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Long-Term Toxicity Test
Many regulatory agencies require that at least two species be used, one a rodent and the other a non-rodent.
Normally, the same number of male and female animals should be used.
Number of animals
In cases of rodents, each group should consist of at least ten males and ten females. In the case of non-rodents, each group should consist of at least three males and three females.
When interim examinations are scheduled, the number of animals should be increased accordingly.
Route of administration
Normally, the expected clinical route of administration should be used.
The period of administration of the test substance to animals will depend on the expected period of clinical use. The period of administration of the toxicity study may vary from country to country, according to its individual regulations.
The following table reflects commonly used ranges of administration periods:
Expected period of clinical use
Administration period for the toxicity study
Single administration or repeated administration for less than one week
2 weeks to 1 month
Repeated administration, between one week to four weeks
4 weeks to 3 months
Repeated administration, between one to six months
3 to 6 months
Long-term repeated administration for more than six months
9 to 12 months
As a rule, the test substance should be administered seven days a week. Administration periods for the toxicity study must be recorded in each result.
Groups receiving at least three different dose levels should be used.
One dose level should not cause toxic changes (no-effect dose) and one dose level that produces overt toxic effects should be included. Within this range the addition of at least one more dose may enhance the possibility of observing a dose-response relationship for toxic manifestations. All studies should include a vehicle control group of test animals.
Observations and examinations
Observations and examinations should be performed on the following items (from 1 to 6):
1. General signs, body weight and food and water intake
For all experimental animals, the general signs should be observed daily and body weight and food intake should be measured periodically. If useful, water intake should also be determined. The frequency of measurements should normally be as follows:
• Body weight: before the start of drug administration, at least once a week for the first three months of administration and at least once every four weeks thereafter.
• Food intake: before the start of drug administration, at least once a week for the first three months of administration and at least once every four weeks thereafter. If the test substance is administered mixed in the food, the intake should be measured once a week.
2. Haematological examination
For rodents, blood samples should be taken before autopsy. For non-rodents, blood samples should be taken before the start of drug administration, at least once during the administration period (for studies of longer than one month), and before autopsy.
For both haematological and blood chemistry examinations, it is desirable to include as many parameters as possible.
3. Renal and hepatic function tests
Since the liver and kidneys are the usual organs of metabolism and excretion, potentially toxic agents easily affect them; their functions should be monitored in long-term toxicity studies.
For rodents, a fixed number of animals from each group should be selected and urinalysis should be performed before the start of drug administration, and at least once during the administration period.
4. Other function tests
If appropriate, ECG and visual, auditory tests should be performed. For rodents, ophthalmological examination should be performed on a fixed number of animals from each group at least once during the administration period; for non-rodents, examination should be performed on all animals before the start of drug administration and at least once during the period of administration.
5. Animals found dead during the examination should be autopsied as soon as possible. A macroscopic examination should be made of organs and tissues. In addition, where possible, organ weight measurements and histopathological examinations should be performed in an attempt to identify the cause of death and the nature (severity or degree) of the toxic changes present.
6. In order to maximize the amount of useful information that can be obtained during the administration period, all moribund animals should be sacrificed rather than allowed to die. Prior to sacrifice, clinical observations should be recorded and blood samples collected for haematological and blood chemical analysis. At autopsy, a macroscopic examination of organs and tissues and measurement of organ weights should be recorded. A full histopathological examination should be performed in an attempt to characterize the nature (severity of degree) of all toxic changes.
All survivors should be autopsied at the end of the administration period or of the recovery period after taking blood samples for haematological (including blood chemistry) examinations; organs and tissues should be examined macroscopically and organ weights measured. Histopathological examinations of the organs and tissues of animals receiving lower dosage should also be performed, if changes are found on gross or macroscopic examination of their organs and tissues of these animals, or if the highest dose group reveal significant changes. On the other hand, histopathological examination of all rodents will further improve the chances of detecting toxicity.
Recovery from toxicity
In order to investigate the recovery from toxic changes, animals that are allowed to live for varying lengths of time after cessation of the period of administration of the test substance, should be examined.