INTRODUCTION:

The amount of pharmacological substances, chemical and herbal drugs being used in the human community today have increased to almost an innumerable amount. These may be presented today in the form or as constituents of food substances medicines, beverages, otherindustrial and household products. However these chemicals or herbal drug may result in chronic toxicity in the living system when used over a long period of time or acute toxicity may also occur when large quantities capable of eliciting immediate toxic effect are used. These effects may be mild or severe depending upon nature of the substance.

Acute toxicity is defined as the unwanted effect that occurs either immediate or at a short time interval after a single or multiple administration of such substance within 24 hours. The unwanted effect is any effect is any effect that produces functional impairments in organs and/or biochemical lesion which could alter the functioning of the organism in general or individual organs ^1-2.

Toxicity is the fundamental science of poisons. The organization for Economic and Development (OECD) mentioned acute toxicity as the advance effects occurring within short time of oral administration. Phychochemical interactions of poisons leads to the injury or death of living tissues. Toxicology is like science and an art like medicine. It includes observational data gathering and data utilization to predict outcome of exposure in human and animals. Acute toxicity is distinguished from chronic toxicity, which describes the adverse health effects from repeated exposures, often at lower levels to a substance over a longer time period^3-4.

Acute toxicity (LD₅₀) test:

Acute systemic toxicity evaluates the adverse effects that occur following exposure of organisms to a single or multiple doses of a test substance within 24 hours by a known route (oral, dermal or inhalation). After administration, the test substance is absorbed and distributed to various parts of the body before it elicits systemic adverse effect. The regulatory body requires the acute toxicity test report for labelling and classification of substances for human use.

The LD₅₀ (median lethal dose) test was introduced in 1927 by J.W. Trevan to estimate the dose of a test substance that produces 50% death in a given species of animals. It is usually the first test conducted for every chemical before further toxicity tests are carried out. It is used for estimating the potential hazards of chemicals on humans. Although its major endpoint is death, non-lethal acute effect may occur as sign of toxicity depending on the chemical being tested.

Assessment of the acute toxic potential of substances is required to determine their adverse effects that might occur due to accidental or deliberate short-term exposure. Result from acute toxicity test serve as a guide in dosage selection for long term toxicity studies are well as other studies that involve the use of animals.

From the result of an acute toxicity test, a conclusioncan be made on the toxicity status of the substance. As depicted in table 1, substance with LD₅₀below 5 mg/kg are classified to be highly toxic while substance with LD₅₀ above 15,000 mg/kg are termed relatively harmless ^5-8.

Table 1: Classification of LD₅₀ based on dose range.

LD₅₀	Classification
<5 mg/kg	Extremely toxic
5-50 mg/kg	Highly toxic
50-500 mg/kg	Moderately toxic
500-5000 mg/kg	Slightly toxic
5000-50000 mg/kg	Practically non-toxic
>15000 mg/kg	Relatively harmless

OECD Guideline for Acute Oral Toxicity:

OECD Guideline for the testing of chemicals are periodically reviewed in the light of scientific progress or changing assessment practices.The conventional acute oral toxicity test (formerly OECD Test Guideline 401)is the most heavily criticized test in terms of animal welfare and this concern was the driving force behind the development of three alternative tests for acute oral toxicity (Test Guideline 420,423,425).Anticipating the presence of validated alternatives, Member countries took the initiative to plan the deletion of Guideline 401^9-12.

A Nominated Expert Meeting (Rome 1998) and an Expert Consultation Meeting, (Arlington 1999) were convened to determine the acute oral toxicity data requirement needs of Member countries and to assess the capabilities of the alternatives to meet these needs. On the basis of these technical discussions, the 29^th Joint Meeting concluded in June 1999 that not all data needs could be met by the alternatives (and not always by Guideline 401). The Joint Meeting decided that Guidelines 420,423 and 425 should be revised to meet regulatory needs of member of countries.

The revision of Guidelines 420, 423, and 425 was completed in 2000 following a second Expert Consultation Meeting (Paris 2000) and process of deletion of guideline 401 was started^13-15.

OECD Guideline 420:

Acute Oral Toxicity-Fixed Dose Procedure:

The method provides information on the hazardous properties and allows the substance to be ranked and classified to the Globally Harmonised System (GHS) for the classification of chemicals which cause acute toxicity.

Principle of the test:

Group of animals of a single are dosed in a stepwise procedure using thefixed doses of 5, 50, 300 and 2000 mg/kg (exceptionally an additional fixed dose of 5000 mg/kg may be considered). The initial dose level is selected on the basis of a sighting study as the dose expected to produce some signs of toxicity without causing severe toxic effects or mortality. Further groups of animals may be dosed at higher or lower fixed doses depending on the presence or absence of signs of toxicity or mortality. This procedure continues until the dose causing evident toxicity or no more than one death is identified or when no effect are seen at the highest dose or when death occur at the lowest dose.

