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EC number: 931-295-2 | CAS number: -
Accuracy of preparation:
The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%).
The slightly higher accuracies may in part have resulted from the higher nominal concentrations, since nominal amounts of test substance and vehicle used for formulation were higher than targeted concentrations. Accuracy was based on target concentrations.
Small responses at the retention time of the test substance were observed in the chromatograms of the Group 1 formulations. It was considered to derive from carry over since similar responses were obtained in the analytical blanks.
The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).
Analysis of Group 2 and Group 4 formulations after storage yielded a relative difference of ≤ 10%. Based on this, the formulations were found to be stable during storage at room temperature under
normal laboratory light conditions for at least 6 hours.
In light of the corrosive properties of the substance, and the possible local nature of the effects that could affect a clear evaluation of systemic effects, dosing by dietary route was recommended for this 90-day study by oral route. However, after various attempts no adequate analytical method could be developed, and it was decided to proceed with dose by gavage. Besides, in view of the low dose levels selected for the study, interference through local effects that are expected to occur especially in the forestomach (serves as a storage reservoir in rodents), were considered to be minimal.
Wistar rats were administered 0, 0.5, 2.2 or 8.8 mg/kg bw/d of the test substance by gavage daily for 90 days. The control group received the vehicle propylene glycol. Examinations were performed according to OECD 408 with additionally rectal temperature and estrous cycle determination.
A total of 12/20 animals at 8.8 mg/kg bw/d were sacrificed in extremis or were found dead between weeks 6 and 10. Additionally, 2/20 animals at 0.5 mg/kg bw/d and 5/20 animals at 2.2 mg/kg bw/d were sacrificed in extremis in week 12 and between weeks 8 and 12, respectively. All remaining animals at 8.8 mg/kg bw/d were sacrificed in week 10. Morbidity was related to marked arthritis of the tarsal joints and/or granuloma(s), with central necrosis of the mesenteric lymph nodes, and at 8.8 mg/kg bw/d also in one case due to pleuritis. The incidence and onset of mortality showed an apparent dose-related trend.
The hindlegs and/or tail of several rats treated at 0.5 mg/kg bw/d and the majority of rats treated at 2.2 and 8.8 mg/kg bw/d were affected, showing clinical symptoms such as abnormal gait/hypotonia/swelling. Occasionally, testicles showed erythema at 8.8 mg/kg bw/d. These clinical signs generally showed an increasing trend with regard to the number of animals affected and earlier onset of symptoms with increasing doses. These animals also displayed supportive macroscopic findings such as thickened hindlegs/tail and/or enlarged popliteal lymph nodes and/or reduced size of gastrocnemius muscle(s). Histopathologically, these findings were supported by a dose-related occurrence of arthritis with or without hyperostosis in the tarsal joints (up to marked severity at 0.5 mg/kg bw/d and up to massive severity at 2.2 mg/kg bw/d). In general, lower severities were related to acute inflammations with edema of the soft tissues surrounding tarsal bones and joints as hallmark, whereas higher severities were more frequently noted in chronic inflammations including hyperostosis of bones and in some cases with fissures of bones. In several animals a similar histopathology was noted in tail vertebrae (2.2 and 8.8 mg/kg bw/d) and/or knee joint (8.8 mg/kg bw/d) which in a few cases was accompanied by atrophy of the gastrocnemius muscle (2.2 and 8.8 mg/kg bw/d). This atrophy was regarded as a feature of inactivity. All these findings in the hindlegs can be correlated with in-life clinical symptoms, such as abnormal gait /hypotonia/swelling. One female treated at 2.2 mg/kg bw/d showed arthritis of the rib-sternal junction.
The draining popliteal lymph node showed increased cellularity of the common cell types of lymph nodes (without a clear predominance) in the cortex and/or medullary cords, similar as noted in the iliac and renal lymph nodes (supported at necropsy by enlargement of these nodes). Increased cellularity was also noted for the liver at 8 mg/kg and correlated to enlargement of the liver.
Granulocytic inflammation (up to marked) observed in the small intestines (especially in the ileum) at 2.2 mg/kg bw/d was considered to be due to a direct exposure of the test item or its metabolite. Furthermore, there were (foamy) macrophages in the lamina propria up to slight degree noted in most rats at 2.2 mg/kg bw/d. At 0.5 mg/kg bw/d, there were no test item-related findings in the small intestines.
At 0.5 mg/kg bw/d and above, the draining mesenteric lymph node showed up to massive granulomas with or without central necrosis (especially at the outer side of the cortex), most times merging into extranodal inflammation and/or necrosis with or without peritonitis (up to moderate). Incidences and severity showed a dose related response. Enlarged mesenteric lymph nodes recorded at necropsy at 0.5 mg/kg bw/d and above correlated to increased cellularity, granuloma(s), central necrosis and/or medullary sinus ectasia.
