Single dose toxicity study of 89Zr-DFO-nimotuzumab in mice

Acute (Day-2: considering Day 1 as the day of injection) and delayed (Day-14) toxicity studies was carried out in male and female (n = 5 males + 5 females), six week old Balb/C mice (Charles Rivers). Mice were monitored for weight and behavior changes. The vehicle for injection was PBS as it was used in the final preparation of ⁸⁹Zr-DFO-nimotuzumab. 74 MBq (10 mg) of immunoconjugates was proposed maximum dosage for human use. 100 mice were divided in to five groups: 20 mice were used for base line study, 40 mice (control group) were treated with vehicle (PBS). 20 of these were sacrificed as a control of the acute toxicity study on Day-2 and 20 mice were sacrificed as a control of the delayed toxicity study on Day-14. 40 mice (treatment group) were injected with 3.2 MBq, 0.065 mg (which corresponds to 168-fold excess radioactivity dose and 25-fold excess mass dose of ⁸⁹Zr-nimotuzumab that is projected for human studies on a weight/weight basis). 20 of these were sacrificed on Day-2 for the acute toxicity study and 20 of them were sacrificed for delayed toxicity study on Day-14.

All animals in this study were observed regularly for signs of mortality, morbidity, injury, and availability of food and water. Individual body weights were determined and recorded during the quarantine period and (every other day) experimental period. Blood was collected via cardiac puncture for hematology and clinical chemistry. A complete whole blood and plasma analyses were done. Mice were sacrificed on, day 2, or day 14 and organs of interest (kidneys, spleen, liver, bone, heart, lungs, brain, and testes/uterus) were harvested and stored in 10% formaldehyde solution. They were further processed and examined for histopathological analyses.