WP 4: Determination of safety of key botanical pesticides through vertebrate toxicity studies Brighton Mvumi & Thokozani Hove UNIVERSITY OF ZIMBABWE

Objectives  Provide vertebrate toxicity data of up to 10 species to assess potential human health and animal risks  Build capacity of SADC scientists to carry out vertebrate toxicity trials for the preliminary screening of pesticidal plant materials

Test Animals Test rodents will be reared under laboratory conditions to sexual maturity and in conformity with local ethics committee procedures For each treatment, use 10 (5 M & 5 F) healthy, normal mice (BALB/c) aged 4-6 weeks The rodents must be kept, and fed on normal diet, in captivity for ≥ 1 wk before tests commence (acclimatisation).

Testing procedure (1) Make serial dilutions of test products –which will be mixed with normal feed/water (oral), or –for use as injectables, if applicable (intravenous/intramuscular) The 1 st route of administration to be tested is the one that is normally used for the product For oral admin, rodents have to be starved and deprived of water overnight. Problem with oral - exact dose taken by each animal cannot be determined unless the mice are fed individually Can use a special blunt needle that is inserted right into the posterior part of the mouth.

Testing procedure (2) Provide or apply products to test animals for a predetermined period using standard protocols. For each product to be tested, clearly mark each rodent for easy identification Ensure adequate water supply to each rat throughout the test period. Maintain a group of 10 rodents (5M, 5F) as controls during the test period, feeding them on the standard feed and water Monitor the desired parameters

Assessment parameters (1) Mortality Effect on subsequent reproduction and the resultant progeny as predetermined using standard protocols. Clinical signs and the day/time of onset, duration & recovery of animal if applicable Necropsies will be done on all animals that die and the survivors Weight will be recorded daily

Assessment parameters (2) Serum/blood will be collected every week for clinical pathology and other analyses (may need specialist labs South Africa/UK) If more than one control rat in any series dies, disregard the results for that series and repeat the test Animals will be sacrificed at the end of the trial (6 weeks) and organs sent for clinical pathology and histology Livers and kidneys will be weighed, measured and examined histologically for cell damage, increased mitosis or other abnormalities Sectioning can be done by Central Vet Lab, then examined by UZ pathologist (may need UK labs)

Equipment Test room – standard rooms, cages and feeders for keeping laboratory animals. Ensure room is kept quiet during the test period. Feeder containers- A metal dish and fastened onto the centre of a metal sheet. Water bottle - Any suitable commercially available water bottle for small rodents that will fit into the rodent cage. Cages –constructed of metal and a strong wire mesh. Microtome - for tissue sectioning

Capacity-building of SADC scientists  One MPhil student from SADC will be recruited for training in vertebrate toxicity trials