Brueghel ’ s Two Monkeys : Passing the Final Exam in the History of Mankind

When I was in school, we didn’t consider the rights or protection of animals an issue. I went to a convent in the Himalayas in Kodaikanal, Southern India, and I gassed animals for dissection class without much thought. looking out at this room, I see some of you are my age or thereabouts and so probably, like me, missed out on the animal rights and ethics courses offered in universities today. In other words, we are late in wrapping our brains around these concepts. the rest of you have no excuse! Are most human beings inherently kind? Well, I don’t have any empirical evidence that many people are unkind, but I do have a lot of anecdotal evidence that some are. And it strikes me that, on the strength of that – as with criminal law, where a collection of circumstantial evidence is allowed to win a conviction – we can pretty much characterize the human race, of which I am a living, breathing part, as being, quite often, “a species behaving badly” – especially when no one is looking. In Amsterdam, for instance, the honor system of putting out public bicycles had to be dissolved, because so many bicycles have been stolen, repainted, and sold. And then there’s the person who stole my wallet. luckily they left the euros and only took the dollars, which are almost worthless. But such is human nature that even friendly, well-educated people who should know better often behave badly, and I’m not just talking about hedge fund managers. In a study of visitors to Antarctica, it was found that people with a university or postgraduate education were significantly more inclined to harass seals and trample plants than those with less formal education. And then there is “pack behavior”: Abu Ghraib isn’t the only place where people have engaged in conduct that would be seen as truly depraved under normal circumBrueghel’s Two Monkeys: Passing the Final Exam in the History of Mankind

1 What kind of animal are we?
When I was in school, we didn't consider the rights or protection of animals an issue.I went to a convent in the Himalayas in Kodaikanal, Southern India, and I gassed animals for dissection class without much thought.looking out at this room, I see some of you are my age or thereabouts and so probably, like me, missed out on the animal rights and ethics courses offered in universities today.In other words, we are late in wrapping our brains around these concepts.the rest of you have no excuse!Are most human beings inherently kind?Well, I don't have any empirical evidence that many people are unkind, but I do have a lot of anecdotal evidence that some are.And it strikes me that, on the strength of that -as with criminal law, where a collection of circumstantial evidence is allowed to win a conviction -we can pretty much characterize the human race, of which I am a living, breathing part, as being, quite often, "a species behaving badly" -especially when no one is looking.
In Amsterdam, for instance, the honor system of putting out public bicycles had to be dissolved, because so many bicycles have been stolen, repainted, and sold.And then there's the person who stole my wallet.luckily they left the euros and only took the dollars, which are almost worthless.But such is human nature that even friendly, well-educated people who should know better often behave badly, and I'm not just talking about hedge fund managers.
In a study of visitors to Antarctica, it was found that people with a university or postgraduate education were significantly more inclined to harass seals and trample plants than those with less formal education.And then there is "pack behavior": Abu Ghraib isn't the only place where people have engaged in conduct that would be seen as truly depraved under normal circum-stances but which became normal and routine, even amusing, as the "group mind" muted out decency.
So, average people are capable of more cruelty than we'd like to believe, sometimes not even seeing their behavior as wrong.And sometimes this cruelty becomes institutionalized and is not even seen as abnormal any more.that means, of course, that animals, elderly people, children, and any others placed in a vulnerable position or in an institution, such as an orphanage, a nursing home, or a laboratory, are far more likely to be abused.let me give some examples of why I say this: look at those who not only have taken a solemn vow to be good but who are expected to set the bar on goodness.the church is an institution more respected than any other.Yet the Roman Catholic Church, not only in the US but in europe, has had to learn that going along with and, indeed, covering up abuse may one day catch up with you.We all know now about the little boys, and some girls, who were too scared to speak up and who had their lives and bodies interfered with.But, if pillars of the community demonstrate no regard for the feelings of children in their care, it would be mad to think that animals in laboratories, who certainly don't enjoy anything like the revered status of children in our society, are being well treated!And no one is naïve enough to believe that research is somehow an exception -that it is the one place on earth where those in charge can be trusted to police themselves.that's like leaving a 5-year old in charge of a chocolate cake.Which, actually, researchers have done and the results are not surprising!let me offer some idea of what our investigators and others have found: When they thought no one who cared was looking: UNC researchers put live animals into the freezer bins and cut off rodents' heads with scissors without any effort to lessen their suffering; researchers who couldn't be bothered to walk to the gas chamber room chose to violate their protocols and kill the animals by breaking their necks against the cage cardholders.technicians at Huntingdon life Sciences in the U.K. were filmed punching beagles in the face and simulating sex with each other as they tried to inject a frightened dog at the same time.Researchers at the largest contract laboratory in the US, Covance, were caught slamming petrified monkeys into steel cages or, having tied them down to the table, stuffing bottles in their mouths and mocking them while whooping it up to loud music.Covance's reaction was not to decry the behavior and fling the abusers and their supervisors out the door, but to sue us -both in the UK and in the US -to get us to stop showing those videos.they lost their cases.
these are dismaying examples of people shamelessly abusing their power over others in their care and then trying to cover it up.And it's not just at Covance.every single time we go into an institution undercover -including at AAAlAC-accredited institutions -we come out with footage of atrocities.
Animals, like institutionalized people, are often simply forgotten; they become wallpaper.Once, I was touring the National Institute of Health (NIH) model facility in Poolesville, Maryland -a "model" facility, mind you -when I found some baboons being kept in small metal isolation cages.I found them because I was walking along one of the corridors and heard a fearful banging noise.that made me look through the little window into their room to see a huge male smashing his head into the stainless steel back wall of his cage.He was a Hamadryus baboon: huge, with a big snout like a dog, and such a colorful coat, sticking out all over, that he looked like a man who had been plucked off the street on his way to a fancy dress ball.I enquired about these baboons, each sitting in a standard, small metal isolation cage in this stark, barren room, with nothing to do or see or touch, no contact with each other, unable to even walk two steps.And I discovered that the researcher who had been using them in a cancer study had accepted another job two years earlier and had moved way!He was living his life in a new town, driving around, shopping, watching tV, talking to his kids, and the baboons were sitting there, day in and day out.they had been plucked from their homes, troupes, and families in Africa seven years earlier, shipped to Russia and then to the US on what could only have been frightening journeys for them, and then locked in see-through boxes in a room, and left to stare at the walls for seven years.Once a day, men in masks entered and hosed down the room and put food in their metal bowls.
except for the absence of water boarding, they might as well have been at Guantanamo Bay.
Who was to blame for this casual oversight that caused these bright animals so much misery?the grant provider?the researcher who left? the technicians?the IACUC?All of the above, surely?Not one of them had thought the baboons important enough to wonder about, even those who had seen these animals every day.this kind of appalling neglect, in which a living being is left to experience needless mental strain, goes on all the time.So, we must be vigilant, each of us in a position to do so, to spot it and stop it.Otherwise, what kind of animal are we?
People in institutions often get used to things that disturb other people.they are like the lighthouse keeper who was so accustomed to hearing a gun go off under his nose, every six minutes, every night, to warn ships at sea that he slept through it.One night, the mechanism failed and the gun didn't go off.the lighthouse keeper woke with a start, sat up in bed and said, "What's that?" We need to be vigilant to what's happening to others around us, because those over whom we have control can only depend on us to notice their circumstances.We cannot allow animals to become the wallpaper we don't see any more.
those of you here who are striving to get animals out of research or at least treated with some understanding of who they are and what they need, have to deal with those who can't empathize (neurophysiologists can debate whether that means that their "mirror neurons" are underdeveloped or not), and with others who think it's perfectly fine to wait until some unspecified time in the future to make changes or that change is just not a priority.And we all have to deal with people on IACUCs who are untrained, don't appreciate the important responsibility vested in them, or who get too busy -personally or professionally -to take a proper look when a protocol is presented that could result in animals being put through painful, uncomfort-able, and worrisome procedures that should have been modified or rejected outright.that is unconscionable.

