Animal tests

Toxicology

The majority of products that you use in your bathroom, your kitchen, garden or for your car, including food products, have been tested on animals at some point to see how much of that product is poisonous (toxic) and what effects it has on the body. These cruel animal tests  can be replaced with advanced technology and also, with predications based on the vast knowledge we already have about the effects of chemicals, from human use.  However, despite the alternatives available, animal tests continue because that is the way that things have always been done.

NAVSweb_HLS arrivalPrimates are used in regulatory tests (toxicology, safety tests), which are required for a licence for a product to be sold on the market. Regulatory testing represents over 50% of the 6,000 primates used in European laboratories each year.

Animal tests have always been used because animals are available, and the animal testing technology is over 100 years old. However, there are no specific legal requirements for the use of primates in regulatory testing – the regulations specify that two species of mammals are to be used and one must be a non-rodent species.  So the idea is that the tests are started with smaller animals such as rats and mice, and then move on to larger mammals like dogs and monkeys. In theory, dogs and monkeys are used at the end of the testing strategy.

For example, safety/toxicity testing of a drug is about a key piece of information – the way a drug travels through the body – the rate and route by which it breaks down, before being excreted. This is crucial and affects how the drug affects the body. However, our genes and other factors influence this process. These key genetic differences between humans and other primates are hugely important in drug development and testing – despite the similarities between humans and the other primates such as use of tools, language, emotions and intelligence.

Case Study:  Huntingdon Life Sciences (now called Envigo) Laboratories

Huntingdon Life Sciences is a major European contract testing laboratory; they perform the standard animal tests required by regulators on behalf of their clients, the manufacturer of the product being tested – drugs, chemicals, and other products (for example things used in the home).

A case study by the National Anti-Vivisection Society (NAVS) provides an insight into the functioning of a typical European commercial testing laboratory, Huntingdon Life Sciences (HLS). The nature of the experiments conducted  on behalf of their clients (including GlaxoSmithKline, the Ministry of Defence and AstraZeneca) was to look for adverse effects or symptoms of test compounds.

During the investigation, the numbers of monkeys (cynomolgus macaques) observed being used in tests ranged between 4-72; just five studies accounted for the lives of 217 animals. Monkeys arrived from breeding colonies in Vietnam and tests typically involved restraint, being captured from their cages and firmly grasped by their arms, legs and tail. Three monkeys suffered rectal prolapses during one study. Rectal prolapse is known to indicate extreme stress in monkeys and tells us that they suffered terribly – probably as much through fear and anxiety as anything else.

NAVSweb_HLS restraintAn incontinence drug was given daily for a year, by forcing it down monkeys’ throats with a pipe (gavage dosing). The monkeys were removed from their cages, restrained and had the tube pushed down the throat to deliver the product being tested. Some of the monkeys vomited every time they were dosed, including ‘control’ monkeys who did not receive the drug, indicating that the procedure itself, not the test substance, was causing vomiting. Gavage is known to be unpleasant for animals; monkeys are observed to “forcefully resist such intervention”.  During this study, several animals suffered prolapses, which appear to be the result of fear due to anticipation of the procedures.

Yet a review of the incontinence drug market found at least 13 other incontinence drugs in development in the US, Europe and Japan. The market for these drugs was set to become “extremely competitive”.

Tests for a blood clotting drug involved 56 monkeys: they were put in restraint chairs and the substance administered through a cannula inserted into the back of their leg over a period of 2-15 minutes. Animals were bled before and during the study and were fasted every night before they were dosed.  Some animals were killed 24 hours after dosing and others were killed two weeks after dosing.

One female was trembling during dosing, indicating distress. It is known that biochemical changes in the body as a result of stress can influence the outcome of a test “which can cause anomalous experimental results”. Almost a decade before these tests on monkeys, a review of animal models of a blood clotting disorder led to the conclusion that differences in the animal and human disorder cannot be avoided, so “…one should be cautious about extrapolating the results from animals to humans” .

Case Study: Inveresk Laboratories

Documents and photographs, including reports of commissioned experiments were leaked to the NAVS. They provide a chilling insight into the world of ‘contract research’ – commercial safety testing. The company stated they would help businesses to comply with the EU’s REACH chemicals regulations by carrying out animal experiments on their behalf.

NAVSweb-InvereskAn asthma drug tested on monkeys involved a cannula being inserted into the leg vein of 16 animals where it remained so that they could be dosed daily for 14 days. The animals were then killed. Monkeys were observed for any signs of ill health or reaction to treatment. In order to obtain samples for analysis, the monkeys were deprived of food and water.

