In 1996, a satellite group of 50 young Wistar rats (12-13 weeks of age) from an oncogenicity study was histologically examined at RCC Itingen in Switzerland. In the high dose group, treatment-related hepatocellular hypertrophy, accompanied by a morphologically distinctive "diffuse follicular hypertrophy", in the thyroid gland was reported. However, the thyroid gland findings were also seen in some control animals without apparent reason. Thereafter, this finding was more often recorded in almost all studies randomly distributed across all groups including the controls. Retrospectively, the first case appeared in RCC studies in 1995.
In 1997, Schering AG Berlin flagged up a problem with Wistar rats derived from BRL Füllinsdorf, Switzerland. Approximately 25% of the offspring were affected by thyroid lesions, and a number of them showed growth retardation. Similarly, studies in other companies, including the breeders of the GALAS programme, were affected by this particular lesion. Rainer Ernst from Schering AG demonstrated a probable hereditary origin for this finding. This, as well as histological details, led to the use of the term "thyroid gland dysplasia". The problem was presented first at the GTP Seminar at Zürich in 1998.
In spring 1999 it was decided between RCC Ltd and the members of the GALAS programme to purge the breeding colony in order to completely eradicate the thyroid alteration from the RCC breeding colony during 2000.
LESION AND RELATED PROBLEMS
By light microscopy, thyroid dysplasia is a mainly diffuse, rarely focal or multifocal, lesion consisting of balloonised follicle cells which typically have apically placed nuclei located on a subcellular structure (colloid containing vesicle) 1). The subcellular vesicular structure can be shown by electron microscopy as dilated endoplasmic reticulum (figs. 3, 4) that has displaced the normal, basally located nucleus into an apical position. It is evident, that only the basally located endoplasmic reticulum structures become dilated, in contrast to the apical portion.
Up to approximately 25% of the offspring were affected by enlarged thyroids along with increased organ weights, and the described histological features. Animals with normal body size did not show any clinical or biochemical alteration. However, some of the animals were growth retarded, reaching only half the size of the normal litter mates. A high proportion of the animals displayed abnormal springing movement. The proportion of dwarfs, normal-size animals with thyroid dysplasia, and unaffected animals was approximately 22%, 52% and 26%, respectively, leading to the assumption of an intermediary hereditary disorder.
In dwarfs, Rainer Ernst from Schering AG Berlin demonstrated the loss of growth hormone in the pituitary gland by immunohistochemistry. In addition, in contrast to normal-sized animals, there was a 3- to 5-fold increase in serum TSH levels along with a severe decrease of T4 leading to pathological TSH/T4 ratios. Morphologically, there was no difference between the thyroid gland of dwarfs and normal sized animals with thyroid dysplasia.
These findings gave rise to the possibility that normal-sized animals with thyroid dysplasia may react differently to normal animals in toxicity studies. Problems that could arise consisted of increased tumor incidences, exaggerated reactions to weak goitrogens or secondary to hepatocellular hypertrophy. It was also unclear if the hormonal homeostasis changed with increasing age. Moreover, animals suffering from hereditary disorder giving birth to a high number of dwarfs were not usable for reprotoxicity studies.
It was proposed to all GALAS members that they examine histologically the thyroid glands from all pups from all litters of numbered breeding pairs. Litter mates should receive the number of the breeding pair. All parental breeding pairs which had affected pups would be eliminated from further breeding. The remaining parental animals should be crossed for a further search for thyroid-dysplasia gene bearers. In addition, a hormone study should be performed to establish the validity of studies already performed with such animals. A number of companies took part in this test, either in co-operation with RCC or they performed inhouse studies.
RESULTS OF PURGING PROCEDURE
The purging procedure was conducted in three steps. In the first phase, all pups of 141 litters were sacrificed. The percentage of all litters affected was 19.6% and the incidence of affected pups within a litter ranged from 12.5% to 64.0%. After rotation breeding, the pups of 46 litters were examined in the second phase and there were no animals found with the thyroid lesion.
However, in a 28-day toxicity study, a new case was observed. Therefore, in a third phase, three pups per litter from a total of 250 litters were sacrificed and examined. Although animals were still found with thyroid dysplasia, no new case has been detected from 2000 onwards. Thus, RCC's purging procedure was successful.
Thyroid dysplasia was recognized and confirmed as a congenital disorder (intermediate heritage). The gene or genes were not identified, but it is deemed that homozygous animals with thyroid dysplasia are runts suffering from reduced pituitary gland GH and are hypo-thyreote. In contrast, heterozygotes are affected by thyroid dysplasia but are euthyreote. Moreover, the heterozygous state did not lead to any changed hormonal homeostasis by increa-sed age or different incidences of preneoplastic or neoplastic lesions in oncogenicity studies.
- (Dr. Klaus Weber, head, Business Unit Pharma, Head Pathology, Business Unit Pharma, RCC Ltd., Switzerland weber.klaus@rcc.ch www.rcc.ch & Rainer Ernst, Head Pathology, Schering AG Berlin, Germany.)