3.1. Subchronic oral toxicity study in rats
Daily gavage administration of MSC to rats for 28 days at doses of up to 2 mg/kg/day (12 mg/m2/day) induced no mortality in either sex. No evidence of treatment-related gross toxicity was identified during clinical observations of male rats exposed to any dose of MSC used in the study. In females, agent-related clinical observations were limited to dose-related alopecia in the middle (4/20 rats) and high (10/20 rats) dose groups.
MSC induced dose-related body weight loss and/or suppression of body weight gain in both sexes. All groups of male rats gained body weight throughout the dosing period (). However, mean terminal body weight in male rats receiving daily oral exposure to the high dose of MSC was reduced by approximately 12% versus sex-matched vehicle controls (p < 0.05). Throughout the study, group mean body weights in male rats receiving the middle or low doses of MSC were slightly, but not significantly, reduced from those seen in male rats in the vehicle control group.
Group mean body weights in male rats receiving daily oral exposure to MSC.
MSC had a much greater effect on body weight in female rats than in males. During the first week of exposure, dose-related body weight loss was seen in groups receiving the middle and high doses of MSC (p < 0.05 versus vehicle control; ). At the end of the second week of MSC exposure, mean body weight in female rats in the high dose group was still below their initial (Day 0) weight; body weight in the high dose group remained significantly below the mean body weight of female vehicle controls throughout the dosing period. Body weights in female rats in the middle dose group were also significantly below sex-matched vehicle controls at all times in the exposure period (p < 0.05; ). Group mean body weights in female rats receiving the low dose of MSC were not significantly different from control.
Group mean body weights in female rats receiving daily oral exposure to MSC.
The effects of MSC on animal body weight were clearly associated with reductions in food consumption. As seen with body weights, significant reductions in mean food consumption in male rats were seen only in the high dose group (). By contrast, statistically significant, dose-related reductions in mean food consumption were observed in female rats in both the middle and high dose MSC groups during all four weeks of the dosing period, and in the low dose group during the first week of exposure ().
Group mean food consumption in male rats receiving daily oral exposure to MSC.
Group mean food consumption in female rats receiving daily oral exposure to MSC.
Clinical pathology evaluations performed at the terminal necropsy also demonstrated that MSC induced greater toxicity in female rats than in male rats. In male rats, statistically significant alterations in clinical pathology parameters were limited to very small (< 4%) reductions in mean RBC volume and mean cell hemoglobin in the high dose group (); all other hematologic, clinical chemistry, and coagulation parameters in male rats exposed to MSC were within normal limits and did not differ from sex-matched vehicle controls.
Selected hematology parameters in male rats exposed to MSC
By contrast to the minimal hematologic effects in male rats, MSC induced mild, dose-related anemia and thrombocytopenia in female rats. Statistically significant, dose-related reductions in hematocrit, hemoglobin, mean RBC volume, and platelet count were seen in female rats exposed to the middle and high doses of MSC (). Furthermore, significant reductions in RBC count and mean cell hemoglobin content, coupled with an apparent compensatory increase in reticulocyte count, were seen in high dose females only. High dose female rats also demonstrated a statistically significant increase in prothrombin time ().
Selected hematology parameters in female rats exposed to MSC
Statistically significant increases in alkaline phosphatase (152%), alanine transaminase (81%), aspartate transaminase (60%), and γ-glutamultranspeptidase (200%) were seen in females in the high dose group only; these changes were not seen in females in the middle or low dose groups, or in males exposed to MSC at any dose level. Small but statistically significant increases in mean cholesterol (38 to 56%) and triglyceride (48 to 81%) levels were seen in females (but not in males) in all MSC dose groups.
Organ weight measurements performed at the terminal necropsy identified a number of significant effects of MSC. Increases in absolute and/or relative liver weights were observed in all dose groups in both sexes. In rats exposed to the high dose of MSC, absolute liver weights were increased by 12% in males and 18% in females, while relative liver weights were increased by 27% in males and 43% in females.
High dose male rats also demonstrated small but statistically significant reductions in the absolute weights of the adrenals, heart, testes, and thymus. In female rats, statistically significant reductions in absolute weights of the adrenals, heart, and ovaries were seen in the middle and high dose groups, while reductions in the absolute weights of the brain, thymus, and thyroids were present in females in the high dose group. However, with the exception of liver weights, none of the changes in absolute organ weights in MSC-treated rats remained significant when organ weights were normalized to terminal fasted body weights. These data suggest that observed reductions in absolute organ weights (other than the liver) reflect reductions in body weight induced by MSC, and do not demonstrate organ-specific toxic effects of the organoselenium compound.
Gross pathology in male rats at the terminal necropsy provided no evidence of MSC toxicity. In females, abnormal pigmentation and rough, irregular surface of the liver were observed in > 50% of rats in the high dose group, providing further corroborative evidence that the liver is primary a target organ for MSC toxicity. Gross pathology in other organs in female rats was unremarkable.