From approximately 5 minutes after dosing, all of the animals suffered reddening of the face and lips; this increased with dose levels, in terms of both incidence and length of time. They also experienced a range of symptoms including diarrhoea, swelling in the stomach, redness of feet and hands, white pigmentation on the feet; the males’ testes increased in weight and they suffered red and swollen penises and scrota. In addition, the monkeys were reported to be subdued and hunched on their branches; body tremors were seen in one; another did not use its right leg throughout the test, apparently due to “an injury”; and females suffered abdominal and umbilical hernias.

The report of one study admits that the client, AstraZeneca, was in possession of information from previous experiments on these monkeys, which indicated “the test compound may affect the cardiac function and produce pericardial effusion in cynomolgus monkeys when given intravenously or via inhalation”. The use of cynomolgus monkeys has also been criticised as being the most misleading laboratory animal model for the study of human cardiotoxicity (i.e. toxic effects on the heart), yet this is one of the key questions that the test intended to address.

The experimental drug was intended for inhalation administration in people, but the drug was given to the monkeys directly into the bloodstream. Yet it is already known that side effects can differ, dependant upon whether a drug is given through the blood stream, or inhaled. Results obtained on the effects of a drug administered through the vein may be misleading if the drug is intended for inhalation.

Case Study – A new disaster despite animal toxicity testing:  BIA 10-2474 Drug Trial – another TGN1412?

Following the severe damage to the human volunteers in the TGN1412 drug trial disaster, there appears to be another case on the horizon, BIA 10-2474. With TGN1412, laboratory primates had been given doses of the drug 500X that given to the human volunteers, with no apparent side effects. The human volunteers suffered life-threatening side effects.  Although the investigation of what happened continues, the BIA 10-2474 case has disturbing similarities.

A French research company, Biotrial, were carrying out clinical trials with healthy human volunteers testing the safety of a drug for Bial, a Portuguese pharmaceutical company. The drug, named BIA 10-2474, was being tested as a potential pain killer among other possible uses.

During the human drug trial, six male volunteers in the highest dose group (100mg) received the drug by mouth. Three days later, a participant in this group was admitted to Rennes University Hospital in France after beginning to feel sick; he was later diagnosed as brain dead and died a week after being admitted. During this time, the other five men in the same dosage group were also hospitalised; four of them showed evidence (by MRI) of deep cerebral haemorrhage and necrosis. In a press conference, the French Minister for Health, stated that 3 of the 4 men who were displaying neurological symptoms “already have a severe enough clinical picture to fear that even in the best situation there will be an irreversible handicap”; and it has since been reported that at least one of the volunteers lost all his fingers and toes.

Three separate investigations are currently in progress. A preliminary report from the first meeting of the Temporary Specialist Scientific Committee reports that no toxicity, especially neurological (central or peripheral) comparable to that observed in the accident in Rennes, appears to have been demonstrated in animals, despite the use of 4 different species and high doses administered over long periods.” [emphasis added].

The Animal Tests

NAVSweb_beaglePrior to the clinical trial, the drug had been tested on mice, rats, dogs, and monkeys for effects on the heart, kidneys, and gastrointestinal tract, and on rabbits and rats (both including their foetuses) to determine its reproductive and developmental toxicity effects. These animals would have inevitably suffered terribly during study of the toxic and analgesic effects of the drug.

Toxicity testing was carried out daily for up to 13 weeks in mice, dogs and monkeys, and up to 26 weeks in rats. Oral dosing for toxicity testing often involves animals being restrained while tubes are pushed down their throat so that drugs can be delivered directly into the stomach. Most procedures carried out on primates require restraint. NAVS investigations have shown the capture of monkeys from their cages involving a technician grabbing the monkeys from their cage. Three workers are usually needed; two to hold the animal, one to perform the procedure. The monkey’s arms, legs and tail may be held with the animal face up or face down.  All terrifying, and extremely stressful, for the monkey.

Species differences

The published Protocol on this drug does not clearly identify exactly what condition or ailment the drug was intended to treat in humans. It reports minimal in-vitro testing (alternative skin, cell or non-animal methods) on human target systems. Up to the date of the Protocol there was “no experience with BIA 10-2474 in human subjects”. Clearly the tests on animals did not predict its effect in humans, nor any indication for the type and severity of potential outcomes to the participants. In animals the Protocol notes that “few adverse clinical findings were observed at the highest dose tested”; it has been estimated that monkeys were given doses of the drug approximately 75 times greater than the highest dose given to patients, with no ill effects. Drug action does not transfer between humans and non-human animals.

Case Study: Testing of a cancer drug

These experiments on monkeys and rats were to test toxicity (potential effects) on the reproductive system of a cancer immunotherapy produced by GSK Vaccines. Additional rat studies were conducted at Huntingdon Life Sciences, UK.

