Reaction mass of 7-azatridecane-1,13-diamine and hexamethylenediamine

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:

From 9 APR 2002 to 30 MAY 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline Study (OECD TG 471)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Remarks:

in compliance e.g. with the U.S. FDA GLP Regulations as published in 21 CFR 58, the U.S. EPA GLP STandards 40 CFR 160, and 40 CFR 792 (with some minor exceptions, e.g. stability of test and control item not tested)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

bis(6-aminohexyl)amine; hexane-1,6-diamine

EC Number:

907-605-7

Cas Number:

68815-47-4

Molecular formula:

C6H16N2 (HMD) C12H29N3 (BHT)

IUPAC Name:

bis(6-aminohexyl)amine; hexane-1,6-diamine

Details on test material:

- Name of test material (as cited in study report):1,6-Hexandiamine, N-(6-aminohexyl) or H-25264; BHMT amine
- Physical state: liquid

Method

Target gene:

histidine auxotroph bacterial strains

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S9 (10% (v/v))

Test concentrations with justification for top dose:

Preliminary test (vehicle DMSO): 6.7, 10, 33, 67, 100, 333, 667, 1000, 3333 and 5000 µg/plate
Experiments were done with and without metabolic activation system

Final Mutagenicity tests (vehicle ethanol):
Experiment B1: 25, 75, 200, 600, 1800 and 5000 µg/plate
All tester strains with and without metabolic activation (10% (v/v) rat liver S9-mix)

Experiment B2: 25, 75, 200, 600, 1800, 2500 and 5000 µg/plate
Strain TA 100 and TA1537 with metabolic activation (10% (v/v) rat liver S9-mix)

Experiment B3 and 4: 25, 75, 200, 600, 1800, 2500, 3333 and 5000 µg/plate
Strain TA1537 with metabolic activation (B3: 5% (v/v) rat liver S9-mix; B4: 10% (v/v) rat liver S9-mix ).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:
Preliminary test: DMSO
Final Mutagenicity tests: ethanol
- Justification for choice of solvent/vehicle: while DMSO would allow the bacterial mutation test to be conducted at the regulatory-required top dose of 5 mg per plate the regulatory -required top doses could not be achieved in the other genetic toxicology assays when using DMSO. For the reason of comparability with other tests, ethanol was subsequently selected as the solvent of choice based on compatibility with the target cells and solubility of the test substance. The test substance was soluble and clear in ethanol at approx. 500 mg/ml, the maximum concentration tested .

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

i.e. DMSO in preliminary test and ethanol in mutagenicity tests

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide (TA 1535 and TA 100), 2-Nitrofluorene (TA 98), 9-aminoacridine (TA 1537), methyl methane sulfonate (WP2 uvrA)

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

i.e. DMSO in preliminary test and ethanol in mutagenicity tests

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation (rat liver S9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: at least 48 hours, up to 72 hours at 37+/-2 °C

NUMBER OF REPLICATIONS: 3 plates per strain and dose level, including the controls

DETERMINATION OF CYTOTOXICITY
The condition of the bacterial background lawn was evaluated for evidence of test substance toxicity by using a dissecting microscope. Precipitate was evaluated by using visual examination without magnification. Toxicity and degree of precipitation were scored relative to the behicle control plate.

Evaluation criteria:

For the test item to be considered positive a dose dependent increase in mean revertants per plate of at least one tester strain over a minimm of two increasing concentrations must be seen.
Moreover the test item is considered as a mutagen if a biologically relevant increase in the number of revertants equaling or exceeding the threshold of twice (strains TA 98, TA 100, WP2 uvrA) or thrice (strains TA 1535, TA 1537) the colony count of the corresponding solvent colony is observed.

Statistics:

Arithmetic means and standard deviation of the counted colonies were calculated.

Results and discussion

Test results

Species / strain:

other: S. typhimurium TA 1535, TA 98, TA 100 and TA 1537, E. coli WP2uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

beginning at 1800 µg per plate up to the highest dose tested

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Non-dose responsive increases (2.4 -fold and 2.9 -fold) were observed with tester strains TA 1537 with S9 mix. To clarify these responses, a retest was conducted using 5 % and 15 % rat liver S9 mix. No positive response was observed in either restest.

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 and without metabolic activation

Under the test conditions the test item did not exert mutagenic activity in the reverse bacterial mutation assay with and without metabolic activation.

Executive summary:

Mutagenic activity of the test item was investigated in Salmonella typhimurium strains TA 1535, TA 1537, TA98 and TA100 as well as Escherichia coli strain WP2 uvrA with (induced rat liver S9 mix at 10 % (v/v)) and without metabolic activation at concentrations of 25, 75, 200, 600, 1800 and 5000 µg/plate in the plate incorporation assay (according to OECD471, GLP, vehicle ethanol).

Restesting of strain TA1537 with metabolic activation at various S9 mix concentrations (i.e. 5, 10 or 15 % (v/v); experiuments B2, B3 or B4 respectively) was done at 25, 75, 200, 600, 1800, 2500, 3333, and 5000 µg/plate (Experiment B2 without concentration 3333 µg/plate) also in the plate incorporation assay. Toxicity was observed beginning at 1800 µg per plate up to the highest dose tested in all strains. No precipitate was observed.

The test item did not reveal any mutagenic activity under the conditions tested. The appropriate reference mutagenes showed distinct positive mutagenic effects.