ASSESSMENT OF CHRONIC TOXICITY AND CARCINOGENICITY IN RATS OF WINGSTAY(R)100, A RUBBER ANTIOXIDANT ANTIOZONANT/

The chronic toxicity and carcinogenicity of Wingstay(R) 100 (W 100), a rubber antioxidant/antiozonant, were studied in Fischer 344 (F 344) r ats in two chronic studies. Earlier genetic studies indicated that the product had weak activity in a bacterial mutation assay, but lacked a ctivity in chromosomal aberration assays. In an one year study, both g enders of F 344 rats were exposed to 53, 310 or 1900 ppm in NIH-07 die t for 52 weeks, and sacrifices were made at 38, 52 and 64 weeks. No te st substance related deaths occurred, although the high dose of 1900 p pm caused a decrease in body weight gain and food consumption in both genders. Red blood cell mean corpuscular volume was significantly incr eased at 38 weeks, accompanied by a significant decrease in mean corpu scular hemoglobin concentration. At 52 weeks, the red blood cell count and hemoglobin values were also significantly decreased in high dose animals of both genders. Total bilirubin and cholesterol were increase d in high dose animals of 38 and 52-week sacrifices. During the 3 mont h recovery, hematology parameters, bilirubin and cholesterol returned to control values. Total protein was reduced in high dose animals of b oth genders, throughout the entire exposure and recovery periods. W 10 0 also produced increases in relative liver, spleen, heart and kidney weights in high dose animals. Both genders of all W 100 groups exhibit ed significant increases in urothelial cell proliferation (measured by PCNA) and adaptive hyperplasia. No regenerative hyperplasia, preneopl asia, or neoplasia were present. There was microscopic evidence of ext ramedullary erythropoiesis in the spleen and liver of high dose animal s in both genders, otherwise no other pertinent microscopic finding wa s evident. In parallel, an accelerated bioassay (ABA) was conducted, w hich is a mechanistic initiation/promotion carcinogenicity study desig ned to assess tumor induction and pro motion potential of a test subst ance in major organs of carcinogenesis. The present study was conducte d in male F 344 rats for 38 weeks. The target sites chosen for the ABA were liver and urinary bladder and the dose for W 100 was 1900 ppm pr eviously established to be a toxic dose. The liver tumor initiator was diethylnitrosamine (DEN), and the urinary bladder initiator was N-but yl N-(4-hydroxybutyl) nitrosamine (BBN). The initiators were administe red during the first 14 weeks followed by the promoters. The promoters , phenobarbital (PB) for the liver and nitrilotriacetate (NTA) for the urinary bladder, were administered during the last 24 weeks of the st udy after the test substance. The study had II test groups including a negative control. The critical comparisons for initiating activity we re conducted between groups 3 (PB) and 6 (W 100 + PB) for the liver an d groups 8 (NTA) and 11 (W 100 + NTA) for the urinary bladder. The cri tical comparisons for promoting activity were conducted between groups 2 (DEN) and 5 (DEN + W 100) for the liver and groups 7 (BBN) and 10 ( BBN + W 100) for the urinary bladder. There were 26 and 38-week sacrif ices. In this study, most body weight reductions were due to DEN. At 2 6 weeks, significant increases in liver weights were present in all PB -exposed groups. Significant increases in renal weights occurred in al l NTA, BBN and DEN groups. A similar organ weight pattern was present at 38 weeks. At 26 weeks, there were hepatocellular (33 %) and urothel ial (67 %) tumors present in positive control groups (DEN/DEN + PB/BBN /BBN + NTA). In contrast, in the DEN + W 100 (5) and the BBN + W 100 ( 10) groups no tumors were present indicating absence of promotion. In addition, no tumors were present in groups 6 (W 100 + PB) or 11 (W 100 + NTA) indicating absence of initiation. At 38 weeks, the incidences of hepatocellular adenomas and carcinomas in positive control group (D EN) was 44 %. The incidence of urothelial adenomas and carcinomas was 67 % in group 7 (BBN). In contrast, groups 5 (DEN + W 100) or group 10 (BBN + W 100) had a significantly lower incidences of 25 % and 33 %, respectively. This also indicates absence of promotion. At 38 weeks, n o tumors were present in groups 6 or 11 indicating absence of initiati on. In addition, the mean number of neoplasms per animals was signific antly reduced from 2.7 per animal in group 2 to 2 in group 5, and from 3.7 in group 7 to 1.5 in group 10. Also, the biggest diameter of carc inomas was reduced in groups 5 and 10 compared to groups 2 and 7. Fina lly, the incidence of animals with carcinoma of the urinary bladder wa s 25 % in group 7 and was only 4.2 % in group 10. In conclusion, under the conditions of these 2 experiments, W 100 caused a chronic macrocy tic anemia, accompanied by body weight reduction, compensatory extrame dullary erythropoiesis (in liver and spleen), cardio-renal overload an d disruption of bilirubin metabolism. These effects are postulated to lead to changes in the renal filtrate and luminal conditions in the ur inary bladder, leading to development of adaptive hyperplasia througho ut 64 weeks of observation. This hyperplasia did not give rise to eith er regenerative hyperplasia, preneoplasia or in situ neoplasia. Rather , W 100 administration was associated with the reduction in incidence, size and multiplicity of neoplasms, initiated by DEN in the liver and BBN in the urinary bladder. Furthermore, at the end of 15 months, the re were significant reductions in the number of rats with neoplasms in the high dose W 100 group, at 64 weeks, compared to controls (from 10 0 % in controls to 67 % in W 100), and absence of multiple neoplasms i n high dose W 100 rats, compared to 40 % incidence in controls. Accord ingly, W 100 did not exhibit either initiating or promoting carcinogen ic activites in the liver or urinary bladder of male rats. The combina tion of a lack of tumor formation in rats chronically exposed to W 100 , with the absence of tumor initiating or promoting activities indicat e that W 100 is noncarcinogenic under the test conditions.