copper sulfate;N'-[2-(2-aminoethylamino)ethyl]ethane-1,2-diamine

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian germ cell study: gene mutation

Type of information:

experimental study planned

Justification for type of information:

TESTING PROPOSAL ON VERTEBRATE ANIMALS: genetic toxicity in vivo
[Please provide information for all of the points below. The information should be specific to the endpoint for which testing is proposed. Note that for testing proposals addressing testing on vertebrate animals under the REACH Regulation this document will be published on the ECHA website along with the third party consultation on the testing proposal(s).]

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out:
Reaction product of copper sulfate and tetraethylenepentamine

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION [please address all points below]:
- Available GLP studies:
• Etude 2017-GQQ-1 (PH-17/0643): Study according to OECD 471 test, GLP-compliant. Positive without metabolic activation system, negative with metabolic activation system.
- Available non-GLP studies:
• Foureman 1994. Chemical Mutagenesis Testing in Drosophila. IX. Results of 50 Coded Compounds Tested for the National Toxicology Program. OECD 477 study with Triethylenetetramine (constituent of TEPA) for Drosophila. Non-GLP study. Negative.
- Historical human data:
No relevant historical human data was available for this substance.
- (Q)SAR:
• VEGA: Mutagenicity (Ames test) CONSENSUS model 1.0.2.: Mutagenic with a consensus score of 0.3 for 4 models. This was confirmed with the OECD 471 test listed under GLP studies.
• The applicability of QSARs for higher tier mutagenicity endpoints is limited and therefore not relevant.
- In vitro methods:
The available in vitro methods have been considered as part of the tiered approach to testing, as specified in REACH Guidance 7a. Additional in vitro mutagenicity tests are deemed unnecessary because (i) the Ames test was positive, which necessitates further in vivo tests irrespective of other in vitro test results, (ii) the Ames test indicates gene mutation effects and no chromosome effects and (iii) neither Cu nor TEPA are classified for mutagenicity, making chromosome effects unlikely and further in vitro tests for chromosome effects unnecessary.
- Weight of evidence:
“Reaction product of copper sulfate and tetraethylenepentamine” was tested in vitro with the OECD 471 study and returned indications of mutagenicity. QSAR predictions also suggested the genotoxic potential of the substance. As outlined in the Integrated Testing Strategy (ITS) for mutagenicity (ECHA, 2017), an appropriate in vivo somatic genotoxicity study should be performed after any positive in vitro genotoxicity test. No relevant in vivo genotoxicity studies were identified for this or related substances. Following the ECHA testing strategy, an in vivo test is now required to conclude on the mutagenicity of this substance.
- Grouping and read-across:
Copper sulfate is not expected to have genotoxic effects but the genotoxic effects of tetraethylenepentamine (TEPA) are unclear. No suitable in-vivo tests are available for TEPA or similar substances to use as read-across information. The in-vivo test used in the TEPA registration dossier is a read-across from an in-vivo micronucleus test for Triethylenetetramine (TETA). The OECD 471 test for “Reaction product of copper sulfate and tetraethylenepentamine” suggest mutagenic effects while the micronucleus test is used to detect clastogenecity. Therefore, no suitable in-vivo tests have been identified to read-across from.

- Substance-tailored exposure driven testing [if applicable]:
Not applicable.
- Approaches in addition to above [if applicable]:
Not applicable.
- Other reasons [if applicable]:
Not applicable.

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
The substance is not classified for carcinogenicity or mutagenicity therefore genetic toxicity testing cannot be waived. As outlined in the Integrated Testing Strategy (ITS) for mutagenicity (ECHA, 2017), “if there is a positive result in any of the in vitro studies from Annex VII or VIII and there are no appropriate results available from an in vivo study already, an appropriate in vivo somatic cell genotoxicity study should be proposed”. No in vivo genotoxicity data were identified for this substance. The observed mutagenicity effects in bacteria therefore necessitate the consideration of further in vivo testing “as a last resort” (ECHA, 2016).

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
- Details on study design / methodology proposed:
To assess the potential to induce gene mutations in vivo, a transgenic rodent gene mutation assay (TGR; OECD Test Guideline 488) is proposed. As specified in the OECD 488 protocol, “an in vivo assay for gene mutations is useful for further investigation of a mutagenic effect detected by an in vitro system”. The principle of the TGR assay is to sample tissues of interest after an administration period, insert genomic DNA in bacterial hosts and subsequently detect mutations in these bacterial hosts. It is proposed to conduct the study in either mice or rats following oral gavage as this is the most likely route of exposure. The glandular stomach (site-of-contact) and the liver (systemic exposure) are proposed as focal tissues.

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 488 (Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays)

Test material

Test material information

Constituent 1

Reference substance name:

copper sulfate;N'-[2-(2-aminoethylamino)ethyl]ethane-1,2-diamine

EC Number:

701-399-0

Molecular formula:

C6H18N4CuSO4

IUPAC Name:

copper sulfate;N'-[2-(2-aminoethylamino)ethyl]ethane-1,2-diamine