Description of the method:

1. Selection of animal species:

The preferredrodent species is the rat. Normally females are used. When the test is conducted in males, adequate justification should be provided. Females should be nulliparous an 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.

2. Housing and feeding condition:

The temperature of the experimental animal room should be 22 c⁰. Although the relative humidity should be at least 30% and preferably not exceed 70%.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.

3. Preparation of animals:

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

4. Preparation of doses:

Test substances should be administer in a constant volume over the range of doses. In case of liquid end product or mixture is to be tested –use undiluted test substance i.e at a constant concentration .In either case,the maximum dose volume for administration must not exceeded .In rodents , the volume should not normally exceed 1ml /100g of body weight however in case of aqueous solution 2ml/100g body weight can be considered. For vehicles other than water the toxicological characteristics of the vehicle should be known.

Procedure:

1. Administration of doses:

By gavage using a stomach tube or suitable incubation canula(unusual circumstance –fraction of doses).Fasted prior to dosing. Weigh animals and administer the test substance. Withheld food for a further 3-4 hours in rats or 1-2 hours in mice (in case of fraction of doses).

2. Sighting study:

The purpose of the sighting study is to allow selection of the appropriate starting dose for the main study. The substance is administered to single animals in a sequential manner following the flow charts.The starting dose for the sighting study is selected from the fixed dose levels of 5,50,300 and 2000 mg/kg as a dose expected to produce evident toxicity. Also, evidence from in vivo and in vitro data from the same chemical and from structurally related chemicals. In the absence of such information, the starting dose will be 300 mg/ kg. At least 24 hours will be allowed between the dosing of each animal. Consideration of use of an additional upper fixed dose level of 5000 mg/kg. In cases where an animal tested at the lowest fixed dose level (5 mg/kg) in the sighting study dies, the normal procedure is to terminate the study and assign the substance to GHS category. For further confirmation use supplementary procedure.

3. Main study:

Numbers of animals and dose levels

Select dose from sighting study. Perform study on a total of five animals of one sex at each level including animals tested in sighting study. Time interval between dosing at each level depends on 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. To check delayed Toxicity-A period of 3 or 4 days between dosing at each dose level is recommended. In case of inconclusive response time interval may be adjusted as appropriate. Fixed dose of 5000 mg/kg procedure outline in Annex 4.

4. Limit test:

Performed when information indicating that the test material is likely to be nontoxic i.e. having toxicity only above regulatory limit doses. How information about the toxicity of the test material can be gained?. Using the normal procedure test will be done ¹⁶.

OECD Guideline 423:

Acute Oral Toxicity-Acute Toxic Class Method:

The acute toxic class method is based on biometric evaluation 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 LD₅₀data obtain from the literature, both nationally and internationally.

Principle of the test:

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 tested using a stepwise procedure, each step using three animals of a single sex. 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.

Description of the method:

1. Selection of animal species:

The preferred rodent species is the rat. When the test is conducted in males adequate justification should provided. 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 with in ±20%of the mean weight of any previously dosed animals.

2. Housing and feeding conditions:

The temperature in the experimental animal room should be 22⁰C. Although the relative humidity should be at least 30% and preferably not exceed 70%. Lighting should be artificial, the sequence being 12 hours light, 12hours dark for feeding conventional laboratory diets may be used with an unlimited supply of drinking water.

3. Preparation of animals:

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 acclimatization to the laboratory conditions.

4. Preparation of doses:

Test substances should be administer in a constant volume over the range of doses. In case of liquid end product or mixture is to be tested-use undiluted test substance i.e. at a constant concentration. In either case, the maximum dose volume for administration must not exceeded. In rodent, the volume should not normally exceed 1 ml/100 g of body weight however in case of aqueous solution 2 ml/100 g body weight can be considered. For vehicles other than water the toxicological characteristics of the vehicle should be known.

Procedure:

1. Administration of doses:

The test substance is administered in a single dose by gavage using a stomach tube or suitable intubation canula. Single dose is not possible, the dose may be given in smaller fractions over a period not exceeding 24 hours. Animal should be fasted prior to dosing. 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.

2. 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.

When available information suggests that mortality is unlikely at the highest starting dose level, 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.

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

3. Limit test:

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 situation where there is little or no information about its toxicity, or in which the test material is expected to be toxic, the main test should be performed.

A limit test at one dose level of 2000 mg/kg body weight may be carried out with six animals. Exceptioally a limit test at one dose level of 5000 mg/kg may be carried out with three animals. If test substance-related mortality is produced, further testing at the next lower level may need to be carried out ¹⁷.

OECD Guideline 425:

Acute Oral Toxicity: Up and Down procedure-

This test procedure is of principal value in minimising the number of animals required to estimate the acute oral toxicity of a chemical and in estimating a medium lethal dose. The medium lethal dose allows for comparison with historical data. In addition to the observation of mortality, LD₅₀ allows the observation of signs of toxicity. The latter is useful for classification purpose and in the planning of additional toxicity tests.

