Pruebas de caja negra

In many situations quick information may be wanted on whether a compound or product should be con-sidered toxic or not, including a rough idea of how toxic it is to animals. For such an examination one

will rarely use highly developed animals such as mice, out of both ethical and financial considera-tions. Instead a battery of tests may be used which could also be employed for the elucidation of any environmental consequence of the spreading of the compound, for the screening of a series of com-pounds extracted and purified from a medical plant, or for the screening of synthetic pesticide candidates, e.g. for insecticidal activity.

One such method uses fruit flies, which may even be wingless mutants to ease the handling.

Other test types use larvae of various species of the Artemia genus (brine shrimps). The Artemia genus is scattered worldwide and the word ‘brine’ means very salty water. After mating, the 8–10 mm long animals lay their eggs. Under favourable conditions these hatch immediately, while in water which is too saline or with a low oxygen level the eggs develop a thick shell, after which they are called cysts. When hatching, either directly after laying or from the cyst stadium, the larvae are released, called nauplii. These are able to live for about 3 days without food. The nauplii develop into full-grown brine shrimps through 15 different stages.

Cysts such as those of Artemia salina can be purchased commercially, since owners of aquari-ums hatch the cysts and use the larvae as feed for their fish. Some of the cysts on the market derive from large salt lakes in the state of Utah, USA (home of the large town Salt Lake City).

The brine shrimp test

The first to point to A. salina as a suitable organ-ism for toxicity tests were A.S. Michael, C.G.

Thompson and M. Abramovitz from the Entomology Research Branch, USDA, Beltsville, Maryland. The three scientists developed the test for their work on the identification of new com-pounds with potential as insecticides. At first dif-ferent setups of the method were used and so too were both fully developed shrimps and several of the different larval stages. One of the methods included observing the time for the adult animal to sink to the bottom due to paralysis of the swim-ming movements.

Since the first article on the usage of Artemia, many other scientists have developed their own pro-tocols for how to test toxicity using this organism.

Meyer et al. used nauplii which they fed with yeast during the test. They determined LC₅₀ for a long list of plant extracts after 6 and 24 h of exposure and

compared the results with those from two tests for cytotoxicity to cancer cells for the same plant extracts. A long line of other experiments confirm that the brine shrimp test is applicable for many dif-ferent types of compound. The first to perform the test in a microtitre plate were Pablo et al. in 1992.

Such tests are today commercially available in kits.

As mentioned above, a large selection of structur-ally very different compounds has been tested for toxicity using A. salina. Hence, when searching on the Internet, it is possible to find a database listing many compounds and their toxic effect(s) on this organism (‘Brine shrimp (Artemia salina) Chemical Toxicity Studies’, available at http://www.pestici-deinfo.org/List_AquireAll.jsp?Species=366).

The brine shrimp test for toxicity:

how to perform the test

On the first day 25 ml of artificial seawater is meas-ured and placed in a Petri dish. Eggs of the species A. salina are used for this test. An appropriate amount of eggs is taken, i.e. about 200 mg, and dispersed on the water surface in the Petri dish and the lid is replaced to protect from pollution, reduce evaporation and ensure a high humidity in the air layer above the water. The Petri dish is then placed under an electric light bulb, ensuring that the tem-perature is between 25 and 28°C. The eggs are left to hatch overnight.

After 24–48 h the majority of the larvae are hatched and ready for use. A funnel is placed in a large plastic centrifuge tube and the content of the Petri dish is transferred to the tube. If neces-sary an additional 5 ml of artificial seawater is used to rinse the Petri dish for any remnants of larvae and eggs.

The centrifuge tube is closed and gently turned upside down a few times, then left to rest for a few minutes. This will give the non-hatched eggs an opportunity to gather on the surface. The lid is removed without shaking the tube. The eggs float-ing on the surface are gently removed usfloat-ing a Pasteur pipette.

Five millilitres of the resulting larvae suspension is transferred to a clean centrifuge tube. The sus-pension is taken from the middle of the liquid column to avoid any eggs from the top or the bottom.

One drop of the suspension with the larvae is placed on a glass slide with a cover slip on top and the larvae can be observed under a microscope.

