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Every time you reach for an eyedrop orreapply a lip salve, you do so confidentthat the chemicals they contain are safe to use. But the toxicology tests on

which regulators rely to gather this informa-tion are stuck in a time warp, and are largelybased on wasteful and often poorly predictiveanimal experiments. Efforts in Europe areabout to change this, and the man chargedwith bringing toxicology into the twenty-firstcentury is a plain-talking German: ThomasHartung. Although Hartung acknowledgesthe immense challenges ahead, he sees this asan opportunity for toxicology “to turn itself atlast into a respectable science”.

Three years ago, when Hartung becamedirector of the European Centre for the Vali-dation of Alternative Methods (ECVAM) inIspra, Italy, he didn’t know that the job wasabout to shift gear dramatically. ECVAM wasset up in 1993 to support European Union policy aimed at reducing the number of ani-mals used in regulatory testing.

The centre, which nestles on the sleepyshores of Lake Maggiore in the Italian Alps,originally had ten members of staff and facedan uphill struggle to cut back the millions ofanimal tests carried out in Europe every year.Then in 2003, two major policy changes wereannounced from above, increasing the pres-sure on the centre’s labs. ECVAM found itselffacing an unexpectedly short deadline fordelivering a slew of animal-free methods fortesting chemical toxicity.

Rule changeThe first change was to the European Union’sCosmetics Directive, which phases out overten years the use of animals in cosmetics test-ing. A short while later, the European Com-mission proposed its controversial REACHlegislation (Registration, Evaluation andAuthorization of Chemicals). Europe pro-duces some 30,000 chemicals for which toxic-ity data have never been registered. REACHaims to make registration mandatory for bothfuture and existing chemicals — even thosethat have been on the market for decades.

If, as expected, the REACH directive isapproved next year, it will come into effect in2007. Animal-welfare groups fear that this willmean millions more animals will be used intests to meet the regulatory requirements. Andindustry claims that the testing process couldcost it billions of euros. Almost overnight,industry’s interest in cheaper, animal-free testing skyrocketed.

Last month ECVAM was put in charge of

developing, with industry and regulatoryagencies, the testing strategies for REACH.Now commanding 50 staff, Hartung is risingto the challenge. The toxicity tests that havebeen used for decades are “simply bad science”,he explains. “We now have an opportunity tostart with a clean slate and develop evidence-based tests that have true predictive value.”

Many of the animal tests used today weredeveloped under crisis conditions. The noto-rious Draize test, which assesses the irritationor damage caused by chemicals simply byputting them into the eyes of rabbits, is a primeexample. It was developed by the US Food andDrug Administration in 1944 after reports inthe 1930s that some cosmetics were causingpermanent eye injuries. One 38-year-oldwoman had gone blind after dyeing her lasheswith Lash-Lure, a product that contained aderivative of coal tar.

Then came the calamity of thalidomide,which was given to pregnant women in the late1950s to control morning sickness, but whichcaused horrific birth defects. By this time, gov-

ernments were highly sensitive to public con-cerns and called on their authorities to developanimal-based tests that would predict all con-ceivable toxic effects of drugs and chemicals.The principles behind most of those testsremain more or less unchanged today.

The battery of tests demanded by Europeanauthorities covers all eventualities, from acuteeffects that are seen shortly after exposure —such as eye and skin irritation — to concernsabout whether in the longer term a compoundmight cause cancer, or brain or birth defects. Inaddition, tests must be carried out to define therisk a chemical might pose to the environment,such as toxicity to fish and other aquatic species.

Safety catchEach chemical that goes through the multipletests required for registration can use up to5,000 animals — or 12,000 if the chemical is apesticide. The cost of doing this for the 30,000unregistered chemicals so that they complywith REACH has been estimated at between€5 billion (US$6 billion) and €10 billion.

In the decade since ECVAM was established,the number of animals used in toxicology test-ing has fallen slightly, although it still hovers atabout one million per year. This reduction is aresult of the refinement of existing tests, andthe introduction of some alternative methodsthat rely on in vitro tests using cell cultures.

ECVAM is still pushing on both fronts. For

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Commercial and political pressures are pushing for a halt to the use of animals in toxicology testsin Europe. This change will also mean a move towards better science, says Alison Abbott.

More than a cosmetic change

Skin deep: animal testing for all cosmetics is being phased out in Europe.

“To test a chemical for its potentialto cause cancer takes five years

and involves 400 rats. More than50% of the results are positive, of

which 90% are false positives.”

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example, the LD50 acute toxicity test, whichinvolves feeding animals with a chemical todetermine the lethal dose, still accounts forone-third of all animal tests worldwide. Butthe numbers involved have fallen from 150animals per chemical in the 1970s to just eightanimals in 2002.

