research

Introduction

It is therefore necessary to take the right measures, even from a legal point of view, in order to protect both man and environment from the risks of marketing these new chemicals. It is necessary to follow the developments of these chemicals and their usage carefully, and to create a catalogue to classify them accurately.

In practice, it is essential to know and label the hazardousness of each new substance before it's marketed.

Consequently, the 92/32/EEC law [1], that will be fully discussed in the next paragraph, provides a system to classify the hazardousness of a substance and to order chemical, physical and toxicological tests. This means that it is imperative to examine as closely as possible every new chemical substance before putting it on the market and in the environment.

According to the afore-mentioned regulation, the hazardousness of a chemical substance currently has to be valued using animal tests.

The 92/32/EEC law does not concern any substance meant either for cosmetics or pharmacological use. These substances are regulated by different laws and subjected to special particular toxicity tests which are not dealt with in this report.

The aim of this report is to explain how and why it is possible, by replacing the current animal tests with the new alternative methodologies, to increase protection for the E.C. citizens.

The commitment to reduce the number of animals used for tests in in line with the duties which have often been underlined by the E.C. legislators. Both the legislative and the scientific aspect will be simplified as much as possible in order to make them easy to understand for those who do not have any particular legal or scientific knowledge.

The legislative aspect

If potentially dangerous chemicals are not rated with precision, if their toxicity is not determined with certainty, the whole directive loses its meaning.

Security issues regarding the preparation and production of dangerous substances, their packing and transport, their use, the safety of the consumer and the environmental impact in general, are based on their dangerousness, as determined by the testing regimen imposed by this law. The better the toxicity tests, the greater the safety for consumers.

At present toxicity tests are carried out using animals. According to the tests results, substances are classified in the following categories:

• very toxic
• toxic
• harmful
• not dangerous

The law accepts different procedures to catalogue substances; however, all of them use animals and are based on the assumption that the results are indicative for human beings too.

The directive explicitly establishes (art. 3, n. 1, cpv.) the faculty of avoiding the experimentation of substances already tested with different methods, to the tests enclosed in the same directive. The necessity of reducing experiments upon vertebrate animals to a minimum, is the criterion used for avoiding conducting the tests in the directive. In consideration of the delicacy and the importance of the matter, the directive imposes the adaptation of the testing modalities, prescribed in the enclosure, to take into account the technical progress.

Animal tests are also regulated by the 86/609/EEC [2] directive in the matter of "protection of animals used for experimental or other scientific purposes", which orders to replace or reduce the use of animals as much as possible.

Number of animals used

The percentage goes from a minimum of 10 per cent to a maximum of 75 per cent [3-8] of all the animals used in Europe for testing purposes. In any case, these are highly significant percentages which translate into several millions animals a year.

This report does not deal with other types of animal tests or vivisection.

The scientific aspect

The toxicity test LD50 (lethal dose 50) was developed in 1927 [9] and is carried out by giving different doses of the substance to be tested, to the animals, by mouth, through the skin or by inhalation.

The LD50 value obtained means the dose of the substance needed to kill 50% of the animals on which it is tested: in practice, the smaller the dose is, the higher the toxicity and the dangerousness of the substance are.

The LD50 value is expressed in milligrams (mg) of substance per kilograms (kg) of body weight.

To simplify the matter as much as possible: if a dog that weighs 40 kilos is killed by a 40 mg dose, the lethal dose for a man of 80 kilos should be 80 mg.

The sorting and labelling of the substances tested, are directly related to the lethal dose 50.

For instance, if a substance given orally has an LD50 value lower than 25 mg/kg, it is labelled as "very toxic"; if its value is between 25 and 200 mg/kg it is said to be "toxic"; if it's between 200 and 2000 mg/kg it's labelled as "noxious". An LD50 value higher than 2000 mg/kg means that the substance is not particularly dangerous.

Since 1927 different procedures have been developed [10-14] to improve this test and reduce the number of animals used, without however modifying the LD50 methodology substantially.

In 1981 Professor Gerhard Zbinden, advisor of the World Health Organization, published an article [15] in which he heavily criticized this methodology, showing that the results obtained by animal tests depend not only on the animal species used, but also on the conditions of the experiment i.e. on the branch, sex, age, housing conditions, the feeding, etc.

For instance, digitoxin should be classified as "very toxic" if tested on young rats (LD50 = 0.1 mg/kg), but it would change its classification if tested on adult rats (LD50 for females = 76 - for males = 56 mg/kg).

On the contrary, amidephrine mesilate would be classified as "harmless" if tested on young rats (LD50 = 3000 mg/Kg), but as "toxic" if tested on adult rats (LD50 = 36 mg/kg).

Isoproterenol results "harmful" on rats of 200 g (LD50 = 800 mg/kg), while it shows 200 times more toxic (LD50 = 0.3 mg/kg) in rats of 600 g.

