Toxicity Testing incl. LD50

Toxicity tests

Toxicity tests examine what happens when a chemical is absorbed into the body, usually via the mouth, but sometimes also through the skin or the lungs. The aim is to find out if the substance has toxic effects. The most common test of acute (short-term) toxicity is the LD50 test. Many different substances are tested in this way, including all drugs, agricultural chemicals, cleaners, and some cosmetics and their ingredients.

This text is one in the set on safety testing. If you want to read shorter and less complicated texts, go to:

Cosmetic testing
Drug testing

Other texts in this set on safety testing are:
Skin and eye irritancy tests
Tests for chemicals that cause cancer or birth defects

LD50 test

LD50 stands for Lethal Dose 50%, in other words, the dose of a substance that will kill half the test animals. The result is usually expressed as mg/kg. For example, if it took 400 milligrams of a test substance to kill half the rabbits weighing 4 kilograms, then the LD50 is 100mg/kg.

A single dose of the test substance is usually placed directly into the stomach of animals via a tube. Different groups of animals are given increasing doses of the test substance to see which dose will kill half of them.

The animals are observed for 14 days, and any that haven't already died are then killed and dissected. Researchers record the number of deaths on each day, and any signs of illness (1).

Obviously animals can suffer terribly in this test. Symptoms of toxic substances include abdominal pain, cramps, convulsions, vomiting (in some species), diarrhoea, paralysis, breathing difficulties and bleeding ulcers.

Various species are used for LD50 tests. Large numbers of rats and mice are used, but sometimes dogs and rabbits are also included (1).

Inhalation test

Substances which may be inhaled and absorbed through the lungs are sprayed into a chamber. Animal are placed in this chamber, usually for 4 hours, and observed for any toxic effects (1).

Dermal test

Some substances can enter the body through the skin. To test for this effect, the back of rabbits is shaved and the test substance is kept in contact with the skin for 24 hours. As in the oral LD50 test, the rabbits are observed for 14 days. If any die, an LD50 dose is calculated, but if no toxic effects are observed with a dose of 2mg/kg, then no further tests are done (1).

Longer term tests

To find out if a chemical has long-term effects, animals are fed smaller doses every day, often for 90 days. Two species are usually tested, a rodent (rats or mice) and a non-rodent (often dogs).

Problems with the LD50 test

Clearly this is a very cruel test. Gerhard Zbinden, one of the world's best known toxicologists, has called it " a ritual mass execution of animals " (2). It gives no information on treating human poisoning. It is an unreliable way of predicting risk to humans because the results are altered by so many factors.

Firstly, there are huge species differences. Ten-fold species differences are common (2). In other words, the LD50 for the same substance is often 10 times higher in one species than in another. Sometimes the differences are much greater. For example, the LD50 for methylfluroactate was 0.15mg/kg in dogs and 11.00mg/kg in monkeys (2).

There can be large differences even between closely related species. For example, the LD50 for paracetamol was 250-400mg/kg in mice and hamsters. Death was caused by liver damage. However, in rats the LD50 was 1000mg/kg, and there was no sign of liver damage (3).

Even within the same species there can be huge differences. The LD50 for thiourea was 4mg/kg in the Hopkins strain of rat, compared to 1340mg/kg in the Norwegian rat (3).

There are many other factors apart from species and strain which affect LD50 results, including age, sex, amount of food, social environment, and physical environment such as temperature and humidity. For example:

It is not surprising, then, that results from different laboratories can vary 8-14 fold (3). In other words, the LD50 result for a substance can be up to 14 times higher in one laboratory than in another.

Dr Bjorn Ekwall, director of the MEIC project (see below), has said:
" Why have such blunt and crude investigative tools been used in the past and are still being used? The obvious answer seems to be that nobody has yet come up with better ideas. The real answer is probably that until recent times nobody has bothered to try to come up with better ideas " (4).

The LD50 test is neither reliable nor useful. As respected toxicologist Gerhard Zbinden concluded: " For the recognition of the symptomatology of acute poisoning in man, and for the determination of the human lethal dose, the LD50 in animals is of very little value " (3).