NAVSweb_NoveprimA total of eight male monkeys, up to four years in age, were used in the CiToxLAB in France. The laboratory received the animals from Noveprim Europe (originally from Mauritius), where a previous Animal Defenders International (ADI) investigation had found appalling conditions. During the study animals were individually caged which would have caused them immense suffering. It has long been acknowledged that the stress from social isolation can cause physiological and behavioural distortions which can affect the data obtained and therefore provide unreliable information.

Six male macaque monkeys were given eight injections of the compound into various muscles on their body, every two weeks; two monkeys received injections of saline. Doses given to the monkeys were the full human dose, which represented approximately 10- to 20-fold the equivalent human dose when taking body weight into account. Thus, the monkeys were receiving a much higher dose than that which would be given to a human.

To evaluate sperm formation, fully awake monkeys underwent electroejaculation a total of nine times each approximately every three weeks. Authors do not note details of this procedure, but guidelines state that it involves “using an electric current to produce an ejaculate”. Researchers commonly use either rectal probe electrostimulation or penile electrostimulation. The latter does not require anaesthesia, but involves restraining the animal in a frightening restraint chair and using defibrillator pads and lubricating gel wrapped around the penis. The animal is stimulated with increasing voltage until ejaculation occurs. The distress caused by restraint and forced ejaculation by elctrostimulation can only be imagined.

Other procedures undertaken on monkeys included measurements of testes and prostate, examined by ultrasound under anaesthetic and blood sampling. The animals were finally killed by tranquillization and then bleeding out. At the time of publication of these experiments, this drug was already being given to human cancer patients in Phase III clinical trials which will have provided much more relevant information.

Replacing animals – the alternatives

The alternatives to the use of animals in safety testing include databases of known information, human tissue culture and sophisticated analytical techniques. Many non-animal techniques are already used before animals are used, but regulations still require animal tests. See more here

Fundamental or ‘basic’ research

Academic research with no direct application to human health.

Case study: Brain research on monkeys – decades of suffering with no human application

The kind of experiments on monkeys’ brains that we describe here, fall into the category of fundamental or academic research, animal experiments to gather information or data, but with no intended application for human health. Such projects can continue for decades, with no end in sight. For example:

At the Institute of Neurology in London a series of experiments has been funded by The Wellcome Trust, the National Centre for the 3Rs (Refinement, Reduction & Replacement of Animals in Research), and the Biotechnology & Biological Sciences Research Council.

A 2011 report describes an experiment where the heads of three macaque monkeys, two male and one female, were cut open to fix a head piece to restrain their heads and implant a recording chamber and electrodes into their brains. Electrical current was delivered to the brain. The monkeys had to grasp different objects for food reward while their brain activity was recorded both with and without electricity being applied to the brain. Two of the animals were killed at the end of the experiment.

The team received funding for this research, despite that they themselves cite a number of similar experiments on primates, and similar work using humans. The researchers don’t specify how this research might be helpful for humans and there is no discussion in the published paper, of the known differences between human and monkey brains.

NAVSweb_ElisaOne of the authors of this paper, Roger Lemon, has been carrying out invasive brain experiments in monkeys for decades. An investigation and case study by the NAVS in 1996 documented that, at that time, Lemon had already been working on experiments involving primates using chronic recording and stimulation techniques for twenty years.

The same group of workers published a similar paper in 2013, where two monkeys also received repeated surgeries and implants in the brains and this time into their arms, again to monitor brain activity.

There are fundamental differences between human and monkey brains. A study comparing the processing of motion in the human and macaque brains commented that “a complete homology between cortical areas of humans and monkeys is highly unlikely” considering anatomical and behavioural differences and the 30 million year evolutionary gap between the two species.  It also highlights how the specific areas of the brain involved in these cruel experiments differs between humans and monkeys.

There is no doubt that these monkeys will have suffered extreme fear and distress, as well as the pain of surgery, and yet Roger Lemon and his team acknowledge that non-invasive studies using human subjects are already carried out.

Case study: French dental study on monkeys

At the National Veterinary School of Lyon, France, researchers simulated human periodontal disease in 8 healthy long-tailed macaque monkeys, in a study funded by Dental manufacturing company, Dentsply.

A 2014 report describes how researchers performed multiple surgeries on the roots of monkeys’ teeth to study the effect of dental cavity material on healing. Monkeys’ gums were cut to expose roots of the teeth and jaw bones. A 3mm cavity was drilled into the tooth base and the outer layers were removed with a hand scaler. Inflammation was induced in the gums. During a second surgery various substances, including one derived from brains, were used to fill in the damaged teeth.  A total of 8 cavities were created in each monkey. The monkeys underwent numerous CT scans until they were killed 12 weeks later by anaesthetic overdose and draining of the blood, and their jaws cut in to pieces for further analysis.

