1,3,3-trimethyl-N-(2-methylpropylidene)-5-[(2-methylpropylidene)amino]cyclohexanemethylamine

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:

2011-06-24 to 2011-08-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study; GLP study without deviations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2005

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: ICH Guideline S2A:'Genotoxicity: Specific Aspects of Regulatory gentoxocoty tests for Pharmaceuticals (CPMP/ICH/141/95)'

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: ICH Giudeline S2B: 'A Standard Battery for Genotoxicity Testing of Pharmaceuticals (CPMP/ICH/174/95)'

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1,3,3-trimethyl-N-(2-methylpropylidene)-5-[(2-methylpropylidene)amino]cyclohexanemethylamine

EC Number:

259-393-4

EC Name:

1,3,3-trimethyl-N-(2-methylpropylidene)-5-[(2-methylpropylidene)amino]cyclohexanemethylamine

Cas Number:

54914-37-3

Molecular formula:

C18H34N2

IUPAC Name:

1,3,3-Trimethyl-N-(2-methylpropylidene)-5-[(2-methylpropylidene)amino]cyclohexanemethanamine

Test material form:

other: liquid

Details on test material:

Dialdimine 98.30%
Isophorondiamine 0.42%
Monoaldimine 0.47%
Isobutyraldehyde 0.11%
Unidentified 0.70%

Method

Target gene:

mutated gene loci resposible for histidine auxotropy

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

other: histidine auxotroph

Metabolic activation:

with and without

Metabolic activation system:

Arochlor 1254 induced rat liver S9; male rats

Test concentrations with justification for top dose:

Experiment I and II:
10.0, 31.6, 100, 316, 1000 and 3160 µg per plate

Vehicle / solvent:

dimethyl sulfoxide (DMSO) , a factor of 1.017 was used to correct for impurities

Controls

Untreated negative controls:

no

Remarks:

solvent test will be used as negative reference item

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

for details see below

Positive control substance:

other: without metabolic activation: sodium azide in aqua ad iniectabilia for TA 1535 and TA 100, 2-nitroflurene in DMSO for TA 98, 9-amino-acridine in ethanol abs. for TA 1537, Cumene hydroperoxide in DMSO for TA 102

Remarks:

with metabolic activation: 2-aminoanthracene in DMSO for TA 98, TA 102, TA 1537, cyclophosphamide in aqua ad iniectabilia for TA 100, TA 1535

Details on test system and experimental conditions:

Bacterial Reverse Mutation Test
SYSTEM OF TESTING
- Pre-Experiment: plate incorporation cytotoxicity test (+/- metabolic activation) with strain TA 100,
10 concentrations ranging from 0.316 to 5000 µg/plate were tested, pronounced cytotoxicity (scarce background lawn and reduction of the
number of revertants by more than 50%) was noted at concentrations of 3160 and 5000 µg/plate (see tables 1 and 2). Hence, 3160 µg
test item/plate was chosen as top concentration for the main study in the plate incorporation test and in the preincubation test, respectively.
- Main test: 1st - Standard plate incorporation method, 2nd - Preincubation method
- Metabolic activation assay: Arochlor 1254 induced rat liver S9 fraction, collected from 20 - 30 rats, the protein content of the S9 fraction was 34.5 mg/mL S9, cytochrome P-450: 0.43 nmol/mg protein.
ADMINISTRATION
- Dosing: 10.0, 31.6, 100, 316, 1000 and 3160 µg per plate
- Data : 2 independent experiments with and without metabolic activation
- Number of replicates: 3 per concentration and experiment
- Positive and negative control groups and treatment:
- without metabolic activation:
10 µg/plate sodium azide in aqua ad iniectabilia for TA 1535 and TA 100
10 µg/plate 2-nitroflurene in DMSO for TA 98
100 µg/plate 9-amino-acridine in ethanol abs. for TA 1537
10 µg/plate cumene hydroperoxide in DMSO for TA 102
- with metabolic acivation
2 µg/plate 2-aminoanthracene in DMSO for TA 98, TA 102, TA 1537
1500 µg/plate cyclophosphamide in aqua ad iniectabilia for TA 100, TA 1535
- negative control: solvent control: DMSO
- Incubation time: 48 h to 72 h at 37 °C in the dark
- Pre-incubation time: 20 min at 37 °C;

Evaluation criteria:

The test item is considered to show a positive response if:
- the number of revertants is significantly increased (p ≤ 0 .05, U-test according to MANN and WHITNEY) compared to the solvent control to at least
2-fold of the solvent control for TA 98, TA 100 and TA 102 and 3-fold of the solvent control for TA 1535 and TA 1537 in both independent
experiments.
- additionally, a significant (p ≤ 0.05) concentration (log value)-related effect (Spearman's rank correlation coefficient) is observed.
- positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking random samples on
histidine-free agar plates.

