Screen, predict toxicity, explore mechanisms

Assessing toxicity: animals → humans

Verification of animal experiment results

1. The effects on experimental animals can be extrapolated to humans

2. Experimental animals must be exposed to high doses

3. The experimental subjects are adult healthy animals.

4. The poisoning route is the possible exposure route for humans.

Extrapolation from animal experiments to humans is uncertain

There is uncertainty in extrapolating high doses to low doses

Extrapolation from a small number of animals to a large number of people is uncertain

Adult healthy animals are usually used, and the reaction is relatively simple.

"3R" principle

Ethical issues in animal experimentation

Ethical Principles of Human Experimentation

Observe the toxic effects and properties of the test substance

Dose-response (effect) relationship studies

Determine target organs of toxicity

Determine reversibility of damage

Sensitive detection indicators for toxic effects

biological markers

poisoning mechanism

Toxicokinetics of test substances

Metabolism of test substance and poisoning rescue measures

Animals that have been artificially bred, and the microorganisms they carry are controlled, with clear genetic background and clear origin. It is mainly used for scientific research, effectiveness and safety evaluation of drugs and biological products, and teaching.

Generally choose two species: rodents and non-rodents

The basic principle

Selection of experimental animal strains

Experimental animals classified by genetic control

Microbiological control parameters for experimental animals

Laboratory animal microbial grade standards

Individual selection of experimental animals

Laboratory animal facility structure

Types of experimental animal facilities

randomization principle

Contrast principle

Repeat principle

Dose Grouping for In Vivo Toxicology Tests

Number of animals in each group

Test period

After purchasing experimental animals, males and females should be kept separately.

Generally, quarantine is conducted for 5 to 7 days.

Gender identification of commonly used experimental animals

Animal grouping

Preparation of test substance

Choice of route of exposure

Poison preparations

When preparing, do not heat the test object to a temperature that would change its physical and chemical properties.

To evaluate the skin toxicity of solid test substances, their shape and particle size must be maintained

Prepare the multi-component test substance according to the formula so that the preparation accurately reflects the original mixture

Preparations should maintain chemical stability and consistency with the test substance

The preparation should reduce the total test volume and do not use too much solvent or excipient.

Preparations should be easy to prepare for exposure

The pH of the preparation is best between 5 and 9

Do not use acids or bases to dissociate the test substance

For parenteral poisoning, the final solution should be as isotonic as possible

Oral (gastrointestinal) exposure

Infection via respiratory tract

skin exposure

Exposure via injection

Urine and feces collection

Organ specimen collection

Bile collection

blood collection

General (sodium pentobarbital) and local anesthesia (procaine, etc.)

The process of killing animals using publicly recognized and humane methods so that they die quietly and painlessly without fear or anxiety.

Pathological examination is an important part of toxicology tests, and pathological studies can help determine harmful effects and target organs. Pathological examination includes gross anatomy and histopathological examination.

Within half an hour after the experimental animals were sacrificed, they were dissected using the combined extraction method of chest and abdominal organs.

Observe the shape, surface condition, color, border, size, texture, and section of the relevant organs.

Weigh the designated organs and calculate the organ coefficients.

1. Fix the animal on the dissecting board and dissect the neck (parotid gland, lymph nodes, etc.)

2. Take one side of the breast and waist skin, open the abdomen and expand the incision (internal abdominal organs)

3. Thoracotomy (chest internal organs, tongue and throat, trachea, thyroid gland, etc.)

4. Peel off the left hind limb (muscles, etc.), scalp, and ears, open the skull, and expose the spinal cord.

Use a sharp knife to cut the designated organ or tissue and unify the parts.

Tissue blocks are usually fixed in 10 times the volume of 10% formaldehyde solution, and then routinely prepared (tissue paraffin embedding, sectioning, and HE staining).

Observe and record under a microscope to make pathological diagnosis.

Pairwise comparison between each treatment group and the negative control group and comparison between multiple treatment groups and the negative control group

dose-response relationship and dose-response relationship

quality control chart

Is it statistically significant?

Is it biologically significant?

Whether it has toxicological significance, that is, whether it has harmful effects

Longitudinal comparison: whether there is a dose-response relationship for changes in this parameter.

Horizontal comparison: whether changes in this parameter are accompanied by changes in other related indicators.

The difference between the means of the treatment group and the control group is more than twice the detection error.

Comparison with historical controls: The experimental animal reference normal value is composed of the results of the negative control of at least 10 independent experiments recently conducted in this laboratory using the same strain of experimental animals and the same solvent, and its mean ±1.96 × standard error is used as the reference value range. . Negative controls should be within ±3× standard error of the mean of historical controls.

①The value exceeds the normal reference value range

②The value is within the normal reference value range, but the difference continues for a period of time after stopping contact

③The value is within the normal reference value range, but if the body is in a state of functional or biochemical stress, the difference will be more obvious.

It is a management regulation formulated for the toxicity evaluation of drugs, food additives, pesticides, cosmetics and other medical items.

It is a regulatory document that regulates a complete organizational management system for non-clinical safety research related to human health and the environment, including experimental planning, implementation, supervision, records, and file management.

The development history of GLP at home and abroad

Purpose and significance

Fundamental contents

The purpose is to ensure operational reproducibility and result credibility

When personnel who have received education and training perform tests according to SOP, the reproducibility of their test operations and operation results is better.

In the same research institution or laboratory, if different people perform operations and tests according to SOP, it can ensure more consistent results.

organizational system

Key points in designing in vivo toxicology experiments

In vitro toxicology test design

Laboratory operating procedures and management

Reagent management

Instrument management

environmental management

Laboratory animal management

Blind management

quality control chart

Statistical analysis of experimental data

Evaluation of experimental results

Since chronic toxicity testing has a long cycle, consumes a lot of animals, and requires a lot of manpower and material resources, how to ensure the reliability of chronic animal testing?