Toxicity Study of Drugs on Animals | Screening | Zoology

Toxicokinetic studies (generation of pharmacokinetic data either as an integral component of non-clinical toxicity studies or in specially designed studies) should be conducted to assess the systemic exposure achieved in animals and its relationship to dose levels and duration of treatment.

Toxicity testing is of paramount importance while screening drugs. From the 1950s to the 1960s thalidomide was sold in Great Britain to pregnant women to combat morning sickness. But inadequate testing was performed to assess the drug’s safety. This led to catastrophic results for the children of the women who had taken thalidomide during their pregnancies. Children were born malformed or with phocomelia (seal-like limbs, congenital absence of upper limbs).

After this thalidomide tragedy, many countries decided to go for toxicity testing and teratogenicity in both the sexes, so as to prevent further tragedies. Although herbal drags are less toxic as compared to synthetic drags, drugs like black nightshade (Solatium nigrum) have shown cardiac toxicity in rabbits after 21 days of administration.

Toxicity studies are conducted with the assumption that man will behave in the same manner as the animals.

Toxicity studies are of the following types:

1. Acute toxicity studies,

2. Sub-acute toxicity studies and

3. Chronic toxicity studies.

1. Acute Toxicity Studies:

Acute toxicity studies are conducted to determine the median lethal dose (LD₅₀ or LD₉₀, the dose required to kill 50% or 90% respectively) of laboratory animals. In addition, such studies may also indicate the probable target organ of the chemical and its specific toxic effect. It provides guidance on the doses to be used in more prolonged studies. Acute toxicity tests form part of a complete programme of toxicity testing that provide the basis on which to design further testing programmes.

Experimental Design:

Selection of Species of Animals:

These studies should be carried out in at least 2 rodent (mice or rats) species. Rat and mice are preferred because they are economical, readily available and easy to handle. When LD₅₀ values in rats and mice are markedly different or when the rate of bio-transformation in humans is known to be significantly different from rats and mice, use of a non-rodent species is desirable.

LD₅₀ determination is done in animals of both sexes; also in both adult and young animals because of their difference in susceptibility. The age of the animals used is of considerable importance. In general, young immature animals are preferable because of the rapidity of their growth, which enables even a slight depression in growth rate to be measurable. On the other hand, the detection of many actions, particularly on the endocrine and reproductive systems, requires the use of sexually mature animals.

Number of Animals:

The above doses can be estimated using 6-9 animals i.e., a small group of animals (3-5 rodents/sex/dose).

Dose:

Toxicity studies are performed in each species at the same dose level as intended to be used in treatment. Three other doses are also to be administered- that amount that would kill half of the animals (LD₅₀), another that would kill more than half (preferably less than 90%), and a third dose that would kill less than half (preferably more than 10%) of the animals.

Route of Administration:

Toxicity studies are performed in each species using the route by which it is going to be used in humans. Oral route is the most commonly used method. Parentral routes are used in accessing the acute toxicity of parentral drugs. In addition, unless the intended route of administration in humans is only intravenous, at least one more route should be used in one of the species to ensure systemic absorption of the drug. This route should depend on the nature of the drug. A limit of 2 g/kg (or 10 times the normal dose that is intended in humans, whichever is higher) is recommended for oral dosing.

Duration of Study:

If the drug is intended to be used only once in the lifetime of an individual such as a general anaesthetic, only 24 h of acute toxicity studies are performed. Otherwise, they are done for 48 h or so.

Evaluation:

The number and time of death should be examined in order to estimate the LD₅₀. The observation period is usually 7-14 days but may be much longer. Signs of toxicity should also be recorded.

Some methods for acute toxicity evaluation are described below:

Graphical Method of Miller and Tainter (1944):

This method is most commonly used for calculating any ED₅₀ value. A special co-ordinate paper — a logarithm-probit paper — is used. It has two decades of logarithmic scale as the abscissa and a scale of probits as the left ordinate. The right ordinate is marked in a scale of per cent corresponding to the probit scale. The probit corresponding to a per cent value must be found in a table of probits.

Arithmetical Method of Reed and Muench (1938):

This method employs cumulative values. It is assumed that an animal killed by a certain drag would have been killed by a large dose, and that a surviving animal would have survived a smaller dose. The cumulative dead and survivors are recorded. The per cent of survival is calculated and the LD₅₀ is computed. This computation is less reliable than the Miller and Tainter method.