Principle of the test:

Animals are dosed, one at a time, at 24 hours intervals. The first animal receives a dose at the level of the best estimate of the LD₅₀. Depending on the outcome for the previous animal, the dose for the text animal is adjusted up or down. If an animal survives, the dose for the next animal is increased; if it dies, the dose for the next animal is decreased. After reaching the reversal of the initial outcome i.e. the point where an increasing dose pattern is reversed by giving a smaller dose, four additional animals are dosed following the same UDP. The LD₅₀ is calculated using the method of maximum likelihood.

Description of the method:

1. Selection of animal species:

The preferred rodent species is the rat. In the normal procedure female rats are used. When there is adequate information to infer that males are more sensitive, they should replace females in the test. The females should be nulliparous and non-pregnant. At the commencement of the study, the weight variation of the animals should be minimal and not exceed ±20% of the mean weight for each sex. The test animals should be characterised as to species, strain source, sex, weight and age.

2. Housing and feeding conditions:

The temperature in the experimental animal room should be 22⁰ C. Although the relative humidity should be at least 30% and preferably not exceed 70%. Lighting should be artificial, the sequence being 12 hours light and 12 hours dark. For feeding conventional laboratory diets may be used with an unlimited supply of drinking water.

3. Preparation of animals:

The animals are uniquely identified and kept in their cages for at least five days prior to dosing for acclimatisation to the laboratory conditions. Animals demonstrating signs of spontaneous disease or abnormality prior the start of the study are eliminated from the study.

4. Preparation of doses:

When necessary, the test substance is dissolved or suspended in a suitable vehicle. It is recommended that, whenever possible, the use of an aqueous solution or suspension be considered first, followed by consideration of a solution or emulsion in oil and then by possible solution in other vehicle. For vehicles other than water, toxicity of the vehicle must be known.

Procedure:

1. Full test:

Individual animals are dosed in sequence at 24 h intervals, one at time and then observed for a minimum of 24 h. However, the time intervals between dosing should not be fixed rigidly and may be adjusted as appropriate, in case of delayed mortality. The first animal is dosed at the toxicologist’s best estimate of the LD₅₀. If the animal survives, the second animal receives a higher dose, unless the limit dose was used as the starting dose. If the first animal dies or appears moribund the second animal receives a lower dose. Moribund state is characterised by symptoms such as shallow, laboured or irregular respiration, muscular weakness or tremors, absence of voluntary response to external stimuli, cyanosis and coma, Criteria for making the decision to humanely kill moribund and severely suffering animals are the subject of a separate Guidance Document. Animals killed for humane reasons are considered in the same way as animals that died on test.

For selecting the starting dose, all available information should be used, including information on structure-activity relationships. When the information suggests that mortality is unlikely then a limit test should be conducted. When there is no information on the substance to be tested, for animal welfare reasons it is recommended to use the starting dose of 200 or 500 mg/kg body weight. The dose for each successive animal is adjusted up or down, depending on the outcome of the previous animal. If feasible, a dose progression factor of 1.3 is used. Other factors may be used, if justified. After reaching the reversal of the initial direction, four additional animals are dosed using the same UDP. This is the end of the normal test.

2. Limit test:

Doses should not exceed 2000 mg/kg which is considered the upper limit dose. When the first animal is dosed with the upper limit dose and survives, the second animal receives the same dose. When total of three animals have been dosed with the limit dose and no deaths have occurred, then three animals of the other sex should be tested at the limit dose level. If there is again no lethality, the test can be terminated.

3. Optional testing:

Information from one sex may be adequate toxicity. However, if found desirable, comparability of response in the other sex can be evaluated by administering to generally not more than 3 animals, dose above and below the estimated LD₅₀. The point intermediate between doses where responses change can be taken as an appropriate estimate of the lethal dose.

4. Administration of doses:

The test substance is administered in a single dose by gavage, using an oral dosing needle or rubberised tubing. The animal should be fasted prior to dosing by withholding food overnight. Fasted body weight of each rat is determined and the dose is calculated according to the body weight. After dosing food may be withheld for a further 3-4 hours. The volume should not exceed 1 ml/100g body weight, except in the case of aqueous solutions where 2ml/100g body may be used¹⁸.

CONCLUSION:

In acute toxicological study, the investigational product is administered at different dose levels, and the effect is observed for 14 days. All mortalities caused by the investigational product during the experimental period are recorded and morphological, biochemical, pathological, and histological changes in the dead animals are investigated. Acute toxicity study is involved in estimation of LD₅₀ and also it is useful to determine the absolute dose given to the species in a row was multiplied by the factor given at intersection of the relevant row and column. OECD Guideline 420, OECD Guideline 423 and OECD Guideline 425 these methods are used to determine acute oral toxicity of chemical substance and herbal products.

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Received on 05.05.2021 Modified on 30.05.2021

Asian J. Pharm. Res. 2021; 11(4):251-256.

DOI: 10.52711/2231-5691.2021.00044