Repeated Administration of Pharmaceutical-Grade Medium-Chain Triglycerides, a Common Pharmacologic Excipient, Confers Dose-Dependent Toxicity by the Intraperitoneal but Not Oral Route in Mice

Experimental design. Animals were split into 4 groups. The 8 µL S IP group received intraperitoneal administration of 8 µL of sterile saline per gram of BW; the 8 µL M IP group received intraperitoneal administration of 8 µL pharmaceutical-grade MCTs per gram of BW (7.56 g/kg); the 4 µL M IP group received intraperitoneal delivery of 4 µL of pharmaceutical-grade MCTs per gram of BW (3.78 g/kg); and the 8 µL M PO group received oral boluses of 8 µL of pharmaceutical-grade MCTs per gram of BW (7.56 g/kg). All groups received administration of their respective treatments every 12 h for 7 d. Animals were then monitored with weight collection and clinical presentation assessment every 12 h for an additional 7 d. All remaining animals were euthanized at day 14. Five animals per group in the 8 µL M IP, 4 µL M IP, and 8 µL M PO groups, as well as 2 animals in the 8 µL S IP control group, were sent for necropsy, histology, and blood collection at time of death or euthanasia.

Weight change. The body weight of each animal was recorded twice daily for 7 d at time of treatment administration and continued for an additional 7 d after the end of the treatment period. Weight change was then calculated as a percentage of starting weight (SW). No animals reached the 30% weight loss threshold set as criteria for euthanasia. Weights of the 8 µL S IP animals remained relatively unchanged. The 8 µL M PO animals showed moderate, intermittent weight loss (down to 94.5% ± 2.3% of SW) between days 2 and 5 before returning to around SW and stabilizing by day 8. Both of the 8 µL M IP and 4 µL M IP groups showed steep weight loss (∼15%) between days 1 and 3. The 4 µL M IP animals steadily regained weight up to ∼94.8% ± 2.4% of SW. Meanwhile, the 8 µL M IP animals more sharply regained weight up to ∼92.1% ± 3.0% of SW between days 6 and 8, at which point weight sharply decreased until day 10. Note that mortalities in the 8 µL M IP group began on day 8 and all animals died by day 11. Weight change curves for 8 µL M IP and 4 µL M IP groups, but not the 8 µL M PO group, were significantly different (P < 0.0001) compared with the 8 µL S IP group. 8 µL M IP, day 1 n = 9, day 11 n = 0; 4 µL M IP, n = 10; 8 µL S IP, n = 10; 8 µL M PO, n = 7.

Clinical presentation. Clinical presentation was assessed based on scoring criteria outlined in Table 1 twice daily for 7 d at time of treatment administration and continued for an additional 7 d after the end of the treatment period. Appearance, clinical signs of decline, behavior, and body condition each received a score between 0 (normal) and 3 (severe pathology). The total score out of 12 is presented here. The 8 µL S intraperitoneal animals had no change in clinical presentation. Average clinical score for the 8 µL M PO group increased slightly between days 2 and 7 before returning to baseline on day 8 through 14. The average clinical score for the 4 µL M IP group increased slightly on days 3 and 8. The average clinical score for animals in the 8 µL M IP group increased to ∼1 between days 3 and 7, after which the average score sharply increased between days 8 and 10, for a maximum individual score of 9. As compared to the 8 µL S IP group, the clinical scores for the 8 µL M IP and 8 µL M PO groups were significantly different with P < 0.0001 and P = 0.0005, respectively. The clinical score in the 4 µL M IP group was not significantly different than that for the 8 µL S IP group. Note that mortalities in the 8 µL M IP group began on day 8 and all animals died by day 11. 8 µL M IP, day 1 n = 9, day 11 n = 0; 4 µL M IP, n = 10; 8 µL S IP, n = 10; 8 µL M PO, n = 7.

Survival curve. Kaplan–Meier plot represents percent survival throughout the 14-d experimental timeline. All animals in the 4 µL M IP, 8 µL S IP, and 8 µL M PO groups survived to the endpoint. Mortality in the 8 µL M IP group began on day 8.5, with the highest number of mortalities between days 9 and 10. The survival curve for the 8 µL M IP group, but no other group, was significantly different (P = 0.0002) than that of the 8 µL S IP group. All animals died by day 11. 8 µL M IP, day 1 n = 9, day 11 n = 0; 4 µL M IP, n = 10; 8 µL S IP, n = 10; 8 µL M PO, n = 7.

Gross necropsy. (A–C) Gross images from 8 µL M IP mice show fluid within the thoracic cavity (A, white arrows), adhesions between the stomach and liver (B, white arrows; adhesions also shown in inset) and between the stomach and enlarged spleen (black arrow), and adhesions between the liver and diaphragm and liver lobes (C, white arrows) are shown. In (C), the scale bar indicates 1 cm. (D–F) Histology images from the retroperitoneal tissues near the kidney from a 8 µL M IP mouse (D), 4 µL M IP mouse (E), and 8 µL S IP mouse (F) are shown. f, perirenal fat; k, kidney. Black arrows show increased fibrous connective tissue and immune cell infiltration associated with the fascia adjacent to the perirenal fat in (D) and (E). Scale bars indicate 100 μm; tissues were stained with hematoxylin and eosin.

Histologic scoring. Graphs of score distributions by group for pulmonary hemorrhage (A), pulmonary inflammation (B), mediastinal inflammation (C), and peritoneal inflammation (D). *, P = 0.01; **, P ≤ 0.005; ***, P ≤ 0.001.

Histologic lesions. Examples of histologic lesions observed in 8 µL S IP mice (top row, A–C), 8 µL M IP (second row, D–F), 4 µL M IP (third row, G–I), and 8 µL M PO (bottom row, J–L) for lung inflammation (left column), mediastinal inflammation (middle column), and peritoneal inflammation (right column) are shown. Black arrows in (D) indicate clear vacuoles surrounded by inflammation in the lung. Black arrows in (G) indicate small foci of inflammatory cell infiltration without clear vacuoles. Black asterisks in B, E, H, and K indicate mediastinal fat, which in the 8 µL/g mouse (E) is effaced by inflammation, acute hemorrhage, and fibrin. ‘l’ indicates lung tissue. Inset in (E) shows histiocyte with an intracytoplasmic circular structure, potentially consistent with a lipid droplet. Black arrow in (H) indicates a mild accumulation of lymphocytes, which can be an incidental finding in mice. The white asterisk in (F) indicates inflammation of the serosal surface of the liver in an 8 µL M IP mouse, which is not observed in the 4 µL M IP and 8 µL S IP mice. Scale bars in (A), (D), (G), and (J) indicate 50 μm; in all other images, scale bars indicate 100 μm. Tissues were stained with hematoxylin and eosin.