Other examples: trifluoroperidol results "toxic" (LD50 = 140 mg/kg) for females but "not toxic" for males (LD50 = 360 mg/kg); thiourea results "very toxic" for rats of the Hopkin branch (LD50 = 4 mg/kg), but almost not harmful for ones of Wild Norway branch (LD50 = 1830 mg/kg).

More and more scientists have begun to criticize this methodology and in 1987 Dr. Ralph Heywood, scientific manager of Huntingdon Research Center, even wrote: "... the correlation among the toxicity data obtained on man and on animals is between 5% and 25%." [16].

Actually Professor Zbinden had already doubted about the toxicity tests on animals many years before [17]: "The greater part of the adverse reactions resulting on men cannot be demonstrated, foreseen or avoided through the traditional experimentations of acute and chronic toxicity on animals."

Even back in 1964 Dr. James D. Gallagher, medical research manager of Lederle laboratories, declared [18]: "The studies on animals are made for legal reasons and not for scientific reasons. The predictive value of these studies for man is meaningless - this means that our research can be meaningless."

These and other criticism coming from the scientific community have brought about the necessity to create and develop new and more effective methodologies in order guarantee human health and safety.

2) Alternative methodologies

During the years 1989-1996, about 50 laboratories all over the world tested, developed and valued more than 150 new methodologies to analyse the toxicity of chemicals [19-21].

Cellular crops obtained from cells coming from man or animals, are the main alternative methods for these types of tests.

These alternative toxicity tests are commonly called "in vitro" to distinguish them from the traditional tests on animals which are called "in vivo".

The aim of this report is not to describe the alternative methodologies in detail. Anyone interested in knowing more about this matter can find all the necessary information in the journal "Alternatives To Laboratory Animals", ATLA [22] or in the enclosed bibliography [23-53].

3) MEIC (Multicenter Evaluation of In Vitro Toxicology) [23-24]: A comparison between traditional tests on animals and the alternative methodologies.

In order to evaluate alternative methods' reliability, it is possible to compare the results obtained from each of the different methodologies for evaluating the toxicity of some substances with the actual toxicity of the same substances for humans.

This makes possible to determine which methods can better evaluete and therefore "predict" the real danger of different chemicals for human beings. By now we have enough reliable data obtained from human beings [54-114] to be able to make a comparison.

The most encouraging alternative methodologies have been selected (about 60) [23-24], [115-120] from animal or human cellular crops, and the comparison between the results obtained from humans, animals and cellular crops has been made using the most advanced computer techniques [121-136]. The results obtained are the following [19-21], [23-24], [30], [36-39], [44-45], [49], [137-161]:

• Animal tests made on mice and rats, the most used animal species, have shown a very modest capability to predict the lethal dose for humans (predictive index Q² = 0.64).

• The most predictive results of the lethal dose for humans, have been obtained from a series of 3 cellular crops of human cells (predictive index Q² = 0.76-0.82)

• The 3 cellular crops are:
  • Hep G2, that measures how much the substance to be tested influences the protein content (test time lasting 24 hours)
  • HL-60, that measures how much the substance to be tested influeces the ATP content (test time lasting 24 hours)
  • Chang Liver, that measures morphology (24 hours) and pH (7 days) variations.

• This series of three different methodologies has been found to be, not only more predictive, but also quicker and cheaper than traditional tests on animals.

These results do not imply that the other alternative methodologies have proven to be invalid. They only imply at present that for this particular there does exist the possibility of replacing the usual methodologies to value substance toxicity with new methods, which are definitely more predictive, and therefor best protect citizens' health.

We should also emphasise that this series of three different cellular crops has resulted more predictive than the "best" results obtained on animals and that this report does not consider the opinions of other researchers giving animal testing an even lower predictive value [162-167].

These and other alternative methodologies can be already used in other studies or researches which are not the subject of this report.

Pratical aspects

It is not necessary to modify the 92/32/EEC normative law, but to modify only the experimental protocol in enclosure 2, replacing the one based on animal tests by a protocol making use of a combination of the 3 suggested methodologies.

Moreover, changes to the technical part have already been made in the past whenever technological updating has been needed [10-15].

Conclusions

A series of 3 cellular crops will protect citizens' health more safely, more quickly and more cheaply.

We also need to underline that the change to the experimental protocol of the 92/32/EEC directive [1] should be an obligation for legislators by virtue of the 86/609/EEC directive [2] in the matter of "protection of the animals used for experimental or other scientific purposes" which orders to replace or reduce the use of animals as much as possible.

Political leaders and the scientific community would be irresponsible if they did not consider these results and start a trial that, even immediately, would improve public health protection.

It is also vital to press forward with this matter so that, whenever any new or improved methodologies are ready, it will be possible to make them valid (i.e. to have them officially acknowledged and in use as toxicity tests, as the normative law requires) as soon as possible.

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