1,6-Hexandiammonium dihydroxide, N,N'-bis-(2-hydroxypropyl)-N,N,N',N'-tetramethyl

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 28 JUN 2002 to 23 SEP 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

442-730-6

EC Name:

-

Cas Number:

35132-93-5

Molecular formula:

C16H38N202.2H0

IUPAC Name:

(2-hydroxypropyl)({6-[(2-hydroxypropyl)dimethylazaniumyl]hexyl})dimethylazanium propane-1,2-diol dihydroxide

impurity 1

Reference substance name:

Propane-1,2-diol

EC Number:

200-338-0

EC Name:

Propane-1,2-diol

Cas Number:

57-55-6

Molecular formula:

C3H8O2

IUPAC Name:

propylene glycol

impurity 2

Reference substance name:

N,N,N',N'-tetramethylhexamethylenediamine

EC Number:

203-842-9

EC Name:

N,N,N',N'-tetramethylhexamethylenediamine

Cas Number:

111-18-2

Molecular formula:

C10H24N2

IUPAC Name:

N,N,N',N'-tetramethylhexane-1,6-diamine

Test material form:

liquid

Details on test material:

Water added up to 100 %.

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: stock cultures in the bank of "Genetic Toxicology", Aventis Pharma Germany, ProTox, prepared from the original bacterial strains
- Methods for maintenance in cell culture if applicable:
- Modal number of chromosomes: approx. 37 °C in an incubator

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: stock cultures in the bank of "Genetic Toxicology", Aventis Pharma Germany, ProTox, prepared from the original bacterial strains
- Methods for maintenance in cell culture if applicable:
- Modal number of chromosomes: approx. 37 °C in an incubator

Metabolic activation:

with and without

Metabolic activation system:

induced rat liver S9 mix (induced with Aroclor 1254)

Test concentrations with justification for top dose:

plate incorporation test: 0, 119, 381, 1190, 3810 and 11905 µg/plate; corresponding to 0, 50, 160, 500, 1600 and 5000 µg active ingredient/plate
preincubation test: 0, 38.1, 119, 381, 1190, 3810 and 11905 µg/plate; corresponding to 0, 16, 50, 160, 500, 1600 and 5000 µg active ingredient/plate

Vehicle / solvent:

Deionized water

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

for strain TA 100 and TA 1535, without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

for strain TA1537, without metabolic activation

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

for strain TA 98, without metabolic activation

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

for strain WP2uvrA, without metabolic activation

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other:

Remarks:

for strain WP2uvrA, with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Experiment 1) plate incorporation assay
Experiment 2) preincubation assay

DURATION
- Preincubation period: 37 °C for 1 h
- Exposure duration: 48 h at 37 °C

NUMBER OF REPLICATIONS: 3 replicates per concentration

Rationale for test conditions:

Test conditions as described in the guideline

Evaluation criteria:

Criteria for a valid assay
The assay is considered valid if the following criteria are met:
- the solvent control data are within the laboratory's normal control range for the spontaneous mutant frequency
- the positive controls induce increases in the mutation frequency which are significant and within the laboratory's normal range

Criteria for a positive response
A test compound is classified as mutagenic if it has either of the following effects:
a) it produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
b) it induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test compound at complete bacterial background lawn
If the test substance does not achieve either of the above criteria, it is considered to show no evidence of mutagenic activity in this system.

Statistics:

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

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 1600 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 1600 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 1600 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 1600 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: results from experiment I and II

Any other information on results incl. tables

Toxic effects, evident as incomplete or no bacterial lawn, were observed at the following concentrations of active ingredient:

 

Strain	Experiment I [μg/ plate]		Experiment I [μg/ plate]
	Without S9 mix	With S9 mix	Without S9 mix	With S9 mix
TA 100	> 1600	> 1600	> 1600	5000
TA 1535	> 1600	5000	> 1600	5000
TA 1537	> 1600	5000	> 1600	5000
TA 98	> 1600	5000	> 1600	5000
WP2uvrA	5000	5000	5000	5000

Applicant's summary and conclusion

Conclusions:

In a guideline study according to OECD TG 471 under GLP conditions, the test item showed no mutagenic activity in a plate incorporation as well as a preincubation experiment with and without metabolic activation.

Executive summary:

In a guideline study according to OECD TG 471 under GLP conditions 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 and without metabolic activation at concentrations of 0, 50, 160, 500, 1600 and 5000 μg/plate (active ingredient). The first experiment was conducted as a plate-incorporation test, the second as a preincubation test.

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