There is no question that these monkeys would have suffered extreme fear and pain during this study.  As well as the direct impact of the invasive dental treatments, the monkeys would have suffered from the associated effects of recovering from the numerous surgeries and scans. At one point the monkeys were only fed soft food, so that food would not interfere with the surgical sites; this gives an indication of how delicate these sites were – and no indication is given as to whether pain killers were administered.

The research was carried out despite a number of previous studies which investigated the healing effects of the same dental materials, including studies in dogs. In addition, studies in humans have already been carried and treatments for halting the progression of periodontal disease in humans are already successful.

The authors also note that “the induced inflammation referred to in the current study is different from the inflammation caused by periodontal diseases”.  Human studies of regeneration in periodontal disease have highlighted that researchers need to take into account the effect of systemic factors in patients, such as metabolic disorder, and risk factors such as smoking, medication and genetic susceptibility.  Such consideration cannot be made in an animal model that has been artificially created in the laboratory.

Overall, this study does not add any new information apart from testing in another non-human species; it reports the same findings and just adds to this existing data.

Case study: Military research on marmosets at Porton Down

In 2013 a report was published detailing British Ministry of Defence funded research at Porton Down for the purpose of ‘military research’. Researchers investigated a potential therapy for human respiratory disease melioidosis, a bacterial disease from contact with infected soil and water in parts of Asia and Australia.

The researchers surgically implanted telemetry devices into 26 healthy marmoset monkeys that were housed in pairs in an isolation unit. All monkeys received daily injections and 16 of these were also forced to inhale the bacteria for 10 minutes in a head-only exposure chamber. Monkeys suffered a drop in body temperature, fever, lethargy, erect fur and subdued behaviour; many of the monkeys had severe damage to the liver, spleen and lungs, undoubtedly causing them pain, and the non-treated monkeys experienced the most severe liver pathology. It is not indicated whether animals received any form of analgesia to relieve their symptoms. All of the monkeys were killed at the end of the experiment with an anaesthetic overdose to cause death by respiratory arrest.

In addition to the confined housing within a biological containment unit and handling within isolator units by experimenters, the monkeys underwent multiple invasive and painful procedures and would have suffered considerably from the rapidly progressing disease which was induced.

The experiments carried out replicated an earlier study in marmosets. Responses of the therapy being tested have also already been studied in other primates and a similar treatment has already reached later stage clinical trials in humans.

There are already a number of therapies (oral and intravenous) to treat the disease in humans, including some antibiotics. Human susceptibility is largely affected by other clinical risk factors with over 70% of cases occurring in people who are suffering from another illness. As well as in vitro studies, research in human patients has taken place in order to study the biology of the disease, including assessing risk factors for the disease.

Marmosets respond differently to the disease compared to humans. Marmosets appear more susceptible to infection and the disease develops rapidly, within a few hours; the human incubation period can vary from less than a day to decades, with incubation periods of more than two months being common.

Case study: HIV monkey tests at St George’s 

An HIV study carried out at St George’s, University of London using 14 macaque monkeys was funded by the Bill & Melinda Gates Foundation, the Wellcome Trust and the Sir Joseph Hotung Trust.

The research, published in 2011, involved pre clinical tests for efficacy of an intravaginal gel. Female macaques were sedated and had an HIV protein and a gel, inserted into their vaginas, to a depth of 2cm, on nine occasions with 2-3 day intervals. Vaginal secretions were also sampled. Some animals underwent three “series” of this treatment. The animals also received a number of injections, determined by the experimental group they were assigned to.

Authors do not acknowledge that monkeys may have suffered discomfort or irritation from the introduction of substances into their vaginas or distress related to the intrusive procedure. Animals in the different groups were used in the experiment for a variety of days up to 281 days. At the end of the experiment the animals were killed so that their spleen, lymph nodes and “marrow washed from the bone” could be analysed.

Researchers immunised the monkeys using the same gel “to that used in a clinical trial run in parallel”. This means monkeys were undergoing procedures at the same time as women volunteering in trials. One paper actually describes a very similar topical gel showing “great promise against vaginal HIV transmission” in women, and highlights the problems of human adherence to drug administration regimes; something which could not be accounted for in experimental monkey models. Data is available from humans, the species of interest, making the use of primates entirely unnecessary and unjustifiable.

The authors admitted that repeated sedation may have influenced immune responsiveness, calling into question any data retrieved from the use of these animals. Additionally, it is unlikely that women using this gel would be sedated beforehand, making this animal model even less valid in its application to humans.

There are also species differences between monkeys and humans. Certain genes create enzymes which are important in the metabolism of drugs, yet there are differences between these genes in humans and the macaque monkey. Scientists have found that one particular gene in the macaque monkey does not have a corresponding version in humans, “and it is partly responsible for differences in drug metabolism between monkeys and humans”.

If you would like to receive the full briefings on the research cited here, including references, please contact us at info@navs.org.uk.

Replacing animals with advanced scientific techniques

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