Effects of male sexual maturity of reproductive endpoints relevant to DART studies in Wistar Hannover rats.

Wistar Hannover rats have been utilized as one of major strains in regulatory toxicology studies. This study was performed to verify the appropriate age of male sexual maturity in the development and reproductive toxicity (DART) study in Wistar Hannover rats (RccHan:WIST) by comparing reproductive endpoints between 8, 10 and 12 weeks of ages. Although fertility showed a tendency toward decrease in 8-week-old males, copulation index was not different among three ages. Testis weights reached a plateau at 10 weeks of age, whereas weights of other reproductive organs developed until 12 weeks of age. Indices of spermatogenesis (sperm motility, number of sperm in the epididymis and testis and contents of morphologically abnormal sperm) showed age-related progress and did not fully develop except for 12-week-old. For histology, epididymal tubules in 8-week-old animals showed immaturity with tall epithelium. At cesarean section, dams mated with 8-week-old males showed high incidence of preimplantation loss and the number of live fetuses was less than 10. In conclusion, although reproductive performance attained maturity by age of 10 weeks, spermatogenesis was not fully established at 10-week-old, which could result in a low fertility index. Therefore, we recommend that Wistar Hannover male rats at 12-week-old or older are used to conduct DART study properly and evaluate any adverse effects on dams and embryo-fetal development.

[Analytical method for carbamate pesticides in processed foods by LC/MS/MS].

A rapid analytical method for the simultaneous determination of carbamate pesticides in processed foods was established by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS). The pesticides were extracted from samples with acetonitrile using accelerated solvent extract equipment, except for the fine powder type spices, which were extracted in an ultrasonic bath. The crude extract was cleaned up with a multi-solvent GPC column (Shodex Asahipak GF-310 HQ) using acetonitrile as a mobile phase. The eluent from the column at the retention time between 13 to 18 min was concentrated under nitrogen gas and dissolved in a mixture of acetonitrile-water-0.2 mol/L ammonium formate buffer pH 6.0 (10 : 9 : 1). An aliquot was injected into the LC/MS/MS using electrospray ionization (ESI) with acquisition in the positive mode. The recoveries of 29 kinds of pesticide from dried fruits (raisin, prune and mango) and spices (turmeric, masala, sage, thyme and red pepper) fortified at levels of 0.1 and 0.01 microg/g were mostly in the range of 50 to 150% and those from soybean paste and soy sauce fortified at 0.01 microg/g were 46.9 to 122.6% (C.V. 3.8 to 37.6%), except for 4 kinds of pesticide. The determination limits (S/N> or =10) corresponded to 0.001 to 0.05 mug/g of the pesticides in red pepper.

Diluted isoflurane as a suitable alternative for diethyl ether for rat anaesthesia in regular toxicology studies.

Despite its explosive properties and toxicity to both animals and humans, diethyl ether is an agent long used in Japan in the anaesthesia jar method of rat anaesthetises. However, in response to a recent report from the Science Council of Japan condemning diethyl ether as acceptable practice, we searched for an alternative rat anaesthesia method that provided data continuous with pre-existing regular toxicology studies already conducted under diethyl ether anaesthesia. For this, we examined two candidates; 30% isoflurane diluted with propylene glycol and pentobarbitone. Whereas isoflurane is considered to be one of the representatives of modern volatile anaesthetics, the method of propylene glycol-diluted 30% isoflurane used in this study was our modification of a recently reported method revealed to have several advantages as an inhalation anaesthesia. Intraperitoneal pentobarbitone has long been accepted as a humane method in laboratory animal anaesthesiology. These 2 modalities were scrutinized in terms of consistency of haematology and blood chemistry with previous results using ether. We found that pentobarbitone required a much longer induction time than diethyl ether, which is suspected to be the cause of fluctuations in several haematological and blood chemical results. Conversely, only calcium ion concentration showed a slight difference from traditional results in the case of 30% isoflurane. Additionally, serum prolactin and corticosterone levels indicated that 30% isoflurane induced less stress than ether, confirming that 30% isoflurane can both provide results consistent with diethyl ether, while at the same time remove its disadvantages. As such 30% isoflurane appears to be a strong alternative anaesthetic agent for future regular toxicology studies in Japan.