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Key value for chemical safety assessment

Additional information

Valid experimental data were available to assess the genetic toxicity of hydroxyethylpiperazine mixture in vitro and vivo. An Ames test is available done with the mixture itself whereas further in vitro and in vivo studies are available from a single components of the mixture (piperazine, CAS 110-85-0) which can be used as read across.

In vitro:

- Gene mutation in bacteria:

The test substance hydroxyethylpiperazine mixture was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay (OECD 471, GLP, BASF 2012). Following strains were used: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvr. A standard plate test (SPT) and a preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats) and a dose range of 33µg - 7000 µg/plate for both tests were used. No precipitation of the test substance was found up to the hightes applied concentration onward with and without S9 mix. A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions at 7000 µg/plate. A relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system. Thus, under the experimental conditions of this study, the test substance hydroxyethylpiperazine mixture is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

 

- In vitro mammalian cell transformation test:

In a study performed according to procedures similar to EU Method B.21 (In Vitro Mammalian Cell Transformation Test), piperazine (CAS 110-85-0) did not induce a significant and dose-related number of transformed foci over the concentration range of 1.0 µg/mL to 300.0 µg/mL (Huntsman, 1980). This concentration range corresponds to a survival range of approx. 90% to 50 % in the preliminary cytotoxicity test. The test material is considered to be inactive in the Balb/3T3 in vitro transformation assay.

- In vitro mammalian gene mutation:

In a mammalian gene mutation assay with mouse lymphoma L5178Y cells, piperazine (CAS 110-85-0) was tested in concentrations 7.8, 125, 300, 400, and 500 µg/mL, with and without metabolic activation (similar to OECD 476, Huntsman, 1980). The test substance induced toxicity-related increases in the mutant frequency at the TK locus in L5178Y mouse lymphoma cells only in the presence of rat liver S9 microsomal activation. Without activation, concentrations up to 500 µg/mL became highly toxic without inducing meaningful changes in the mutant frequency. With activation, mutagenic activity was observed in the 125 to 500 µg/mL concentration range, and 500 µg/mL induced a 9.7-fold increase over background. Piperazine was therefore considered to be mutagenic in the Mouse Lymphoma Forward Mutation Assay with microsomal activation but not mutagenic without metabolic activation.  

 

- Chromosome aberration:

In an in vitro mammalian chromosome aberration assay similar to OECD guideline 473 the clastogenic potential of piperazine phosphate (CAS 14538-56-8) was observed (Allen et al., 1986 cited in EU risk assessment of piperazine). Chinese hamster ovary cells were treated with piperazine phosphate at concentrations ranging from 1.73 to 110 mg/mL with and without metabolic activation. Piperazine phosphate did not cause structural chromosomal aberration in this in vitro test system. Since piperazine is the active ingredient in this substance it is assumed that piperazine shows the same effects.

 

In vivo:

- Micronucleus test:

In a micronucleus assay CD-1 mice were dosed once orally with 5000 mg piperazine phosphate (CAS 14538-56-8) per kg bodyweight (Marshall, 1987 cited in EU risk assessment of piperazine). No significant increase in the level of micronuclei of polychromatic or normochromatic erythrocytes of the bone marrow could be detected. Since piperazine is the active ingredient in this substance it is assumed that piperazine shows the same effects. Taking all results together, only the in vitro gene mutation test was positive with metabolic activation. All other in vitro tests and the in vivo micronucleous test were negative, therefore a negative potential concerning genotoxicity for hydroxyethylpiperazine mixture is assumed.

Short description of key information:
in vitro:
Ames Test: negative (OECD 471, GLP, BASF 2012)
Mammalian cell transformation test: negative (CAS 110-85-0, guideline study, Huntsman 1980)
Mammalian gene mutation: negative without metabolic activation, positive with metablic activation (CAS 110-85-0, similar to OECD 476, Huntsman 1980)
Chromosome aberration: negative (CAS 14538-56-8, similar to OECD 473, Allen et al., 1986)
in vivo:
Micronucleus test: negative (CAS 14538-56-8, Marshall, 1987)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available studies are considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance is not considered to be classified for genotoxicity under Directive 67/548/EEC, as amended for the 28th time in Directive 2001/59/EC.

 

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for genotoxicity under Regulation (EC) No. 1272/2008.