The Center for Alternatives to Animal Testing is an academic center affiliated with the Division of Toxicological Sciences in the Department of Environmental Health Sciences of the Johns Hopkins University Bloomberg School of Public Health.

 

Johns Hopkins School of Public Health

Proceedings for CAAT 20th Anniversary Symposium

Decreasing our Dependence on Animals: Opportunities for Harmonisation?

Herman B.W.M. Koëter
Organization for Economic Cooperation and Development

Concern about animal welfare in the life sciences has a history as long as that of the life sciences themselves. However, for many years any consideration of animal welfare issues was judged by leading experts as incompatible with progress in science and, consequently, dismissed or, at best, put on the back burner. Even today, when the reduction, replacement and refinement of animal studies have become fashionable, politically correct and widely accepted research considerations, the 3Rs often are perceived as a nuisance and an impediment to progress. Approaches to toxicity testing which place an emphasis on animal welfare considerations are viewed by many as less safe and less reliable.

It is interesting to see how "alternatives" research has developed over the years. Publications from the late 1970s and early 1980s were written, almost without exception, in the same format and approach as any article on an animal study (1), i.e. starting with the introduction followed by method description, results of the test with a series of chemicals (usually between 10 and 50), discussion and conclusions. Surprisingly, whereas this format historically was considered sufficient for any animal study, it did not work for alternative approaches. Alternatives were generally considered to lack the "fidelity" of an animal model and could, at most, provide a "high discrimination power." Although the assumed high fidelity of animal models themselves had been disputed and considered a fallacy by Rusell and Burch as early as 1959 (2), alternatives were viewed nonetheless as less safe. Thus, it was argued, their reliability and relevance should be proven before they could be used. With that notion the modern concept of validation was born! The subsequent complaint of the animal welfare community that validation was just another hurdle erected to avoid the regulatory use of an alternative quickly faded and, instead, respected (inter)national centers for alternatives to animal testing started to develop comprehensive and detailed validation processes (3)(4)(5). At an OECD Workshop in January 1996, the essential elements of these various concepts were summarised in a harmonised set of principles for a valid test and criteria for regulatory acceptance (6). An essential element in the campaign for validation is that formal validation should not be limited to alternative (non-animal) methods but should apply to all new and revised test methods.

Although the internationally agreed-upon principles and criteria for validation and acceptance are undisputed and should indeed apply to all new and substantially revised test methods, the specific requirements of the various approaches often are difficult if not impossible to meet. As a consequence, the discussion of whether or not every new test should be subjected to exactly the same rigid validation procedure may continue, thus wasting costly time and energy of people who should, in fact, be leading the way towards a truly substantial reduction of use and suffering of animals. Alternatively, one could agree on a pragmatic, yet transparent approach based on two principles. The first principle, which is ready for immediate implementation, is that combining (available) alternative approaches with a limited number of "traditional" animal tests is making best use of both types of tests. The application of testing strategies combining animal with non-animal tests would lead to a substantial reduction in animal use without the need for exhaustive validation work. The second principle focuses on long-term goals: Investing in a fundamental understanding of mechanisms of (toxic) action as the basis for new test methods will provide better science and will result in more reliable hazard and risk assessments with fewer or no animals (7). It is so much better to replace the "old" jigsaw puzzle with a new one, showing a brighter and more beautiful picture, than to patch up the old one, replacing a few pieces here and there (8). Development of methodology that provides an insight into mechanisms of action, of course, needs to be followed by a critical scientific appraisal but, as "gold standards" are lacking, the approach will have to be tailor-made.

After a general introduction, the presentation will focus on approaches for harmonised testing and assessment strategies for acute/local effects of toxicity, such as skin and eye effects, as well as for more complicated systemic toxicity such as reproductive toxicity.

References

  1. Goldberg, A.M. (Editor) (1983) Alternative Methods in Toxicology I: Product Safety Evaluation. Mary Ann Liebert, Inc., New York, New York.
  2. Russell, W.M.S. and Burch, R.L. (1959) The Principles of Humane Experimental Technique. Methuen, London.
  3. Balls, M. et al. (1995) ECVAM Workshop Report No.5: Practical Aspects of the Validation of Toxicity Test Procedures, ATLA 23 129-147.
  4. Goldberg, A.M.et al. (1993) Framework for Validation and Implementation of In Vitro Toxicity Tests: Report of the Validation and Technology Transfer Committee of the Johns Hopkins Center for Alternatives to Animal Testing. J.Am.Coll.Tox. 12:23-30.
  5. NIEHS (1997) Validation and Regulatory Acceptance of Toxicological Test Methods: A Report of the Ad hoc Interagency Co-ordinating Committee on the Validation of Alternative Methods. NIH Publication No.97-3981 [link].
  6. OECD (1996) Final Report of the OECD Workshop on Harmonisation of Validation and Acceptance Criteria for Alternative Toxicological Test Methods. OECD Document ENV/MC/CHEM/TG(96)9 [http://www.oecd.org/ehs/test/background.htm].
  7. Frazier, J.M. (1990) Scientific Criteria for Validation of In Vitro Toxicity Tests. OECD Environment Monograph No.36.
  8. Koëter, H.B.W.M. (1991) Current Guidelines and Regulations. In: Animals in Biomedical Research: Present Possibilities and Future Prospects. Eds. Hendriksen, C.F.M. and Koëter, H.B.W.M., pp 35-48. Elsevier, Amsterdam.

