Makoto Miyagawa

The in vivo-in vitro replicative DNA synthesis (RDS) test with hepatocytes prepared from male B6C3F1 mice as an early prediction assay for putative nongenotoxic (Ames-negative) mouse hepatocarcinogens.

To assess the efficacy of the in vivo-in vitro hepatocyte replicative DNA synthesis (RDS) test as a short-term assay, 41 putative nongenotoxic (Ames-negative) mouse hepatocarcinogens, as well as 31 noncarcinogens, were examined using male 8-week-old B6C3F1 mice and an in vitro [methyl-3]thymidine-incorporation technique. Animals were exposed to the maximum tolerated dose (MTD) and 1/2 MTD of each chemical by gavage and after 24, 39 or 48 h, hepatocytes were prepared with a collagenase-perfusion technique. Assessment of the distribution of spontaneous RDS in a total of 337 control mice gave an average incidence of 0.15 +/- 0.08% within the range of 0 to 0.39% (mean +/- 3 x SE) with a 99.7% probability. Values of 0.4% or more for RDS incidences induced by test samples were therefore judged as indicating a positive response in our RDS test. Under the experimental conditions applied, 32 of 41 putative nongenotoxic hepatocarcinogens gave clear positive responses (positive sensitivity: 78%), and of 31 noncarcinogens 25 samples gave negative responses (negative specificity: 81%), thus giving an overall concordance for the RDS test with long-term findings of 79%.

An in vivo-in vitro replicative DNA synthesis (RDS) test using rat hepatocytes as an early prediction assay for nongenotoxic hepatocarcinogens screening of 22 known positives and 25 noncarcinogens.

To evaluate the applicability of an in vivo-in vitro replicative DNA synthesis (RDS) test using rat hepatocytes, we conducted the RDS test with 22 nongenotoxic (Ames-negative) hepatocarcinogens and 25 noncarcinogens under our standardized conditions and judgement criteria. Compared to controls (RDS incidence of under 1.0%), the RDS test gave positive results for 18 hepatocarcinogens (positive sensitivity: 82%), and negative results for 20 noncarcinogens (negative specificity: 80%), and thus the overall concordance was 81%. These findings strongly suggest that the RDS test is an extremely useful method for early detection of nongenotoxic hepatocarcinogens.

Rat liver in vivo replicative DNA synthesis test for short-term prediction of nongenotoxic (Ames-negative) hepatocarcinogenicity: a collaborative study of the Nongenotoxic Carcinogen Study Group of Japan.

A collaborative study was conducted to evaluate whether a replicative DNA synthesis (RDS) test using the rat liver can detect nongenotoxic (Ames-negative) hepatocarcinogens with three or seven daily administrations at dose-levels effective in long-term bioassays. The assay methods were well-validated by the 14 participants. Of six compounds tested, carbon tetrachloride (50 and 100 mg/kg), clofibrate (125 and 250 mg/kg), diethylstilbestrol (0.125 and 0.25 mg/kg) and urethane (100 mg/kg) gave positive results, methyl carbamate (200 and 400 mg/kg) exerted equivocal effects, and D,L-ethionine (125 mg/kg) failed to elevate RDS. These findings suggest that the RDS test can detect many nongenotoxic rat hepatocarcinogens with short-term administration at dose-levels used in long-term bioassays.