Report No DWI0024

Jun 1987


Earlier work carried out at WRc and other laboratories has shown that concentrated extracts prepared from chlorinated drinking waters derived from surface sources (rivers and reservoirs) give positive results in bacterial mutagenicity assays. The mutagens present in these extracts appear to be direct-acting and when tests are carried out in the presence of an in vitro metabolising fraction (ie rat liver S9), activity is reduced. The compounds responsible for the observed activity have not been identified but they appear to be formed as unwanted reaction products between chlorine and naturally-occurring organics in the water. In order to follow up these findings, it was decided that concentrated drinking water samples should be tested for mutagenic activity in a range of higher test systems. The results from such studies would give a better indication as to whether the presence of these substances in drinking water represents a health risk to consumers. In 1983, WRc obtained a 3-year contract from the Department of the Environment to carry out this work and this report summarises the principal findings of the contract.

Concentrated drinking water extracts, prepared by adsorption onto XAD-2 resin, have been shown to induce chromosome aberrations in Chinese hamster ovary (CHO) cells treated in vitro. Positive results have been obtained using drinking waters derived from a lowland river and an upland reservoir. The compounds responsible for most of this activity were shown to be generated during chlorination, although there was some very weak activity in raw water samples. Chromosome damage was also observed following treatment with a laboratory chlorinated humic acid sample, indicating that naturally-occurring humic substances in water are probably an important precursor material for mutagen formation. When CHO cells were treated in the presence of foetal calf serum the clastogenic activity of the extracts was greatly reduced. This presumably indicates that the mutagens bind readily to serum proteins and preliminary experiments with bacterial assays have indicated that albumin may be one of the proteins involved.

In the cytogenetic studies using CHO cells, there were some indications that the extracts produced effects on the mitotic spindle. Studies were therefore carried out to analyse specifically for spindle damage. Raw water extracts derived from a lowland river appeared to induce a small but reproducible increase in the frequency of aberrant mitoses. With final drinking water extracts, the results obtained were inconclusive due to high within-group variability.

Clastogenic activity was also observed in human lymphocytes treated with drinking water extracts, but only when separated lymphocyte cultures were used. In whole blood cultures, the presence of red blood cells appeared to suppress the activity of the extracts. Although evidence of clastogenic activity was observed in the in vitro assays, when a drinking water extract was dosed orally to groups of mice, there was no indication of induced chromosome damage in bone marrow cells sampled 6, 24 or 48 h after administration.

In mammalian cell point mutation assays, drinking water extracts showed only limited evidence of activity. In the mouse lymphoma L5178Y TK+/- assay, two of the three samples examined produced a small increase in trifluorothymidine-resistant clones. However, in Chinese hamster V79 and CHO cells the extracts did not appear to induce 6-thioguanine or ouabain-resistant mutants, even at dose levels that produced high frequencies of chromosome damage in parallel cytogenetic studies. There was no evidence of mutagenic activity in the Drosophila sex-linked, recessive, lethal assay when flies were allowed to feed on sucrose solutions prepared from drinking water extracts.

It is concluded that the mutagens produced during the chlorination of drinking water are capable of inducing genetic damage (ie chromosome aberrations) in mammalian cells in vitro. The mutagens appear to bind readily to exogenous protein, are deactivated by liver enzymes, and negative results were obtained in an in vivo mouse bone marrow cytogenetic study: thus the mutagens in chlorinated drinking water probably do not represent a genetic hazard to man, since they are unlikely to reach the gonads in their active form. However, the possibility of a local carcinogenic action by direct exposure cannot be discounted. The most likely target site for such an effect is the epithelial lining of the gastro-intestinal tract, since these cells may be exposed to the mutagens before they become bound to other substances or are enzymatically detoxified. Recommendations for further work to evaluate this hypothesis are given.

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