1The opinions presented in this paper do not necessarily represent the opinions of the OECD or its Member countries and should therefore be viewed as those of the author.

Estimated Numbers of Chemicals. (EPA estimations, 1995)

How Many Animals in Regulatory Risk Assessment?

How Many Animals in Regulatory Risk Assessment?

OECD and Animal Welfare

Objectives (1):

Objectives (2):

Test Guideline Adopted or draft Animal welfare relevance
Number Title
TG 401 Acute Oral Toxicity 1987 Smaller number of animals: lowering of the limit dose level
TG 402 Acute Dermal Toxicity 1987 Smaller number of animals: lowering of the limit dose level
TG 404 Dermal Irritation/Corrosion 1992 Allowing in vitro methods for corrosivity; recommend to start with one animal
TG 404 Dermal Irritation/Corrosion Draft Adoption of a comprehensive testing strategy, including in vitro screens
--- In vitro Skin Corrosivity 1987 True alternative for in vitro test for skin corrosion
TG 405 Acute Eye Irritation/Corrosion 1987 Allowing in vitro methods for corrosivity; recommend to start with one animal
TG 405 Acute Eye Irritation/Corrosion Draft Adoption of a comprehensive testing strategy, including in vitro screens
TG 406 Skin Sensitization 1992 Reduction of the number of methods allowed; reduction of animal use by 50%
--- In Vitro Phototoxicity Draft First non-animal test for an endpoint not yet covered
TG 407 Repeated Dose Oral Toxicity 1995 Refinement: more information on best dosing practice, more information from the same animal
TG 414 Developmental Toxicity 2001 Smaller number of animals (20% reduction), more information from the same animal
TG 420 Fixed-Dose Procedure (Acute Oral Toxicity) 1992 Alternative animal test to the conventional TG 401. Less suffering, smaller number of animals
TG 420 Fixed-Dose Procedure Draft Further reduction of animals compared to the 1992 version
TG 422 Reproduction/Developmental Toxicity Screening Test 1995 New screening test provides essential information with a minimum number of animals
TG 422 Combined Test of 407 and 421 1995 Combines the new screening test on reproduction toxicity with TG 407 and further reduces the number of animals to an absolute minimum for these combined endpoints
TG 423 Acute Toxic Class Method (Acute Oral Toxicity) 1996 Alternative animal test to the conventional TG 401. Much smaller number of animals (10% of TG 401)
TG 423 Acute Toxic Class Method (Acute Oral Toxicity) Draft Further reduction of animals compared to 1996 version
TG 425 Up-and Down Procedure (Acute Oral Toxicity) 1998 Alternative animal test to the conventional TG 401. Smaller number of animals, provides closer estimate of the LD50 than 420, 423
TG 425 Up-and Down Procedure (Acute Oral Toxicity) Draft Further reduction of animals compared to the 1996 version, better accuracy
TG 428 Skin Absorption In Vitro Method Draft True Alternative to the in vivo method
TG 429 Local Lymph Node Assay (LLNA) Draft Alternative animal method to TG 406, allowing more information and less suffering
TG 471 Bacterial Reverse Mutation Test 1997 In vitro test for point mutations (genetic toxicity)
TG 473 In Vitro Chromosome Aberration Test 1997 True in vitro alternative to the in vivo test
TG 474 Mammalian Micronucleus Test 1997 Allowing a smaller number of animals compared to 1983 version
TG 476 In Vitro Gene Mutation Test 1997 In vitro test for gene mutations (genetic toxicity)
TG 479 In Vitro Sister Chromatid Exchange 1986 In vitro test for DNA exchanges between sister chromatids (genetic toxicity)
TG 480 In Vitro Gene Mutation Assay 1986 In vitro test for gene mutations in Saccharomyces (genetic toxicity)
TG 481 In Vitro Mitotic Recombination Assay 1986 In vitro test for mitotic recombination in Saccharomyces (genetic toxicity)
TG 482 In Vitro DNA Damage and Repair 1986 True alternative to the in vivo method for DNA damage

Objectives (3):

Objectives (4):

The AWA -- 1985 amendments

A lot has been achieved, a lot is going on, but...

the bare truth:

Some personal thoughts, observations...

Development of harmonised test methods

Is there a way forward?

Testing/Assessment Approaches

Weight of Evidence Approach

Figure 1

Sequential Testing Strategy

Sequential Testing Strategy. Level of detail:

I. Endpoint/Hazard Specific, e.g.:

Sequential Testing Strategy for Endpoint/Hazard Specific Assessments

Sequential Testing Strategy for Endpoint/Hazard Specific Assessments

27th Joint Meeting decided:

"Testing strategies should be published in the same format and colour as the guidelines they refer to and as additions to these guidelines. Publication of testing strategies alongside the appropriate guidelines would maximise their visibility and status without being part of the MAD."

Skin and Eye Effects (TG 404,405)

Testing and Evaluation Strategy for Eye Irritation/Corrosion

Figure 2a
Figure 2b
Figure 2c

Sequential Testing Strategy - Level of detail:

II. Multi-Endpoint/Hazard Assessments, e.g.:

Sequential Testing Strategy for Multi-Endpoint Assessment

Reproduction Toxicity (1)

Reproduction Toxicity (2)

Figure 3

Neurotoxicity

Figure 4

OECD Policy:

Figure 5

Figure 6

Figure 7