A dried sludge product resulting from the treatment process of domestic wastewater. The exact processes followed for the production of the dried sludge are: Preliminary + secondary treatment of the wastewater stream, thickening, dehydration and drying of the excess sludge.

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The mutagenic and genotoxic potential of the UVCB -Dried Sludge from domestic wastewater was assessed in the basic battery of in vitro test:

- bacterial reverse mutation assay (OECD TG 471)

- an in vitro micronucleus test (OECD TG 487)

- In Vitro Mammalian Cell Gene Mutation Tests using the Hprt and xprt genes (OECD 476)

 

The item did not show any mutagenic or genotoxic potential up to the highest dose that was tested in each assay (1000μg/plate – Ames test, 100μg/ml – cell micronucleus &mammalian cell gene mutation) .

All three studies indicated no mutagenic or genotoxic potential of Dried sludge from domestic wastewater and therefore, according to EU CLP Regulation (EC No. 1272/2008), the substance is not classified for mutagenesis.

 

Bacterial reverse mutation assay (OECD Guideline No. 471)

The study was conducted using TA98, TA100, TA1535 and TA1537 strains of Salmonella typhimurium and WP2uvrA (pKM101) strain of Escherichia coli. The study consisted of a preliminary toxicity test and two mutation assays comprising three independent experiments. The bacterial tester strains were exposed to the test item in the presence and absence of a metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver). In the preliminary toxicity test, Salmonella typhimurium, TA 100 was exposed to test item at 7.8125, 15.625, 31.25, 62.5, 125, 250, 500 and 1000 μg/plate along with a DMSO control using direct plate incorporation method, for the selection of test doses for the mutation assay. Based on these observations, it was decided to test highest test dose of 1000 μg/plate in the mutation assay. The study indicated that Dried Sludge From Domestic Wastewater was not mutagenic in this Bacterial Reverse Mutation Assay up to the highest dose of 1000 μg/plate based on cytotoxicity, under the conditions of testing employed.

 

In vitro mammalian Cell micronucleus test in human peripheral blood lymphocytes OECD Guideline No. 487

The potential of the test item, Dried Sludge From Domestic Wastewater to induce micronuclei was evaluated using cultured human peripheral blood lymphocytes. Human lymphocytes in whole blood cultures, stimulated to divide by addition of Phytohaemagglutinin (PHA) 48 hours prior to treatment, were exposed to the test item in the presence and absence of an exogenous metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver). The study consisted of a preliminary cytotoxicity test and a definitive micronucleus assay. In a preliminary cytotoxicity test carried out to select the test concentrations for the definitive micronucleus assay, blood cultures were exposed to the test item up to 100 μg/mL. Test item did not show precipitation in the test medium till 50 μg/mL, both in the presence and absence of metabolic activation. In the presence and absence of metabolic activation, at the beginning and end of treatment, there was slight precipitation of the test item at the highest test concentration of 100 μg/mL. The test item did not cause any appreciable change in the pH and osmolality of the treatment medium at any of the tested concentrations compared to the DMSO control. The test item did not exhibit the desired level of cytotoxicity (45±5 %) up to the highest tested concentration of 100 μg/mL, when compared to the DMSO control, both in the presence and absence of metabolic activation with the 3-hours exposure and in the absence of metabolic activation with the 24-hours exposure. Based on these observations, it was decided to test up to 100 μg/mL in the presence and absence of metabolic activation with the 3-hours exposure and in the absence of metabolic activation with the 24-hour exposure in the definitive micronucleus assay. The definitive micronucleus assay consisted of three independent experiments: Experiments 1 and 2, with a 3-hour exposure, in the presence and absence of metabolic activation, respectively, and Experiment 3 with a 24-hour exposure in the absence of metabolic activation. Blood cultures were exposed to the test item in duplicate at concentrations of 25, 50 and 100 μg/mL in Experiments 1 and 2 and 3. There was no evidence of statistically significant induction of micronuclei, either in the presence or in the absence of metabolic activation in any of the tested concentrations. In each of these experiments, under identical conditions, the respective positive control substances produced a large and statistically significant increase in micronuclei.

 

IN VITRO MAMMALIAN CELL GENE MUTATION TEST IN CHO-K1 CELLS USING THE Hprt GENE (OECD Guideline No. 476)

The mutagenic potential of the test item Dried Sludge from Domestic Wastewater to induce gene mutation in mammalian cells was evaluated using Chinese Hamster ovary (CHO) cells. The study consisted of a preliminary cytotoxicity test and a definitive gene mutation test. The gene mutation test comprised of two independent experiments, one each in the presence and absence of metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver). In the gene mutation test, CHO-K1 cells were exposed to the test item in duplicate at concentrations of 12.5, 25, 50 and 100 µg/mL of the medium for 3 hours in the presence (Experiment 1) and absence (Experiment 2) of metabolic activation. In a similar way, a concurrent vehicle control (DMSO) and a positive control, 3-methylcholanthrene (Experiment 1) were also tested in duplicate. There was no evidence of induction of gene mutations in any of the test item treated cultures either in the presence or absence of metabolic activation. The positive control in experiment 1 produced a statistically significant increase in the frequencies of mutants, under identical conditions. The results of the forward gene mutation test at the hprt locus with Dried Sludge from Domestic Wastewater indicated that the test item was non-mutagenic under the conditions of this study

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

All three in vitro tests in genetic toxicity showed negative results. Therefore, there is no need to carry out in vivo studies in genetic toxicity. There is no reason to believe that the negative results would not be relevant to humans.

Justification for classification or non-classification

 Τhe studies indicated no mutagenic potential of Dried sludge from domestic wastewater and therefore, according to EU CLP Regulation (EC No. 1272/2008), the substance is not classified for mutagenecis.