The centrifuge tube is closed and gently turned upside down to remix the suspension. The tube is opened and 0.1 ml is extracted and placed on a glass slide; this is repeated four times. With a mag-nifying glass each of the five drops is observed and the number of living larvae is counted and noted. If this is not possible due to a high number of living larvae, a small amount of 0.5 M sulfuric acid is added. When the larvae are lying still (are dead) counting can be done. The average number of lar-vae per 0.1 ml based on the five drops is calculated.

Artificial seawater is added to the examined sus-pension to dilute this to a resulting concentration of approximately 10 larvae per 0.1 ml.

For the purpose of this discussion we may think of the determination of LC₅₀ after 24 h of exposure to potassium dichromate. First, tubes are prepared with the concentrations to be used. A zero concen-tration is taken directly from the seawater bottle and the highest concentration to be tested (1600 ppm potassium dichromate) is made by pre-paring a solution at the concentration of 3200 ppm, from which 100 ml will be used. The serial dilution is then started by adding 1 ml from the stock solu-tion to produce a concentrasolu-tion of 1600 ppm. Then 800 ppm is produced by taking 1 ml from the 800 ppm tube and adding 1 ml of seawater. This line of dilution is continued until the lowest con-centration has been made.

An eight-channel pipette is prepared with tips for transfer of the larvae suspension to a microtitre plate with 96 wells. The larvae suspension is poured into a 30 ml reservoir called a ‘cradle’, from where it can easily be sucked up with the eight-channel pipette and distributed in the wells. The suspension should be stirred gently before each round of transfer with the pipette. A volume of 100ml is added to each well. Then the number of dead larvae is counted with a magnifying glass and

noted for each well, as these should be excluded in the final calculation of results. The substance to be tested is then added, in this case the potassium dichromate of which the serial dilution has been prepared. Each concentration is tested in six wells. Starting with the lowest concentration so that the pipette does not need to be changed, 100 ml is added to each well and the final test concentration is thereby half of the dilution concentration.

After the addition of the test compound the microtitre plate is covered/sealed. The plate is then placed in an incubator at 25°C overnight. After incubation for 24 h the microtitre plate is removed from the incubator, placed on a coloured back-ground and the sealing membrane removed.

The number of dead larvae in each well is again counted using the magnifying glass and noted on a prepared form. Then from 100 to 150 ml of 0.5 M sulfuric acid is added to each well with the eight-channel pipette. After 15 min all larvae should be immobile (dead) and the total number of larvae in each well is counted and noted. Now the number of live larvae in each well before and after the 24 h of incubation can be calculated. The information needed for determination of the LC₅₀ has been obtained.

Analysing the results:

the Reed–Muench method

When using the Reed–Muench method it is assumed that an animal which survives a given dose would also survive any lower dosages of that compound and an animal which dies at any given dose would also die at any higher dosages. The method is demonstrated with the results shown in Table 13.2.

According to Table 13.2, 15 out of 30 larvae survive a concentration of 1000 ppm. At 800 ppm,

Table 13.2. Example of how to calculate LD₅₀ from the results of a brine shrimp test using the method of Reed and Muench.

Dose (ppm)

Dosage

(log dose) Dead Alive

Accumulated dead

Accumulated alive

Accumulated total

Ratio accumulated

dead/total

Mortality (%)

1000 3.00 15 15 42 15 57 42/57 73.7

800 2.90 8 22 27 37 64 27/64 42.2

600 2.78 8 22 19 59 78 19/78 24.4

400 2.60 7 25 11 84 95 11/95 11.6

200 2.39 4 26 4 110 114 4/114 3.5

22 of 30 larvae survive. According to the Reed–

Muench method the number of surviving larvae at 1000 ppm and 800 ppm are added to one another, meaning that 37 larvae survive at 800 ppm. At 600 ppm a further 22 larvae survive, so the total number of surviving larvae is then 59. The number of dead larvae is calculated by starting with the low-est concentration, i.e. the four dead at 200 ppm are added to the seven dead at 400 ppm and so forth.

The total number of dead larvae then equals 42 at 1000 ppm. So, at 1000 ppm, the accumulated number of dead larvae is 42 and the accumulated number of live larvae is 15, i.e. 42 out of 57 are dead, which equals 73.7%. The concentration which sta-tistically will kill 50% of the animals can then be determined on a graph, where the percentage dead (mortality) is plotted as a function of the logarithm of the concentration. According to the literature, the LC₅₀ for potassium dichromate towards A. salina is somewhere between 500 and 800 ppm at 6 h expo-sure and between 20 and 40 ppm at 24 h expoexpo-sure.