ECVAM believes that it can halve the totalnumber of animals used for regulatory testingwithin a decade. It has just completed its firstlarge-scale validation study of an in vitro cyto-toxicity test, which monitors death of culturedcells following short-term exposure to a chem-ical. Chemicals shown to be harmful in thistest would be excluded from any LD50 animaltests. At least 70% of the chemicals registeredin the past two decades fall into this category,says Hartung. And this is just the beginning.

“We’ve made a lot of progress with the low-hanging fruits, but many of the remaining ani-mal tests — particularly the tests for toxicity inthe long term — are much more challenging toreplace,” says Hartung.

Poor predictionThis is despite the acknowledged poor qualityof most animal tests, which have never under-gone the rigours of validation that in vitroalternatives now face. Most animal tests over-or underestimate toxicity, or simply don’t mirror toxicity in humans very well.

Take the embryotoxicity test in which chem-icals are fed to pregnant animals and the fates of their embryos, and the progeny of two subsequent generations, are studied. “Animalembryotoxicity tests are not reliably predictivefor humans,” says Horst Spielmann, a toxicolo-gist at the Federal Institute for Risk Assessmentin Berlin. “When we find that cortisone isembryotoxic in all species tested except human,what are we supposed to make of them?”

The same goes for cancer. To test a singlechemical for its potential to cause cancer takes

five years and involves 400 rats, each of which istreated with the maximum tolerated dose. It isdramatically over-predictive: more than 50% ofthe results are positive, of which 90% are falsepositives1. Yet the number of compoundsproved to be carcinogenic to humans is very low— the International Agency for Research onCancer in Lyons, France, has identified just 95proven and 66 probable human carcinogens.

As their experience grows, ECVAM staff arefinding out just how complicated it is todevelop persuasive alternative tests. A highlystrategic approach is required, says Hartung.“In most cases it is not just a question ofreplacing one animal test with one in vitrotest,” he explains. Instead, a number of testswill be required, each of which has been

shown to match data on toxicity in humans,assuming such information is available.

For example, several in vitro alternativeshave been developed to replace the Draize test,each with its own advantages and limitations.Among these, some are better at predictingmild irritation than physical damage. Othersare more suited to a particular chemical class,such as detergents.

Life or deathScientists also cannot assume that in vitroalternatives are automatically better, says Spiel-mann. In 1971, a comparison of animal Draizetests in different labs revealed the test to behopelessly non-reproducible2. But Spielmann’s1995 study of animal-free alternatives to theDraize test showed that they were equallyunreliable3. Since then the in vitro tests havebeen standardized, and they are intrinsicallymore reproducible. “Although reproducibilityand relevance are not the same thing,” Spiel-mann cautions.

Relevance requires a good match betweenthe test results and human data. At an ECVAMworkshop in February, 30 industrial scientistsmet to develop the most effective strategy forusing the alternative Draize tests, so that thefalse negatives and false positives of each testcompensate for each other. This strategy is nowgoing through the crucial validation procedure,in which human data, often from occupationalhealth databases, will be used as points of refer-ence (see ‘The validation game’, overleaf).

ECVAM has so far seen 17 alternative teststhrough validation — 11 use in vitro methods,another six involve refining in vivo tests toreduce the number of animals used. An addi-tional 40 or so tests are under peer review, withmore to come.

Most of the new tests assess acute toxicity,

Tests that put chemicals into the eyes of rabbits have changed little since the 1940s.

Life-savers: researchers at ECVAM are developing alternatives to animal toxicity tests.

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but animal use is highest when testing for thetoxic effects of prolonged exposure to chemi-cals for long-term consequences such as cancer and reproductive toxicity. These costly procedures are harder to mimic in vitroand may never be completely replaced.

Sounds familialThis is why, apart from the €30 million it usesto support ECVAM annually, the EuropeanCommission is funding three multimillion-euro ‘Integrated Projects’. Under these, dozensof labs will collaborate for five years to tacklemore difficult issues, such as allergic reactionsor widespread toxicity resulting from chemi-cals entering the bloodstream.

Scientists know that they are likely to find ithardest to convince regulators about alterna-tive tests for highly emotive issues such as cancer and birth defects. More than half of allanimals that will be needed to supportREACH legislation are likely to be used inreproductive toxicology testing. The €9-mil-lion Integrated Project called ReProTect has 27labs dedicated to developing alternatives tothese tests. The ReProTect consortium hasbroken down the human reproductive cycleinto smaller elements, from male and femalefertility to implantation, to pre- and postnataldevelopment, and is trying to develop a mean-ingful package of tests4.

