First draft prepared by P.J. Abbott

    Food Science and Safety Section, National Food Authority, Canberra,


    Biological data





         Alitame was considered by the Committee at its forty-fourth
    meeting (Annex 1, reference 116). At that meeting, the Committee
    evaluated a large number of studies of toxicity, and specifications
    and a toxicological monograph were prepared. The Committee concluded,
    however, that an ADI could not be allocated because of concerns about
    the deficiencies of the studies of carcinogenicity. Specifically, in a
    study with Long-Evans rats, poor survival at 24 months and an
    increased incidence of hepatocellular adenomas in females at the
    highest dose were noted. While it was considered unlikely that the
    adenomas would have progressed to carcinomas, the data were
    inconclusive in this regard. In a study with Sprague-Dawley rats, low
    survival rates were seen at 22 and 24 months, and the study was
    considered inadequate for drawing conclusions about the potential
    carcinogenicity of alitame.

         At its present meeting, the Committee reconsidered the adequacy
    of the carcinogenicity studies in rats in the light of general
    considerations about the survival of animals in contemporary long-term
    studies (Annex 1, reference 122) and on the basis of a further
    statistical analysis of the data from the two studies, which
    specifically assessed the power of the study in Sprague-Dawley rats to
    detect hepatic lesions.

    2.  BIOLOGICAL DATA: Carcinogenicity

         Two two-year studies were performed in rats with alitame, one in
    Long-Evans rats and the other in the Sprague-Dawley strain. The
    details of the design of these studies were described previously
    (Annex 1, reference 117). Further information has been provided that
    allows comparison of the two studies (Pfizer, 1996).

         Table 1 shows the numbers of surviving animals in the two studies
    at 18, 22, and 24 months. The initial numbers of animals were 50 per
    group in the study with Long-Evans rats and 70 per group in the study
    with Sprague-Dawley rats. At 24 months, the number of surviving
    Sprague-Dawley rats was only slightly smaller than that of Long-Evans
    rats, despite the difference in survival rates.

         In the original report, data on hepatic lesions were analysed by
    the statistical model of Peto  et al. (1980). In the more recent
    report, the data on the incidence of hepatocellular adenomas and
    eosinophilic foci in the study on Long-Evans rats were re-examined
    using the model of Dinse & Haseman (1986), which allows estimation of
    the predictive importance of time on study and dose. The model
    developed for the data from the study of Long-Evans rats was used to
    analyse the survival data for the Sprague-Dawley rats, and the 

    Table 1.  Numbers of surviving animals in studies with Long-Evans and
              Sprague-Dawley rats

    Sex       Dose          At 18 months    At 22 months    At 24 months
                             LE      SD      LE      SD      LE      SD

    Male      High           35      49      19      24      14      18
              Mid            41      44      23      11      15       6
              Low            35      43      23      15      17       7
              Controlsa      37      85      24      24      20      10

    Female    High           42      51      25      27      17      12
              Mid            45      49      25      21      21      17
              Low            40      56      28      32      20      22
              Controlsa      46     111      32      60      23      42

    LE, Long-Evans; SD, Sprague-Dawley
    a  Average of two control groups in the study of Sprague-Dawley rats

    probability of developing an adenoma or eosinophilic foci was
    determined. This predicted incidence was then compared with the
    observed incidences. The model predictions and the observed outcomes
    are shown in Table 2.

         The author concluded that (i) the study with Sprague-Dawley rats
    was of adequate length to assess the incidence of hepatic lesions, and
    (ii) the process of hepatocellular adenoma formation in Long-Evans
    rats is specific to that strain.


         The Committee noted that while the survival of Long-Evans rats at
    24 months was low, it was comparable to that seen in contemporary
    studies using this strain, and no hepatocellular carcinomas were seen.
    It also noted that the survival of Sprague-Dawley rats was comparable
    to that in contemporary studies in rats of this strain and that
    because there were more animals per group initially (70) than in the
    study using Long-Evans rats (50), the number of surviving animals was
    comparable between the two strains. On the basis of a logistic
    regression model developed using data from the study with Long-Evans
    rats, an increased probability of developing hepatocellular adenomas
    and eosinophilic foci would have been expected in the Sprague-Dawley
    rats, given the doses and time on study, but no increase was observed.
    On the basis of these considerations the two studies were considered
    adequate to assess carcinogenicity.

    Table 2.  Observed and predicted incidences of hepatocellular adenomas
              and eosinophilic foci in studies of Long-Evans and
              Sprague-Dawley rats

    Lesion              Strain           Dose      Observed   Predicted

    Hepatocellular      Long-Evans       Control        0        0.32
    adenoma                              Low            0        0.43
                                         High          12       12.25
                        Sprague-Dawley   Controla       0        0.60
                                         Low            0        0.49
                                         High           0       12.72
    Eosinophilic foci   Long-Evans       Control        2        3.98
                                         Low            3        4.33
                                         Mid           11        6.47
                                         High          18       19.23
                        Sprague-Dawley   Controla       8        8.22
                                         Low            4        5.41
                                         Mid            4        6.43
                                         High           5       22.13

    a  Average of two control groups


         The Committee concluded that there was no evidence that alitame
    is carcinogenic. The Committee therefore allocated an ADI of 0-1 mg/kg
    bw, on the basis of the NOAEL of 100 mg/kg bw per day identified at
    the forty-fourth meeting in the 18-month study in dogs, using a safety
    factor of 100.

         The Committee noted that, although not specifically requested, a
    further study of tolerance to repeated doses of alitame in diabetic
    subjects is under way, and, as is customary, the results of this study
    should be forwarded to the Committee when available.


    Dinse, G.E. & Haseman, J.K. (1986) Logistic regression analysis of
    incidental tumor data from animal carcinogenicity experiments.
     Fundam. Appl. Toxicol., 6, 44-52.

    Peto, R., Pike, M.C., Day, N.E., Gray, R.G., Lee, P.N., Parish, S.,
    Peto, J., Richards, S. & Wahrendorf, J. (1980) Guidelines for simple,
    sensitive significance tests for carcinogenic effects in long-term
    animal experiments. In:  IARC Monographs on the Evaluation of the
     Carcinogenic Risk of Chemicals to Humans, Suppl. 2,  Long-term and
     Short-term Screening Assays for Carcinogens: A Critical Appraisal,
    Lyon, International Agency for Research on Cancer, pp. 311-426.

    Pfizer (1996) Alitame 2 year chronic studies with  in-utero exposure
    in Long-Evans and Sprague-Dawley rats: Comparative discussion of
    findings related to survival. Unpublished report submitted to WHO by
    Central Research Division, Pfizer Inc., Groton, Connecticut, USA.

    See Also:
       Toxicological Abbreviations
       Alitame (JECFA Food Additives Series 50)
       Alitame (WHO Food Additives Series 35)
       ALITAME (JECFA Evaluation)