Humic acids (Fluka), chlorinated to
carbon:
chlorine (C:Cl) ratios of 1:1 and 1:0.3, were administered to B6C3F1 mice, 50 males and 50 females per group, in the
drinking water at a total organic
carbon (TOC) level of 0.5 g/L. The mice were 6 to 8 weeks old at the beginning of the bioassays. The doses used were based on short-term (8 weeks) evaluations for toxicity, palatability, and
weight gain. The chronic bioassays included the following control groups: unchlorinated
humic acids (0.5 g/L), no-treatment (100 males and 100 females), dibromoethane (DBE, 2.0 mM in
drinking water; positive control) and 0.44%
sodium chloride in
drinking water, i.e., at the same concentration as those receiving chlorinated
humic acids. The chlorinated
humic acids were prepared freshly and chemically assayed once per week. All chemicals were, with the exception of DBE, administered for 24 months; DBE was administered for 18 months. The volumes of solutions consumed were measured once weekly. All treatment groups showed normal
weight gain except the DBE group. At the completion of exposure, the animals were sacrificed and necropsied, and tissue sections were taken for histopathology. No markedly significant increases in
tumor incidences were evident in any of the organs and tissues examined in the chlorinated
humic acid groups compared to unchlorinated
humic acids and the no-treatment control groups. DBE caused the expected high incidence of
squamous carcinomas of the forestomach. The chlorinated
humic acids tested contained direct-acting
alkylating agents, based on their reactivity with p-nitrobenzylpyridine (PNBP), and showed mutagenic activity in S. typhimurium.