II. MATERIALS AND METHODS 



species to assess their health status. Rats were 

 placed on study at 6-7 weeks of age and mice at 

 8-9 weeks of age. The health of the animals was 

 monitored during the course of the studies ac- 

 cording to the protocols of the NTP Sentinel Ani- 

 mal Program (Appendix F). 



Animal Maintenance 



All animals were housed five per cage. F'eed and 

 water were available ad libitum. Further de- 

 tails of animal maintenance are given in Ta- 

 ble 5. 



Clinical Examinations and J'athology 



All animals were observed two times per day. 

 Body weights were recorded once per week for 

 the first 12 (rats) or 13 (mice) weeks of the stud 

 ies and once per month (hereafter Mean body 

 weights were calculated for each group Ani- 

 mals found moribund and those surviving to the 

 end of the studies were humanely killed A nec- 

 ropsy was performed on all animals, including 

 those found dead. Some tissues were excessively 

 autolyzed or cannibalized, and thus, the number 

 of animals from which particular organs or tis- 

 sues were examined microscopically varies and 

 is not necessarily equal to the number of ani- 

 mals that were placed on study. 



During necropsy, all organs and tissues were ex- 

 amined for grossly visible lesions. Tissues were 

 preserved in 10% neutral buffered formalin, em 

 bedded in paraffin, sectioned, and stained with 

 hematoxylin and eosin. Tissues examined mi- 

 croscopically are listed in Table 5. 



When the pathology evaluation was completed, 

 the slides, paraffin blocks, and residual wet tis- 

 sues were sent to the NTP Archives for inven- 

 tory, slide/block match, and wet tissue audit. 

 The slides, individual animal data records, and 

 pathology tables were sent to an independent 

 quality assessment laboratory. The individual 

 animal records and tables were compared for ac- 

 curacy, slides and tissue counts were verified, 

 and histotechnique was evaluated. All tumor di- 

 agnoses, all target tissues, and all tissues from a 

 randomly selected 10% of the animals were eval- 

 uated by a quality assessment pathologist. The 

 quality assessment report and slides were sub- 

 mitted to the Pathology Working Group (PWG) 



Chairperson, who reviewed all target tissues 

 and those about which there was a disagreement 

 between the laboratory and quality assessment 

 pathologists. 



Representative slides selected by the Chairper- 

 son were reviewed by the PWG, which included 

 the laboratory pathologist, without knowledge of 

 previously rendered diagnoses. When the con- 

 sensus diagnosis of the PWG differed from that 

 of the laboratory pathologist, the laboratory pa- 

 thologist was asked to reconsider the original di- 

 agnosis. This procedure has been described, in 

 part, by Maronpot and Boorman (1982) and 

 Boorman et al. (1985). The final diagnoses rep- 

 resent a consensus of contractor pathologists and 

 the NTP Pathology Working Group. For subse- 

 quent analysis of pathology data, the diagnosed 

 lesions for each tissue type are combined accord- 

 ing to the guidelines of McConnell et al ( 1986) 



Slides/tissues are generally not evaluated in a 

 blind fashion (i.e., without knowledge of dose 

 group) unless the lesions in question are subtle 

 or unless there is an inconsistent diagnosis of le- 

 sions by the laboratory pathologist. Nonneo- 

 plastic lesions are not examined routinely by the 

 quality assessment pathologist or PWG unless 

 they are considered part of the toxic effect of the 

 chemical 



Additional histologic sections of the right and 

 left kidney of male rats in each dose group and 

 the controls were prepared and reviewed by a 

 special PWG. Three or four longitudinal sec- 

 tions were prepared from the remaining half of 

 each of the right and left kidney at approxi- 

 mately 1-mm intervals by standard procedures. 

 All lesions observed during this special PWG re- 

 view were evaluated in a "blind" fashion. 



Statistical Methods 



Data Recording: Data on body weight and feed 

 consumption for this experiment were recorded 

 in the Carcinogenesis Bioassay Data System 

 (Linhart et al., 1974). Other data elements were 

 recorded in the Toxicology Data Management 

 System. The data elements include animals, ex- 

 perimental design, survival, and individual 

 pathologic results, as recommended by the Inter- 

 national Union Against Cancer (Berenblum, 

 1969). 



Nitrofurantoin, NTP TR 341 



32 



