Report No DWI0013
REPORT OF RESEARCH UNDERTAKEN AT LIFE SCIENCE RESEARCH TO DEVELOP A METHOD FOR TESTING CONCENTRATED WATER EXTRACTS IN SEPARATED HUMAN LYMPHOCYTE CULTURES (EHT 9154 SLD) DoE 1442-M
Ten separate experiments were performed, involving method evaluation and validation, and also including cytogenetic assays on two concentrated water samples, X055 and X098.
Initial method evaluation led to the selection of RPMI 1640 as a suitable growth medium for separated human peripheral lymphocytes. Further experiments revealed that total serum deprivation for periods up to and including four hours in no way affected survival of separated human lymphocytes, and that addition of donor serum to cultures during both the establishment and post-treatment periods was beneficial to cell growth. A 72 hour pre-treatment establishment period was selected in order to ensure that sufficient cells had accumulated prior to treatment. Therefore, cultures were established in RPMI 1640 complete culture medium containing 5% donor serum and incubated 37ºC for 72 hours prior to treatment. Treatment was for a period of three hours in serum-free medium and, following treatment, cultures were re-established in complete culture medium (with 5% donor serum) for a 21 hour post-treatment recovery period.
Investigation of the known direct-acting clastogens chlorambucil and ethyl methanesulphonate resulted in the selection of a chlorambucil concentration of 2.5µg/ml for use as the positive control in separated lymphocyte cultures.
Toxicity and cytogenetic assays on X055 showed gross toxicity at concentrations of 2.0 and 1.5 litres/ml (no metaphases present for analysis), and at 1.0 litre/ml 24% of metaphases seen contained damaged chromosomes. This sample was considered unduly toxic by the Study Sponsor, and further testing was performed on a replacement sample, X098.
Toxicity and cytogenetic assays showed that X098 showed some limited evidence of clastogenic potential at both 1.0 and 2.0 litres/ml when gap-type aberrations were included in the total aberrations seen. However, no real increase in damage other than gaps was seen over control values at any X098 concentration.
Consideration of mitotic indices showed a dose-related cytotoxic effect in response to X098 treatment, with reductions in mitotic index (compared to the control value) of 52, 84, 95 and 100% at concentrations of 0.5, 1.0, 2.0 and 3.0 litres/ml respectively.
As a result of these procedural evaluation assays, a definitive study has been initiated; this will be fully described in a future report.Copies of this report may be available as an Acrobat pdf download under the 'Pre 2000 Reports' heading on the DWI website.