Introduction

1. The proper conduct of carcinogenicity studies in rats is an important part of the evaluation and prediction of potential human carcinogens. Significant reductions in the number of control rats surviving to termination have been widely reported in the scientific literature.(1-5) This is a matter of concern since inadequate carcinogenicity studies could be important in decisions regarding the identification of potential human carcinogens and in particular the failure to identify such compounds. In addition there is a possibility that inadequate studies could be rejected by regulatory agencies with the consequent need for use of further animals to obtain a valid result. For a negative result from a rat carcinogenicity bioassay to be considered acceptable, survival at 24 months should be 50% or greater in all groups.(6,7)

2. The Committee reviewed the evidence for application of dietary restriction techniques in 2000 and a statement was published (COC/00/S3). It was concluded that the available information supports the view reached by the COC in its guidelines published in 1991 that dietary restriction in carcinogenicity studies should be applied with caution and is the responsibility of the toxicologist undertaking the study. The COC agreed that the subject of dietary restriction should be reviewed when more information is available.

3. Some investigators have also proposed that reducing the duration of carcinogenicity bioassays undertaken in rats would have the desired effect of terminating studies before survival was reduced to below 50% in tests groups.(8) The Committee reviewed two published papers which had evaluated published data on the duration of carcinogenicity studies. These two papers (see paras 4 and 5 below) came to contrasting views. The Committee was asked to provide generic advice on the desirability of reducing he minimum duration of carcinogenicity studies in rats.

Review of two selected investigations

Davis et al, 2000(8)

4. Davis et al studied IARC chemical Monographs (Vols 1-70) to determine the time of onset to 'treatment-related' tumorigenicity in long-term rodent studies for chemicals classified by the IARC as showing evidence of carcinogenicity in animals. The chemicals were categorised as producing tumours at <12m, 12-18m, or >18m. The analysis excluded studies on metals and their salts, studies on particulates, studies by parental routes of administration that resulted in tumours only at the site of exposure, and studies that did not approximate to the current standard long term rodent carcinogenicity bioassay e.g. transplacental or multigeneration studies, initiator-promoter studies, lung tumour assays in 'Strain A' mice and studies in new born animals. Davis et al considered that from a total of 210 chemicals, overall, evidence of treatment related tumorigenicity was first apparent within 12 months for 66% of the chemicals and that studies longer than 18 months were necessary for 7%. All IARC Group 1 chemicals were detected in animals within 18 months and most within 12 months. Most of the tumour types that required more than 18 months for detection were considered by Davis et al to be of "dubious" relevance to human risk assessment On this basis Davis et al concluded that termination of rodent carcinogenicity studies at 18 months or earlier was justified, and would greatly reduce the complications that arise in interpreting findings in aged animals.

Kodell et al, 2000(9)

5 Data from bioassay studies in rats using selected pharmaceuticals were used to formulate biologically based dose-response models of carcinogenesis based on the 2-stage clonal expansion model. These dose response models, which were chosen to represent 6 variations of the initiation-promotion-completion cancer model were employed to generate a large number of representative bioassay data sets using Monte Carlo simulations. The six variations of the model were based on data:

For a variety of tumour dose-response trends, tumour lethality and competing risk-survival rates, the power of age-adjusted statistical tests to assess the significance of carcinogenic potential was evaluated at 18 and 21 months and compared to the power at the normal 24 month termination time. Kodell et al results showed that termination at 18 months would reduce statistical power to an unacceptable level for all 6 variations of the 2-stage clonal expansion model, with the pure-completer models being most adversely affected.

COC Discussion

6. The committee agreed that some rat strains, namely, Sprague-Dawley (in certain labs) have inadequate survival at 24 months. Members noted the argument put forward by Davis et al that the pathology associated with old age might mask important cancer pathology in animals terminated at 24 months. Davis et al had also argued that it is possible that an earlier onset of the incidence of a common spontaneous tumour type could be detected at 18 months and missed at 24 months. However, members considered that in 24-month studies, autopsy of the dead animals and analysis of tumour incidence in deceedents would pick this up.

7. The committee considered that a single study would not be looked at in isolation and that consideration of the mechanism of an effect was crucial in the overall evaluation. Members were also concerned about modifying an already imperfect lifetime model, and agreed that possible dietary methods of extending life span, such as by caloric restriction, needed to be considered on a case-by-case basis with regard to laboratory historical control data on tumour incidence.

8. The committee did not agree with the conclusions drawn by Davis et al that carcinogens detected after 18 months were unlikely to be relevant to human health assessment. Members were concerned that such shortened studies might not be sufficiently sensitive to detect some human carcinogens. The Committee agreed that the approach taken by Kodell et al to the modelling of carcinogen dose-response was satisfactory.

COC Conclusion

9. The COC concluded that there was insufficient evidence to recommend a change to the international guidelines for the conduct of long term carcinogenicity bioassays, that for a negative result to be acceptable in a rat carcinogenicity bioassay, survival should be at least 50% in all groups at 24 months. The Committee reaffirmed that it was the responsibility of the study director to use rat strains that would ensure adequate survival at 24 months.

February 2002

References

1. Allaben WT, Turturro A, Leakey JEA, Seng JE and Hart RW (1996). FDA Points to consider Documents: The need for dietary control for reduction of experimental variability within animal assays and the use of dietary restriction to achieve dietary control. Toxicologic Pathology, 24, 776-781.

2. Roe FJC, Lee PN, Conybeare G, Kelly D, Matter B, Prentice D and Tobin G (1995). The Biosure study: Influence of composition of diet and food consumption on longevity, degenerative diseases and neoplasia in Wistar rats studied for up to 30 months post weaning. Fd Chem Tox, 33, suppl 1, 1S-100S.

3. Keenan KP et al (1996). The effects of diet, ad-libitum overfeeding, and moderate dietary restriction on the rodent bioassay: The uncontrolled variable in safety assessment. Toxicologic pathology, 24 (6), 757-768.

4. Haseman JK (1995). Statistical considerations in long-term dietary restriction studies. In Dietary Restriction. Implications for the design and interpretation of toxicity and carcinogenicity studies. Edited Hart R, Neumann DA and Robertson RT. Published ILSI Press, Washington DC, U.S.A., pp141-153.

5. Keenan KP, Ballam GC, Soper KA, Laroque P, Coleman JB and Dixit R (1999). Diet, caloric restriction and the rodent bioassay. Toxicological Science, 52, (supplement), 24-34.

6. OECD (1981a) Guideline 451. Carcinogenicity studies (17 pages; adopted 12 May 1981). In: OECD Guidelines for the testing of chemicals (1993) Section 4: Health effects. Vol. 2. Paris, Organisation for Economic Cooperation & Development.

7. OECD (1981c) Guideline 453. Combined chronic toxicity/carcinogenicity studies (15 pages; adopted 12 May 1981). In: OECD Guidelines for the testing of chemicals (1993) Section 4: Health effects. Vol. 2. Paris, Organisation for Economic Cooperation & Development.

8. Davis TS, Lynch BS, Monro AM, Munro IC, Nestman ER (2000) Rodent carcinogenicity Tests Need be no longer than 18 months: An analysis based on 210 chemicals in the IARC monographs. Food and Chemical Toxicology 38 (2000) 219-235.

9. Kodell RL, Lin KK, Thorn BT, Chen JJ (2000). Bioassays of shortened duration for drugs: Statistical Implications. Toxicological sciences 55, 415-432.

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