Today's conduct as future "past" horrific behavior
It is said that the only thing we learn from history is that we don't learn from history.let me examine with you how history applies to our behavior toward animals today.
One of the most studied cases in research ethics is, as you know, the tuskegee experiment, in which poor black men in the southern US were purposely not told by their doctors that they had syphilis and were simply used as research subjects.this is a good example because it involves a marginalized group -individuals with no recourse, who didn't seem to count for much, and whom the dominant group did not understand or consider important.And because the researchers may indeed have had some sort of good intentions but didn't realize that, at some later point they'd be condemned for such a lack of empathy.
But it's not only the tuskegee men.Human orphans were used in tuberculin tests and trials of low levels of radiation; poor Irish women immigrants to the US were used in gynecological practice surgeries which, when perfected, were performed on the rich, paying classes.even human neonates were operated on without anesthesia until quite recently.
In the US, enlisted men (GIs) were used in lSD experiments without their knowledge; some thought they were going mad and killed themselves.GIs were used because they are a pool of often low-income men considered cheap, disposable, and replaceable.Sound familiar?
It is easy to be appalled by what has been done in the past, but callous behavior seen as acceptable just yesterday is now seen for what it is: ignorant and wrong.
For truly uncomfortable reading, there is "the Nazi Doctors," by Robert J. lifton.In his study of what made doctors able to live with themselves while doing to prisoners exactly the sorts of ghastly things that are done to animals in today's laboratories, including drowning experiments and teratogenicity experiments, he discovers that the comfortable idea that they were just "some madmen" ruled by another madman is absolutely unsupported.In fact, vast numbers of people somehow rationalized these extreme cruelties and killings in a variety of ways and not only accepted it but participated in it.
lifton's conclusion is that these experimenters were ordinary people like us.One of them, Dr. Siegmund Rascher, even felt comfortable enough to put on paper a formal request to Berlin to move his experiments on inmates from Dachau to Auschwitz because, he wrote: "In Auschwitz, the freezing process is faster because it is colder there.Moreover, the camp is bigger, so that the subjects' howling can hardly be heard." the analogies are clear.Nobel laureate Isaac Bashevis Singer, whose family fled the Nazis, became a vegetarian because he looked out of his window above a slaughterhouse in Chicago, watched the cattle shackled together, being prodded and poked down the ramp to their deaths and wrote, "to animals, all men are Nazis."lucy Kaplan Rosen, who wrote the introduction to Eternal Treblinka, tells the story of her father, who was transported in a cattle car to Birkenau-Auschwitz in 1944.this was after he had witnessed the murder of his wife and two daughters.He survived six camps.Ms. Rosen says that what she loved most about her father was that when, in 1945, at only 100 lbs and bearing the injuries of years of Nazi abuse, he emerged with his previous compassion for animals enhanced precisely because he realized that he had been treated like one.
So, we see that past atrocities weren't necessarily an aberration, and ordinary people can do hideous things if they don't discipline themselves not to or are not stopped by someone else.
the trick, however, is not in simply looking backwards but in figuring out what is being done today that will be looked back upon in the future with disgust.the trick is to be one of the people who finds a way to reach those who can't or won't relate to the being on their hotplate.
lifton's study is also instructive in another way.It suggests that -if the same observations of human nature apply todayof every three people hired as animal caretakers, two will not object, even silently, when an animal is abused or neglected, and one of the three will be easily capable of joining in flagrant abuse.let's ask ourselves then, of every ten people appointed to an IACUC, how many will speak up when an unnecessary or unnecessarily cruel experiment is proposed?
On the encouraging side, we teach our children that "Might Does Not Make Right," and we ask them to obey the Golden Rule of "Do Unto Others as You Would Have them Do Unto You." Presumably, we mean those things.But when you think about it, only "might" allows someone to pick up a rat, a mammal every bit as sentient as any dog or cat or me, and bleed him from the eye and then toss him back into a shoebox-sized container, as if the rat's experience, his fears and sensations aren't real.And only a lack of empathy would allow someone to joke that now the animal will need a white stick when he goes out on the town.
We all know that scientists want to be thought of as precise and particular.Perhaps you know the joke about two scientists driving along in Australia when they pass a flock of sheep."look," said one, "those sheep have all been sheared.""Well," says the other scientist, "On one side of their bodies, anyway."like most stereotypes, I'm not sure this business about particularity is true, and I'll tell you why: A few years back, we ran a photo in PetA's Animal Times magazine that showed rats stuffed into narrow plastic inhalation tubes like so many socks.You can imagine how frightening it must be to have giant aliens, whose intentions are not benign, stuff you into a tube so tightly that your nose and ears are squashed against the sides.the picture was from an article published in the journal of the National Institute of environmental Health Sciences, and the "cute" caption under the original photo read: "Nosing Around."It reminded me of a caption I saw on a photograph of a group of black males, unable to find work in a South African township, with the caption, "lounging Around." the article in the NIeHS magazine described an experiment in which rats were forced to breathe mercury vapors in an attempt to duplicate the reproductive effects already seen for years in dental hygienists exposed to mercury in fillings.Incidentally, the Principal Investigator concluded: "We weren̓t able to reproduce any of those effects in our animal model." When we showed this photo to the head of ICCVAM, he shook his head and said, "that's terrible."But it turns out he didn't mean that the experiment was terrible or what was done to the rats was terrible; he meant that it was terrible that the photo had been put in the magazine for everyone to see! PetA researchers attend many toxicology conferences.they hear the jokes, the references to boondoggles, the acknowledgements that certain experiments have no value whatsoever.On a tour of the USUHS, the US Uniformed Health Services facility, the chief veterinarian in charge pointed to the desert tortoises being used to study tMJ (temporo mandibular joint pain).Now, the desert tortoise's jaw is not like a human jaw at all; it is especially arranged for a fibrous, plant-based dietthere are no teeth!People chew their food, tortoises do not, and there are many other critical differences.Furthermore, the NIH says that tMJ treatment should not be surgical, rather it begins with simply changing jaw movement and avoiding teeth-grinding.Considering that a desert tortoise cannot be reasonably expected to speak our language, and we don't understand hers, it will be difficult to explain this to her and to know when she feels better.
PetA did a double take and got the funding on that experiment pulled.If we hadn't, who knows how many other tortoises would have had their jaws damaged and how many more tax dollars would have been wasted.
I was once invited to lecture at the USUHS and made the mistake -or clever move -of arriving early enough to sit unnoticed in the back before the earlier session ended.the laboratory chief told his students: "When you fill out the government form as to why you chose to use rats or mice, do not write 'because they are cheap, easy to handle, and few people care about them.' the form is supposed to show there is a good science-y sounding reason for your choice."But the truth is, there is no "science-y" reason.
At a conference on neurotoxicity, a panelist discussing the ePA's developmental neurotoxicity test (which uses at least 1,300 animals every time it is conducted) joked that the "FOB" -which is the acronym for the "functional observation battery" that is used in neurotoxicity testing -really stands for, and I quote: "functional observation bullshit."In that government scientist's words, "we do it because the ePA tells us to," regardless of relevance.In my words, "They die for our sins." The EPA official on the panel acknowledged: "We know the rat isn't the right model.But it's like being in a bad marriageyou know you should get out but you don't because there's so much history there."that would be funny, but it isn't if you are the subject of a painful experiment.
So, the gig is up on pretending that results from one species apply across the board to others.I've always said that "When it comes to feelings, like hunger, pain, and thirst, a rat is a pig is a dog is a boy." that's just a plain old fact.What isn't a fact is that when it comes to physiology, a rat is a pig is a dog is a boy.Meaning that it's time for the so-called "gold standard" of animal testing to be recognized as the lump of coal it is.
One more example of how casually the animals' suffering is viewed: Several years ago, at the National Academy of Sciences' Institute for laboratory Animal Research workshop on federal reporting requirements for pain and distress in animals used in laboratories, panelists who included animal researchers from the US Department of Agriculture (USDA), the NIH, and various universities and professional associations, were often seen yukking it up over animal suffering.empathy was in short supply, if there at all.One of the panelists spoke about the importance of proper training.Reciting a "can-ya-believe-thisone" story, he gave a litany of botched jobs by under-trained or under-skilled lab staff, culminating in a story of someone improperly restraining a mouse so that, as the animal's skull was being drilled into, his body spun around with the drill-bit.throughout the list of examples, other members of the panel chuckled in recognition, and at this last example, several panelists laughed openly.
I read a story in the newspaper about a group of young people who had been caught standing around a BBQ pit, poking a live kitten into the burning coals and laughing as the kitten cried.Who wouldn't be upset at the mental health of these individuals?But, when you think about it, the place is unimportant.Whether suffering is seen as a joke in the lab, at a conference, or at the barbeque pit, it must stop.
Cruelty doesn't have to mean being the person who pokes the kitten deeper into the coals, or being the person who wields the scalpel or syringe.It also means being the person who ignores the plight of the pain-wracked or lonely animal in the cage.Where there is acceptance of the perpetuation of wrong, there is complicity.Perhaps the Nazi doctors could not have spoken up without being shot, but we are not in that position -not any of us.
What allows this kind of jocular, cruel nonsense to go on is a lack of empathy (that undeveloped "mirror neuron") or the group acceptance of unacceptable behavior.It will continue until every one of us who cares -and we are many -gets a moral backbone, gets truly interested in real science, and speaks up, complains, refuses to accept this kind of thinking, and puts an end to it.
It's history again, isn't it?Someone gave me a book of parlor games from the Southern US.One game in it is described this way: "A distinguished lady is chosen to address the group.She must explain that a baby has been orphaned, and she will suggest that the club rise to the occasion by chipping in to raise the child for its first year.Of course, everyone will agree, and one member must be asked to volunteer to be the first to take the child home.As the volunteer comes forward, everyone claps with appreciation.Have the maid bring a swaddled baby into the room.When the lucky volunteer is handed the infant, all will howl with laughter when the cloth is pulled back and the lady finds she is holding a Negro child."The book continues: "If a Negro child is not readily available, you can achieve the same effect by using a baby pig." We find that beyond horrific now, but it was all good fun down South in that bastion of civil liberties, the United States of America, not so long ago.And it is a great illustration of why change must keep come right on coming.
3 The capacity for pleasure and fear I'm not so much condemning the people who didn't "get it" then and who don't "get it" now; rather, I'm offering it as a blunt reminder to ourselves that we are making history every day that we live and breathe.today's conduct can be seen as future "past" horrific behavior, if you know what I mean.It was just 30 or so years ago, when I was already smoking cigarettes, that physicians in white coats appeared on tV, advising us to smoke low tar cigarettes to soothe a sore throat.It was just 30 years ago that, as Dr. Jane Goodall points out, scientists openly ridiculed the idea that chimpanzees were intelligent, had social needs, engaged in tool making and use, and had language.Or more recently, take the octopuses commonly used in laboratories: It is only after years of electro-shock experiments on them that experimenters have conceded that these bizarre -to us -animals are so emotionally upset by their loss of control over their destiny and their inability to flee the pain meted out to them, that they commit suicide by pecking themselves to death.Jacques Cousteau first revealed how dolphins in captivity would sometimes take their own lives; now we see that behavior in other captive species, including cephalopods.Yet these animals have been treated in laboratories as if they were inanimate.Or actually, that's not so, because if they were thought to be inanimate, no one could design an experiment to hurt them and see what they would do, could they?So, people recognize that octopuses have feelings but are deliberately ignoring the fact.
Recently, an extensive study of pleasure in the animal kingdom showed that "from tickling to playing catch, animals engage in certain behaviors just for fun, even enjoying sensations that are unknown to humans."The author of the findings, published in Applied Animal Behavior Science, believes scientists, conservationists, and others should not overlook animal joy."the capacity for pleasure," the author writes, "means that an animal's life has intrinsic value, that is, value to the individual independent of his or her value to anyone else, including humans."1And what isn't mentioned here, is that one of the most overlooked areas of animal suffering is fear.We talk about caging size and other considerations, but the "Fear Factor" isn't just an American tV game show.
this animal at this podium knows a bit about fear.My father was a very daring man, quite an adventurer who went out in fierce storms and into war zones and was at Bikini Atoll to help set up nuclear testing.He once took my mother in a jeep across the little Rani of Kutch in India.this is an area that, at certain times of the year, is extremely dangerous, pitted with pockets of quicksand that are impossible to see but which can gobble you up.My mother only found that out, and found out that no one else had had dared accompany my father on this expedition, when they were well out in the middle of it.She remembers yelling at him and my father saying, "Oh come on, where's your spirit!" to which she replied, "At home in the drinks cabinet, but damn you, if I'd known that we might die, I'd have brought some with me." I know what she meant, because when I was a tiny tot, he took me up a glacier many thousands of feet high.Despite the physical pain of the cold, which was intense (we didn't have polar fleece back then), what was far worse was the fear: the fear of tumbling thousands of feet down the side of that vast wall of ice, the fear that something terrible was about to happen to me. that is the experience animals in laboratories live with all the time: the mother monkey clutching her baby to her chest in her small metal box, wondering if that giant, powerful animal entering the room to take her blood is also going to steal her child.And he is, just as if he were taking a box off a shelf, ignoring her fear grimace, her desperately chattering teeth and her little begging sounds.Yet her love for her child is indisputably as strong as any human mother's love for her infant.What must her suffering be like when she cannot protect her own child?Who will speak up for her?
In a BBC documentary, scientists refer to cuttlefish as "aliens from inner space."It's a fascinating term, because, of course, our species is out there in space, spending a great deal of time, money, and effort searching for intelligent life, yet it is all around us: from those cuttlefish who communicate in waves of color (able to create a magnificent pattern on one side of his body that lures a prospective mate while creating another pattern on the other side that wards off a competitor); to the smallest desert mouse who rolls a stone in front of her burrow to collect dew; to the Indigo buntings who navigate by learning the constellations, fix their position by the height of the sun and, if blown off course, reset their paths by the phases of the moon and the rising and setting of the stars; to the rhino who communicates by altering his breathing.
And should any Cartesians be among us and laugh such things off as programming, they must also laugh off their own loves, desires, and fears, their own programmed behaviors.the Cartesians also would have to ignore studies like the one this very month reporting how five crows all were able to figure out how to use a short stick to get hold of a medium one and the medium one to reach a large one -the only one that could allow them to retrieve a food reward.All five figured it out without training, and four of the birds did it in the first try.