The following table shows that the human lethal dose, how much it takes to kill a person, can't be predicted from animal studies (2):

Substance Human lethal dose Rat LD50 Mouse LD50 Rabbit LD50
Lindane 840mg/kg 125mg/kg - 130mg/kg
Caffeine 192mg/kg 192mg/kg 620mg/kg -
Boric acid 640mg/kg 2660mg/kg 3450mg/kg -
Amytal 43mg/kg 560mg/kg - 575mg/kg

In the case of non-toxic substances, absolutely ridiculous amounts have been fed to animals to make half of them die. For example, doses of up to 32gr/kg of tartaric acid, a food preservative, were fed to animals. It killed them, not by poisoning, but by corrosion of the stomach lining (3). The equivalent for an adult human would be to eat 2.25kg of tartaric acid in one sitting!

Up to 25g/kg of lipstick has been fed to animals. The equivalent amount for an adult human would be 1.75kg (3)!

To avoid these ridiculous situations a limit test, or fixed dose test, can be done. If a substance is likely to be non-toxic, a dose of up to 10g/kg is tested on animals. If there is no effect, the substance is considered non-toxic (2). This is a more sensible approach than the LD50 test. However, it doesn't avoid the ethical problem of using other individuals for experiments, or the scientific problem of species differences.

Alternative 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 blood stream of people who died with the concentration that produced a 50% reduction in the growth of cell cultures (IC50). Results from the final evaluation show that human cell culture tests are more accurate than animal LD50 tests (5):

Rat LD50 - Human LD 60% accurate
Mouse LD50 - Human LD 66% accurate
Human cells IC50 - Human forensic LC 71% accurate

The 71% is the combined results of 18 tests using human cells. When only the single best test was considered, a test using MRC-5 cells and 6 weeks exposure to chemicals, it predicted the human lethal blood concentration with 75%-80% accuracy (6).

For the best 3 tests combined, there was 78% agreement between the cell culture results and the human lethal concentration (7). Human cell cultures were more accurate than animal cell cultures (6).

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 6 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 (8).

Human cell cultures have several advantages in predicting toxicity:

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 cell culture tests discussed so far have been used to measure general cytotoxicty, that is, how toxic chemicals are to cells in general. However, cell culture tests can also be used more specifically to show how a chemical affects cells from a particular organ. The following are just a few examples.

Skin

Some drugs make the user very sensitive to ultra-violet (UV) radiation, as in sunlight. This is called phototoxicity. Two major groups of antibiotics known to have this effect were tested on human skin cells taken from hospital operations. The cells were exposed to the test substances and then to UV radiation. The damage to the cells closely reflected human experience (9).

It is important to know whether a substance can pass through the skin and have a possible toxic effect in that way. Three ingredients used in medical skin creams were tested on human skin from cadavers. This skin predicted the absorption rate in humans better than rat studies (10).

Blood

Some drugs have a depressing effect on the immune system, which is called immunotoxicity. Human white blood cells (lymphocytes) were used to test the effect of 2 AIDS drugs. The cells produced an antibody response to foreign proteins, but this response was reduced when the drugs were added to the cell culture. The effect was similar to that seen in humans taking the drugs, so the cell culture can be used to predict this effect in new drugs (11).

People exposed to nickel in industry appear to have reduced resistance to infection. To test this effect on the immune system, human white blood cells (lymphocytes) were exposed to nickel compounds. The number of natural killer cells, an important part of the immune system, was significantly reduced in these cell cultures (12).

Lungs

Many chemicals are inhaled, often as industrial or agricultural fumes or dust, but sometimes also as aerosol products. A continuous line of rat lung cells were grown on a nutrient gel rather than in a liquid, so that they could be exposed to airborne pollutants. The concentration of test substances required to kill 50% of the cells was a good indication of their toxicity (13).

There are now human cell lines available to test inhalable materials. Cells from the alveoli have been used to test the toxicity of mineral dusts, such as asbestos (14).

Cell lines from the alveoli (15) and the bronchi (16) have been used to study the effect of cigarette smoke, as in passive smoking. The smoke produced clear cell damage in as little as 1 hour, including reduction in cell metabolism and reproduction, as well as damage to the cell membrane.

