Purpose and objectives

Acute toxicity studies are conducted in animals to ascertain the total adverse biological effects caused during a finite period of time following the administration of single, frequently large doses of an agent (or several doses repeated over a short interval of time)

Death, sudden or otherwise is frequent;y the most important end-point measured

Acute toxicity studies are frequently designed to express the potency of the toxicantin terms of the MEDIAN LETHAL DOSE or the LD50: The value representing the estimated dose causing death of 50% of the universal population of the species exposed under the defined conditions of the test In situations where the agent is not administered directly to the animals, toxicant potencyis presented as the MEDIAN LETHAL CONCENTRATION or the LC50: The estimated concentration in the environment to which the animals are exposed, that will result in 50% mortality of the population of animals under the conditions defined for the study

The LD50gives a comparative indication of the immediate toxicity of an agent in a strain, age group, and sex of a particular species of animals, permitting only a comparison of the toxicity of one agent relative to other agents tested in the same manner in the same species (strain, age, and sex )

Protocol Designs:
Certain principles and criteria should be universally applied
First A wide variety of factors can influence the outcome of a lethality test such as:

Species Strain, or sub-strain

Age, weight, and sex

Housing practices Food and water quality, nutritional state Volume and vehicle of test substance delivery, etc Various strains of laboratory mice or rats are used routinely in determining the LD50because they are:

Inexpensive-minimal variation between animals-ready availability Ease of handling-economics of housing and caring

The animals should be: Placed in a quarantine area for at least 10-14 days Examined to ensure that they are healthy by choosing randomly selected animals for gross and histopathological examination of organs, as well as blood chemistry

Second

Great care must be given to the preparation and delivery of the test articles Investigators routinely triple check dosage calculations and concentration validation of the mixing procedures and mix only in calibrated and precise glassware

Third

We must make sure that all animals are successfully dosed. Permanent numbers of animals are not assigned until we are certain that the dose has been successfully delivered Depending on administration route we must pay close attention to dosing techniques and volume limitations. For example when using a feeding needle (by gavage), the volume of solution administered must be kept constant (in ml/kg)

For the rodent species, volumes of solutions of the order of 2-5 ml/kg can be administered without undue stress to the animals.

Accidental deaths should be identified as such. Animals found dead should be examined for evidence of accidental trauma Deaths that are clearly accidental should not be considered in the final conclusion.

Fourth

Lethality protocols dont specify all dosages. This can sometimes result in a study in which absurdly high doses are administered. Hence, all protocols should clearly state what the ceiling or limit dosage will be and the reasons for selection

Animals of the same species/strain, age and sex are divided into groups. All the animals in the same group receive the same dosage Different groups are treated at different dosages. All animals are treated by the same route. The animals are then held and observed for a set and consistent period of time

Mortality (animals that die during the observation period) = number dead/ number dosed x 100

Sigmoidal dose-response curve The LD50 simply the dosage observed or yields 50% thatcalculated mortality.

Traditionally FDA and foreign regulatory guidelines protocols have frequently been designed to include both sexes of 2 species (generally rats and mice) and 2 routes of administration

At least one route must be the intended or the most probable human exposure route

In the drug industry (where this approach is common) the 2 routes are generally:
Oral and intraperitoneal---------- oral drug Oral and intravenous---------- for an intravenous drug

I-Number and size of dosage groups:

The precision with which the curves are described will depend on the number of groups (hence, dosages) and the number of animals in each group. Between 1940 and 1980, the standard was to use from 4-6 dosages with 10 animals / dosage The number and size of dosage groups will depend, to an extent on the methods of statistical analysis

The classic statistical methods for analyzing lethality data:

1. The probit method: - Requires at least 2 groups of partial responses (mortality greater than 0% but less than 100%)

- Doesnt deal effectively with groups that had either 0 or 100 % lethality (substitute 0.1 for 0 % and 99.7 for 100 %)

2. The moving average method:

This method requires that the dosages be separated by a constant geometric factor (e.g 2, 4, 8 mg/kg) and that groups be of equal size

3. The Litchfield-Wilcoxon method:

It is certainly a valid method. Modern handheld calculators and the ready availability of simple computer programs have made other methods more convenient to run

4. The normit X 2method :

Like, the probit method, this method does not absolutely require equally spaced dosages or equal group sizes, but does require at least one partial response