It was remarkable that there were rats with granulomas of the mesenteric lymph nodes without a clear relation with histopathology of the small intestines (granulocytic inflammations). This started in males at 0.5 mg/kg bw/d with moderate and marked degrees of granulomas of the mesenteric lymph node without any finding in the small intestines.
Granulomas were also noted at low incidence in the spleen at 8.8 mg/kg bw/d (up to marked, only from rats with a macroscopically enlarged spleen) and liver at 2.2 and 8.8 mg/kg bw/d. Capsules of these abdominal organs were unaffected (one spleen at 8.8 mg/kg bw/d excluded), making it less plausible that the liver and spleen findings were caused by peritonitis. The higher spleen weight recorded for females at 2.2 mg/kg bw/d were attributed to leucocytosis and granulomas in the spleen.
In the thoracic cavity, major findings were present in the lung, consisting of adhesions seen at necropsy at 2.2 and 8.8 mg/kg bw/d correlating to inflammation of the pleura (up to marked), and a clear increase in incidence and severity of perivascular eosinophilic/lymphocytic infiltrates. Furthermore, there was a slight increase in severity and/or incidence of alveolar and/or granulomatous inflammation (correlating to reddish/gray-white foci at necropsy at 0.5 and 2.2 mg/kg bw/d). Secondary changes to the pleural inflammation consisted in a few cases of serosa infiltrates with inflammatory cells of thymus or aorta and in one rat (at 8.8 mg/kg bw/d) of watery clear fluid in the thoracic cavity.
Myeloid hyperplasia of the sternal bone marrow (up to slight) and leucocytosis (mainly detectable in spleen and liver, and correlating with the macroscopic finding enlarged) started at 0.5 mg/kg bw/d and are considered to be a consequence of the inflammatory processes. The neutrophilia recorded for males and females at 0.5 and 2.2 mg/kg bw/d was regarded to be related to several inflammatory processes, most severely present in mesenteric lymph nodes and tarsal joints. Myeloid hyperplasia in the sternal bone marrow could account for this increase in neutrophils.
Inactive uterus (cervix) epithelium and/or atrophy/mucification of the vagina epithelium in 1/1 females at 0.5 mg/kg bw/d and 4/10 females at 2.2 mg/kg bw/d, and increased incidence and/or severity of acinar atrophy in the pancreas and/or sublingual salivary glands at 2.2 mg/kg bw/d were considered to be related to the poor health condition, affecting normal estrous cycling behavior.
Summarizing, it remained unclear whether most findings were caused systemically by direct exposure to the test item or its metabolite (either by vascular or lymphatic circulation) or indirectly (e.g. by an elicited immune-mediated response, such as release of cytokines/chemotaxis from the inflammatory processes in primarily ileum and/or mesenteric lymph node). Overall, it was considered that exposure of intestines lead to (necrotizing) inflammation and granulomas up to massive degrees of draining mesenteric lymph nodes with possible breakthrough of granulomas into the abdominal cavity. However, this possibly induced peritonitis cannot explain the several findings (which are not restricted to surfaces of organs) in many organs at several locations.
At 2.2 and 8.8 mg/kg bw/d, a lower body weight/body weight gain and lower food consumption was noted for males and/or females, essentially during the second half of the treatment period.
Next to changes in (differential) white blood cell counts, haematological changes consisted of lower haemoglobin, haematocrit, mean corpuscular volume and mean corpuscular haemoglobin, and higher platelet counts in males and/or females at 0.5 and/or 2.2 mg/kg bw/d. Changes in clinical biochemistry parameters consisted of lower total protein, albumin, cholesterol, glucose and calcium, and higher chloride in males and/or females at 0.5 and/or 2.2 mg/kg bw/d. Additionally, a higher urinary volume was recorded for both sexes at 2.2 mg/kg bw/d.
The apparent trend towards a reduction in grip strength of both fore-and hindlegs across dose groups of both sexes may be secondary to the observed clinical signs and resulting impairment of movements. Other functional observation parameters remained unaffected.
There were no indications of possible reproductive toxicity based on the parameters determined in this study. Estrous cycle length/regularity appeared unaffected during the period in which estrous cycle length was determined (Day 76-91), and histopathological examination of the male and female reproductive organs did not show lesions that were directly related to treatment.
No toxicologically relevant changes in rectal temperature and ophthalmology findings were noted.
Based on these results no NOAEL could be established and the LOAEL was determined to be 0.5 mg/kg bw/d.
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