A slippery slope is just another term for progress
So, what if we do come across intelligent life during our space explorations?If it is stronger than we are, we will undoubtedly beg for mercy and understanding and insist that "We come in Peace."But if we find intelligent life out there that is not as strong as we are, what will happen to those noble protestations?Our governments will want to do to them what we've done to all the intelligent life forms on this planet: Capture them, cage them, dissect them, and deny them any consideration.Perhaps snack on a few of them just as we snack on the sea slug who, like a fat opera diva, so gracefully dances among the rocks, her cloak floating behind her, her mind on who knows what.But we don't have to be like that computer Pacman, gobbling up everything in our path; we can be considerate of those with less power than we have.
I hear people who want to cling to the status quo say, "Don't concede that primates need social enrichment or that rats and mice must be afforded protection, it's a slippery slope."And of course it is, but isn't a "slippery slope" that leads us away from treating others badly just another term for progress?A society can't evolve if it is afraid of the slippery slope.looking back, it was a blink ago in time that a noted Harvard surgeon was deeply worried about the "absurd" idea that a woman might be allowed into the operating room, let alone be trained as a physician.In the time of the Suffragettes it was said that "If you give women the right to vote, you might as well give asses the right to vote."Today, we have fine female physicians and scientists and -as for women voting -well, we should be entitled to make the same mistakes at the polling booth as any man!And frankly, looking at who we elect sometimes, one wonders if asses couldn't do a better job than the lot of us.
So, what can we do?You may think that I want all animals out of the laboratories now and you'd be right.I do think it is morally indefensible -given what we know in this day and age -for us to inflict pain and suffering and fear on any other living being simply because we can.that is the lesson I take from history.And if you don't "get it," look at the animals, learn about the animals, and if you still don't "get it," look again.
However, one can still help enormously without having to embrace that belief.
5 Regulatory testing: the obstacles and the movement forward I polled the PetA staff who work on these issues, asking them to give me basic starting points.I am only focusing here on regulatory and toxicity testing.let me go through the list and I hope you will agree: 1.Where there is a non-animal alternative, use it.2. Don't automatically default to animal testing: profiling a chemical's biological activity using a suite of non-animal methods will allow for thoughtful toxicology by identifying the most hazardous chemicals and providing information that can guide further testing and, of course, NO experiment using animals should eVeR be carried out if the information is available or can be derived elsewhere.3.Where there isn't an alternative yet, work to find one and keep an eye on what's going to be available soon.Alternatives for many biological endpoints are in sight, and for the more complex endpoints in the areas of toxicokinetics, chronic toxicity, and carcinogenicity, there are efforts that ur-gently need promoting, including PetA's planned workshop focusing on replacing the rodent cancer bioassay.4. the ePA and NIH are beginning to act on the vision set forth in 2007 by the National Academy of Sciences, which recognizes that the near-exclusive reliance on animal testing that has characterized chemical testing programs to date are costly, time-consuming, and not up to the task of accurately and adequately assessing the toxicity of tens of thousands of chemicals.Both the NAS report and the 2009 ePA strategic plan recommend moving away from dependence on animal tests to a process that relies more heavily on in vitro assays to predict human health effects.ePA and NIH have created formal collaborations to develop and implement this approach, such as the toxCast and tox 21 initiatives.
It is encouraging to note that federal agencies and some industry consortia are stepping up to the plate to help fund these initiatives, in addition to PetA which, up till now has tried to step into the void by providing massive donations to in vitro laboratories and QSAR experts, even though our budget is mere manicure money to federal agencies such as the ePA.
May I solicit your support to encourage and fund the use of these technologies in current and future testing programs?there is still so much work to be done as current testing programs have been slow to incorporate this new approach.
For example, there is the hideous mess that is the ePA's endocrine Disruptor Screening Program.The first phase could kill more than 40,000 animals -and not one of them will die quickly or painlessly -to test just 67 chemicals, all of which are either pesticides or High Production Volume chemicals that have already been heavily tested.the additional testing is highly unlikely to provide any useful information for additional regulation of these chemicals, especially when the ePA still can't say how it is going to use the resulting information!this program is so out of tune with where we are now that it is like designing an iPhone app using carrier pigeons.this program needs to be redesigned from the bottom up to take advantage of the latest technology and new approaches.Please take a look at our poster, number 539, that describes an integrated approach to endocrine testing and see how you Recently, ICCVAM rejected the work of a consortium of companies that worked together to develop a non-animal method for assessing eye irritation.the work was so promising that the ePA launched its own pilot program, accepting data using this method.So, now the ePA is making more progress than the federal entity whose job it is to do so.
Another shameful example is that nine years ago, an international workshop concluded that in vitro cytotoxicity could be used immediately as a dose setting measure to reduce the number of animals poisoned in lethal dose tests.the experts also concluded that, with interest and funds, the test could be validated as a complete replacement method for lethal dose tests within 2-3 years.Yet it took until 2008 for ICCVAM to issue formal recommendations to agencies to use the cytotox method and then only as a reduction method to set the starting dose for poisoning animals.
NIeHS has not made appropriate funding of ICCVAM a priority, and ICCVAM does not do the sort of independent research performed by eCVAM and ZeBet.It appears that ICCVAM members, who are drawn from the federal agencies, are being allowed to misuse ICCVAM to perpetuate their antiquated biases in favor of animal tests.this is evident through ICCVAM's continued presumption that animal tests are the "gold standard" of toxicology to which all non-animal assays must measure up (or down as the case may be) and through emails leaked to PetA in which IC-CVAM representatives discuss circling the wagons against evidence-based toxicology.It should therefore come as no surprise that the US lags so far behind europe and some other countries, not only in real football but in implementing non-animal testing methods too.We documented that disgrace in an extensive report last year that ended up on the front pages of the Washington Post. 5. the current US toxic chemicals legislation, the toxic Substances Control Act, is about to be revised.While this is sure to lead to requirements for information similar to ReACH in the eU, it also provides an unprecedented opportunity to incorporate these new approaches and new technology into toxics legislation -something we at PetA are working hard to do and which you will hear about in the next session on chemicals and pesticides.6.In europe, there is much work to be done as well.In spite of the deadlines imposed by the Cosmetics Directive to eliminate animal testing and a number of eU initiatives, there are still no accepted alternatives for eye irritation nor any completely non-animal methods for acute toxicity, and nonanimal replacements for chronic and developmental toxicity will not be in place for the 2013 deadline.ReACH will have an enormous impact on the number of animals used in testing.there are some animal reduction provisions in ReACH, but with thousands of chemicals requiring base data sets by 2010, there will be animal suffering on an unprecedented scale.even without ReACH yet in full swing, the numbers of animals used in the UK has risen every year since 2000, with a dramatic increase of 42% in the past decade.Most of this is due to the increasing use of transgenic mice -with thousands of them being used to breed and maintain each lineeven though these so-called "models" of human disease are of questionable relevance.And in spite of public support for a ban on primate experiments, the use of primates in the UK actually rose 16% in 2008 over 2007.Surely, intelligent and dedicated people can find a way to reverse this trend.
While the continuing revision of Directive 86/609 provides an opportunity to address long-standing problems with the use of animals in european experiments, there is a worrisome de-regulatory agenda that threatens to gut the proposal of its most progressive measures.europe must not miss the opportunity to put in place a rigorous and comprehensive system of regulation that reduces animal suffering; fosters the development, validation, regulatory acceptance, and uptake of replacement methods; and, most importantly, provides the basis on which meaningful progress will be made towards the goal of eliminating all animal experiments as soon as possible.that is the end of the list.

Passing the final exam in the history of mankind
One good thing that came out of the 1960s, and I'm not talking about tie-dye, was the expression, "If you aren't part of the solution, you are part of the problem."Society is deeply indebted to each of you who are part of the solution.those of you who are in government, it is vital to rock the boat; those of you who are on committees and in funding agencies who opine that animal tests are not only ethically flawed but often conducted out of habit, obstinacy, laziness, and because no one has spoken up, thank you for living.Now, let me return to where I started.If we believe what social scientists have told us -that ordinary humans are capable of extraordinary cruelty -we must recognize that laboratories are exactly the place where such things will occur.It is not enough to regulate vigorously, although we must do at least that.As a group, human beings are far too tolerant of cruelty and far too unpredictable to be entrusted with the lives of truly vulnerable beings, yet we are in that position and must be vigilant about our conduct.
And finally, if you are thinking, "What was that bit about Brueghel's two Monkeys?" -it refers to the poem by Wislawa Szymborska, who wrote: This is what I see in my dreams about final exams; Two monkeys, chained to the floor, sit on the windowsill, The sky behind them flutters, the sea is taking a bath.
The Exam is the History of Mankind I stammer and hedge.
One monkey stares and listens with mocking disdain, the other seems to be dreaming away -But when it's clear I don't know what to say He prompts me with a gentle Clinking of his chain.the animals are all around us, intelligence and emotions shining from their eyes.they are prompting us -with the clinking of their chains -to lead a life we will be proud of when the time comes, as it always does to mortal beings, to take that final exam.
No one knows when that will be, but a time will come to all of us -those of us who answer to a god and those of us who answer to ourselves -when we look back on our careers and our lives with pride or with regret.
Society's ethical values expand as we come to understand that we not only have the capacity but are duty-bound to extend consideration beyond just ourselves, our families, our races, and, without a doubt, our species.to understand, as Dr. Albert Schweitzer said, "ethics are complete, profound and alive only when addressed to all living beings." to put it in practical perspective, I was in england recently and, as I was reading the Sunday paper, I came across a column written about dogs. the columnist wrote: "Contrary to what Buddhists would have you believe, remember, a dog is just a dog: he will never write a great book or compose a great symphony."I thought "Hang on a minute!"I'm going to bet that this columnist will never write a great book or compose a great symphony, and one thing I know is that he will never detect a cancerous tumor with his nose, and he certainly wouldn't be able to find his way home over hundreds of miles without the benefit of a GPS, a map, a street sign or advice from another human being.Perhaps what separates humans from other ani-mals is the desperate quest that our species has to find something that distinguishes us from the other animals.
Maybe the question should be "When will we all start seeing ourselves as just one of the many musicians in this vast orchestra of life, one no more special than the others?" When we take those final exams, may we all be able write that we contributed to the History of Mankind by bravely confronting our biases and by helping our species evolve from undisciplined bully to compassionate citizen.May we be able to say that each of us had the nerve, the backbone, the principle, and the vision to say what needs to be said about the use of animals, the suffering of animals, and the appropriateness of the behavior of those around us.I wish you all the best in everything you do to pass that exam.

