N,N-dimethyl-N'-(2,2,6,6-tetramethylpiperidin-4-yl)propane-1,3-diamine

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian germ cell study: gene mutation

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study planned

Study period:

The study will be conducted after a decision on the requirement to carry out the proposed test has been taken according to the procedure laid down in Regulation (EC) 1907/2006, and a deadline to submit the information required has been set by the Agency.

Justification for type of information:

TESTING PROPOSAL ON VERTEBRATE ANIMALS

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out: Triacetonetriamine

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Available GLP studies: none concerning in vivo gene mutation
- Available non-GLP studies: none in vivo gene mutation
- Historical human data: none in vivo gene mutation
- (Q)SAR: none concerning in vivo gene mutation
- In vitro methods: no valid methods availablein vivo gene mutation
- Weight of evidence: no data available
- Grouping and read-across: no data on strucutral analogues available

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
Please refer to principles of method if other than guideline

Materials and methods

Test guideline

Qualifier:

no guideline available

Guideline:

other: standard protocols of PigA assay

Principles of method if other than guideline:

According to Regulation (EC) No. 1907/2006, Annex VIII, point 8.4, appropriate in vivo mutagenicity studies shall be considered in case of a positive result in any of the genotoxicity studies in Annex VII or VIII. In the Ames test with triacetonetriamine positive results were obtained in one test strain without metabolic activation. The test item induced gene mutations by base pair changes in the genome of the strain TA 1535 in the absence of metabolic activation. In the available cell mutation assay in mouse lymphoma cells no mutagenic activity with or without metabolic activation was observed. The available in vitro micronucleus test in human lymphocytes also showed clearly negative results.
The registrant has decided to propose an appropriate test in vivo to clarify the genotoxicity observed in vitro. The positive result occured in an in vitro mutagenicity study, therefore the in vivo test needs to focus on mutagenicity as the genotoxic mode of action.
For animals welfare reasons it would be preferable to include the in vivo mutagenicity assessment into another in vivo study which needs to be proposed to fulfil the standard data requirements. The tonnage band of 100 tonnes/year is exceeded. Thus, the performance of a sub-chronic (90 days, OECD TG 408) toxicity study is being proposed according to Annex IX, 8.6.2. The registrant has been evaluated the possibilities to include the in vivo mutagenicity assay into the subchronic toxicity study.
A novel assay, the in vivo PIG-A (endogenous X-linked phosphatidylinositol glycan, Class A, Pig-a in rodents and PIG-A in humans) gene mutation assay, shows promise for regulatory applications as a reporter of in vivo mutation, by integration of gene mutation measurement into repeat-dose toxicology studies. [1, 7] The measurement of PIG-A mutants by counting cells with the GPI-negative (glycosylphosphatidyl inositol) phenotype has proved to be effective to measure mutant frequency in peripheral blood cells of humans and of others animals. [2] Currently the PIG-A assay has been extended for its use in human erythrocytes. Its advantages are the applicability across species, its simplicity and statistical power, and the relatively non-invasive nature. [3] A collaborative international trial was conducted to evaluate the reproducibility and transferability of an in vivo mutation assay based on the enumeration of CD59-negative rat erythrocytes across 14 laboratories. The methodology was demonstrated to be reproducible and a good transferability was evident from the similar kinetics and magnitude of the dose-related responses that were observed among different laboratories. The results of the trial demonstrate that the method is a robust in vivo mutation assay that is readily transferable across laboratories. [4] The erythrocyte-based Pig-a mutation assay was successfully used to assess the ability of to discriminate between genotoxic and non-genotoxic modes of action, for methyl carbamate (MC) and ethyl carbamate (EC). [8] It has been demonstrated recently that the utility and sensitivity of the Pig-a in vivo gene mutation assay, can be easily integrated, along with other standard genotoxicity endpoints, into e.g. 28-day rodent toxicity studies. [5] It has been concluded that the PIG-A assay could be a useful and sensitive endpoint for a repeat dose protocol and complements other genotoxicity endpoints. [6]
A committee entitled “Relevance and Follow-up of Positive Results from In Vivo Genetic Toxicity Testing” (IVGT), initiated by the Health and Environmental Sciences Institute (HESI), recommends the Pig-a assay (in combination with the micronucleus assay (MN)) in repeat-dose toxicity studies “as it would allow for the first time the simple assessment of aneugenicity, clastogenicity, and mutagenicity” [9]
The US EPA has concluded that although the Pig-A gene mutation assay does not have an OECD Test guideline yet, it is a promising new in vivo mutation test. US EPA encourage the incorporation of genotoxicity endpoints into routine toxicology studies where scientifically feasible. [http://www.epa.gov/pesticide-science-and-assessing-pesticide-risks/advances-genetic-toxicology-and-integration-vivo]
Taking into account the relatively simple integration of the PIG-A assay into repeated-dose toxicity studies, the low volume of blood needed, the reduction of animal use and the fact that no expensive transgenic animals are required, the consideration of the PIG-A assay being a suitable methodology for the in vivo verification of a mutagenic potential observed in vitro is reasonable.

