The IACUC must evaluate both phases of the proposed procedure: the induction of disease and the investigation of potential therapies. Concerns regarding variability in the development of cancer in this model over the anticipated year-long period need to be addressed, as an early onset could result in pain and distress. If advanced disease states become apparent, the IACUC needs to be informed about what steps are planned to respond to the symptoms. If an animal begins to suffer from signs of cancer metastases, will the surgical procedure be performed earlier than planned or will the rabbit be euthanized (in essence wasting an animal)? Additionally, very little information is provided on either the toxic effects of diethylnitrosamine in this model or the frequency of post-treatment observations. The IACUC needs to be informed about the dose and route of administration of this chemical and any expected adverse outcomes. The PI needs to provide this information based upon prior personal experience and/or relevant reference literature.

Furthermore, Lee needs to present broader, and in some areas more specific, information describing humane endpoints. Although listing some signs of pain and distress in the rabbit is a good start, the list is incomplete. Ideally, the protocol should state that intervention will be taken if pain or distress is indicated, as defined by specific examples of signs that will be monitored. By defining pain and distress in general and specific terms, it allows both performance standards and specific criteria to be used in the evaluation of the status of the animal. Examples that should be added to the narrative include: decreased appetite, dehydration, twitching, hunched posture, self-mutilation, increased or decreased temperature, and anorexia, among others. Decreased fecal output, soft feces or night stool, or lack of feces are also often first signs of pain and distress in rabbits. The IACUC needs to ask the investigator whether weight loss is a good indicator of pain or distress in this model, as it could be masked by the additional weight of growing tumors or ascites. If weight loss is an appropriate indicator, then in order to avoid potential disagreements or misinterpretations, a specific percentage should be provided as a weight-loss endpoint. Increased heart rate is not reliable or practical in this model and should be removed from the list.

Regarding the treatment/surgical phase of the protocol, the investigator should be advised to consult the institutional veterinarian for the most appropriate analgesics to use for this procedure. Buprenorphine or other nonsteroidal anti-inflammatory drugs (NSAIDs) are more effective than aspirin. Additionally, pre-emptive analgesics should be administered in order to get pain relief on-board during the recovery phase. Although the investigator attempts to address general indicators of pain, means to recognize localized pain at the incision site need to be included in the protocol narrative. Signs of redness, swelling, dehiscence, and infection are all indications that veterinary intervention is necessary.

Lastly, the protocol should state the qualifications of those working with the rabbits, specifically addressing their experience in surgery and in identifying pain in rabbits. Poor surgical techniques can lead to prolonged healing times, infection, and unnecessary pain. Experience administering anesthetics to rabbits is also crucial. Depending on the extent of tumor development, the animals' health and tolerance of anesthesia may be compromised.