Clinical Observations and Mortality

The proper conduct of carcinogenicity studies is an important part in the evaluation and prediction of potential human carcinogens. Significant reductions in the number of animals surviving to termination have been widely reported in the scientific literature. This is a matter of concern since inadequate carcinogenicity studies could result in studies being rejected, and hence large numbers of animals being needlessly used, or in the failure to identify potential human carcinogens. 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 [73, 74, 75, 79]

In our studies the mortality of animals were checked twice a day during the treatment period, and the data were recorded in the appropriate record sheet. The significance of survival was estimated by Log Rank, Breslow and Tarone Ware methods.[52]

The following table shows the survival data and the results of statistical analysis Table 8 Survival of Rats in the 2-Year Feeding Studies

Male Normal Diet Low Energy Diet

Animals initially in study 50 50

Moribund and death animals: 22 17

Survival: 28 33

Mean survival (days): 639.7 661.,9

Statistical evaluation NS NS

Female Normal Diet Low Energy Diet

Animals initially in study 50 50

Moribund and death animals: 23 26*

Survival: 27 24

Mean survival (days): 653.4 658,9

Statistical evaluation NS NS

Remarks:

* = The last animal died during the necropsy period, so it was fully evaluated.

NS = Not significant

No statistically significant difference of survival of male and female rats fed with different diets occurred during the comparisons, but important observations were taken. The details of lethality and statistical analysis see in Appendix 1.

Considering the results obtained in the studies, the mean survival of male and female groups fed with Low Energy Diet was longer than the groups fed with Normal Diet. In male groups the difference was more obvious. Less male animal died during the 2-year period fed with Low Energy Diet, the survival was 66% in Low Energy Diet group, and 56% ind the Normal Diet group. It is very important, since usually the survival problems occur in male rats. In females, in spite of the higher number of animals died during the study the mean survival was longer in the Low Energy Diet group than in the Normal Diet group. Overall we can state, that the gross of death appeared later in the Low Energy Diet group, than in the Normal Diet Group.

The survival graphs of male and female rats see in Figure 7 and 8.

Figure 7 Survival Graph of Male Rats Fed with Different Diets

20 25 30 35 40 45 50 55

0 31 60 91 121 152 182 213 244 274 305 335 366 397 425 456 486 517 547 578 609 639 670 700

Days

Surviving

Normal Diet Low energy

Figure 8 Survival Graph of Female Rats Fed with Different Diets

The most frequent clinical signs were the followings:

Table 9 Percentage Distribution of Clinical Signs Observed during the 2-year Period

Symptoms / Percent of

Animals Male Normal

Diet Male Low

Energy Diet Female

Normal Diet Female Low Energy Diet

The observed clinical signs are mostly in connection with aging of animals and with tumour development. The total number of clinical symptoms were lower in the male animals in the Low Energy Diet group than in the Normal Diet group.

The general health state of animals was better in the Low Energy Diet group.

20

4.2. Body Weight

Body weight was weighed weekly during the first 12 weeks of the study and once every four weeks thereafter with precision of 1 g.

Evaluating the mean body weight of the groups, significant difference occurred in the first year between the two male and the two female groups fed with different diet. The difference between the groups reached the 10 percent limit in the eighth week in both sexes. The animals fed with normal diet gained much faster, showed almost exponential body weight gain. The difference exceeded the twenty percent to the 40th week in males and to the 36th week in females. The difference increased slightly to the end of the first year. After one year the animals were housed individually in the first study, and the differences in the mean body weight showed slow decrease from the 60th week in males and 64th week in the female groups. After this change in the housing of animals, the mean body weight of male groups became slowly similar, the difference went below the 10 percent to the 68th week. This change of body weight gain may be in connection with the individual housing of the animals.

In females the difference between the mean body weights of two groups fed by different diet decreased below 10 percent to the 72nd week.

At the end of the two-year period the mean body weight of the males fed with normal diet was higher with 6%, than the mean body weight of the males fed with low energy diet. In females the mean body weight of the rats fed with low energy diet was higher with 5 %, than the mean body weight of females fed with normal diet on week 104.

The observed changes of body weight gain were not in close correlation with the food consumption of animals.

See Figures 9, 10 and Appendix 2.

Figure 9 Mean Body Weight of Male Rats Fed with Different Diet during 2 Years

Figure 10 Mean Body Weight of Female Rats Fed with Different Diet during 2 Years

MEAN BODY WEIGHT OF MALE ANIMALS

0.0

MEAN BODY WEIGHT OF FEMALE ANIMALS

0.0

4.3. Food Consumption

The quantity of the food consumed by the animals of each cage was weighed weekly during the first 13 weeks of study and biweekly thereafter with precision of 1 g. In the first study the animals were housed individually after the end of the first year, in the second study two animals were kept in one cage during the whole observation period.

In male rats the food consumption of animals fed with Low Energy Diet was tendentiously and significantly higher, than the food consumption of animals fed with Normal Diet. However, the initial body weight gain was much higher in the animals fed with Normal Diet. The expected effect of the energy restriction of the diet on the body weight gain was observed.

In female animals similar tendency occurred that in male rats except for the period between weeks 28 and 46, when the food consumption of animals fed with Normal diet was higher significantly (reached the 25% difference) than the food consumption of animals fed with Low Energy Diet. After this period the food consumption of animals fed with Low energy diet increased significantly, it was in some cases more than 70% higher, than the food consumption of animals fed with Normal Diet. In correlation with it the mean body weight of animals fed with normal diet was higher during the major part of the observation period, but the terminal mean body weight of the female animals fed with Low Energy Diet was higher than the mean body weight of females fed with Normal Diet. The effect of changing the housing occurred in the food consumption of animals, too.

The results see in Figures 11, 12 and in Appendix 3

Figure 5 Mean Food Consumption of Male Rats in 2-year Period Fed with Different Diet.

Figure 12 Mean Food Consumption of Female Rats in 2-year Period Fed with Different Diet.

FOOD CONSUMPTION OF FEMALE RATS (g/animal/day)

0,00 FOOD CONSUMPTION OF MALE RATS (g/animal/day)

0,00

4.4. Laboratory Examinations

4.4.1 Haematology

In the first study, when the animals were fed with normal diet, only limited data set is available. Blood smear examinations were performed on the basis of the OECD Guideline for Testing of Chemicals [75]

On the basis of the available haematological data similar tendencies are observed in both groups, the feeding with different energy content diet did not influence significantly the haematological parameters. As aging signs, the rate of segmented cells showed continuous increase with the age of animals, the rate of lymphocytes decreased with the age in similar extent in both sexes. These are normal physiological changes.[65, 70, 72].

In second study detailed haematological examinations were carried out.

Significant increase of WBC count was observed in both sexes compared the 24-month values to the base level. The cause of this increase can be explained by the tumourous state of some of the old animals.