II-Timing of doses:
It is best if all the animals used for determining a specific curve are dosed on the same day, and if possible at the same time of day However, if the test substance is of unknown lethality A single dosage group is treated on the first day, the doses for the second and third groups are adjusted depending on the result of the first group

III-Duration of acute toxicity studies:

24 hours regardless of the route of administration

Survivors are returned to their cages for further observation for 14 days
LD50 represents the median lethal dose for deaths occurring in the first 24 hours LC50------------96 hours

IV-Selection of Dosages:

I
In setting the dosages, a few common sense rules have to be applied 1. Factors such as the intrinsic biological and chemical activity of the chemical, known pharmacological profile, chemical and physical characteristics should be considered 2. It is best advised to pick widely spaced rather than closely spaced dosages

Many protocols are available for selection of doses: 1. Dose Probes 2.The Up-and-Down Method 3. Pyramiding Studies 4. Limit Tests

5. Fixed Dose Procedure

 Animals are weighed and a narrow range of body weights are distributed between and within subgroups Each animal is identified in some fashion and is housed individually in a proper cage In most cases the animals will be fasted overnight prior to oral administration of the test agent---------no food related impediment for absorption of the chemical The animals should be weighed just prior to dosing

The test substance is applied to the skin in graduated doses to several groups of experimental animals (one dose/group) What is frequently determined in the study is the LC50

The animals are prepared 24 hours in advance by shaving the fur from and area on the back or the abdomen equivalent to approximately 10 % of the total body surface area Rats----------3 x 4 cm Rabbits------5 x 6 cm

Applying the test agent: Liquids

Administer evenly over the shaved area

Dissolve in 0.9 % saline in a relatively inert organic solvent Dry or water-moistened paste for solid insoluble material are applied.

Solids

Powders

Groups of experimental animals, rodent and non-rodent males and females of each will be exposed for a defined period, usually 4 hours to the test substance in graduated concentrations, one concentration / group

Essential Equipment for such studies:

A negative-pressure, dynamic inhalation system with controllable airflow
A suitable system to generate the concentration of the material required Instrumentation to continuously monitor the actual levels attained in the system

A trapping system to capture the toxicant in the exhausted air

The animals may be accommodated within the inhalation chamber (whole body exposure), or alternatively head-only, or oro-nasal exposure to the agent may be used

Several problems in conducting a well-designed, whole body exposure inhalation study: 1. Physicochemical properties of the toxicant will dictate the active amount of the ingredient that can be suspended in air 2. The particle size of aerosols must be in the respirable range (< 2 um) for effective inhalation 3. The animals curl up and go to sleep decreasing their respiratory rate

4. Surviving animals will groom themselves

5. Irritant substances----------mucosecretory response

IV-Observations: Organ system

Autonomic Behavioral Sensory Neuromuscular Cardiovascular Respiratory Ocular GIT Gastro-urinary Cutaneous

Toxic signs
Salivation, nasal discharge, diarrhea, etc Sedation, drooping head, panting, staring, Reflexes, sensitivity to pain, nystagmus.. Fasciculations, tremors, muscle tone, . Alteration of heart rate, cyanosis, v.c, . Gasping, dyspnea, apnea, Lacrimation, ptosis, mydriasis, miosis,. Salivation, retching, diarrhea, bloody stools,. Urinary frequency, bloody urine. Piloerection, swelling, edema, necrosis,

Following the acute phase of the study (24 hrs), the animals at the point of death and normal appearing animals should be examined for 14 days to note delayed effects or secondary toxicity During this time animals are weighed daily and weight changes are plotted against time The following tests are performed: -Blood samples for full biochemistry -Urine and stool analysis -Neurological assessment by non-invasive techniques -Histopathological examination of selected organs -Necropsy of all animals dying

Provides a measure of the relative toxicity of an unknown agent compared to other agents given to the same species, strain, age and sex of animals

During the acute phase of the study, observation of animals shows:

- Signs and symptoms of acute toxicity

- Possible target organs

- Duration and intensity of the toxic effects

 This information is of considerable use to occupational health physicians and the staff of emergency treatment units in cases of accidental poisoning, environmental disaster and suicidal overdose The results will also assist investigators in designing subsequent sub-chronic and chronic studies, estimating the potential hazard to health and well-being, as well as planning therapeutic trials of drugs in humans