Correspondence to
Ingrid e. Newkirk, President People for the ethical treatment of Animals 501 Front Street Norfolk, VA 23510 USA e-mail: jessicas@peta.org

Introduction
On 21 August 2007, in tokyo, Japan, during the Opening Ceremony of the 6 th World Congress on Alternatives and Animal Use in the Life Sciences, we paid tribute to the rich and varied life of W. M. S. Russell, who, with R. l.Burch, gave us the three Rs concept, in their book, The Principles of Humane Experimental Technique (Russell and Burch, 1959;Balls, 2008).Now, two years later, in Rome, Italy, toward the end of the 7 th Congress, we are celebrating the 50 th anniversary of the publication of The Principles.
I firmly believe that The Principles contains timeless insights into how we should think about the use of laboratory animals for research and testing, which are as relevant today as they were in 1959, and which can guide us as we seek to achieve genuine progress, whilst maintaining the highest standards in terms of both scientific methodology and animal welfare.The book also contains warnings about how fundamental mistakes can be made, which compromise the value of the science and threaten the welfare of the animals.
My concern is that, although a large number of people say they are committed to supporting the three Rs concept

Michael Balls
FRAMe, Nottingham, UK

Summary
In the Principles of Humane experimental technique, Russell and Burch said that "the central problem is that of determining what is and what is not humane, and how humanity can be promoted without prejudice to scientific and medical aims".They then explained how the Three Rs can be used to diminish or remove direct inhumanity ("the infliction of distress as an unavoidable consequence of the procedure employed") and contingent inhumanity ("the infliction of distress as an incidental and inadvertent by-product of the use of a procedure").They concluded that "Replacement is always a satisfactory answer, but Reduction and Refinement should, whenever possible, be used in combination".Many of the commonsense insights in the Principles are no less relevant today than they were in 1959.However, their warnings about the limited value of models and, in particular, the danger of succumbing to the high-fidelity fallacy (whereby it is assumed that the best models for humans are always placental mammals, because they are more like humans than other animals), appear to have largely gone unheeded.Of particular importance is their discussion on toxicity testing, which they saw as one use of laboratory animals "which is an urgent humanitarian problem, for it regularly involves considerable and sometimes acute distress".How, then, can it be that mammalian models are still routinely used in attempts to detect chemical carcinogens and reproductive toxins, despite the fact that the relevance to humans of the data they provide has not been, and perhaps could never be, satisfactorily established?Nevertheless, there are signs that some significant changes in attitude are taking place, particularly in the USA, which could be more in line with the main thrust of The Principles, the belief that good science and human technique inextricably go hand-in-hand.Keywords: animal experimentation, reduction, refinement, replacement, Russell & Burch, Three Rs, toxicity testing of Reduction, Refinement and Replacement, as put forward by Russell and Burch, most of them are unaware of the detailed implications of these insights and warnings, because they have not read the book itself.the result is that I am disappointed that the great benefits afforded by a careful consideration and dedicated application of The Principles have not been achieved.I therefore hope that this Congress will mark a new beginning -a much-needed, renaissance of the three Rs.
As one of the initiatives to celebrate its own 40 th anniversary, FRAMe has made an abridged version of The Principles available, with the cooperation and support of Cleo Paskal, W. M. S. Russell's literary executor.the principal aim of The Three Rs and the Humanity Criterion (Balls, 2009) is "to seek to retain the remarkable concepts and flavour of the original, whilst clarifying some of the english language employed, as well as reducing some of the lengthy discussions based on uses of animals in the 1950s which are no longer practised".
What I plan to do here, is to list some of the insights, then say why the failure to heed some of the warnings severely limits the impact of the three Rs, and, as a result, compromises the high standards of scientific practice and animal welfare which Russell and Burch sought.Quotations from The Principles are shown in italics in the sections 2 to 4.

The insights
the main principle on which Russell and Burch based their analysis is that it is widely recognised that the humanest possible treatment of experimental animals, far from being an obstacle, is actually a prerequisite for successful animal experiments.they considered the central problem to be that of determining what is and what is not humane, and how humanity can be promoted without prejudice to scientific and medical aims.
they began with the concept of inhumanity and its relation to those of pain and distress, then turned to the positive aspect -the analysis of methods of diminishing inhumanity in experimentation.
they said that we must first distinguish direct and contingent inhumanity.By the former, we mean the infliction of distress as an unavoidable consequence of the procedure employed.By the latter, we mean the infliction of distress as an incidental and inadvertent by-product of the use of the procedure, which is not necessary for its success.
their thesis was that inhumanity can be, and is being, diminished or removed under the three broad headings of Replacement, Reduction, and Refinement -the Three Rs of humane technique: -Reduction means reduction in the numbers of animals used to obtain information of a given amount and precision.-Refinement means any decrease in the incidence or severity of inhumane procedures.-Replacement means the substitution for conscious living higher animals of insentient material.It is always a satisfactory answer, but reduction and refinement should, wherever possible, be used in combination.

The warnings
The Principles also contains a number of important warnings, but it will only be possible to discuss two of them here.First, the tendency to misunderstand the nature of models, and especially, the use of animals as models for man: A perfect model of the human organism would obviously be indistinguishable by any test from its original.Any other in vivo model must depart in some degree from the original.There are two factors governing the way in which the model differs from the original.

Fidelity means overall proportionate difference, and discrimination means the extent to which the model reproduces one particular property of the original.
the point is that, however great the overall similarity between the original and a model may be, if there are significant differences in the specific properties being studied, the model will not be useful.Also, however, great the differences between the original and a model may be, if there are sufficient similarities in the specific properties being studied, the model may be a useful one.Clearly, high fidelity/high discrimination models are most useful, but, where this is not possible, a low fidelity/ high discrimination model is preferable to a high fidelity/low discrimination one.
Russell and Burch go on to say that Progress in replacement has been restricted by certain plausible, but untenable assumptions about models, which have led to the high-fidelity fallacy.the major premise is that the highest possible fidelity is always desired in medical research and testing, and that, for man, a member of another placental mammal species would be a model of higher fidelity than a bird or a microbe.this assumption can have disastrous consequences in terms of the data produced, and can also lead to unnecessary, and therefore unacceptable, animal suffering.
Most of the macaques used in the UK are involved in toxicity testing for the pharmaceutical industry.When asked about the 16% increase in 2008, a senior scientist from Global R&D of a leading pharmaceutical company said that this was driven by a move toward more biological medicines (Gill, 2009): "these treatments need to be tested in a human-like model, and old world primates are closer relatives of humans than new world primates."But what about the tGN 412 scandal, where the "human-like model" did not reveal the acute adverse effects of this humanised product, which later occurred in human volunteers?Macaques should not be used merely because of their overall similarity to humans, but only when it has been established, in advance, that they are appropriate models for use in a particular study.
Russell and Burch were concerned about toxicity testing on more-general grounds, since they considered it to be an urgent humanitarian problem, for it regularly involves considerable and sometimes acute distress, and to be an activity where the highfidelity fallacy may be more prevalent and influential at the legal level, rather than at the laboratory level.they clearly foresaw the problem of persuading regulators to accept the use of scientifically-advanced, replacement alternative methods instead of the animal tests with which they are more comfortable.

Reduction
At the 5 th World Congress, in Berlin in 2005, I said that "the progressive reduction in the numbers of animal experiments which had been foreseen when the new legislation was passed in the 1980s seems to have come to an end, especially as more and more mice are sacrificed on the altar of genetic exploitation.Also, far from working together toward the zero option of the use of non-human primates, there is pressure to build more and more primate research centres (Balls, 2006)."Sadly, the situation has worsened since 2005, rather than improved.
In Britain, the number of scientific procedures on living animals in 2008 was higher than the number in 1987 (Anon, 2009), the first year after the Animals (Scientific Procedures) Act 1986 came into force (Fig. 1), and the number of procedures applied to old world primates (macaques) in 2008 (4230) was much higher than in 1987 (2470; Fig. 2).
the situation in Britain is no doubt mirrored in other countries, and the production and use of genetically modified mice is widely used to excuse the overall situation.the chilling prospect is that there is much worse to come, given a recent report in Nature (Abbot, 2009): "european investment could see knockout rats catching up with mutant mice in medical research.the European Commission has approved the world's first major systems-biology programme to study the rat.Known as eURAt-RANS -for european large-scale functional genomics in the rat for translational research -the multimillion-€ project includes collaborators in the United States and Japan."Will this be followed by a move to set up programmes for producing large numbers of transgenic non-human primates?
One of the main scientific points of emphasis in The Principles, the need for high quality experimental design and statistical anal-ysis, has been largely ignored.Indeed, there is much evidence to support the contention that scientists, regulators, universities, industries, governments and grant-giving bodies are content to tolerate bad science.there are some praiseworthy efforts to redress this situation, such as the training schools run by the FRAMe Reduction Steering Committee, in collaboration with the University of Manchester, and with the support of the european Commission's COSt programme (Howard et al., 2009).
Virtually no time has been specifically devoted to Reduction at this 7 th World Congress, so it is impossible to avoid the conclusion that it is the forgotten R, even though Russell and Burch saw it as of great importance, and of all the modes of progress, it is the one most obviously, immediately, and universally advantageous in terms of efficiency.

Refinement
there has been considerable progress concerning the husbandry and use of laboratory animals, not least because of greater recognition of the importance of laboratory animal technicians and laboratory animal veterinarians.
That is to be welcomed, but there is also a danger that refinement can be used as a convenient way of showing commitment to the three Rs, whilst ensuring that animal experimentation is seen as respectable and can be allowed to continue, while the fundamental ethical questions raised by it are avoided.
this is not my area of expertise, so I will not dwell on it further.However, I do wonder whether the activities linked to ethical review processes and institutional animal care and use committees, however positive they may be in terms of refinement, have any significant effects in relation to reduction and replacement.We should remember that Russell and Burch said that, in general, refinement is never enough, and we should always seek further for reduction and if possible replacement.

Replacement
the position adopted by academia and research-funding bodies has long been that new techniques emerge during the natural development of a science, so deliberately seeking replacement alternatives for the vast array of procedures applied to laboratory animals in the basic sciences is not necessary.However, it could be argued that the legislation which regulates animal experimentation imposes legal and ethical obligations on all concerned, which should not be so easily avoided.From the scientific point of view, the high-fidelity fallacy deserves far greater recognition and resultant action, especially in the case of animal models of human disease, where insufficient about the disease is known for sound judgements to be made about the relevance, or otherwise, of the model.Nevertheless, it is toxicity testing where, as Russell and Burch recognised, the greatest concerns arise.For example: Why is it believed that the rodent bioassay can tell us what chemicals are likely to be carcinogenic in humans, when dosing is based on the maximum tolerated dose, and the mouse is a poor model for the rat and vice versa?Why is it believed that the current regulatory reproductive toxicity tests can tell us what chemicals are likely to be reprotoxic in humans, when so many false positives and false negatives occur that it is impossible to judge whether the test procedures can even identity reprotoxins in the animal models themselves?Why are animal data still widely regarded as the "gold standard" to be matched by non-animal tests, when the reliability and relevance of the tests concerned cannot be established, even for the animals? the Draize rabbit eye irritancy test data are so variable that the test cannot reliably be used to identify potential eye irritants in rabbits, so why is it believed that the data have any relevance to humans?

