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.
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.
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.
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 irritancy tests
Many different in vitro (test tube) systems have been suggested as alternatives to animal irritancy tests. The following are only a few examples.
Shiseido and the Kao Group’s human Cell Line Activation Test (h-CLAT) has been adopted as a globally recognised official test method for skin sensitisation by the Organisation for Economic Co-operation and Development (OECD), providing an alternative to animal testing.
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.
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.
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).
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.
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.
alternative toxicity tests
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.