Arithmetical Method of Karber (1931):

The interval mean of the number of dead in each group of animals is used in this method, as well as the difference between doses for the same interval.

Observations and Examinations:

Daily water and food intake of the animals have to be monitored. The initial weight and weight after the study has to be recorded.

Laboratory Tests:

List of some of the biochemical parameters, body organs and systems that might be affected by the drug and which have to be accessed during the study given below:

1. Hematological Parameters ― Haemoglobin, Total RBC count, Haematocrit, Reticulocyte, Total WBC count, Differential WBC count, Platelet count, Terminal bone marrow examination, ESR (non-rodents only), General blood picture – A special mention of abnormal and immature cells should be made, Coagulation parameters (non-rodents only) – Bleeding time, coagulation time, prothombin time, activated partial thromboplastin time.

2. Urinalysis Parameters ― Colour, Appearance, Specific gravity, 24-hour urinary output, Reaction (pH), Albumin, Sugar, Acetone, Bile pigments, Urobilinogen, Occult blood, Microscopic examination of urinary sediment.

3. Blood Biochemical Parameters ― Glucose,Cholesterol, Triglycerides, HDL cholesterol (non-rodents only), LDL cholesterol (non-rodents only), Bilirubin, SGPT (ALT), SGOT (AST), Alkaline phosphatase (ALP), GGT (non-rodents only), Blood urea nitrogen, Creatinine, Total proteins, Albumin, Globulin (calculated values), Sodium, Potassium, Phosphorus, and Calcium.

4. Gross and Microscopic Pathology ― Brain* – Cerebrum, cerebellum, mid-brain, Spinal cord**, Eye, Middle ear, Thyroid, Parathyroid**, Spleen*, Thymus, Adrenal*, Pancreas**, Trachea**, Lung*, Heart*, Aorta, Oesophagus, Stomach, Duodenum, Jejunum, Terminal ileum, Colon,Rectum**, Liver*, Kidney*, Urinary bladder, Epididymis, Testis*, Ovary, Uterus*, Skin, Mammary gland, Mesenteric lymph node, Skeletal muscle ― (a) Organs to be weighed, (b) Organs should be examined as the ding might have an external effect on it.

OECD Guidelines for Acute Oral Toxicity Studies:

The Organization for Economic Co-operation and Development has set down various guidelines for the testing of chemicals. These guidelines are based on the procedure adopted by the American Society of Testing and Materials (ASTM) in 1987 and revised in 1990. They help in minimising the number of animals required to estimate the acute oral toxicity of a chemical.

Definitions as per OECD Guidelines:

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 h.

Dose:

Refers to the amount of test substance administered. Dose is expressed as weight (g or mg) or as weight of test substance per unit weight of test animal (e.g., mg/kg).

The preferred rodent species, as per this guideline, is the rat, although other rodent species may be used. Normally female rats are used since females are generally slightly more sensitive than males. Females should be nulliparous and non-pregnant. When the test is conducted on males, adequate justification should be provided.

Each animal should be between 8 and 12 weeks old and its weight should fall in an interval within ± 20% of the mean initial weight of any previously dosed animals.

The temperature in the experimental animal room should be 22°C (± 3°C). The relative humidity should be at least 30% and preferably not exceed 70%. Lighting should be artificial—the sequence being 12 h light and 12 h dark. The animals should be 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 laboratory conditions.

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. 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 1 ml/100 g of body weight; however, in the case of aqueous solutions, 2 ml/100 g body weights can be considered.

The test substance should be administered in a single dose by gavages using a stomach tube or a suitable incubation cannula. Animals should be fasted prior to dosing (e.g., for the rat, food but not water should be withheld overnight; for the mouse, food but not water should be withheld for 3-4 h).

Following the period of fasting, the animals should be weighed and the test substance administered. The fasted body weight of each animal is determined and the dose calculated according to the body weight. After the substance has been administered, food may be withheld for a further 3-4 h in rats or 1-2 h in mice.

Limit Test – Guideline 425 (Acute Oral Toxicity – Updated Guidelines Adopted, December 20, 2001):

A limit test is generally performed before the main test. As per the 425 guidelines, one animal is dosed at the test dose i.e., 2000 mg/kg. If the animal dies, the main test is conducted to determine the LD₅₀. If the animal survives, four additional animals are dosed sequentially so that a total of five animals are tested. If three animals die, the limit test is terminated and the main test is performed.