Moving backward in Europe
Despite much positive talk by politicians and senior officials about the importance of the three Rs and their commitment to them, it could be argued that, in actual fact, europe is going backwards.the great promises of the 1990s have not been delivered.Three examples will suffice, although this is not the place to discuss them in detail.
1.The REACH system: totally unworkable, proposed by ill-informed ambitious civil servants, taken up by ill-informed ambitious politicians, and then by ill-informed ambitious governments.It was clear from the early drafts of the Commission's White Paper that nobody had any coherent or defensible idea of the numbers of chemicals that would need to be registered, the number of additional animal tests that would be required, or how human health and the environment would be afforded greater protection.there was no mention of non-animal tests or their validation.later on the potential value of replacement alternatives was grudgingly accepted, and, as it became clear that the numbers and costs of various aspects would be much, much higher than had been expected, they came to be seen as a way of saving face and reducing embarrassment.We now have an expensive agency in Helsinki, which is producing thousand upon thousand of "guidance" documents.What is the value of accumulating so-called "missing" data, if its value and genuine usefulness have not been established?there is a likelihood that, since the science is being driven by the politics, the validation process itself will be corrupted.Instead of waiting until they have been independently shown to be reliable and relevant for their stated purposes, replacement alternative tests may come to be accepted because they are "suitable" (i.e.politically convenient).But aren't plausibility and suitability, based on the high-fidelity fallacy, among the reasons why we have so many useless animal tests?What will be the consequences, if "suitable" tests are found, in time, to have been "unsuitable" after all, and who will accept the responsibility for their failure?
2. The 7 th Amendment to the Cosmetics Directive: a ruse of no value, seemingly designed to convince politicians and a gullible public that something is being done.the situation with regard to cosmetic ingredients is no less unsatisfactory.Many of the chemicals used in cosmetics are also used for other purposes, and the ReACH system will apply to them.If the testing of cosmetic ingredients in compliance with the Cosmetics Directive comes to be banned, which companies will admit to doing any animal testing for that purpose?Won't they say that the testing was done for compliance with the ReACH system, and won't some of them try to stick to "not tested in animals for cosmetics purposes" labelling, while conveniently and dishonestly omitting the last three words?In addition, the definition of a "cosmetic" used in Europe is increasingly unsatisfactory, as cosmetic products are produced which actively alter the biological properties of the components of the skin.

Draft proposals for a Directive to replace
Directive 86/609/ EEC: one of the worst pieces of draft legislation ever published, which even foresees circumstances in which Member States could permit experiments on Great Apes. the Commission's proposals were produced after years of discussion with all kinds of stakeholders, but what emerged was not a draft directive at all.Rather than having its intentions spelled out clearly, in a way which could be implemented as a law, the result was a curate's egg mishmash of ideas which were either ill-conceived or in need of further discussion and development.As a result, hundreds of amendments have been put forward, and the result is a threat to both good science and sound animal welfare.Despite this totally unsatisfactory situation, there is great political pressure to get something in the statute book.
I mention these three points, because I fear that they illustrate the fact that europe is going down a slippery slope as far as the three Rs and a sensible balance between science and animal welfare are concerned.In the long run, fine words and catchy slogans count for nothing -it is sustainable actions of high quality that matter.As Jesus said, "Ye shall know them by their fruits" (Matthew's Gospel, 1611 translation).the problem is, who has the power and the desire to intervene and see that the downward trend is reversed?6 Moving forward in the USA?Meanwhile in the USA, a number of very promising developments are taking place, and in particular, the follow up to the publication by the US National Academy of Sciences of Toxicity Testing in the 21 st Century -A Vision and a Strategy (National Research Council, 2007) and by the US Food and Drug Administration of the Critical Path Initiative (FDA, 2004).What these two documents have in common is the recognition that animal models can no longer be relied on in drug development and in toxicity testing in general, and that more effort should be put into the development, evaluation, acceptance and use of what we would describe as replacement alternative methods and strategies, particularly when they are of direct relevance to humans.
to be fair, I must recognise that promising developments are also taking place in europe in relation to speed and safety in drug discovery, as was shown, for example, at a symposium held in london in 2008 (Gard and Clotworthy, 2009).

Concluding remarks
the published proceedings of the 7 th World Congress will reveal a wealth of activity, mainly focused, perhaps, on the possibility of replacing animal procedures by more-modern methods, based on the remarkable progress being made in cellular and molecular biology.In a way, then, the three Rs concept is to the fore as we celebrate the 50 th anniversary of the publication of The Principles.
Nevertheless, as I have pointed out, there are grave causes for concern, especially as the number of animal procedures conducted each year continues to increase, and legislative changes, especially in europe, threaten to perpetuate and expand that increase even more.
I hope that many of the grandiose statements made in apparent support of good and ethical science based on the three Rs will lead to identifiable and excellent outcomes, which will demonstrate a genuine renaissance in line with Russell and Burch's outstanding concept.
In particular: significant reduction in animal use should be achieved, without further delay, through better experimental design and statistical analysis; refinement, however welcome, should not be seen as an end in itself; and much greater resources should be invested in the dedicated search for replacement alternatives.Meanwhile, Russell and Burch's warning about the high fidelity fallacy should be taken much more seriously and acted upon.
the way in which Russell and Burch put it cannot be repeated too often: If we are to use a criterion for choosing experiments, that of humanity is the best we could possibly invent.The greatest scientific experiments have always been the most humane and attractive, conveying that sense of beauty and elegance which is the essence of science at its most successful.
So, let us all take this opportunity to renew our commitment to live up to this ideal, with total sincerity, then go home, and get on with the job.

Introduction
On this the 50 th anniversary of Bill Russell and Rex Burch's book, The Principles of Humane Experimental Technique (The Principles), I share the honor, with Michael Balls (FRAMe), of presenting my thoughts on the relevance of this landmark publication to the conduct of science today.In brief, these Principles are more important than ever.
I had the pleasure of meeting Bill Russell and his wife Claire in their home in Reading, england in 1992.I met Rex Burch a few days later in Sheringham, england.With each of them, I shared stories, books, news clippings, and other publications illustrating the profound impact of their book, The Principles of Humane Experimental Technique, over the 30-plus years since its publication.
I visited Bill and Rex shortly after I was awarded the Russell and Burch Prize by the HSUS in 1991.At this point, Bill and Rex had not seen each other since the publication of their book.the two scientists went their separate ways, and neither had any idea of the impact of their work until Martin Stephens of the HSUS called them to get approval to use their names on the award.
Many of us subsequently had the pleasure of getting to know Bill, and those interactions have been amply recorded in two sets of publications -both of which are readily accessible in ATLA.The first is a series of obituaries of Bill, collected shortly after he died (Balls et al., 2006).the second is a collection of personal reflections given as part of FRAME's 40 th Anniversary celebration (2009, in press).
Rex Burch, on the other hand, is less well known.Unfortunately, few of us had the opportunity to spend much time with him.When I met Rex, he was the sole owner and sole employee of a small microbiology testing laboratory in the basement of the town Hall of Sheringham, england.Since it was a one-person operation, Rex worked 7 days a week, 52 weeks a year.He was an exceedingly gracious host.
Rex's contribution to The Principles of Humane Experimental Technique lay primarily in conducting interviews and collecting data.We have little knowledge of his role in the planning of the book, organization of material, or actual writing of the manuscript.Perhaps, as the Russell archive is evaluated, we may learn more.
Michael and I each will share how we see the impact of the Principles.I have chosen to address the relevance of Bill and Rex's thinking and why their book is a monumental contribution that anticipates where we are today.
to answer the question, is The Principles relevant today, I will focus on four areas of activity essential to the future of in vitro sciences as they apply to toxicology and Risk Assessment.these areas are: 1) training of scientists; 2) the NAS report: toxicity testing in the 21 st Century; 3) the transatlantic think tank for toxicology (t 4 ): working together; and 4) humane sciences: the art of the question.

The Principles of Humane Experimental Technique: Is It Relevant Today? Alan M. Goldberg
CAAt, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA

Summary
In the section on replacement in their 1959 book, The Principles of Humane Experimental Technique, Bill Russell and Rex Burch state: "As new fields of biology open in the future, it may become a matter of routine to apply the lessons of the past and turn as soon as possible to the techniques of replacement."They foresaw in vitro techniques, in their infancy at that time, as the science of the future.Today, in the US, the National Academy of Sciences publication of Toxicity Testing in the 21 st Century: A Vision and a Strategy proves their point.This pivotal publication recognizes that the future of toxicity testing lies in the use of human cells in culture and in methods that Bill Russell and Rex Burch could not have possibly conceived of in 1959 but which they identified generically as "the future."To truly establish this 21 st Century approach will require very specific training in translational toxicology (the use of clinical observations to develop in vitro methods to understand pathways and systems biology), the development of transnational programs, and ways to evaluate the accuracy, validity and importance of new and/or traditional studies.These evaluations are known at evidence-based toxicology (EBT).Science is "the art of the question."The concepts identified above are the tools to answer these questions -and to lead us to the next round of questions.The principles that Bill Russell and Rex Burch developed during the 1954-59 writing of "The Book" may be more important today than ever before.They argued that the newest science, the most humane science is also the very best science.This hypothesis is being proven now, as each of us contributes to the world's body of knowledge.

Training of scientists
the principles of humane experimental technique reside in two concepts: humane science is the best science, and to practice humane science one must incorporate the 3Rs: reduction, replacement, and refinement.I list them alphabetically, rather than in the order suggested by The Principles, to emphasize that all three Rs are critically important to good science (Goldberg, 2004).
Most scientists have not been taught the 3Rs or the principles one must follow to practice humane science.For most, humane science is the untaught standard that the scientist strives for.Bill Russell and Rex Burch provided the tools; now we must make sure these tools are made available to all scientists.We also must make sure that we teach our students that failure to practice the most humane science can and will compromise the quality of their results.
I have the privilege of co-teaching a course titled "Animals in Research: Science, Policy and law."It is offered twice per year -once as a classroom course and once as a distance education course with electronic communication and live talks.In the past two years, about 50 students have taken the course.these students get it.their questions, their discussions, and their defense of proper use of animals in research all tell me that animal research will only be accepted if humane science is practiced.NAS, 2007).It truly looked into the future and suggested a clear and uncompromising path to fulfill the vision.The publication has been widely recognized prior to this meeting, and at this meeting several awards have been presented to the Academy and the Authors.the report has four basic conclusions: -Animal research is expensive -Animal research is not always predictive of human consequences -the future of toxicology lies in the use of human cell systems in culture -to understand toxicity we must understand pathways (mechanisms) and systems biology these are the same concepts that the Johns Hopkins Center for Alternatives to Animal testing (CAAt) has focused on since our founding in 1981.From the outset, we funded research on mechanisms and human cells in culture.I wish I could take credit for all of it, but the Advisory Board of CAAt was more than a co-equal partner.
two additional aspects of a 21 st Century toxicology warrant consideration here: translational toxicology and evidencedbased toxicology (eBt).the NAS report does not directly ad-dress these topics, but both are important as we complete the paradigm shift in toxicology.
Translational toxicology involves, first, using observations in clinical or toxicological events and designing in vitro and/ or mechanistic approaches to understand the observation; then, using the newly developed in vitro methods to evaluate different chemicals with these methods; and finally, developing approaches to predict in vivo consequences from in vitro data.Clear examples of this approach have been developing over the last few years (Sawada et al., 2005).
evidence-based toxicology is an extension of evidencedbased medicine.It is a structured approach to literature evaluation.evidenced-based approaches serve to eliminate studies that do not meet the criteria for inclusion in summary evaluations and to strengthen those studies that do meet the criteria.Studies or methods that do not have scientific validity or scientific rigor are eliminated.In clinical medicine this allows one to identify "best" treatment options.In toxicology it will allow better risk assessment and management of chemicals.One example of an evidenced based approach is the Cochrane Collaboration (the Cochrane Collaboration, founded 1993).the Cochrane Collaboration provides a wonderful example of the depth of information that can be achieved with evidence-based approaches.
The Principles anticipated what has become the toxicology of the 21 st century.It states, "As new fields of biology open in the future, it may become a matter of routine to apply the lessons of the past and turn as soon as possible to the techniques of replacement" (p.104).Bill and Rex recognized that in vitro techniques, in their infancy at that time, would become the science of the future.

