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of NR678 administered to rats homozygous for

FMO2*1 (Welsh

rats)

The general increase of FMO activity toward the test molecule observed in the Welsh rat lungs compared to the Wistar strain may result in increased susceptibility of this organ to the toxic effects of NR678. In order to confirm this hypothesis, the Welsh rat colony genotyped as described above was used in a toxicity study (study 4), where male Welsh rats received a dose of 0.5 mg/kg of NR678 and were euthanased at 3, 6 and 24 h after dosing. The objective of the study was to assess drug-related morphological changes and compare the development and dynamics of tolerance in rats homozygous for FMO2*1 (Welsh) with those obtained from Wistar rats (homozygous for FMO2*2) in study 3; for this reason, the selected dosage and time points in study 4 were identical to those employed in the low dose group of study 3.

3.4.4.1Clinical assessment

All rats administered a dose of NR678 of 0.5 mg/kg survived until the scheduled euthanasia. Most animals were asymptomatic, however, two rats (12L-2257 and 12L- 2258) exhibited mild or moderate dyspnoea, respectively, together with piloerection and decreased motor activity, starting at 5 h after dosing. Both rats were euthanased at the scheduled time point (6 h). Another animal (12L-2262) showed mild dyspnoea and decreased motor activity at 6 h post dosing. These symptoms were no longer present on the following day at the time of scheduled euthanasia (24 h).

3.4.4.2Gross post mortem findings

The two rats that had shown mild dyspnoea (scheduled euthanased at 6 h after dosing), exhibited mild hydrothorax (characterised by the presence of 1.5 mL clear fluid in the thoracic cavity) and moderate diffuse hyperaemia in the lungs. No macroscopic findings were observed in the remaining rats.

histological finding was a moderate or marked alveolar and interstitial oedema (Figure 3.40), which had similar features to that observed in Wistar rats receiving a high dose (5 and 10 mg/kg; see paragraph 3.1.3). Pulmonary oedema occurred in rats euthanased at 3 h (1/3 animals) and 6 h after dosing (2/3 animals) and was not detected in the three rats euthanased at 24 h. Scattered apoptotic cells (severity: slight) were present in the alveolar lining in one rat euthanased at the 3 h time point. A mild or moderate increase in the number of alveolar macrophages occurred in all rats euthanased at 3 and 6 h post treatment and in 1/3 rats at 24 h. This finding had been seen previously in Wistar rats receiving NR678 at low (see paragraph 3.2.2.1.3) and high (see paragraph 3.1.3) doses.

Table 3.10. Summary of the key histological findings in the lungs of Welsh rats administered a low dose (0.5 mg/kg) of NR678 (study 4).

G ro up NR678 dose (mg/kg) Time of death post treatment Histological findings Alveolar and interstitial oedema Increased alveolar macrophage Alveolar cell apoptosis 1 0 24 h 0/3 0/3 0/3 2 0.5 3 h 1/3 (4) 3/3 (2.6) 1/3 (1) 3 6 h 2/3 (3.5) 3/3 (2) 0/3 4 24 h 0/3 1/3 (2) 0/3

Results are expressed as number of animals showing the histological finding/ number of animals per group. The average severity of each finding (in brackets) was calculated by summing the severity grades and dividing the total by the number of animals affected by that finding.

In addition to the treatment-related findings described above, there was evidence of an inflammatory reaction in the lungs of the Welsh rats (Figure 3.41), with an incidence and severity that was similar in all different groups, including the controls. This was represented by a slight to moderate multifocal perivascular and interstitial granulomatous inflammatory infiltration, composed of lymphocytes, fewer macrophages and plasma cells and occasional neutrophils, with scattered multinucleated giant cells; and moderate to severe proliferation of the bronchus- associated lymphoid tissue (BALT). These findings were considered spontaneous, i.e. not treatment related, and were likely caused by an infectious agent affecting the Welsh rat colony, which is conventionally, rather than barrier, -maintained. Although serological tests were not undertaken in these animals or other animals in the colony and the specific aetiologic agent involved in the inflammatory process could not be

identified, such histological lesions, in particular the BALT hyperplasia and the lymphocytic-dominated inflammatory infiltrate, are highly suggestive of the early stage of Mycoplasma pulmonis infection (Percy and Barthold, 2007; Schoeb et al., 2009).

Figure 3.40. Histological features of alveolar and interstitial oedema in the lungs of Welsh rats that had received a dose of 0.5 mg/kg of NR678, compared to concurrent controls. (a) Rat 12L-2251 (control, euthanased at 24 h post dosing). The alveolar lumen (*) is filled with air and appears as a clear space. There is virtually no separation between the outline of vessels (double-headed arrow: perivascular space) and the adjacent parenchyma. (b) Rat 12L-2257 (0.5 mg/kg of NR678, 6 h after dosing). Alveolar lumina (*) contain abundant eosinophilic homogenous proteinaceous fluid. The perivascular space (double-headed arrow) is markedly distended by the presence of fluid (interstitial oedema). (c) Rat 12L-2251. Closer view of the normal alveoli. (d) Rat 12L-2257. Higher magnification of the pale eosinophilic proteinaceous fluid filling the alveoli. There is an increased number of macrophages within the alveolar lumen (arrows).

Figure 3.41. Histological features of the spontaneous inflammatory changes occurring in the lungs of Welsh rats from study 4, including the controls. (a) Rat 12L-2260 (0.5 mg/kg of NR678, euthanased at 24 h post dosing). There is a periarteriolar aggregate of lymphocytes (solid arrowhead) and a more diffuse interstitial infiltration of mononuclear cells (open arrowheads), composed predominantly of macrophages, with fewer lymphocytes and plasma cells (bar: 20 µm). (b, c and d) Rat 12L-2253 (control, 24 h post dosing). (b) Closer view of the inflammatory infiltrate in a control animal. Macrophages (open arrowhead) are large and characterised by abundant eosinophilic cytoplasm (bar: 20 µm). (c) Surrounding a bronchus (*), there is a large accumulation of lymphocytes, consistent with hyperplasia of BALT (bar: 200 µm). (d) At higher magnification, lymphocytes infiltrate the bronchial wall and there is marked lymphocytic exocytosis in the epithelium (arrow). Bar: 50 µm.