“Quite correctly everyone feels uneasy about

but, wanting to be safe rather than sorry, theyaccept it because it is believed to throw up fewfalse negatives. They prefer to let industryprove the innocence of any compound thatshows up positive. Any replacement tests willneed to reassure both regulators and industry.

With so much yet to be achieved, is Europeon target to deliver toxicity tests to meet thenew regulations? The Cosmetics Directivephases out animal use in acute toxicity testingin 2009, and in testing for long-term effects in2013. “We are on target for the first deadline,but the second deadline may be more diffi-cult,” says Hartung. Fortunately, the 2013deadline can be renegotiated.

The REACH legislation is yet to be finalized,and alternatives to tests that present the highestfinancial and animal burden, such as repro-ductive toxicology and carcinogenicity, will notbe in place when REACH first becomes law.But the longer-term picture should see a reduc-tion in animal suffering going hand in handwith use of better science. ■

Alison Abbott is Nature’s senior Europeancorrespondent.

1. Gold, L. S., Manley, N. B., Slone, T. H., Rohrbach, L. &Garfinkel, G. B. Toxicol. Sci. 85, 747–808 (2005).

2. Weil, C. S. & Scala, R. A. Toxicol. Appl. Pharmacol. 19,276–360 (1971).

3. Balls, M., Botham, P. A., Bruner, L. H. & Spielmann, H.Toxicol. In Vitro 9, 871–929 (1995).

4. Hareng, L., Pellizzer, C., Bremer, S., Schwarz, M. & Hartung, T. Reprod. Toxicol. 20, 441–452 (2005).

The number of toxicity testsrequired by regulatory authoritiesmushroomed in the 1960s and1970s. But tests accepted byauthorities in one country wererarely identical to those acceptedelsewhere. This forced companiesto repeat the same test with smallvariations in every country theywanted to market their product,wasting both money and animals.

So in the early 1980s, theOrganisation for Economic Co-operation and Development(OECD) launched a majorharmonization initiative. Nationalauthorities met regularly underOECD auspices to negotiatemodifications to their testschedules that would make themcompatible with those in othercountries. Most of the differenceshave now been resolved.

But at the same time there wasgrowing pressure from the public toreplace or reduce animal testing, sothe OECD expanded its role tooversee global acceptance of newanimal-free, or animal-lite, tests.Validation — the proof that a testaccurately predicts a specific effectin humans — is the biggest

challenge for alternative methods.At the European Centre for the

Validation of AlternativeMethods (ECVAM) in Ispra,Italy, validation typicallyrequires testing a newprotocol in three or fourdifferent externallaboratories. The testchemicals are analysed bypersonnel who do not know thecompounds’ identities. If a testproves reproducible, it is sentfor peer review by ECVAM’sScientific Advisory Committee,whose members include scientists,representatives from all memberstates of the European Union andrelevant industrial and animal-welfare groups.

Once approved by the committee,the test is adopted by the EuropeanChemicals Bureau, also based atIspra, and then sent to the OECD forthe all-important global validation.Most of the labs involved in ECVAMtesting are European, but the centrehas been careful to involve otherregulatory authorities in earlystages of the development processto speed OECD validation.

In 2000, the 3T3 Neutral Red

Uptake Phototoxicity Test becamethe first replacement test to bevalidated by European authorities.The test identifies whether achemical becomes toxic whenexposed to light. Chemicals areadded to a culture of skin cells,which is then irradiated withultraviolet light. The rate at whichthe cells die before and afterirradiation is monitored. The test was developed by HorstSpielmann from the FederalInstitute for Risk Assessment in Berlin.

Ideally, the predictive value of a

new protocol is tested againsthuman rather than animal data.

Human toxicity data are, ofcourse, hard to find, so

Spielmann was gratified to be invited to join in a

clinical trial assessing thepotential phototoxicity of

drugs to which physicians hadraised concerns. He was able

to compare his test’s data for some standard chemicalswith the irritation they caused

on a patch of human skin. He found a perfect match. The testwas accepted by the OECD last year.

One of the 40 or so tests nowgoing through validation is the newcytotoxicity test to help replace theanimal lethal-dose (LD50) test (see main story). It was the firstvalidation study to involve both USand European groups from thestart. It is also the first to use datafrom the records at national poisoncentres. The predictions of the in vitro test provided a better match than the rat LD50 test when compared with the toxicityinformation on 42 chemicals listedas having poisoned people.

“We all want to besure that there isreal evidence thatalternative testsare predictive ofhuman toxicity.”

— Thomas Hartung

The validation game

taking risks where stakes are so high and issuesso emotive,” says Hartung. “We all want to besure that there is real evidence that alternativetests are predictive of human toxicity.”

For example, regulators know the weak-nesses of the rat cancer test as well as scientists

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Rats are widely used to assess whethercompounds can cause cancer.

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