depending on the length of the period.

Number of animals and dose levels
Three animals are used for each step. The dose level to be used as the starting dose is selected from one of four fixed levels, 5, 50, 300 and 2000 mg/kg body weight. The starting dose level should be that which is most likely to produce mortality in some of the dosed animals. The flow charts of Annex 2 describe the procedure that should be followed for each of the starting doses.

19.
When available information suggests that mortality is unlikely at the highest starting dose level (2000 mg/kg body weight), then a limit test should be conducted. When there is no information on a substance to be tested, for animal welfare reasons it is recommended to use the starting dose of 300 mg/kg body weight.

20
The time interval between treatment groups is determined by the onset, duration, and severity of toxic signs. Treatment of animals at the next dose, should be delayed until one is confident of survival of the previously dosed animals.

21.
Exceptionally, and only when justified by specific regulatory needs, the use of additional upper dose level of 5000 mg/kg body weight may be considered (see Annex 3). For reasons of animal welfare concern, testing of animals in GHS Category 5 ranges (2000-5000mg/kg) is discouraged and should only be considered when there is a strong likelihood that results of such a test have a direct relevance for protecting human or animal health or the environment.

22.
The limit test is primarily used in situations where the experimenter has information indicating that the test material is likely to be nontoxic, i.e., having toxicity only above regulatory limit doses. Information about the toxicity of the test material can be gained from knowledge about similar tested compounds or similar tested mixtures or products, taking into consideration the identity and percentage of components known to be of toxicological significance. In those situations where there is little or no information about its toxicity, or in which the the test material is expected to be toxic, the main test should be performed.

23.
A limit test at one dose level of 2000 mg/kg body weight may be carried out with six animals (three animals per step). Exceptionally a limit test at one dose level of 5000 mg/kg may be carried out with three animals (see Annex 3). If test substance-related mortality is produced, further testing at the next lower level may need to be carried out.

24.
Animals are observed individually after dosing at least once during the first 30 minutes, periodically during the first 24 hours, with special attention given during the first 4 hours, and daily thereafter, for a total of 14 days, except where they need to be removed from the study and humanely killed for animal welfare reasons or are found dead. However, the duration of observation should not be fixed rigidly. It should be determined by the toxic reactions, time of onset and length of recovery period, and may thus be extended when considered necessary. The times at which signs of toxicity appear and disappear are important, especially if there is a tendency for toxic signs to be delayed (12). All observations are systematically recorded with individual records being maintained for each animal.

25.
Additional observations will be necessary if the animals continue to display signs of toxicity. Observations should include changes in skin and fur, eyes and mucous membranes, and also respiratory, circulatory, autonomic and central nervous systems, and somatomotor activity and behaviour pattern. Attention should be directed to observations of tremors, convulsions, salivation, diarrhoea, lethargy, sleep 5/14 and coma. The principles and criteria summarised in the Humane Endpoints Guidance Document (9) should be taken into consideration. Animals found in a moribund condition and animals showing severe pain or enduring signs of severe distress should be humanely killed. When animals are killed for humane reasons or found dead, the time of death should be recorded as precisely as possible.

26.
Individual weights of animals should be determined shortly before the test substance is administered, and at least weekly thereafter. Weight changes should be calculated and recorded. At the end of the test surviving animals are weighed and humanely killed.

27.
All test animals (including those that die during the test or are removed from the study for animal welfare reasons) should be subjected to gross necropsy. All gross pathological changes should be recorded for each animal. Microscopic examination of organs showing evidence of gross pathology in animals surviving 24 or more hours may also be considered because it may yield useful information.

28.
Individual animal data should be provided. Additionally, all data should be summarised in tabular form, showing for each test group the number of animals used, the number of animals displaying signs of toxicity, the number of animals found dead during the test or killed for humane reasons, time of death of individual animals, a description and the time course of toxic effects and reversibility, and necropsy findings.

29.
The test report must include the following information, as appropriate: Test substance: − physical nature, purity, and, where relevant, physico-chemical properties (including isomerisation); − identification data, including CAS number.

Vehicle (if appropriate):
− justification for choice of vehicle, if other than water.
Test animals: − species/strain used; − microbiological status of the animals, when known; − number, age, and sex of animals (including, where appropriate, a rationale for the use of males instead of females); − source, housing conditions, diet etc.

Test conditions:
− details of test substance formulation including details of the physical form of the 6/14 material administered; − details of the administration of the test substance including dosing volumes and time of dosing; − details of food and water quality (including diet type/source, water source); − the rationale for the selection of the starting dose.

DEFINITIONS
Acute oral toxicity refers to those adverse effects occurring following oral administration of a single dose of a substance, or multiple doses given within 24 hours.
Delayed death means that an animal does not die or appear moribund within 48 hours but dies later during the 14-day observation period.
Dose is the amount of test substance administered. Dose is expressed as weight of test substance per unit weight of test animal (e.g. mg/kg). Impending death: when moribund state or death is expected prior to the next planned time of observation. Signs indicative of this state in rodents could include convulsions, lateral position, recumbence, and tremor (See the Humane Endpoint Guidance Document (9) for more details).

GHS: Globally
LD50 (median lethal oral dose) is a statistically derived single dose of a substance that can be expected to cause death in 50 per cent of animals when administered by the oral route. The LD50 value is expressed in terms of weight of test substance per unit weight of test animal (mg/kg).
Limit dose refers to a dose at an upper limitation on testing (2000 or 5000 mg/kg).
Moribund status: being in a state of dying or inability to survive, even if treated (See the Humane Endpoint Guidance Document (9) for more details).
Predictable death: presence of clinical signs indicative of death at a known time in the future before the planned end of the experimen ; for example: inability to reach water or food. (See the Humane Endpoint Guidance Document (9) for more details).

1.
For each starting dose, the respective testing schemes as included in this Annex outline the procedure to be followed.