Intraperitoneal pentobarbitone

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The text on this page is taken from an informal compilation of opinions of contributors to the online VOLE List. As such, they are not peer reviewed and may contain differences of opinion. Those wishing to contact the list may contact Adrian Smith.

Is intraperitoneal pentobarbitone considered unacceptable/painful I? Here, researchers generally use commercial pentobarbitone euthanasia solution (325mg/ml), with dose rate of 160mg/kg, given i.p. for euthanasia in rodents, but we have heard that 60mg/ml concentration is less painful. Does dilution help (we have checked the pH and it doesn't changed much when diluted to 60 mg/ml, i.e. it decreases from 11 to 10, which would suggest that perhaps it is not a pH issue). Also, others have suggested mixing lignocaine with pentobarbitone prior to i.p. injection - but given the delay to onset of lignocaine, we are unsure that this would have  the desired pain reduction effect, unless lignocaine was give as a separate injection several minutes prior to pentobarbitone.

Is use of Zoletil, followed by pentobarbitone (particularly by intracardiac injection) acceptable?

Are all i.p. injections painful? We have one new committee member (a retired vet) who is of the opinion that all i.p. injections (even of saline) are painful (and hence should be avoided). This will be difficult to implement in a lab animal research setting and I wondered what your view of this is?

Is intraperitoneal Ketamine particularly painful?

As a supplement to this, I would like to know if lignocaine is painful when administered i.p.?

See also Intraperitoneal injection

My own personal opinion is that I would like to see i.p. injections per se reduced to an absolute minimum. In addition to the continual worries about pain, there is the real chance of injecting into viscera rather than into the abdominal cavity. I consider it to be one of the most difficult routine injections for that reason, particularly in small mammals where it is used most! I am now, in addition, working part-time in a small animal veterinary practice, and i.p. injections there are reserved for situations where the animal is already anaesthetised. Likewise, we virtually never give i.m. injections, because of the risk of pain. Is it about time we reconsidered our injection policy and made a "positive list" of those few situations where an i.p./i.m. injection is scientifically justifiable - or am I being naively unrealistic? The argument that most drugs act too slowly if given s.c. is in my experience not valid. A good time to discuss this, at any rate, with the new Directive's severity classification system looming on the horizon!

I agree and from a scientific viewpoint there is a need to question administration of substances by this route.  It was shown 2 decades ago that even with skilled technicians it was very unreliable: Walwoort NC (1991) Assessment of distress through pathological examination.  In: Replacement, Reduction and Refinement: Present Possibilities and Future Prospects.  eds. Hendriksen CFM & Koeter HBWM. Elsevier, Amsterdam: pp 265 - 271.

However, when I was in general veterinary practice I did find that in very small kittens that needed re-hydrating an i.p. cannula (with a drip rubber bung in the Luer mount for preventing bacterial entry) sewn into the skin was better tolerated than an i.v. drip.  It also avoided repeated saline injections.

When Ambrose did work on euthanasia with he found that it was difficult to add lignocaine without it coming out of solution and precipitating over time (not sure whether it was the lignocaine or the barbituric acid or both), but that can be overcome using a solvent (such as propylene glycol, I think it was), and it was also available commercially: Ambrose, N, Wadham JJB and Morton, DB (1999) Refinement of Euthanasia. In: Programme and Abstracts of the 3rd World Congress on Alternatives and Animal Use in the Life Sciences. Page 156  Aug 29 to Sept. 2 Bologna 1999.  Special issue of ATLA.

Welfare can also be measured by observing writhing (drawing in of the abdominal muscles) in the animals afterwards (Roughan and Flecknell have published this, post-laparotomy).  Writhing is a well known sign of pain in analgesic tests for visceral pain using i.p. acetic acid.  Nembutal (60mg/ml Sodium PentoBarbitone) has been found to be OK and I think that is because it has a far lowering buffering power than 200mg+ solutions. pH has also to be seen in the light of the pK of the buffer and its molarity.  The higher the molarity and osmolarity, the greater amount of peritoneal effusion will be needed to counteract it and bring the pH back to biological limits and to make it isotonic.

I think that lignocaine acts fairly fast and it certainly reduces writhing.


The lignocaine effect first reported by Ambrose has since been replicated[1]. Diluting pentobarbitone solutions 1:1 with a 20 mg/mL lignocaine solution reduced abdominal writhing. The pentobarbitone/lignocaine solutions were made up the same day with no additional excipients. Bupivicaine also seemed to work. But dilution didn't seem to reduce the pH too much either.

Another approach to the abdominal writhing problem with pentobarbitone is to give a very high dose[2].

  1. Khoo, Shaun Yon-Seng; Lay, Belinda P. P.; Joya, Josephine; McNally, Gavan P. (2017). "Local anaesthetic refinement of pentobarbital euthanasia reduces abdominal writhing without affecting immunohistochemical endpoints in rats". Laboratory Animals. 52 (2): 152–162. doi:10.1177/0023677217721260. PMID 28758534.
  2. Zatroch, Katie K.; Knight, Cameron G.; Reimer, Julie N.; Pang, Daniel S. J. (2017). "Refinement of intraperitoneal injection of sodium pentobarbital for euthanasia in laboratory rats (Rattus norvegicus)". BMC Veterinary Research volume. 13: 60. doi:10.1186/s12917-017-0982-y. PMID 28222732.