The LD₅₀ is less than the test dose (2000 mg/kg) when three or more animals die. If a third animal dies, the main test is conducted. The LD₅₀ is greater than the test dose (2000 mg/kg) when three or more animals survive.

If an animal unexpectedly dies late in the study, and there are other survivors, it is appropriate to stop dosing and observe all animals to see if other animals will also die during a similar observation period. Late deaths should be counted the same as other deaths.

Main Test – Guideline 425:

Single animals are dosed in sequence usually at 48 h intervals. However, the time interval between dosing is determined by the onset, duration and severity of toxic signs. Treatment of an animal at the next dose should be delayed until one is confident of survival of the previously dosed animal.

The first animal is dosed a step below the best preliminary estimate of the LD₅₀. If the animal survives, the second animal receives a higher dose. If the first animal dies or appears sick, the second animal receives a lower dose.

Dosing continues depending on the fixed-time interval (e.g., 48 h) outcomes of all the animals up to that time.

The testing stops when one of the following stopping criteria is met:

a. 3 consecutive animals survive at the upper bound;

b. 5 reversals occur in any 6 consecutive animals tested;

c. At least 4 animals have followed the first reversal and the specified likelihood ratios exceed the critical value.

Animals are observed individually at least once during the first 30 min. after dosing, periodically during the first 24 h (with special attention given during the first 4 h), and daily thereafter, for a total of 14 days.

The times at which signs of toxicity appear and disappear are important, especially, if there is a tendency for toxic signs to be delayed. All observations are systematically recorded with individual records being maintained for each animal.

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, somatomotor activity and behaviour patterns. Attention should be directed to observations of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

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 then humanely killed.

All animals (including those which die during the test 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.

2. Sub-Acute Toxicity Studies:

Sub-acute toxicity studies are conducted to determine the organs affected by different dose levels. These studies access the nature of toxic affects under more realistic situations than the acute toxicity studies.

Experimental Design:

Selection of Species of Animals:

These studies should be carried out in at least 2 phylogenetically different species, of which one should be a non-rodent and the other a rodent (mice, rats) species. Ideally, the animals chosen should bio-transform the chemical in a manner essentially identical to humans.

Number of Animals:

Equal number of male and female animals should be used. Generally 10-50 rats are used in each dose group as well as in the control group so as to provide the data, which is statistically analysable.

Dose:

It is advisable to select three doses- a dose that is high enough to elicit definite signs of toxicity but not high enough to kill many of the animals, a low dose that is expected to induce no toxic effect, and an intermediate dose. A control group should also be included.

Doses are generally selected on the basis of information obtained in acute toxicity studies, using both the LD₅₀ and the slope of the dose-response curve.

Route of Administration:

Toxicity studies are performed in each species using the same route as that intended in humans.

Duration of Study:

The duration of the study in rats is generally 90 days. In dogs, the duration is often extended to 6 months or even 1 year. The duration of sub-acute toxicity studies depends on the intended duration of the test drugs in therapeutics (Table 9.8).

Evaluation:

They provide an indication of the effect on the target organs, dose-effect, and dose-response relationships. Autopsy of the animals and gross pathological examination provides useful information on the toxicity of the chemical under test.

Observations and Examinations:

Body Weight and Food Consumption:

This should be determined weekly. Decreased body weight is an index of toxic effects. Food consumption is also a useful indicator.

General Observations:

These include monitoring of general appearance, gross behaviour and any other abnormalities.

Postmortem Examination:

Lists of organs, which are to be histopathologically examined, are ― (Urinalysis Parameters) – Colour, Appearance, Specific gravity, 24-hour urinary output, Reaction (pH), Albumin, Sugar, Acetone, Bile pigments, Urobilinogen, Occult blood, Microscopic examination of urinary sediment. The organs isolated should be weighed; the number of organs and all the gross lesions has to be determined as they serve useful indicators of toxicity.

3. Long-Term Toxicity Studies (Chronic Toxicity Studies):

In these studies, the animals are exposed over a long period of time to the toxic effects of the drug in order to mimic more realistic situations. On the basis of information obtained in sub-acute toxicity studies, the main aim of these long-term studies is to determine the organs affected and determine whether the drug is potentially carcinogenic or not.

These tests may be conducted concurrently with the initial studies in humans. The procedures involved in both sub-acute and chronic studies are similar except for their duration. The duration of long-term studies in rats is generally 1-2 years, and it can extend to 7 years in case of dogs.