N,N'-bis(2,2,6,6-tetramethylpiperidin-4-yl)hexane-1,6-diamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07.07.2015 to 04.09.2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-bis(2,2,6,6-tetramethylpiperidin-4-yl)hexane-1,6-diamine

EC Number:

262-679-1

EC Name:

N,N'-bis(2,2,6,6-tetramethylpiperidin-4-yl)hexane-1,6-diamine

Cas Number:

61260-55-7

Molecular formula:

C24H50N4

IUPAC Name:

N1,N6-bis(2,2,6,6-tetramethylpiperidin-4-yl)hexane-1,6-diamine

Test material form:

solid: flakes

Method

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Periodically checked with regard to membrane permeability (rfa), UV sensitivity (uvrA and uvrB), ampicillin resistance (amp), as well as spontaneous mutation frequencies.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

5000; 2500; 1000; 316; 100; 31.6 and 10 µg/plate (Range finding test)
5000; 1581; 500; 158.1; 50; 15.81; 5 and 1.581 µg/plate (Main tests)

Vehicle / solvent:

Based on the results of a solubility test, the test item was formulated in Acetone.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

4 µg/plate

Positive control substance:

other: 4-nitro-1,2-phenylene-diamine (NPD)

Remarks:

TA98 without activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Distilled water

Positive controls:

yes

Remarks:

2 µg/plate

Positive control substance:

sodium azide

Remarks:

TA100 and TA1535 without activation

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

50 µg/plate

Positive control substance:

9-aminoacridine

Remarks:

TA1537 without activation

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Distilled water

Positive controls:

yes

Remarks:

2 µL/plate

Positive control substance:

methylmethanesulfonate

Remarks:

WP2 uvrA without activation

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

2 µg/plate

Positive control substance:

other: 2-aminoanthracene

Remarks:

all Salmonella strains with activation

Controls 6

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

50 µg/strain

Positive control substance:

other: 2-aminoanthracene

Remarks:

WP2 uvrA with activation

Details on test system and experimental conditions:

Preliminary Concentration Range Finding Test and Initial Mutation Test:
- plate incorporation method
- incubation: 48+/-1 hours at 37°C (plates)
Confirmatory Mutation Test:
- pre-incubation method
- incubation: 20 at 37°C (direct contact between bacteria and test item), 48+/-1 hours at 37°C (plates)
In the main tests, each sample (including the controls) was tested in triplicate.

Evaluation criteria:

Criteria for Validity:
The study was considered valid if:
- the number of revertant colonies of the negative (vehicle/solvent) and positive controls were in the historical control range in all strains of the main tests;
- at least five analyzable concentrations were presented in all strains of the main tests.

Criteria for a Positive Response:
A test item was considered mutagenic if:
- a dose–related increase in the number of revertants occurred and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.

An increase was considered biologically relevant if:
- the number of reversions was more than two times higher than the reversion rate of the negative (vehicle/solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
- the number of reversions was more than three times higher than the reversion rate of the negative (vehicle/solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.

Criteria for a Negative Response:
A test item was considered non-mutagenic if it produced neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Cytotoxicity (absent/reduced/slightly reduced background lawn development and pinpoint colonies, in some cases reduced revertant counts):
- Initial Mutation Test in S. typhimurium TA98, TA100 and TA1537 bacterial strains at 5000 μg/plate with and without metabolic activation and in Escherichia coli WP2 uvrA strain at 5000 μg/plate without metabolic activation;
- Confirmatory Mutation Test in all examined bacterial strains at 5000 and 1581 μg/plate with and without metabolic activation; and in S. typhimurium TA 98, TA100 and TA1535 strains at 500 μg/plate without metabolic activation.

Higher numbers of revertant colonies compared to the vehicle control were detected in the main test in some sporadic cases. However, no dose-dependence was observed in those cases and they were below the biologically relevant threshold value.
The numbers of revertant colonies were within the historical control range in each case, so they were considered as reflecting the biological variability of the test.

No precipitate was detected on the plates in the main tests in any examined bacterial strains with and without metabolic activation.

The examined concentration range was considered to be adequate as the highest examined concentration was the highest recommended concentration (5000 μg/plate). At least five analyzable concentrations were presented in all strains with and without metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

In conclusion, the test item Hexamethylene-bis-triacetone diamine (HMBTAD) had no mutagenic activity in the examined bacterial strains with and without metabolic activation under the test conditions of this study.