The pesticides and other chemicals used during the agricultural production mean hazard either for the plants or animals, and through the food-chain, or during the work for the peoples. So the stipulation of use and circulation of these chemicals is authority registration.
The determination of the risk or hazard represented by the chemical product is performed by the toxicology, as interdisciplinar science.
The potential carcinogenic effect of the chemical product during a long-term, even life-span repeated exposition can be estimated on the basis of the carcinogenicity study.
This assay summarize the observations, development and incidence of spontaneous tumours of the control groups of two carcinogenicity studies in Crl:CD Br rats performed under similar conditions, in the same laboratory and by the same staff. The only difference between the two studies was the composition of the diet. The control group in the first study received „normal” diet, the control group of the second study was fed with „low energy” diet. Our results can be used as historical control data during the evaluation of carcinogenicity studies performed in the investigated rat strain, and reveal the effect of dietary energy intake on tumour development in carcinogenicity studies.
The presence and evaluation of the historical control data are also very important during the evaluation of the carcinogenicity study in dividing the spontaneous and the treatment-related tumours and expressing an opinion about the test item, which is the basis of the risk assessment during the registration procedure and releasing for trading.
During the 2-years studies in the groups evaluated in this assay detailed clinical observations, body weight measurements, food consumption measurements, clinical pathological examinations were performed. The moribunds and animals found dead were dissected as soon as it was possible after the death. At termination necropsy was performed on all surviving animal, the weights of selected organs were measured and the organs and tissues were fixed in formaldehyde solution. Total histological processing was performed on all animals and about 10 000 slides were evaluated by two independent
histopathologists.
In the first study the animals received SSNIFF R/M-Z+H 15 MM AUTOKLAVABLE COMPLET DIET FOR RATS AND MICE - BREEDING + MAINTENANCE. In the assay this diet was called as Normal Diet.
In the second study the animals were fed with SSNIFF R/M-H, ERED LOW ENERGY COMPLETE DIET FOR RATS AND MICE. In the assay this diet was called as Low Energy Diet.
Both diets were produced by Ssniff Spezialdiäten GmbH, D-59494 Soest Germany. The food were offered ad libitum.
The data collected during the 2-year observation periods of male and female test animals fed with Normal Diet were compared to the data of animals received Low Energy Diet.
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. The survival was 66% in males of Low Energy Diet group, and 56%
in the Normal Diet group.
In females, in spite of the higher number of animals died during the study the mean survival in days was longer in the Low Energy Diet group than in the Normal Diet group.
The observed clinical signs are in connection with aging of animals and with tumour development. The total number of clinical symptoms was lower 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.
The mean body weight of the male and female animals fed with Normal Diet were significantly higher the mean weight of test animals fed with Low Energy Diet in the major part of the observation period. During this period the food consumption of male and female animals was significantly higher in the Low Energy Diet group, thain in the Normal Diet Group, except for the females, where this tendency changed between weeks 28 and 48.
On the basis of available haematological parameters the feeding with different energy content diet did not influence significantly the haematological parameters, the rate of segmented cells showed continuous increase with the age of animals, the rate of segmented cells increased, the rate of lymphocytes decreased with the age in similar extent in both sexes. These are normal physiological changes.
With macroscopic examination alterations of tumour-origin (masses, foci, enlargements, etc.) could be observed in various organs of the animals of both groups. They showed a relatively higher incidence in the hypophysis and skin of the males. In the males hypophysis tumours could be detected most frequently in both groups, and the historical control data showed, that this type of tumour is typical to this strain. Tumour under the skin occurred in all groups too, but with lower incidence. Tumours were observed in different groups with low frequency in the thymus, spinal column, omentum, kidneys, testes, prostate, abdominal lymph node, liver, mammary gland, Tumour-like alterations (enlargement, focus) occurred in the thyroid, salivary gland, lungs, adrenals, kidneys, spleen and in different lymph nodes as well.
In the female animals tumour-like alterations occurred in the hypophysis, brain, lung, liver, adrenal gland, kidneys, in the genital organs, in the skin and mammary glands. The tumour-like alterations occurred sporadically with low incidence in each group.
Other tumours caused moribund state or death sporadically with very low frequency. The shock was indicated as cause of death with increased frequency in male animals, as well.
With the macroscopic examination several non-neoplastic alterations were observed in the different organs of the experimental animals in both sexes and in both groups fed with different energy content diet.
