Masao Horimoto

Rat epididymal sperm motion changes induced by ethylene glycol monoethyl ether, sulfasalazine, and 2,5-hexandione

Epididymal sperm was examined using the Hamilton-Thorne Sperm analyzer (HTM-IVOS, version 10.6) in male rats treated with known male reproductive toxicants that act by different mechanisms to detect effects on sperm motion. Three agents known to produce changes in sperm motion at high exposure levels were administered at lower levels. Ethylene glycol monoethyl ether (EGEE), sulfasalazine (SASP), and 2,5-hexandione (2,5-HD) were administered by oral gavage to adult male Sprague-Dawley rats at 250 or 500 mg/kg/day, at 300 or 600 mg/kg/day, or at 100 or 250 mg/kg/day, respectively. The males were treated with EGEE, SASP, and 2,5-HD for 35, 28, and 28 days, respectively. The males treated with EGEE and SASP were mated with untreated females to assess male fertility. All males were examined for body weight, testicular and epididymal weight, epididymal sperm count, and sperm motion. The sperm motion parameters included percentage of motile sperm, percentage of progressively motile sperm (progressive motility), curvilinear velocity (VCL), average path velocity (VAP), straight line velocity (VSL), amplitude of lateral head displacement (ALH), beat cross frequency (BCF), linearity (LIN), and straightness (STR). For the male rats treated with SASP, no treatment-related effects on percentages of motile sperm or sperm count were observed despite impaired male fertility. However, abnormal motion of epididymal sperm from the SASP treated males was detected by a significant reduction in mean progressive motility, VAP, and ALH, and an increase in BCF and STR. For the males treated with 2,5-HD for 4 weeks, most parameters generated by the HTM-IVOS indicated decreased sperm motion despite no remarkable changes in testicular weight, epididymal weight, or sperm count. In the EGEE-treated males at 250 mg/kg/day for 5 weeks, abnormal motion of epididymal sperm was detected by decreased progressive motility and increased BCF, although there were no treatment-related effects on testicular weight or male fertility. Progressive motility was decreased in all treated groups and the difference from the control value was of the greatest magnitude among the sperm motion parameters generated by the HTM-IVOS. Velocity parameters (VAP, VSL, VCL) responded sensitively to abnormal sperm motion in the SASP and 2,5-HD studies. In spite of decreased sperm motion, BCF values were significantly increased in all treated groups except the 7-week EGEE high-dose group, where there were no motile sperm to evaluate. ALH was significantly decreased in the treated groups in which remarkable effects on sperm motion were noted. There were no significant changes in ALH at the low-dose of EGEE at which only mild effects on sperm motion were observed. STR was increased for epididymal sperm from the males treated with SASP when compared with the controls. For the males treated with EGEE and 2,5-HD, however, STR was decreased when compared with the controls. There were no significant differences in LIN in any of the groups treated with SASP, in which remarkably reduced sperm motion was detected by the other parameters. In conclusion, among the parameters generated by the HTM-IVOS, progressive motility was significantly decreased in all treated groups and the most valuable for detecting slight changes in sperm motion induced by these three different target toxicants. Further investigation with a larger set of compounds is needed to evaluate which IVOS parameters are the most sensitive in detecting motion changes.

Historical control data on prenatal developmental toxicity studies in rabbits

Historical control data on rabbit prenatal developmental toxicity studies, performed between 1994–2010, were obtained from 20 laboratories, including 11 pharmaceutical and chemical companies and nine contract laboratories, in Japan. In this paper, data were incorporated from a laboratory if the information was based on 10 studies or more. Japanese White rabbits and New Zealand White rabbits were used for prenatal developmental toxicity studies. The data included maternal reproductive findings at terminal cesarean sections and fetal findings including spontaneous incidences of morphological alterations. No noticeable differences between strains or laboratories were observed in the maternal reproductive and fetal developmental data. The inter‐laboratory variations in the incidences of fetal external, visceral, and skeletal alterations seem to be due to differences in the selection of observation parameters, observation criteria, and classification of the findings, and terminology of fetal alterations.

Historical control data on developmental toxicity studies in rodents.

Historical control data on rodent developmental toxicity studies, performed between 1994 and 2010, were obtained from 19 laboratories in Japan, including 10 pharmaceutical and chemical companies and nine contract research organizations. Rats, mice, and hamsters were used for developmental toxicity studies. Data included maternal reproductive findings at terminal cesarean sections and fetal findings including the spontaneous incidences of external, visceral, and skeletal anomalies. No noticeable differences were observed in maternal reproductive data between laboratories. Inter‐laboratory variations in the incidences of fetuses with anomalies appeared to be due to differences in the selection of observation parameters, observation criteria, classification of the findings, and terminology of fetal alterations. Historical control data are useful for the appropriate interpretation of experimental results and evaluation of the effects of chemical on reproductive and developmental toxicities.

Timing of implantation and closure of the palatal shelf in New Zealand white and Japanese white rabbits.

Two specific developmental events, namely implantation and palatal shelf closure, are of specific interest because they define, respectively, the beginning and the end of the treatment period in embryo-fetal developmental toxicity studies for pharmaceutical products. Thus, a detailed evaluation of the timing of implantation and closure of the hard palate is necessary to assure use of the proper exposure window in developmental toxicity studies in rabbits, the nonrodent species most commonly evaluated in regulatory developmental toxicology studies. The purpose of this study was to determine the timeline for implantation and closure of the hard palate in the New Zealand White rabbit, and to determine if this timeline differed in the Japanese White rabbit. To describe the timing of implantation, the uteri from does of the New Zealand White rabbit and the Japanese White rabbit were examined on gestation days (GDs) 5 through 8 for macroscopic evidence of implantation. To assess palatal shelf closure, fetuses were removed on GDs 17, 18, and 19 and fixed in Bouins solution. The fetuses were then categorized into five stages of palatal shelf closure: open (Stage I); approach of the palatal shelves (Stage II); partial closure of the hard palate (Stage III); full closure of the hard palate (Stage IV); and full closure of the soft palate (Stage V). In both the New Zealand White and Japanese White rabbit strains, implantation was initiated on GD 6.5 and was completed on GD 7. Partial closure of the palate began on GD 17.5, and by GD 19, closure of the hard palate was completed in all fetuses, and closure of the soft palate was completed in 75–96% of the fetuses. The timing of implantation and palatal shelf closure were comparable between the New Zealand White rabbit and the Japanese White rabbit. Therefore, treatment beginning on GD 7 and continuing until GD 19 encompasses the period of major organogenesis and is considered appropriate for use in developmental toxicity studies using either of these two strains of rabbits.