الرحمن الله بسمالرحيم

Toxicology Course

Toxicological Testing in vivo

In vivo testing,

Use of laboratory animals,

Animal testing,

Animal experimentation,

Animal research.

Benefits

Scientists use whole living systems, animal models, in

experiments to understand the relation-ships between

exposure and effect.

Data from these studies provide the essential foundation

to distinguish and recommend which level of exposure is

safe and which is harmful to people, animals and the

environment.

Toxicologists perform research in whole animals to learn

how various chemicals and dosages interact with living

systems and to ensure the short-term and long-term safety

of such products before they are brought to market.

Guiding Principles in the use of animals in toxicology:

1. The use, care and transportation of animals for toxicological research and testing for the purpose of

protecting human and animal health and the environment must comply with all applicable animal welfare laws.

2. When scientifically appropriate, alternative procedures that reduce the number of animals used, refine the use of whole animals or replace whole animals (e.g., in vitro models, invertebrate organisms) should be considered.

3. The species should be carefully selected and the number of animals kept to the minimum required to achieve scientifically valid results.

4. All reasonable steps should be taken to avoid or minimize discomfort, distress or pain of animals.

5. Appropriate aseptic technique, anesthesia and postoperative analgesia should be provided if a surgical procedure is required.

6. Care and handling of all animals must be directed by

veterinarians or other individuals trained and experienced in the proper care, handling and use of the species being maintained or studied.

7. Investigators and other personnel shall be qualified and

trained appropriately for conducting procedures on living

animals, including training in the proper and humane care

and use of laboratory animals.

Rat Mice

Laboratory Animals

Animal house

LD50 (The median lethal dose)

Estimation of lethality

Definition:

The LD50 (mean or average lethal dose) is the dose of a chemical needed to produce death in 50% of treated animals and statistically calculated.

The lower the LD50, the more toxic the

chemical.

Importance:

It is an initial step in the assessment and evaluation of

the toxicity of a substance (new substance).

To determine the therapeutic index (TI), i.e. ratio

between the mean lethal dose and the effective dose in

the same strain and species (LD50/ED50). The greater

the index, safer is the compound.

The test is a measure of acute systemic toxicity.

Test procedure

All available information on the test substance should be considered :

Chemical structure of the substance.

Physical and chemical properties.

The results of any other in vitro or in vivo toxicity tests on the

substance or mixtures.

Toxicological data on structurally related substances or similar

mixtures.

The anticipated use (s) of the substance.

Initial Considerations:

All factors should be fixed.

Animal species, sex, age, average weight and

environmental conditions (temperature,

bedding, cages, water and food supply…….,)

Animals1- Species: the animals used most often in the LD50 Test are rats and

mice; others include rabbits, guinea pigs, dogs, monkeys, birds, and

fish.

2- Age: between 8 and 12 weeks old and its weight should fall in an

interval ± 20 % of the mean initial weight of all previously dosed

animals.

3- Sex.

4- Females should be nulliparous and non-pregnant

Housing:

Animals were group-caged by sex, but the number

of animals per cage must not interfere with clear

observation of each animal.

The biological properties of the test substance or

toxic effects (e.g. morbidity, excitability, etc.) may

indicate the need for individual caging.

Preparation of doses:

Solvent : When necessary, the test substance was

dissolved or suspended in a suitable solvent. Dosing

preparations must be prepared shortly prior to

administration.

Doses are typically measured in terms of

milligrams of the chemical per kilogram of the

animals’ body weight (mg/kg b.w.).

Volume: In rodents, the volume should not

exceed 1 ml/100g of body weight, maximum of 5

ml/100g .

Administration of doses:

Oral– gavage: dosing is typically through force-

feeding.

Inhalation: forcing the animal to breathe the substance.

Dermal :applying the substance directly to the animals

shaved skin.

Intraperitoneal (IP) : injecting the substance into the

animal’s abdomen.

Intravenous (IV): injection.