Liver

The liver is a very important organ which breaks down chemicals. As a result, liver cells are often among those that are damaged by a toxic chemical, and liver cell cultures are particularly important to test substances.

Human liver can be used for testing either as very thin slices or as isolated cells (hepatocytes). Normal cells taken directly from livers do not live very long, but there are now continuous cell lines that have been growing for several years and still have all the functions of liver cells (17).

There are large species differences in the way chemicals are broken down, as shown in a test of diazepam (Valium). The drug was broken down much more quickly in liver cells from rats, rabbits, guinea pigs and dogs than in human liver cells. The longer a drug stays in the body, the more likely it is to have toxic effects, so this kind of difference is extremely important. It will only be known if human cells are used (18).

Kidney

The kidney is exposed to chemicals as it filters the blood, so kidney cells are also among those that can be damaged by toxic substances. The kidney is a complex organ made up of many different cell types, so it is important to choose the right type for particular tests.

It is known that exposure to cadmium in the workplace leads to kidney problems. Human kidney cell (proximal tubule cells) removed during operations were used to investigate the toxicity of cadmium. The cell cultures were able to demonstrate what had previously been observed in people, namely that there is a large variation between individuals in their sensitivity to this heavy metal (19).

People with kidney failure need dialysis to remove waste products from their system. Sometimes this dialysis is done through the lining of the abdominal cavity (peritoneal mesothelium). The mesothelium is exposed to dialysis fluids and may be damaged as a result. Human mesothelial cell cultures were used to test the toxicity of different fluids, and so to be able to provide the safest treatment (20).

Nervous system

Toxic effects on the nervous system can be extremely serious and produce effects such as convulsions , paralysis, numbness, trembling, hallucinations, anxiety, and depression. Some of these symptoms are difficult, if not impossible, to detect in animals, such as sleep disorders, memory impairment, mood changes or loss of drive. In addition, there are major species differences. As one toxicologist has concluded:

" There are many examples where species differ markedly in their responses to neurotoxic chemicals, and this makes the extrapolation of animal data to man a risky procedure " (21).

The study of the nervous system will always be complex and difficult, but at least the use of human cells eliminates species differences. Human nervous system cells (neuroblastomas) have been used to study the effects of the organophosphate group of pesticides, which are known to be neurotoxic (22).

Human neuroblastoma cells have also been used to investigate why some patients given chloroprocaine anaesthetics had nervous system side effects. The anaesthetic solution significantly reduced the growth of neuroblastoma cell colonies, but the researchers could show that this neurotoxic effect was due to one of the additives to the anaesthetic solution, rather than the drug chloroprocaine itself (23).

Toxic effects specific to nerve cells can be observed to study reasons for neurotoxicity. For example, neurotoxic chemicals caused a decrease in neurites and changes in calcium concentration in neuroblastoma cells (24).

Bone

One toxic effect of drugs is agranulocytosis, where there is a sudden drop in the number of white blood cells produced by the bone marrow. To examine this effect, cells from the bone marrow of different people was exposed to a range of drugs. The growth of cell colonies was significantly reduced in the bone marrow of people who were likely to develop agranulocytosis in response to particular drugs (25).

Various types of cell cultures, including human cells, are already being used to test materials for medical implants, such as metal pins for broken bones. The same cell type as will be in contact with the material (eg bone cells) can be used to make sure the implant material will not damage the cells. Cell culture tests are good predictors of what will happen in the body (26).

Cell culture technology has already come a long way, and will continue to develop in the future. It allows researchers to investigate not only whether a substance is toxic, but also how the toxic effect comes about, and how the substance is likely to affect humans. For all these reasons, human cell culture tests are more useful, and more humane, than LD50 and other animal tests.

LD50 tests in Australia

LD50 tests are restricted but not banned in some states of Australia, including Victoria, NSW and South Australia. In Victoria, lethal dose testing may only be done in potentially lifesaving research, and with the approval of the Minister. In NSW, ministerial approval is required when the LD50 is used for product testing. In South Australia, lethal dose testing may only be done in research which benefits humans or animals, and when there is no alternative that is less painful.

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