ANIMAL FREE

ALTERNATIVES

A simple change.

A serious impact.

In the case of cosmetics, the manufacturer could choose ingredients that have been used for a long time, and so are likely to be safe. If the chemical is a new one, the first test could be the QSAR computer analysis to predict its likely irritancy.

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In the next stage, a number of in vitro (test tube) tests could be used. Some tests are better than others for substances of a certain form, for example liquid rather than solid, and for substances of a certain chemical class, for example alcohols rather than oils. The aim would be to choose the best set of tests for a particular substance.

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Doing more than one test is not a problem because in vitro tests are so much faster and cheaper than animal tests. For example, an Agarose Diffusion Test takes 24 hours per product, whereas a Draize test takes at least three days per product and costs 10 times more to carry out.

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If the product is shown to be safe by this set of tests, it can then be trialed by human volunteers. This is the final and best test of all. By going through this series of steps products can be guaranteed to be safe without the suffering that is currently inflicted on animals.

 

Alternative Toxicity Tests

The studies

In 1989 the Scandinavian Society for Cell Toxicology organised a large international study of alternative methods. This study was called Multicenter Evaluation of In Vitro Cytotoxicity (MEIC), and involved laboratories in many different countries. A list of 50 chemicals was selected for testing by the Swedish Poison Information Centre. Chemicals for which there was good human data were chosen. In other words, for these chemicals it was known how much it would take to kill a human from the results of accidents or suicides. When the MEIC project closed in 1996, 59 laboratories from all over the world had submitted results. There were 29 laboratories that had tested all 50 chemicals. In total, 61 different in vitro (test tube) methods were used. The researchers compared the lethal doses (LD) taken by humans with the LD50 in animals. They also compared the lethal concentration (LC) in the bloodstream of people who died with the concentration that produced a 50% reduction in the growth of cell cultures (IC50). Results from the final evaluation showed that human cell culture tests were more accurate than animal LD50 tests. As MEIC director, Dr Bjorn Ekwall, has commented, cell culture systems can still be further improved, but animal tests can’t. By the year 2003 a new project, called EDIT, aims to develop and publish a set of about six in vitro tests that will predict human toxicity with 90% accuracy. EDIT also aims to develop a set of tests to predict long-term toxicity. Tests are being worked on where repeated doses of a chemical are added to the cell culture for 6 weeks.



Human cell cultures have several advantages in predicting toxicity:

  • They are human and so avoid species differences;
  • They can be taken from the tissue that a particular test chemical is most likely to affect, for example the skin, or the liver;
  • They allow researchers to study how a substance causes damage to the cells, that is, why it is toxic;
  • They avoid causing pain and death to animals.
  • Human tissues for testing are becoming more available in the USA and Europe, although less so in Australia.
  • There are companies that market cell lines from normal tissues and from tumours. For example:
  • The American Type Culture Collection has available over 2300 animal and human cell lines.
  • Companies such as Clonetics market cell cultures derived from human skin, cardiovascular system, brain, respiratory system, kidneys and muscles (visit the Clonetics web site).
  • Researchers can collect their own human tissue, for example, blood from volunteers or skin from plastic surgery. For many tissues, though, it is easier to use tissue banks.
  • In the USA, the National Disease Research Interchange in Philadelphia collects tissues removed during operations and from donors, and distributes them to researchers (visit the NDRI web site and look at About Us and Our Mission and History).
  • In the UK, the University of Leicester is doing the same. They use organs that are not suitable for transplantation and would otherwise be destroyed.



Alternative Irritancy Tests

h-CLAT (Human Cell Line Activation Test)

This in vitro method quantifies the changes seen in the cell surface markers following exposure to a test chemical. These changes are measured by flow cytometry after cell staining with fluorochrome-tagged antibodies. The level of fluorescence intensity is calculated and used in a prediction model to discriminate between sensitizers and non-sensitizers.



Eytex

This test for eye irritancy uses a vegetable protein extracted from jack beans. Like the cornea of the eye, this clear protein gel becomes cloudy when in contact with an irritating substance. In the Draize test, people have to estimate the degree of damage caused, that is, how swollen or red part of the rabbit’s eye is. This system isn’t very accurate. In the Eytex test, the degree of cloudiness (“damage”) can be measured by a machine, a spectrophotometer, which is much more reliable.



Mutagenicity

There are few validated alternative tests relating to mutagenicity/genotoxicity; referring to the mutation of cells causing degenerative conditions and heritable damage. The in vitro Mammalian Cell Micronucleus test uses cultured human cells to detect micronuclei in the cytoplasm of cells which have undergone cell division either during or after exposure to the test substance. The presence of micronuclei can demonstrate chromosomal instability and thus the hazardous nature of the test substance.



Reconstructed Human Epidermis

This is a multi-layered human skin grown in the laboratory, which can be used to test skin irritancy. It is sold commercially under trade names such as Skin Squared and Episkin. There are various ways of measuring damage when an irritating product is applied to this test skin. For example, cells can be examined under the microscope, membrane damage can be assessed by leakage of enzymes, or inflammation can be determined by release of interleukins. Whatever method is used, the result can be measured accurately, unlike in animal studies where observers estimate the degree of swelling or redness. In October 2014 after five years in development, Australia-based Baxter Laboratories launched an in-vitro skin compatibility and safety test. Read more here.



