Time for alternative action

The use of the LD50 test for measuring the potency of botulinum toxin preparations is unacceptable. FRAME and HSUS are calling for an improvement

The bacterium Clost-ridium botulinum was first isolated in 1895 during an investigation into an outbreak of food poisoning that had resulted in the paralysis and eventual death of a number of victims. But it was not until 1946 that the toxin which caused the symptoms was isolated and crystallised. In the early 1970s, the therapeutic value of the paralytic potential of botulinum toxin for treating problems involving the extraocular muscles was first recognised and in 1989 the US Food and Drug Administration (FDA) approved the use of Botox, produced by Allergan Inc,[1] for treating squinting (strabismus), involuntary and uncontrollable closure of the eyelids (blepharospasm) and some hemifacial spasms. Botulinum toxin now has at least 50 different therapeutic applications.[2]

In 1987, ophthalmologist Jean Carruthers noticed that the use of the toxin in patients with blepharospasm resulted in the disappearance of frown lines between the eyes, and informed her husband, dermatologist Alastair Carruthers, of this. Their 1992 publication[3] is recognised as being of crucial significance in the explosive development of the cosmetic use of botulinum toxin for dealing with crow’s feet, the shaping of eyebrows, dimpling and various other types of wrinkles. Allergan now produces Botox for therapeutic use and Botox Cosmetic for cosmetic applications, and by 2004 Botox Cosmetic accounted for net sales of 40% of the company’s botulinum toxin production, representing $295m for 2.8 million treatments.

The FRAME campaign

In 2003 Krys Bottrill, a scientist at the Fund for the Replacement of Animals in Medical Experiments (FRAME), revealed that the current definition of the potency of botulinum toxin for therapeutic or cosmetic purposes is based on a test to determine the amount required to kill 50% of a group of mice injected intraperi-toneally with the preparation; ie an LD50 test.[4] At least 100 mice are used to test each batch of the toxin, which results in diffuse muscular paralysis, impaired vision and paralysis of the diaphragm followed by suffocation and death. Some institutional animal care and use committees insist that the mice are killed immediately after the onset of severe symptoms, but there can be no relief from the earlier toxin effects.

The thrust of Bottrill’s article was that, paradoxically, the general public had enthusiastically adopted the use of botulinum toxin as an anti-wrinkle treatment against a background of moves to stop the animal testing of cosmetic ingredients and cosmetic products, and in particular Ipsen Ltd UK was producing and testing its product, Dysport, in the UK, although the government claimed to have banned all testing of cosmetics. The government’s response was that Dysport was only licensed for therapeutic use, and individual clinicians and their patients had to accept the responsibility themselves for using the product for other purposes.

Since the suffering of mice was a matter of great concern, whatever the eventual use of the product, Bottrill also raised the question of why an LD50 test was still being used. After all, this kind of procedure has been replaced by more humane methods, some of which avoid lethality as an endpoint, in the OECD Health Effects Test Guidelines for oral systemic acute toxicity testing. And there are a number of proposals for reducing the numbers of animals used, refining the procedure in order to minimise the suffering caused to them and eventually replacing the need for animal testing altogether. This situation was put to the Home Office, the government department responsible for regulating animal experimentation in the UK, and was discussed with the National Centre for Biological Standards and Control (NIBSC), which is responsible for the batch testing of various biological products, including botulinum toxin products, before their sale and use is authorised.

Although FRAME first met with denials that there was a problem, and questions to government ministers received unsatisfactory answers,[5] it is now clear that various kinds of action have resulted from, or been further encouraged by the Bottrill revelations. The Home Office appears to have encouraged Ipsen to be more active in seeking alternative approaches to batch potency testing, although it appears that the company and its sub-contractors are still permitted to use the LD50 test. Meanwhile, the NIBSC continues to be active in the development and use of refinement and replacement alternatives. In vitro methods are used there on a routine basis and a non-lethal in vivo test is used only rarely when the results of an in vitro test are inconclusive or are close to pass/fail specifications.