Histopathologic evaluation of tissues identified the liver as the primary site of MSC toxicity in both sexes of rats (). In females, hepatocellular degeneration was identified in 3/20 females in the low dose group, 7/20 females in the middle dose group, and 18/20 females in the high dose group; lesion severity also increased with increasing MSC dose (Fig. 5). Female rats in the high dose group also demonstrated peliosis hepatis (15/20 rats; Fig. 6) and hepatic subacute inflammation (9/20 rats); these changes were not identified in female rats in the middle or low dose groups. In males, microscopic changes in the liver were limited to hepatocellular degeneration (6/20 male rats in the high dose group; no changes at lower doses); peliosis hepatic and inflammation were not seen. In addition to being identified in lower incidence, degenerative liver lesions seen in male rats in the high dose group were less severe than were comparable lesions seen in high dose females.
Exposure-related microscopic lesions in rats exposed to MSC
In male rats, testicular degeneration and arrested spermatogenesis were seen in 11/20 and 10/20 rats in the high dose group, respectively. These changes were not seen in the middle or low dose group, but did correlate with oligospermia observed in the epididymis of 11/20 high dose rats.
In female rats, MSC induced atrophy of the corpora lutea in 10/20 high dose and 7/20 middle dose rats; no ovarian changes were seen in female rats treated with the low dose of MSC. In the hair follicle, MSC induced atrophy (8/20 high dose females) and epithelial necrosis (4/20 high dose females); these microscopic changes were note seen in male rats, and were correlated to gross clinical observations of alopecia in females (only) in the high dose group. Microscopic changes in the hair follicle were not seen in female rats exposed to the middle or low doses of MSC.
Histopathologic evaluations demonstrated remaining tissues to be within normal limits in all dose groups in both sexes.
3.2. Subchronic oral toxicity study in dogs
Daily oral (gavage) administration of MSC to beagle dogs for 28 days at doses of 0.15, 0.3, or 0.6 mg/kg/day (3, 6, or 12 mg/m2/day) induced no mortality in either sex. Clinical signs were unremarkable in all groups of female dogs and in male dogs treated with the low or middle doses of MSC. One male dog in the high dose group was observed to be hypoactive at several days during the second half of the four-week dosing period.
Oral administration of MSC to dogs induced no statistically significant effects on group mean body weight, body weight gain, or mean daily food consumption in either sex in any dose group (data not shown). Group mean body weights and body weight gains in male dogs receiving the middle or low doses of MSC, and in female dogs exposed to all doses of MSC were comparable to those of sex-matched vehicle controls at all times during the study. In comparison to a mean four-week weight gain of 0.69 kg in male dogs in the vehicle control group, male dogs exposed to the high dose of MSC lost 0.02 kg during the four-week dosing period; however, this reduction in body weight gain was not statistically significant.
MSC induced a mild, but dose-related and statistically significant anemia in both sexes of beagle dogs; the effects of MSC were slightly more severe in males than in females. In male dogs, statistically significant reductions in RBC count and hematocrit were observed at the middle and high doses (), while hemoglobin concentration was reduced at the high dose only. Female dogs in the high dose group demonstrated significant decreases in hematocrit and hemoglobin concentration; no changes in these parameters were seen in female dogs exposed to the middle or low doses of MSC. Other hematology evaluations, as well as the results of clinical chemistry, coagulation, and urinalysis tests failed to identify any additional effects of MSC exposure in either sex.
Selected hematology parameters in dogs exposed to MSC
The results of ophthalmic examinations and electrocardiographic evaluations provided no evidence of MSC toxicity in any treated animal. Similarly, gross pathology at the terminal necropsy was unremarkable.
As was the case in the rat study, histopathologic evaluation of tissues from the canine toxicity study identified the liver as a primary site of MSC toxicity (). It is of interest, however that the sex distribution of hepatic lesions in dogs differs from that observed in rats: whereas hepatic changes induced by MSC in rats were more severe in females than in males, microscopic alterations in liver morphology were seen in males only. In male dogs, midzonal necrosis was identified in 4/4 dogs in the high dose group, 4/4 dogs in the middle dose group, and 3/4 dogs in the low dose group; this change was present in 0/4 vehicle controls. Other hepatic changes (peliosis hepatis and midzonal degeneration; Figure 7) were identified in 1/4 male dogs in the high dose group only, and were not seen in groups exposed to the middle or low dose of MSC. No microscopic evidence of MSC toxicity was identified in the liver of any female dog in the study.
Exposure-related microscopic lesions in dogs exposed to MSC
Also consistent with the findings of the rat toxicity study was the observation of an increased incidence of degenerate spermatocytes in the epididymis of male dogs.
Microscopic alterations were also identified in several other tissues of MSC-treated dogs (); however, these lesions were interpreted as being of less toxicologic significance than the liver lesions described above. Increased incidences of acute intestinal inflammation and crypt dilatation were seen in dogs of both sexes exposed to MSC. Similarly, thymic atrophy and depletion/necrosis of gut-associated lymphoid tissue were also observed in higher incidences in both male and female dogs exposed to MSC than in animals in sex-matched vehicle control groups. The results of histopathologic evaluations demonstrated that all other tissues from dogs in all dose groups in both sexes were within normal limits.