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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: positive in one tester strain (Salmonella th. TA 1535) without metabolic activation

Mouse lymphoma assay: negative with/without metabolic activation

Micronucleus test: negative with/without metabolic activation

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (rat)

Test concentrations with justification for top dose:

3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

deionised water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (4-NOPD), 2-aminoanthracene (2-AA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: 1st main experiment: plate incorporation; 2nd main experiment: preincubation

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: determination of background lawn

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA 98, TA 100, TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Positive without metabolic activation in strain TA 1535
Negtaive without metabolic activation in strains TA 1537, TA 98, TA 100 and E. coli WP2 uvrA
Negative with metabolic activation all strains tested

The test item induced gene mutations by base pair changes in the genome of the strain TA 1535 in the absence of metabolic activation.

Executive summary:

This study was performed to investigate the potential of TAT (Triacetonetriamine) to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

3; 10; 33; 100; 333; 1000; 2500; and 5000µg/plate

The plates incubated with the test item showed normal background growth up to 5000 Ng/plate with and without metabolic activation in both independent experiments.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

A substantial and dose dependent increase in revertant colony numbers was observed following treatment with TAT (Triacetonetriamine) in strain TA 1535 in the absence of metabolic activation. The threshold of thrice the number of the corresponding solvent control was exceeded at 2500 Ng/plate. A dose dependent increase in revertant colony numbers was also observed in strain TA 100 without metabolic activation. However, the threshold of twice the number of the corresponding solvent control was not reached.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Additional information

Additional information from genetic toxicity in vitro:

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

Justification for classification or non-classification

Based on the currently available experimental in vitro data, no conclusion on the in vivo relevance of the positive results obtained in the Ames test can be made. Re-evaluation of the classification will be performed after in vivo data has become available.