The results for screening an unknown com-pound or extract are regarded as acceptable if a simultaneous determination of LC₅₀ for a positive standard (such as potassium dichromate exempli-fied here) is within the expected – formerly deter-mined – confidence interval for this standard and if the mortality in a negative control is low, i.e. less than 5%. The positive standard is included in the examination to ensure that, among other things, the larvae used are of a suitable quality.

A report would include doing a graphical presen-tation of the results and the calculations, along with an evaluation of the compound or extract tested in relation to the results.

13.10 Conclusion

Mixtures of compounds (products) as well as pure compounds are today tested in a number of

different tests for toxicity using live animals, iso-lated organs, primary cell cultures or continuous cell cultures. Such testing will in most cases be structured so that simple (short-term, inexpen-sive) tests come first and are followed by more long-term and expensive tests planned on the basis of the results from the previously performed tests.

Notes

1 Lu, F.C. and Kacew, S. (2002) Lu’s Basic Toxicology:

Fundamentals, Target Organs and Risk Assessment.

Taylor & Francis, London.

2 The Encyclopedia of Earth (2008) Dose–response relationship. Yuill, T. and Miller, M. (eds). EIC Secretariat, National Council for Science and the Environment, Washington, DC; available at http://www.eoearth.org/

article/Dose-response_relationship

Further Reading

Board on Environmental Studies and Toxicology, Institute for Laboratory Animal Research, Division on Earth and Life Studies, National Research Council (2007) Toxicity Testing in the 21st Century:

A Vision and A Strategy. National Academies Press, Washington, DC.

Ecobichon, D.J. (ed.) (1997) The Basis of Toxicity Testing, 2nd edn. CRC Press, Boca Raton, Florida.

Gad, S.C. (2002) Drug Safety Evaluation. John Wiley &

Sons, New York, New York.

Hodgson, E. (ed.) (2004) Modern Toxicology, 3rd edn.

John Wiley & Sons, Hoboken, New Jersey.

Lu, F.C. and Kacew, S. (2009) Lu’s Basic Toxicology:

Fundamentals, Target Organs, and Risk Assess-ment, 5th edn. Informa Healthcare, New York, New York.

Mongelli, E., Martianez, J., Ananya, J., Grande, C., Grande, M., Torres, P. and Pomillo, A.B. (2003) Bolax gummifera: Toxicity against Artemia sp. of Bornyl and iso-Bornyl Esters. Molecular Medicinal Chemistry 1, 26–29.

14.1 Introduction

In vitro (Latin: in a glass) refers to biological experi-ments outside the intact live organism using cultures of isolated organs, primary cells or cell lines and subcel-lular fractions. The development of in vitro methods really took off in 1986 as the Animal Welfare Guidelines were implemented and the EU institutions declared policy was to support the development of alternative methods that can reduce, replace or refine (RRR) the use of animal experiments. The use of in vitro methods is therefore increasingly favoured in the EU but similar developments have also been seen in both the USA and Japan. The developments are coordinated by OECD and in 1991 the European Centre of Validation of Alternative Methods (ECVAM; http://ecvam.jrc.it) was founded. Other similar institutions, such as the US Interagency Coordinating Committee on the Validation of Alternative methods (ICCVAM; http://iccvam.niehs.

nih.gov/) and the National Toxi cology Program Interagency Center for the Evaluation of Alternative Toxicological Methods (NICICEATM; http://iccvam.

niehs.nih.gov), also exist. The validation of an alterna-tive method such as an in vitro test consists of several stages. First of all the following questions are asked: (i)

‘Is the method optimized?’ and (ii) ‘Can the same results be obtained in another laboratory using the same method?’ The next step is to validate its relevance, i.e. how well the in vitro test results correlate with the results obtained in vivo. If the method is both reliable and relevant, a formal validation is carried out and if the method is scientifically accepted it may be adopted as a regulatory guideline by the EU or OECD.

In this chapter the following in vitro methods are described:

● in vitro testing of metabolism/toxicokinetics;

● in vitro acute toxicity testing;

● in vitro genotoxicity testing; and

● in vitro testing of developmental toxicity.

14.2 Metabolism/Toxicokinetics Testing