IP injection

Observation period:

1–2 h, and thereafter at intervals. Maximum for 14 days.

increased motor activity, tremors, arching and

rolling, colonic convulsions, tonic extension,

lacrimation, salivation, muscle spasm,

opisthotonus, hyperesthesia, depression, ataxia,

stimulation, sedation, hypnosis, cyanosis and

analgesia.

Signs recorded during acute toxicity studies:

Experimental design :

1. Preliminary test:Before the actual LD50 determination. A-pilot study :

conducted on a small group of mice mainly to select the dose ranges for the subsequent study.

B- Up and down or the ‘staircase’ method:

The subsequent doses were then increased or decreased.

Once the maximum non-lethal dose (kill none of the exposed animals) and the minimum lethal dose (kill all the animals) were thus determined using only about ten mice.

Final and more reliable LD50 assay was planned using at least three or four dose levels within this range.

From the previous responses we can select some doses where:

- There is a fixed range between these doses

- At least three or four dose levels

- The obtained responses from these doses must be clearly different.

The data should be sufficient to produce a dose-response curve and permit an acceptable estimation of LD50.

2- The test proper:

Record all results in a table.Prepare the dose effect curve on a logarithmic

paper.From the curve detect the average dose which

causes 50% lethality.LD50 will be calculated statistically from the

table .

Compare the obtained value of LD50 with the Hodge and

Sterner table to detect either the tested compound is

toxic or not. If it is toxic, detect the degree of toxicity.

Note: -A chemical with a small LD 50 is highly toxic

than a chemical with large LD50.

Detection of the toxicity degree:

Oral LD50 in male albino rat Degree of toxicity

Up to 5 mg/kg b.w. Super toxic

More than 5 - 50 mg/kg b.w. Extremely toxic

More than 50 – 500 mg/kg b.w. Very toxic

More than 500 – 5000 mg/kg b.w. Moderately toxic

More than 5000 – 15000 mg/kg b.w. Slightly toxic

More than 15 g/kg b.w. Practically non toxic

Hodge and Sterner table

Example:

Preliminary toxicity studies ( IP - LD50) of

the methanol extract of the stem bark of

Garcinia kola (Heckel) in mice.

1 - Preliminary test

4 mice per pilot dose level,

With the pilot tests it was possible to establish the highest dose of the extract that killed none of the exposed animals (200mg/kg) and the lowest dose that killed all the animals (500mg/kg).

The dose levels used in the acute toxicity study ranged between these two dose extremes 200 and 500mg/kg.

2 - Proper test

30 mice weighing between 20 - 25 g were kept in 5 cages (6 per cage) and handled according to standard guidelines for the use and care of laboratory animals.

The animals were maintained on standard animal diet and water.

food was withdrawn 18 h before the start of the experiment.

The five groups of mice were administered

intraperitoneally with 250mg/kg, 300mg/kg, 350mg/kg,

400mg/kg & 450mg/kg of the extract respectively.

Animals were observed for mortality for 24 hours.

Results

850

LC50 (median lethal concentration)

The concentration of a chemical in the air or water that causes death to 50% of the animals.

This concentration is usually quoted as parts per million (ppm) or milligrams per cubic metre (mg/m3).

Number of used animals

“Classical” LD50 Test : approximately 100 animals.

The Organization for Economic Co-operation and Development (OECD) called for at least 30 animals divided into 3 dose groups of 10 animals.

The OECD calling for fewer animals per group, resulting in 20 animals used per test.

The 3Rs of humane experimental technique:

Replacement, some involve taking tissues from animals euthanized for this purpose, so these are not ideal.

Reduction : use cell-based tests to better estimate the starting doses for the animal-based tests

Refinement: less animal suffering Each uses fewer animals and refined the

endpoint from death to “evident signs of toxicity.”

use cell-based tests (tissue culture) to better estimate the starting doses for the animal-based tests.

Consequently, the number of animals used in these LD50 substitutes should drop.

Tissue culture