The HSUS campaign

In the light of Bottrill’s revelations, the Humane Society of the United States (HSUS) took up the issue with Allergan and sought disclosure of the details of its current potency testing and of its efforts to develop and use alternatives to mouse LD50 testing. Allergan revealed that it does use the LD50 test and claimed to have an active alternatives research programme, but provided few details and refused to collaborate with the HSUS. HSUS supporters then sent thousands of e-mails, faxes and letters to the company, but Allergan remained non-cooperative. The HSUS therefore turned to the FDA, but received no answers to its initial queries. So in 2004 the HSUS filed two requests under the US Freedom of Information Act, followed by legal action in 2005, the outcome of which is still pending. The HSUS is encouraged that scientists at the US Centers for Disease Control and Prevention (CDC) have developed the Endopep-MS assay, a potential replacement for the mouse LD50 assay for assessing the potency of a wide range of botulinum toxin products.[6] The HSUS is in the process of nominating this and other potential alternatives to the US Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) for review and possible follow-up work.

Replacement alternatives

Bottrill listed a number of approaches which could partly or fully eliminate the need for in vivo testing, the most promising being molecular assays to determine the action of the toxin on its target molecule, SNAP-25 (synaptosomal-associated protein with a molecular mass of 25kDa). An immunoassay for detecting botulinum toxin Type A activity toward recombinant SNAP-25 substrates was shown to be more sensitive than the mouse assay, and a good correlation with results in the in vivo bioassay was demonstrated by the assay’s developers at the NIBSC, where the assay is now in routine use.[7]

It has now become possible to use the Endopep-MS assay to detect and differentiate a wider range of types of botulinum toxin (Types A, B, E and F), “by linking the highly specific activities of the toxins with target peptide specific for each toxin serotype. The product peptides derived from the activities of the toxins are detected by matrix-assisted laser-desorption ionisation of time-of-flight mass spectrometry (MS)”. This method can detect toxin equivalents of as little as 0.01 of the mouse LD50 dose. “An HPLC-tandem MS method for quantifying active toxin, where the amount of toxin can be correlated to the amount of product peptides”, is also available.[6]

FRAME and the HSUS urge the relevant players, including manufacturers, government departments and control authorities, including NIBSC, the European Centre for the Validation of Alternative Methods (ECVAM), the European Directorate for the Quality of Medicines (EDQM), CDC and ICCVAM, to cooperate to secure the rapid and independent validation of replacement assays such as the SNAP-25 and Endopep-MS assays, so that the use of painful animal procedures in botulinum toxin potency testing can be eliminated.

The role of advocacy

FRAME, the HSUS and other similar organisations engage in advocacy of the Three Rs because they want to accelerate the application of the Three Rs of Russell and Burch[8] - namely reduction, refinement and replacement - in all aspects of laboratory animal use in research, education and testing.

In the case of botulinum toxin testing, it is clear that some progress was being made prior to the launch of the FRAME and HSUS campaigns. However, FRAME and HSUS are confident that their activities have focused the attention of those with the power to bring about an end to LD50 testing and the use of other in vivo procedures.[9] They are convinced that the challenges to be faced in this, as in many other spheres, are best met by collaborative effort which is open to scrutiny rather than by unverifiable statements about what is claimed to be happening behind closed doors.

Sadly campaigns so far have aroused little interest in the media. This may be partly because of the perception that mice don’t matter very much, because of the complexity of the issues, or because manufacturers and governments find it convenient not to be able to define whether the batches of botulinum being tested will be used for therapeutic or cosmetic purposes. FRAME and HSUS remain positive and encouraged that substantial changes for the better may well be under way. Meanwhile, those who use botulinum toxin products for cosmetic purposes should be made fully aware of the real costs involved.

Source

Professor Michael BallsFRAME, UK Tel +44 115 9584740E-mail michael.balls@btopenworld.comDr Martin L StephensHSUS, USTel +1 301 258 3040E-mail mstephens@hsus.org

Refs

1. “Botox” is often used as a generic term for botulinum toxin products, but BOTOX is a registered trademark of Allergan Inc
2. Jost, W H & Kohl A, Journal of Neurology 248, suppl 1, 39-44 (2001)
3. Carruthers, J D A & Carruthers J A, Journal of Dermatological Surgery and Oncology 18, 17-21 (1992)
4. Bottrill K, ATLA 31, 381-91 (2003)
5. Balls M, ATLA 31, 611-5 (2003)
6. Barr J R, Moura H, Boyer A E et al, Emerging Infectious Diseases 11, 1578-83 (2005)
7. Ekong T A N, Feavers I M & Sesardic D, Microbiology 143, 3337-47 (1997)
8. Russell W M S & Burch R L, The Principles of Humane Experimental Technique, Methuen, London (1959)
9. Stephens M L & Balls M, ALTEX, in press (2006)