3Rs Alternatives: Reduction, Refinement, and Replacement in Animal Testing
The 3Rs in Animal Research
Reduction: Fewer animals used to obtain information of a given amount and precision.
Refinement: Less painful procedures applied to animals necessarily used.
Replacement: Substitution of conscious living vertebrates by non-sentient material.
Reduction Alternatives
Good planning of studies:
- Rational & efficient use of animals:
- No wasting
- Pilot studies
- Screening tests
- Proper statistical design
- Use of inbred strains (for some study types)
Refinement Alternatives
Minimized potential for pain or distress; Enhanced animal well-being; Improved housing conditions & experimental techniques.
Replacement Alternatives
Efficient use of existing information; In silico methods (computer simulations, mathematical models, QSAR); In vitro methods:
- Isolated organs
- Tissue slices
- Tissue cultures
- Cell cultures
- Subcellular fractions
Lower organisms; Early stages of development.
Source of Materials for In Vitro Systems
Subcellular fractions; Primary cell cultures; Cell lines; Organs; Slices
Types of Replacement Alternatives
Absolute replacement: No need to use animals (cell lines, human or invertebrate cells & tissues). Example: No need to test for skin irritation if pH < 2.0 or > 11.5; No need to test for eye irritation if the chemical is a skin irritant.
Relative replacement: Humane killing of animals to provide cells or tissues for in vitro studies.
Partial replacement: Use of non-animal methods as prescreens in toxicity testing.
Direct replacement: Human or guinea pig skin is used in vitro to replace guinea pig tests in vivo.
Indirect replacement: Limulus amoebocyte lysate (LAL) test or a test based on whole human blood is used to replace rabbit pyrogen tests.
Advantages and Disadvantages of In Vitro Systems
Advantages:
- No interactions with other organs
- Increased sensitivity
- Better control conditions
- Better experimental flexibility
- Clearer interpretation
- Large sample capacity
- Small amounts of test substance needed
Disadvantages:
- No interactions with other organs
- Biokinetics
- Metabolism
- Possible change of properties
- More difficult extrapolation
Validation of Replacement Alternatives
Qualities of the alternative:
- Minimally as good as the replaced animal test
- Being validated/ evaluated
- Accepted by users, legislators & public
Alternatives have to be validated. Validation is the scientific process by which the reliability & relevance of a procedure are established for a specific purpose.
Validation steps:
- Test development (Standardisation)
- Prevalidation (Protocol refinement; Protocol transfer; Protocol performance)
- Validation (Interlaboratory assessment *reproducibility; Test database; Independent assessment)
Specific In Vitro Methods
BCOP (Bovine Corneal Opacity-Permeability): Cornea for assessing ocular irritation. BCOP provides a useful parallel for possible human exposure. BCOP can be used in combination with other alternatives such as HET-CAM to further determine ocular irritancy classification.
HET-CAM (Hen’s egg test on chorioallantoic membrane): Qualitative method of assessing the potential irritancy of chemicals. CAM is complete tissue containing arteries, veins & capillaries & technically easy to study. Responds to injury with an inflammatory process similar to the conjunctival tissue of a rabbit’s eye.
ICE (Isolated Chicken Eye): Three parameters are evaluated to measure the extent of eye damage following exposure to a chemical substance: corneal swelling, corneal opacity, and fluorescein retention.
IRE (Isolated Rabbit Eye): In vitro alternative to the in vivo Draize rabbit eye test method for assessment of eye irritation. Liquid test substances are spread using a syringe & solids are pulverized & applied as a powder over the corneas of enucleated rabbit eyes. Principal advantages are that animals are euthanized prior to ocular irritancy testing.