Statistics:

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

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxicity was noted at concentrations of 3160 and 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

GENTOXIC EFFECTS:
- With metabolic activation: negativ
- Without metabolic activation: negativ

CYTOTOXICITY EFFECTS:
- Cytotoxicity was noted at concentrations of 3160 and 5000 µg/plate

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

The following chemicals served as positive control items:

a) without metabolic activation
sodium azide^[1]inaqua ad iniectabilia^[2] (10 µg/plate)	TA 1535, TA 100
2-nitro-fluorene^[3]in DMSO^[4] (10 µg/plate)	TA 98
9-amino-acridine^3in ethanol, abs.^[5] (100 µg/plate)	TA 1537
Cumene hydroperoxide^[6] in DMSO^6(10 µg/plate)	TA 102
b) with metabolic activation
2-amino-anthracene^3in DMSO^6 (2 µg/plate)	TA 98, TA 102, TA 1537
cyclophosphamide^3inaqua ad iniectabilia^4 (1500 µg/plate)	TA 100, TA 1535

^[1]     SIGMA-ALDRICH Chemie GmbH, 82024 Taufkirchen, Germany

^[2]     DeltaSelect GmbH, 63303 Dreieich, Germany

^[3]     Riedel de Haën AG, 30926 Seelze, Germany

^[4]     DMSO, spectrometric grade; E. MERCK, 64293,

^[5]     Ethanol spectrometric grade; E. MERCK, 64293,

^[6]     E. MERCK, 64293,

Applicant's summary and conclusion

Conclusions:

In conclusion, under the present test conditions 1,3,3-trimethyl-N-(2-methylpropylidene)-5-[(2-methylpropylidene)amino]cyclohexane-
methylamine tested up to a cytotoxic concentration of 3160 µg/plate, caused no mutagenic effect in the Salmonella typhimurium strains TA 98,
TA 100, TA 102, TA 1535 and TA 1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with
metabolic activation.

Executive summary:

The purpose of this study was to evaluate 1,3,3 -Trimethyl-N-(2-methylpropylidene)-5-[(2-methylpropylidene)amino]cyclohexane-

methylamine for mutagenic activity (gene mutation) in bacteria without and with the addition of a mammalian metabolic activation system as originally described by AMES et al. (1973, 1975) and revised by MARON and (1983).

The test item was examined in the 5 Salmonella typhimuriumstrains TA 98, TA 100, TA 102, TA 1535 and TA 1537 in two independent experiments, each carried out without and with metabolic activation (a microsomal preparation derived from Aroclor 1254-induced rat liver). The first experiment was carried out as a plate incorporation test and the second as a preincubation test.

The test item was completely dissolved indimethyl sulfoxide (DMSO). A factor of 1.017 was used to correct for impurities.

Preliminary test

The test item was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA 100. Ten concentrations ranging from 0.316 to 5000 µg/plate were tested. Pronounced cytotoxicity

(scarce background lawn and reduction of the number of revertants by more than 50%) was noted at concentrations of 3160 and 5000 µg/plate.

Hence, 3160 µg test item/plate was chosen as top concentration for the main study in the plate incorporation test and in the preincubation test, respectively.

Main study

Six concentrations ranging from 10.0 to 3160 µg test item/plate were employed in two independent experiments each carried out without and with metabolic activation.

Cytotoxicity

In the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation cytotoxicity (scarce background lawn and reduction of the number of revertants) was noted at the top concentration of 3160 µg/plate in all test strains.

Mutagenicity

No mutagenic effect (no increase in revertant colony numbers as compared with control counts) was observed for the test item, tested up to a cytotoxic concentrationof 3160 µg/plate,in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation and preincubation test).

In conclusion, under the present test conditions the test item tested up to a cytotoxic concentration of 3160 µg/plate, caused no mutagenic effect in the Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.