Corneal cell lines/strong

The SIRC is a continuous cell line of rabbit corneal cells. These are cells that are now grown in the laboratory, and no further rabbits are killed. When six shampoos were tested on these cells, there was very good agreement with Draize results. The test assessed how much of a substance was needed to kill half the cells. Obviously, the less of a substance that is needed to produce this result, the more damaging it is. However, to avoid species differences it would obviously be preferable to use human cells. One problem with using cells from human corneas has been that these cells don’t live for very long. Now researchers have found a way of not only increasing the number of these cells, but also extending their life span so that they can be studied in more detail. Researchers used human corneas from an eye bank to grow the cells. This cell culture can be used not only to study eye irritation, but also wound healing, parasite infection, and radiation damage in the eye.



Neutral Red Uptake Test

Normal cells in culture readily absorb and hold this neutral red dye. When the cell membrane, or the lysosomes inside the cell are damaged by an irritating chemical, dye will be lost through the leaky membranes. Less dye will remain in the cell. A spectrophotometer is used to accurately measure how much has been lost.



Agarose Diffusion Test

The problem with cell cultures such as those in the Neutral Red Uptake Test is that the cells are in fluid, so only soluble substances can be tested. In the Agarose Diffusion Test a small amount of agarose (a seaweed extract) is added to form a gel layer. Some of the test substance is placed on a small piece of filter paper, which is then placed on the agarose. The substance diffuses through the agarose into the cell culture below. The irritancy of the substance is assessed by measuring the area, in millimetres, of dead cells under the filter paper, that is, cells that have lost their neutral red dye.



Microphysiometer

An irritating product will produce changes in the functioning of cells. The microphysiometer is an instrument that detects very small changes in cell metabolism by measuring changes in the pH of the cell culture nutrient fluid (changes in lactate, CO2 production).



Computer modelling

Expert computer systems can be used to predict the irritancy of new substances on the basis of what is already known about the irritancy of substances with a similar chemical structure. This approach is known as Quantitative Structure-Activity Relationship, or QSAR for short. The molecular structure of known substances is entered into a computer database. Particular chemical structures are linked to particular kinds of chemical activity, in this case irritancy. When a new substance is entered, the expert system tries to match its molecular structure to others in the database. If it finds a close similarity, it predicts that the new substance has the same level of irritancy.



Human Studies

Some cosmetics companies already use human volunteers to test new formulations. This is the most reliable test of all. Human irritancy can be assessed through patch testing, where test substances are placed on small areas of the upper back and covered with a patch for two days.

Microdosing is another method of testing on humans. Volunteers are given an extremely small dose of the drug before sophisticated imaging techniques are used to monitor how it is metabolised within and affects the body. This is effective in screening out drug compounds which will have no effect on humans, thus eliminating the governmental requirement for animal testing.

Functional Magnetic Resonance Imaging (fMRI) is another technique which utilises humans rather than animals in the study of the brain. fMRI uses brain imaging and recording techniques to safely study the brain. Researches are even able to temporarily and reversibly induce brain disorders through transcranial magnetic stimulation; thus eliminating the archaic method of inducing brain damage on rats, cats and monkeys.



Organ Chips

Wyss Institute’s ‘Organs-on-chips’ is an in vitro alternative test that utilises human cells which have been grown to mimic the structure and function of human organs and their systems. The chip is made from a clear, flexible polymer which contains hollow microfluidic channels which are lined by specific living human organ cells and interfaced with a human endothelial cell-lined artificial vasculature. Mechanical forces are then applied to mimic the physical microenvironment of living organs, such as the motion of breathing for lung cells. Therefore they are able to be exposed to disease and drug testing while accurately replicating human physiology responses.

Organoids are also similar in vitro methods. These are miniature and simplified versions of human organs which are grown in a lab. They allow for the study of disease and treatment, and have been used successfully in the Netherlands to show how certain drugs affect individuals being treated for cystic fibrosis. As a specific organoid can be grown from a biopsy of an individual patient, this allows for extremely accurate testing.



CATMoS - Collaborative Acute Toxicity Modelling Suite

CATMoS is an in silico predictive modelling method which tests for acute toxicity, and can replace in vivo testing. It utilises reference animal test data and international consensus predictions of how a substance may react following oral consumption. This free resource for screening organic chemicals for acute toxicity has been effective in reducing the number of animals used for toxicity testing.



QSAR - Quantitative Structure-activity Relationships

These are in silico computer modelling techniques. Past research and our knowledge of human biology are combined to develop mathematical algorithms which are integrated with computer code to formulate the predicted behaviour and effects of a substance. This method of alternative testing is heavily supported due to its accuracy and ability to significantly reduce the use of animals in scientific testing.



KeratinoSens and LuSens Assay

These are also validated alternative skin sensitisation tests.

KeratinoSens uses a human keratinocyte cell line (HaCaT) which are transfected with a plasmid which contains the luciferase gene, which is known to be regulated by contact sensitizers. The luciferases are oxidating enzymes which emit light that can be quantified by luminescence detection. These cells are then exposed to a substance and the luciferase concentration can be used to predict sensitisation.

The LuSens assay is based on the same concepts. It utilises the same HaCaT cells and employs the same luciferans reporter gene.

It is recommended that these tests are used in combination with other in vitro and in chemico models.



DPRA – Direct Peptide Reactivity Assay

DPRA is a skin sensitisation test. This in chemico method quantifies the reactivity of test chemicals towards model synthetic peptides which contain either lysine or cysteine. Evaluating the protein reactivity by measuring the peptide depletion values allows the categorisation of a substance into one of four reactivity classes; thus determining whether the substance is a skin sensitizer or not.



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