t -Transatlantic Think Tank for Toxicology
In the early 90s, when I was a trustee of the Doerenkamp-Zbinden Foundation, I pushed the foundation to establish endowed chairs in in vitro toxicology to guarantee the future of the field and to enhance the impact of the foundation's work.The first chair was awarded to Marcel Leist at the University of Konstanz, Germany.My term as a trustee with the Foundation was completed, but the idea was in place and well supported.the next chair was established at the University of Utrecht in the person of Bas Blaauboer, who was followed most recently by thomas Hartung at the Johns Hopkins University.
Prior to his arrival at Hopkins, thomas had already established the transatlantic think tank for toxicology (t 4 ) to serve as an incubator for new ideas and to provide a forum where concepts can be discussed at an international level.Its agenda is to examine ideas and concepts in light of changing science and regulation.thomas asked me to be a principal member of t 4 , along with the Doerenkamp-Zbinden Professors. the initial activities were defined at the time of the 2009 SOT meeting in Baltimore, as Thomas arrived and was installed officially as the Doerenkamp-Zbinden Professor and endowed Chair for evidence-based toxicology.
The first publication identified as coming from the t 4 , "Reevaluation of animal numbers and cost for in vivo tests to accom-plish ReACH legislation requirements for chemicals -a report by the transatlantic think tank for toxicology (t 4 )," written by Constanza Rovida and thomas Hartung, was published in the 3/09 issue of ALTEX.A two-page version of the paper appeared in Nature (27 August 2009).this paper cogently argues that, since the initial evaluation of cost and numbers of animals required to meet the requirements of ReACH, many intervening circumstances have resulted in significantly higher figures on both counts.Rovida and Hartung demonstrate that the actual costs of meeting the REACH requirements will require some 54 million animals and $ 9.5 billion.Both figures are unacceptable and undoable.this makes it all the more imperative that we fulfill the NAS Vision and Strategy.
the t 4 also will participate in the implementation of the NAS report, Toxicity Testing in the 21 st Century.these related but separate activities form the basis of the paradigm shift in toxicology.In vitro toxicology has been established on a solid foundation of activities that span the globe.the t 4 is a major building block of this foundation.

Humane science: the art of the question
As we move from animal studies to in vitro studies, the goal of humaneness is generally achieved.As Michael Balls points out in his companion piece, The Principles require that refinement and reduction be used Science can be defined as the art of the question.The better the question, the better the science.Humane science -the 3Rs in practice -requires a very different set of questions.In the US, the responsibility for posing the appropriate questions is shared between the investigator and the Institutional Animal Care and Use Committee (IACUC).the investigator has the responsibility to ask whether the experiment will answer the question asked, whether the species selected is the best choice, and whether the experimental design, including the number of animals, is planned so as to maximize the benefits of the question.the IACUC, by law, does not look at the science.Its role is to examine whether the above identified parameters are fulfilled.It asks such questions as: Are methods available that would make these experiments more humane?Are non-invasive methods available?Are they being used?Have humane endpoints been considered?Is this the best approach to answer the question being addressed?
In The Principles, Bill and Rex hypothesize that humane science is the best science.Over the last 50 years, the scientific community has proven their hypotheses to be true.the title of this talk: The Principles of Humane Experimental Technique: Is It Relevant today?
the answer: A resounding yes.

Introduction
this year marks the 50 th anniversary of the publication of The Principles of Humane Experimental Technique by William Russell and Rex Burch.their contribution focused attention on the 3Rs -replacement, reduction, and refinement.In toxicity testing, the primary initiative with the 3Rs in the intervening decades has arguably focused on reduction of animal usage while holding firm the belief that results from animal studies provide a "Gold Standard" for making decisions about possible human health risks of compounds.The very wording, "alternatives", has often been regarded by many in toxicology as those test methods that will reduce animal usage even though the result from the tests are not necessarily optimal for risk assessment decision-making.
A second challenge in reduction of animal use through mechanistically-based testing arises from the idea of validating "alter-natives".The process of validation with alternatives, in general, focuses on the ability of a test or a series of tests to give results consistent with those that would be obtained through testing in animals.In this context, all alternatives will fall short of the mark of complete concordance with in vivo outcomes.Are all efforts to reduce animal use significantly doomed to disaster as they are dashed against the "gold standard" barrier? the recommendations of a recent report (NRC, 2007) from the US National Academy of Sciences, Toxicity Testing in the 21 st Century: A Vision and A Strategy argues that it is time to redefine the toxicity testing paradigm, moving away from high dose studies in animals to in vitro assays assessing perturbations of toxicity pathways by environmental agents.In essence, the report supports a sweeping redefinition of our "gold standard."The author of this present paper was a member of the NAS toxicity testing committee.Since the publication of the NAS report in June 2007, Calling on Science: Making "Alternatives" the New Gold Standard1

Melvin E. Andersen
The Hamner Institutes for Health Sciences, Research Triangle Park, NC, USA

Summary
All of life's great journeys start with a goal in mind!The 2007 NAS report, Toxicity Testing in the 21 st Century -A Vision and A Strategy, has proposed a clear goal.This report envisions a not-so-distant future where all routine toxicity testing for environmental agents will be conducted in human cells in vitro evaluating perturbations of cellular responses in a suite of toxicity pathway assays.Dose response modeling would utilize computational systems biology models of the circuitry underlying each toxicity pathway; in vitro to in vivo extrapolations would use pharmacokinetic models, ideally physiologically based pharmacokinetic models, to predict human blood and tissue concentrations under specific exposure conditions.Results from these toxicity pathway assays and associated dose response modeling tools rather than those from high dose studies in animals would represent the new gold standard for chemical risk assessment.This talk focuses on some of the scientific challenges required to make this vision a reality, including characteristics of assay design, prospects for mapping and modeling toxicity pathways, assay validation, and biokinetic modeling.All of these tools necessary for this transformation of toxicity testing to an in vitro platform are either available or in advanced development.Science must lead the transformation.The scientific community, animal alternatives groups, regulatory agencies, and funding organizations will also have to muster the resolve to work together to make this vision a reality.Keywords: gold standard, toxicity pathways, in vitro biology, computational systems biology, toxicity testing transformation PL8 several of the NAS committee members have presented aspects of the report at more than 40 venues in North America and europe.These presentations and the lively debate engendered on these occasions have sharpened ideas about the use of results from in vitro toxicity pathway assays in risk or safety assessments.the NAS report, although published in 2007, was essentially completed in fall 2006.Advances in several key technologies in the past three years -especially stem cell biology, computational systems biology, and pathway mapping and modeling -appear likely to be key catalysts for moving the vision forward.Finally, the transformation from current, traditional approaches to new in vitro methods based on human biology will not come easily.Who will step up to assist in the transformation to a new approach to testing and risk assessment?Several initiatives within the United States, both in federal government research organizations and in the private sector, look likely to accelerate implementation.these topics -(1) the recommendations from the NAS report, (2) the manner in which the in vitro toxicity pathway data can be organized for risk/safety assessments, (3) the call to the alternatives community to embrace 21 st century computational and bioinformatics methodologies in designing and interpreting in vitro results, and (4) the institutional opportunities to accelerate implementation of the NAS vision -are discussed in turn in this current paper.

Toxicity Testing in the 21 st Century: a Vision and a Strategy
The US Environmental Protection Agency and the US National Institute of environmental Health Sciences asked the US National Research Council (NRC) to provide guidance on new directions in toxicity testing, incorporating emerging technologies such as genomics and computational systems biology into a new vision for toxicity testing.In 2004, the NRC convened a 22 person committee for this purpose (tab.1). the committee produced two reports.the committee's interim report (NRC, 2006) provided an overview of testing methods and approaches that could incrementally improve traditional toxicity testing.This report noted that health protection agencies and the public had experienced increasing frustration with the failure of current approaches to toxicity testing to provide timely, relevant information to support informed regulation of environmental agents.These toxicity testing strategies relied primarily on the observation of adverse health responses in laboratory animals treated with high doses of these agents.Estimating risks to human populations based on high dose animal studies require difficult extrapolations, first from high doses to environmental levels that are usually ordersof-magnitude lower than those used in the animal studies, and then from animals to humans.These traditional toxicity testing approaches and methods for their interpretation date back some 30 to 60 years, and were developed at a time when knowledge of biology -and of the manner in which chemical exposures perturbed biological processes -was primitive.While there have been steady, incremental improvements in toxicity testing over the years, there has been no comprehensive evaluation of the manner in which advances in cellular and molecular biology might improve toxicity testing practices.
The final report of the toxicity testing committee (NRC, 2007) outlined design criteria that needed to be considered in any revisions of practices for toxicity testing.In choosing among various toxicity testing options, four criteria are important: (1) achieving broad coverage of chemicals, chemical mixtures, outcomes, and life stages, (2) reducing the cost and time required for toxicity testing, (3) developing a better scientific basis for assessing human health effects of environmental chemicals, including knowledge of modes of action, and (4) minimizing use of animals in testing.The consideration of how these criteria should guide a modern approach to toxicity testing led the committee to propose a new framework for toxicity testing that would entail a major overhaul of current practice.