Therefore the registrant proposes to perform a PIG-A assay as an integral part of the also proposed subchronic oral repeated-dose toxicity study (OECD 408) according to Annex IX, 8.6.2 in order to verify the positive results obtained in the Ames test and fulfilling the data requirements according to Regulation (EC) No. 1907/2006, Annex VIII, point 8.4.

[1] Dobrovolsky VN et al., The in vivo Pig-a gene mutation assay, a potential tool for regulatory safety assessment, Environ Mol Mutagen. 2010 Oct-Dec;51(8-9):825-35,
[2] Peruzzi et al., The use of PIG-A as a sentinel gene for the study of the somatic mutation rate and of mutagenic agents in vivo, Mutat Res. 2010 Jul-Sep;705(1):3-10,
[3] Dertinger SD et al., Human erythrocyte PIG-A assay: an easily monitored index of gene mutation requiring low volume blood samples, Environ Mol Mutagen. 2015 May;56(4):366-77,
[4] Dertinger SD et al., International Pig-a gene mutation assay trial: evaluation of transferability across 14 laboratories, Environ Mol Mutagen. 2011 Dec;52(9):690-8,
[5] Stankowski LF et al., Integration of Pig-a, micronucleus, chromosome aberration and comet assay endpoints in a 28-day rodent toxicity study with urethane, Mutagenesis. 2015 May;30(3):335-42,
[6] Gunther WC et al., Evaluation of the Pig-a, micronucleus, and comet assay endpoints in a 28-day study with ethyl methanesulfonate, Environ Mol Mutagen. 2014 Jul;55(6):492-9,
[7] Bhalli JA et al., Sensitivity of the Pig-a assay for detecting gene mutation in rats exposed acutely to strong clastogens, Mutagenesis (2013),
[8] Bemis JC et al., Rat Pig-a mutation assay responds to the genotoxic carcinogen ethyl carbamate but not the non-genotoxic carcinogen methyl carbamate, Mutagenesis (2015)
[9] Schuler M et al., Need and potential value of the Pig-a in vivo mutation assay – a HESI perspective, Environ. Mol. Mutagen., 52 (2011), pp. 685–689

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: PigA assay

Test material

Test material information

Constituent 1

Reference substance name:

N,N-dimethyl-N'-(2,2,6,6-tetramethylpiperidin-4-yl)propane-1,3-diamine

EC Number:

278-817-9

EC Name:

N,N-dimethyl-N'-(2,2,6,6-tetramethylpiperidin-4-yl)propane-1,3-diamine

Cas Number:

78014-16-1

Molecular formula:

C14H31N3

IUPAC Name:

N-[3-(dimethylamino)propyl]-2,2,6,6-tetramethylpiperidin-4-amine

Test material form:

other: liquid

Test animals

Species:

rat

Administration / exposure

Route of administration:

oral: gavage