In the males dark-red, plethoric lungs, pulmonary oedema, heart dilatation, hyperaemic liver, pale liver, pale kidneys, atrophy of genitals, enlargement of testes and the thoracic cavity filled with serous liquid were observed
In the females pale liver, enlargement of spleen and adrenals were observed. The plethoric lungs and the pulmonary oedema occurred in less case than in the males.
Most of the non-neoplastic alterations were likely in connection with extermination and agony, or parts of them were age-related, or sporadic, toxicologically meaningless findings (congestion, discoloration, cysts, abscess, etc.).
The available organ weight data showed that the absolute and relative liver weights in male and female animals fed with Low Energy Diet were higher than this parameter of the Normal Diet group. The differences were more obvious in the male animals.
In this study - according to the historical data - hypophysis adenomas were the most common tumours in both groups in both sexes.
The malignant tumours which caused death of animals were the sarcoma polymorphocellulare, fibrosarcoma, lymphoma malignum, adenocarcinoma, and hepatocellular carcinoma, which were infrequent in both males and females.
Undifferentiated sarcoma in the mammary gland, heart, vagina, undifferentiated carcinoma in the skin, vagina or the abdominal cavity, myxosarcoma in the skin, uterus or in the abdominal cavity, follicular carcinoma in the thyroid gland, plexus carcinoma and astrocytoma in the brain, osteosarcoma in the bone, carcinoma planocellulare in the eye were detectable only sporadically.
The most common benign tumour was in this study the mammary gland adenoma in the female animals. The incidence of this tumour was higher in the Low Energy Diet group than in the Normal Diet group.
The other types of benign neoplasms (fibroma in the mammary gland, skin, thoracic or abdominal cavity, myxoma, fibro-adenoma, adeno-papilloma in the skin, adenoma in the kidney, adrenal gland, thyroid gland, parathyroid gland, salivary gland, pancreas, uterus, lipoma in the wall of stomach, thymoma in the thymus, pheochromocytoma in the adrenal gland, myxoma, fibro-papilloma, leiomyoma in the uterus, haemangioma in the spleen and lymph node) had generally low frequency and had the same incidence in both groups.
The non neoplastic hyperplastic lesions (hyperplastic nodules and bile duct hyperplasia in the liver, C-cell hyperplasia in the thyroid gland, islet-cell hyperplasia in the pancreas, cystic hyperplasia in the seminal vesicle) Occurred in low frequency in both groups in both sexes.
Hyperplastic nodules in the liver were rare and occurred in both groups and in both sexes. Bile duct hyperplasia is a common aging lesion in control rats often accompanied by perilobulary fibrosis.
The C-cell hyperplasia in the thyroid gland the cystic hyperplasia in the seminal vesicle and the islet-cell hyperplasia in the pancreas were detectable only sporadically.
Considering the tumour incidences obtained by histopathological examinations the total number of tumours were slightly higher in male and female animals fed with Low Energy Diet, than in the male and female groups fed with Normal Diet.
It can be explained with late onset of mortality and longer survival of animals fed with Low Energy Diet.
Summarising the results of our evaluations, the advantage of the feeding the experimental animals in the long term and carcinogenicity studies with Low Energy Diet occurred in the better survivals, mainly in male animals, in the better general health state of animals. The body weight gain of animals fed with Low energy diet was balanced in spite of the higher food consumptions. The higher food consumption can help to reach higher dose levels in the feeding studies. The feeding with the Low Energy Diet did not influence the measured haematological parameters, but the liver weights were higher than in the Normal Diet groups.
The total number of tumours was slightly higher in male and female animals fed
with Low Energy Diet, than in the male and female groups fed with Normal Diet.
The use of moderate dietary restriction will provide the scientist with a better con-trolled animal model that will have a lower incidence or at least a delayed onset of background diseases and tumours. Moderate dietary restriction induces no adverse effects in physiology, metabolism, clinical biochemistry, or pathology and has the great advantage of reducing the acceleration of age-related diseases. This operationally simple procedure significantly improves survival, exposure time, and statistical sensitivity for these bioassays to detect a true treatment effect in a 2-year period.
Acknowledgement
I wish to acknowledge the help of Dr. habil. Várnagy László DSc, who served as my supervisor but also encouraged and challenged me throughout my PhD program.
I also wish to acknowledge the collaboration of Dr. Róbert Glávits and Dr. László Rácz pathologists, who performed the histopathological evaluations and gave me explanations in the most important part of this work.
This dissertation could not have been written without the contribution of Dr.
Gábor Hirka, managing director of former Toxicological Research Centre Ltd., who granted me the access to the data.
I thank them all.
25 August 2008 .