Toxicity testing and targeted in life studies
The NAS committee vision consisted of several key technology areas (Fig. 1).While also including in silico methods for assessing structure activity relationships and population assessments, the transformative parts of their new toxicity testing paradigm was the types of toxicity testing and the manner in which results from these tests could be organized to support human health risk assessment.This vision centers on defining dose-response relationships for toxicity pathway perturbations that would be expected to lead to adverse health outcomes if the perturbations were maintained in vivo at a sufficient level of intensity and for a sufficient duration of exposure.The key component of the vision is assaying perturbations of toxicity pathways, which are simply normal biological signalling pathways that may be perturbed by chemical exposures.Toxicity pathway testing would require a suite of in vitro tests that could identify the range of significant perturbations of human pathways that might occur as a result of chemical exposure (Fig. 2).Biologic responses are viewed as re-sults of an intersection of exposure and biologic function.The intersection results in perturbation of biologic pathways.The circuitry affected by the chemical is expected to determine shapes of dose response relationships for these perturbations.Ideally, these assays would be conducted in human cells, cell lines or in engineered human tissues.The committee believed that the use of a comprehensive array of in vitro tests with human cells would markedly reduce the need for whole animal testing, and provide much stronger, mechanistically-based tools for human health safety assessment.It was recognized that the conversion to an in vitro basis had challenges and the committee also suggested that targeted in vitro testing was also likely to continue for some time where such studies could provide information about metabolism, possible metabolite toxicity, toxicity pathways, etc. Metabolism has been recognized as a particular challenge for developing in vitro testing alternatives (Coecke et al., 2006).

Dose-response and extrapolation modeling
How will results from a comprehensive suite of toxicity pathways inform quantitative risk/safety assessments for environmental agents?In this new toxicity testing strategy, in vitro concentration response curve would cover multiple orders of magnitude (Inglese et al., 2006(Inglese et al., , 2007) ) and evaluate responses in cells/tissues from humans, the species of primary interest.The broad range of concentrations permit the definition of dose ranges resulting, or not resulting, in significant alterations of normal biological function.While low dose and interspecies extrapolations are not as problematic, new challenges arise in understanding the mechanistic bases for dose-response behaviors of the toxicity pathway assays, in calibrating expected blood/ tissue concentrations in humans against the vitro concentrations used in the toxicity pathway assays, and in understanding the linkages of early perturbations to adverse responses expected in exposed people.The report identified key technologies that will assist dose response and in vitro-in vivo extrapolations, includ- Adapted from Andersen et al. (2005).
ing (1) empirical dose-response models based on results from the in vitro, mechanistically based toxicity pathway assays, (2) mechanistic dose-response models based on knowledge of toxicity pathway circuitry and dynamics of pathway function, and (3) physiologically based pharmacokinetic (PBPK) models to equate tissue-media concentrations with tissue dose in exposed people.two recent perspectives on the NAS report provide good overviews of the report and directions for implementation (Krewski et al., 2009;Andersen and Krewski, 2009).

The new gold standard in practice
Over the past three years, there has been continuing discussions about the NAS report with diverse stakeholder audiences.During these discussions, many questions were directed at the manner in which the pieces of the new test paradigm would integrate together to provide quantitative approaches for risk or safety assessment.the NAS report did outline two hypothetical cases of "assessments" that might arise from a battery of in vitro test using examples of a reactive gas and of a compound with estrogenic activity.These examples were cursorily developed, but indicated how various parts of the testing and analysis would likely contribute to health assessments.It is possible today to provide a more complete picture of how these pieces might be integrated (Fig. 3).
The core component of the testing will be the suite of toxicity pathway assays (Fig. 3; section i).These assays would be developed for human cells, human cells in culture, or human three-di-mensional tissue surrogates.The toxicity test assays themselves need to be capable of evaluating the progression from initial activation of the pathway on through degrees of perturbation that would be considered sufficiently large to be associated with likely toxicity if maintained over a period of time in an intact organism.For most, if not all assays, concentrations are expected to range from sub-threshold through those causing initial pathway activation, on to regions of adaptation, and finally to those causing adverse cellular consequences.To cover these various degrees of response, each assay would likely provide different levels of biological readout as a function of concentration and duration of treatment.
Each pathway assay is expected to have specific dose response characteristics depending on the organization of the circuitry that determines the action of compounds on the toxicity pathway.the dose response behaviors should arise from the underlying biology of the circuitry.These core signaling processes include the initial signal recognition and then the larger scale network through which the initial perturbation progresses to generate toxicity in the test system.Computational systems biology (Alon, 2006(Alon, , 2007) ) provides the tools for describing these circuits and the differential behavior of the circuits with increasing degrees of perturbation.
The process of validating toxicity pathway assays would be to study its behavior for positive control compounds and to extract the network structure and network dynamics that determine dose response.The sequential passage from sub-threshold, to adaptive, to toxic conditions represents dose-dependent transitions in modes of action in an in vitro system.Dose-dependent  Adapted from Andersen et al. (2005).transitions in in vivo toxicology studies are well known (Slikker et al., 2004a(Slikker et al., , 2004b)).this type of response cascade has been described in vitro by xiao et al. (2003) and by Nel et al. (2006) in work on hierarchical oxidative stress.Feedback process control and dose response have also been examined more theoretically for anti-stress gene regulatory networks (Zhang and Andersen, 2007).Alon (2007) has provided a good overview of network motifs in signaling pathways and in what were termed "developmental" networks.In practice, toxicity pathway characterization would optimally include standard operating procedures (SOPs) for preparing cells, conducting specific assays, generating read-outs, and the detailed process by which the pathway structure, circuit, and dynamics had been evaluated to support dose-response modeling.The detailed pathway characterization (essentially the process of validation of the pathway behavior) would be the mainstay of dose response analyses.For the safety assessment, primary attention would focus on pathways affected at the lowest concentration (Fig. 3; sections ii and iii).
How do we relate concentrations affecting cells in vitro with exposures in human populations likely to cause similar responses in an intact individual?Human biomonitoring for chemicals in blood and excreta is becoming more widespread.In some instances, concentrations of exogenous compounds in humans may be available.Comparisons could be made between those concentrations seen in exposed populations and those affecting cells in the toxicity pathways assays in order to estimate a "margin of safety" or "margin of exposure."This comparison is unlikely to be possible with very many compounds.A more general methodology would be development of biokinetic models (DeJongh et al., 1999) to determine the human exposure situations expected to give cell and tissue concentrations similar to those affecting the human cells in the in vitro pathway assay test (Fig. 3; iv).These approaches are extensions of the physiologically based pharmacokinetic types of models that have been of interest both with toxic substances and pharmaceuticals (Reddy et al., 2005;Bouvier d'Yvoire et al., 2007).A coordinated effort is required to develop a larger suite of PBPK models and to enhance efforts in reverse dosimetry, i.e., estimating the exposure levels in a human population that produce specific blood/tissue concentrations (Clewell et al., 2008).Current efforts to improve dosimetry methods are also advancing in vitro-in vivo extrapolation tools (Gulden and Seibert, 2003;Heringa et al., 2004).
The risk assessment process would entail running the suite of assays for a compound to see the pattern of activation of pathways and the concentrations at which effects were noted in various pathway assays.the most sensitive hits from the suite of assays would then be organized to support both dose response modeling and in vitro-in vivo extrapolation. the pattern of activity across the suite of assays could also provide signatures to indicate the types of toxic endpoints that might be observed in vivo (Dix et al., 2007).For example, specific signatures might indicate a high likelihood of reproductive toxicity or of hepatic toxicity in a qualitative manner.
Risk assessments completed based on results from these toxicity pathway assays are likely to be quite different from those arising from current approaches.Today, we see effects in animals, usually at fairly high doses, and estimate the likely inci-dence of response at lower doses in exposed populations.For cancer, we might try to estimate the expected concentration estimated to give a 1/1,000,000 level of population risk.This process has two less than desirable outcomes -first, labeling compounds based on high dose hazard studies and (2) providing a false sense of precision regarding our ability to extrapolate across doses and species.For instance, if Compound A causes cancer, at a maximally tolerated dose, it becomes labeled as carcinogen regardless of considerations of exposure levels.Secondly, the public is led to believe that the estimates of the low dose extrapolations are scientifically valid without any appreciation of the uncertainties about these estimates.In contrast, the assessments based on the in vitro toxicity pathway assays would be more directed at safety assessment, estimating regions of exposure where no appreciable perturbations are expected in human cells or human tissues in culture.

Calling on 21 st century science
The NAS committee discussed a variety of key technology areas for toxicity testing in the 21 st century.While the broad suite of new tools are likely to influence many areas of toxicology research and to greatly improve understanding of cell signaling pathways, it is important to ask more narrowly how specific technologies and advances will contribute to the four components noted in Fig. 3.The three areas most likely to benefit immediately are in assay design, using stem cell technology, pathway mapping and modeling, and computational systems biology for assessing expected dose response behaviors.

Assay design
A frequently voiced concern after publication of the report was the difficulty in obtaining and working with primary human cells and the caveats associated with use of human cell lines.the past few years have provided optimism in the ability to obtain tissuespecific human and rodent stem cells from which more mature cell types can be generated (Alonso and Fuchs, 2003).The stem cells can be stored and grown as needed for assays and will likely become available for a wider and wider suite of tissues (Reya and Clever, 2005;Gaudio et al., 2009).Embryonic and fetal amniotic fluid stem cells can be used and differentiated through frequently tedious, multi-step processes to multiple cell types (DeCoppi et al., 2007).With tissue-specific stem cells, the route to mature cells is shorter and requires less manipulation (Wang et al., 2009).
In addition to availability of tissue specific stem cells, other advances bringing biomedical and small-scale manufacturing processes offer opportunities to utilize human 3-dimensional tissue in higher throughput contexts.For instance, Khetani and Bhatia (2008) discuss the application of semiconductor manufacturing microtechnology for fabrication of microscale tissues.A miniaturized, multiwall culture system for human liver cells with optimized microscale architecture maintained phenotypic functions or several weeks.These organotypic cultures could be useful in insuring better correspondence between in vitro tests and expected behaviors in vivo.
A major emphasis is required to produce appropriate assays with the right level of detail and an ability to provide appropriate read-outs across different responses levels.For risk/safety assessments with a single compound, rapid, in vitro testing for the suite of pathways is essential.High throughput and high data content methods were emphasized in the NAS report.In this usage, high throughput assays allow evaluation of hundreds or thousands of compounds across multi-point dose response in a period of just a few days.Some assays such as the organotypic liver assay above may not be amenable to high throughput.For toxicity testing, it is useful to distinguish the need for high throughput methods for testing large numbers of compounds from efficient in vitro tests that can be done over the course of days but may not be easily scalable to the ultra-high throughput.For evaluating the chemical space active for a particular pathway, high throughput permits evaluation pathway perturbations for large compound libraries, leading to better in silico modeling of structure activity relationships.

Mapping and modeling toxicity pathways
Assay outputs can be diverse as clearly evident from the US EPA ToxCast group of assays (Dix et al., 2007).Nonetheless, the area where the diverse array of new technologies has the greatest possible for contribution is in mapping and modeling the underlying signaling networks for specific toxicity pathways.the vast majority of perturbations are associated with networks that affect transcriptional control.Such a conclusion is obvious for so-called receptor-mediated toxicants, such as dioxin and the aryl hydrocarbon receptor, but is equally valid for stress response pathways.Antioxidant response signaling starts with oxidants reacting with cellular sensors -primarily Keap1.The modification of Keap1 leads to its dissociation from a complex with Nrf2, allowing Nrf2 and other partnering proteins to form a promotional complex altering expression of genes controlling cellular anti-oxidants (Motohashi and Yamamoto, 2004).
As toxicity pathway circuitry becomes better understood over time, it will be possible to create computational systems biology models for expected dose-response relationships for each of the assays used for toxicity testing following similar principles.Over the past decade, tools for mapping and modeling have blossomed.In a recent paper, Bromberg et al. (2008) described the network by which cannabinoid receptor (CB1R) controls neurite outgrowth.Activation of several hundred transcription factors within the nucleus after cell stimulation was measured to understand the logic of the signaling network.Bioinformatic methodologies connected CB1R to 23 activated transcription factors.Experiments with pharmacological inhibitors of kinases revealed a network organization of partial "OR" gates regulating kinases stacked above AND gates that control transcription factors.As in most instances of current research in systems pharmacology and network modeling, the goal of these studies was not dose-response as would be a primary interest for toxicity pathway analyses.This example provided a glimpse of the structure of the network without attempting a quantitative computational model.The epidermal growth factor (EGF) signaling network is particularly well studied.Amit and col-leagues (2007) used a suite of experimental and bioinformatic tools to determine the forward signaling and feedback processes controlling the EGF network.The network was dissected by transcriptional profiling coupled with reverse phase protein lysate assays that assessed phosphorylation states of proteins within the EGF pathway.The analysis provided the structure of the logic of the circuitry for the early, immediate and later stage portions of the network.

Computational systems biology
It appears likely that a major contribution of 21 st century science will be the application of an array of technologies to elucidation, mapping and modeling the behavior of the test systems for assessing toxicity pathway dynamics.The tools would include mRNA, transcription factor and phospho-protein time course profiling, coupled with bioinformatic technologies to extract network structure.the outcome would provide dynamics of the signaling networks and the dose-and time dependence of expected consequences of perturbations by test compounds, including positive controls for each of the pathways.Dynamic behavior of signaling networks have been described quantitatively using computational systems approaches focusing on models of transcriptional control (Alon, 2007;Aldridge et al., 2006).Theoretical descriptions of networks leading to better understanding of modular design elements in biological circuits have refined our vocabulary -concepts of ultrasensitivity, bistability, network gain, feedback and feed forward motifs, noise, stochasticity, and sequential levels of early, mid-term and late gene expression -to allow discussion of network behaviors with some commonality of terminology.These concepts are more extensively elaborated in a course text on "Computational Systems Biology and Dose Response" available at the Hamner Institutes web-site (http://www.thehamner.org/education-and-training/drm_workshop.html).

Creating the transformational mindset
In a Figure (5-1) in Chapter 5, the NAS report discussed a strategy for implementation, including ballpark estimates of the time (1 to 2 decades) and costs ($ 1-2 billion) for transitioning from current animal intensive toxicity testing to a toxicity pathway based approach.the report stressed the need for an organization to have the lead responsibility for overseeing the technology development to support the transition -a role that could eventually be played by an appropriate laboratory within the US National Institutes of Health.the overall timeline was shown in the report in a linear fashion leading to a transition to new approaches after completion of technology development for assays and achieving some confidence that the suite of assays would provide adequate coverage of possible pathway perturbations.In the current global economic climate and with a variety of competing interests for biomedical research, is it reasonable to expect federal agencies or the private sector to support such a long-term, expensive initiative?Some aspects of the NAS vision are embedded in other programs.Three federal US agencies with responsibilities for health-related research -the Environmental Protection Agency (EPA), the National Institute of Environmental Health Sciences' National Toxicology Program (NTP), and the National Institutes of Health Chemical Genomics Center (NCGC) -have a memorandum of understanding to conduct research necessary to advance the NRC committee vision for the future of toxicity testing.Collaboration among these organizations in the US will be essential in establishing a national commitment to develop the scientific foundation of the vision.This collaboration (Collins et al., 2008) focuses on research (1) to develop high throughput test methods, (2) to identify toxicity pathways, (3) to pursue targeted testing in short-term in vitro tests, and (4) to develop dose-response and extrapolation models.New approaches for in vitro toxicity testing and toxicity profiling are key parts of several federal programs in the US (Dix et al., 2007;National Toxicology Program, 2004).The US EPA ToxCast program (Dix et al., 2007) is using a variety of high throughput tests and computational methods to enhance prioritization of compounds for targeted testing in animals.A professed goal of the new interagency collaboration is predicting high dose results in animals.Prioritization and predicting high dose results are not part of the NAS vision.Nonetheless, the tools and approaches being developed in this collaboration will be important for achieving the long-term vision for transforming toxicity testing.Other tools will mature from efforts that are today primarily focused on animal alternatives (e.g., Spielmann et al., 2000).
In the past year, the Humane Society of the United States (HSUS) and its affiliates, the Humane Society Legislative Fund (HSLF) and Humane Society International (HIS) have taken steps to enlist partners to a stakeholder consortium -the Human Toxicology Project Consortium.The goal of this group is to facilitate the global shift to a cell response pathway paradigm for chemical safety assessments.This shift, in the words of the consortium, holds great promise for more rapid predictions of human health outcomes while superseding traditional animal testing for environmental agents and pharmaceuticals.The goals of this consortium is to (1) promote dialogue, information sharing and establishment of a research and development roadmap, (2) lobby for, coordinate and provide resources to support transatlantic efforts necessary to fulfill NAS vision, (3) engage in collaborative outreach to legislative, regulatory, corporate, academic and public interest audiences, and (4) to urgently develop a targeted research program to jump-start the transformation.
This targeted research plan, focusing on proof of concept efforts, would first focus on prototype compounds and provide examples of the application of results from toxicity pathway assays for risk/safety assessments sequentially rather than waiting 10 to 20 years to bring a totally new risk assessment paradigm on line.the proposed research over a 5 to 10 year period would provide examples with ten to fifteen pathway assays and generate opportunities for diverse stakeholders to gain experience in collecting and using these results for safety assessments.The outline of steps for this more targeted research program includes several components.-Select about 10 prototypes compounds/pathway.these compounds would be chosen based on the breadth of information about animal toxicity and of the expected toxicity pathway targets, serving as a test bed for examining relationships be- tween in vitro toxicity test assays and historical information regarding in vivo results.-Design appropriate cell-based toxicity assays.For these prototype compounds, test assay systems would focus on both rodent and a human assay, preferentially using stem cells or mature cells derived from these stem cells.With one or more of the prototypes, 3-dimensional tissue systems could be used for the assays.-Develop the next generation quantitative risk assessment tools.These assays would be subjected to mapping and modeling analysis to uncover pathway circuitry, the dynamics of pathway responses to positive controls, and the dose response behaviors expected from different levels of perturbation.-examine relationships between perturbations and toxicity for prototypes.The assay design would require consideration of cascades that contain initial target activation, adaptive responses, and adverse responses with prolonged levels of perturbation.
-Integrate results from studies to provide representative health risk/safety assessments.the outcome of each of the prototypes would be risk/safety assessments that would be compared to more conventional approaches from animal toxicity data sets.-Within the first 3-5 years expand from the first 10-prototypes to a larger suite of pathways/compounds.This transition should also allow some mid-course correction in the strategy, stemming from a continuing evaluation of successes and challenges in applying the new science in assisting human health safety assessment.-With success in getting the program jump-started through the consortium, other partners, including toxicity testing organizations, regulatory agencies, and federal research organizations, could be enlisted as partners in moving forward with the transformation.Regardless of which organization seizes leadership for the efforts to create the technology base for shifting to a new "Gold Standard," the central question is whether such an initiative is a good public health investment.From the point-of-view of sparing animal use and a more humane infrastructure for testing the answer has to be yes.Is it also likely to be a good investment in terms of its likely scientific value?The answer here is also a resounding yes!Our primary investment in toxicity testing today is simply box-checking, becoming a bit more mechanistically oriented for high value chemicals that show responses in animal toxicity tests.the in vitro, human biology approach, elaborated here and arising from the NAS vision, has a much reduced emphasis on rote testing and much increased emphasis on generating detailed understanding of the signaling pathways affected by chemicals and how perturbations/modulations in these pathways affect biological outcome.These tools and approaches will be just as valuable in drug safety/drug development, in evaluation of safety of food and consumer products, and in ecotoxicology (Watanabe et al., in press).In addition to the broader applicability for human health outcomes for modulation of these pathways (Fig. 4), the organization of information on pathway structure and function is a natural post-genome program that could provide a better understanding of health, dis-ease and susceptibility within the human population -a much preferred investment compared to today's approach of in vivo testing and the cataloging of testing results.

Conclusions
Toxicity testing and much of the discipline of toxicology have reached a tipping point.Old practices focusing primarily on high dose studies to evaluate end-organ toxicity in animals are giving way to modern practices that assess how chemicals are likely to affect human biology and the concentrations under which these effects might be expected in exposed humans.This change will not occur easily.Even though the current toxicity testing is far from optimal, it is difficult to move away from entrenched traditional practices to a new footing.Change of this magnitude is discomfiting for most everyone.There are, of course, serious challenges to consider in such a transformation.They should not be dismissed or diminished.Chemical toxicity may relate to metabolism.How will the in vitro tests adequately assess metabolites with new compounds undergoing in vitro screening?Which of the observed perturbations will be considered appropriate for the safety assessment -will it be target activation, adaptive responses, or only some clear definition of overt toxicity in the cells?Will it be possible to describe circuitry for most toxicity pathways in enough detail to be confident in expected dose response behaviors?Finally, where will we find the scientists and regulators with the training and background to be comfortable with new practices?These issues are all important, legitimate questions that need to be considered.Yet, they should not divert us from the goal -to move toward a redefinition of the toxicity testing gold standard that focuses on human biology and perturbations of human toxicity pathways in vitro.We must bear in mind all the challenges, but push relentlessly toward the goal of a modern approach to human safety assessment.
can use it.the US National toxicology Program continues to kill thousands of animals every year to test well characterized chemicals, even natural substances such as ginseng and green tea, and it does so if the substance is nominated by anyone, even by a single anonymous person.And what of ICCVAM?In a decade, ICCVAM has gone from beloved baby to Frankenstein monster.Instead of doing the job the US Congress intended it to do -namely to facilitate the incorporation of non-animal methods into government regulatory programs -it has become the chief obstacle in the US to the use of non-animal testing methods.

3
Toxicity Testing in the 21 st Century: a Vision and a Strategy the US environmental Protection Agency (ePA) contracted with the National Academy of Sciences (NAS) to address the question of what toxicology testing will look like in the 21 st century.The resultant final publication is remarkable (Committee on toxicity testing and Assessment of environmental Agents,

Fig. 2 :
Fig. 2: The progressive activation of toxicity pathways from perturbation of initial targets, through activation of stress controlling pathways, to overtly toxic responses (apical endpoints).Adapted fromAndersen et al. (2005).

Fig. 3 :
Fig. 3: The progressive activation of toxicity pathways from perturbation of initial targets, through activation of stress controlling pathways, to overtly toxic responses (apical endpoints).Adapted fromAndersen et al. (2005).

Fig. 4 :
Fig. 4: Commonality of systems approaches to examining perturbations/modulations of normal biology for safety testing with environmental agents, pharmaceuticals, consumer products, and foods and for drug development.SETAC: Society for Environmental Toxicology and Chemistry.