1,3-Cyclohexanedimethanamine, N1,N3-bis(2-methylpropylidene)-

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:

2010-11-09 to 2011-01-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is conducted in accordance with GLP regulations and OECD/EU guideline.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

31 May 2008

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

August 1998

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: – ICH Guidance S2A: Guidance on Specific Aspects of Regulatory Genotoxicity Tests For Pharmaceuticals, 1996 – ICH Guidance S2B: Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals, 1997

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:

(2-methylpropylidene)[(3-{[(2-methylpropylidene)amino]methyl}cyclohexyl)methyl]amine

EC Number:

619-764-7

Cas Number:

173904-11-5

Molecular formula:

C16H30N2

IUPAC Name:

(2-methylpropylidene)[(3-{[(2-methylpropylidene)amino]methyl}cyclohexyl)methyl]amine

Test material form:

other: liquid

Method

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- to his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA 1535, TA 100) and frameshift (TA 1537, TA 98) mutations. The Escherichia coli WP2 uvrA (trp) reversion system measures trp– to trp+ reversions. The Escherichia coli WP2 uvrA detect mutagens that cause other base-pair substitutions (AT to GC).

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:

Phenobarbital (PB) and ß-naphthoflavone (BNF) induced rat liver S 9-mix

Test concentrations with justification for top dose:

5000, 1581, 500, 158, 50 and 15.8 μg/plate

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: good solubility in DMSO (standard vehicle as recommended by guideline)

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

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

Remarks:

Without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without metabolic activation

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without metabolic activation

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2AA)

Remarks:

With metabolic activation

Details on test system and experimental conditions:

Standard plate incorporation procedure was performed, as an initial mutation test. Bacteria (cultured in Nutrient broth No.2) were exposed to the test item, both in the presence and absence of an appropriate metabolic activation system.
Molten top agar was prepared and kept at 45 °C. 2 mL of top agar was aliquotted into individual test tubes (3 tubes per controls or concentration levels). The equivalent number of minimal glucose agar plates was properly labelled. The test item and other components were prepared fresh and added to the overlay (45 °C).
This solution was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 Mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions (without S9 Mix and with addition of S9 Mix) and each of them with the addition of negative and positive controls.
For the pre-incubation method, before overlaying the test item, the bacterial culture and the S9 Mix or phosphate buffer was added into the appropriate tubes, providing direct contact between bacteria and the test item (in its solvent). These tubes were gently mixed and incubated for 20 min at 37 ºC using a shaker. After the incubation the content of the tubes was added to the molten top agar prior to pouring onto the surface of minimal agar plates.
After solidification the plates were inverted and incubated at 37 °C for at least 48 hours in the dark.

Evaluation criteria:

A test item is considered mutagenic if:
- a dose-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in strain TA 100 the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain TA 98, TA 1535, TA 1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.
Criteria for a Negative Response:
A test article is considered non-mutagenic if it produces 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.

Statistics:

NA

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

Five bacterial strains, Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2 uvrA were used to investigate the mutagenic potential of Incorez 397 in two independent experiments, in a plate incorporation test (experiment I, Initial Mutation Test) and in a pre-incubation test (experiment II, Confirmatory Mutation Test). In general, the pre-incubation method is more sensitive than the plate incorporation assay. Each assay was conducted with and without metabolic activation (S9 Mix). The concentrations, including the controls, were tested in triplicate (positive and negative controls were run concurrently). Following concentrations of Incorez 397 were tested in experiment I (the concentrations were chosen based on results obtained in the pre-experiment for toxicity): 5000; 1581; 500; 158; 50 and 15.8 μg/plate.

In the Initial Mutation Test the revertant colony numbers were higher than the revertant colony numbers of the vehicle control plates, furthermore the obtained higher revertant counts were above the corresponding historical control data ranges in the case of S. typhimurium TA 98 at 5000 μg/plate, without metabolic activation (-S9 Mix), and in E. coli WP2 uvrA at 5000 μg/plate (±S9 Mix) and at 1581 μg/plate (-S9 Mix). Higher revertant counts were obtained, however within the historical control data ranges in S. typhimurium TA 100, at the concentration of 5000 μg/plate (-S9 Mix), in TA 98 at 158 μg/plate (-S9 Mix), and in E. coli WP2 uvrA at 1581 μg/plate (+S9 Mix). The higher revertant colony counts remained the threshold for being positive in all cases.

In the Initial Mutation Test lower revertant colony counts were observed in the corresponding historical control data ranges in S. typhimurium TA 98 at the concentration range of 1581-50 μg/plate (+S9 Mix), in TA1535 at the concentration of 500 μg/plate (±S9 Mix), in TA 1537 at the concentration range of 1581-15.8 μg/plate (-S9 Mix) and at 1581 and 15.8 μg/plate (+S9 Mix). The revertant colony numbers were lower than the revertant colony numbers of the vehicle control plates and below the historical control data range in S. typhimurium TA 100 at 15.8 μg/plate (-S9 Mix).

The test item concentrations of Incorez 397 tested in experiment II were the same as in the experiment I. The test concentrations were chosen based on results of the experiment I after discussion with the sponsor. In the Confirmatory Mutation Test the revertant colony numbers were higher than the revertant colony numbers of the vehicle control plates in S. typhimurium TA 98, at the concentration range of 1581-158 μg/plate (-S9 Mix), in TA 1537 at the concentration range of 1581-15.8 μg/plate (-S9 Mix). These revertant colony number increases remained in the historical control data ranges. The revertant colony number increases were above the historical control data ranges in E. coli WP2 uvrA, at 500 μg/plate (-S9 Mix) and at 1581 μg/plate (+S9 Mix). The revertant colony numbers were in the range of the vehicle control, however above the historical control data range in the case of E. coli WP2 uvrA at 1581 μg/plate (-S9 Mix).

In the Confirmatory Mutation Test unequivocal inhibitory toxic effect of the test item was observed (indicated by lower revertant colony numbers than the revertant colony numbers of the vehicle control plates, below the corresponding historical control data ranges and/or reduced or slightly reduced background lawn development) in S. typhimurium TA 100 and in E. coli WP2 uvrA at the concentration of 5000 μg/plate (±S9 Mix), in TA 98 (+S9 Mix). The revertant colony numbers were slightly lower than the revertant colony numbers of the vehicle control (however within the historical control data range) and slightly reduced background lawn development was observed in S. typhimurium TA 1535, at 5000 μg/plate (±S9 Mix), furthermore at 1581 and 500 μg/plate (+S9 Mix). No revertant growth and reduced (or slightly reduced) background lawn development was obtained at 5000 μg/plate in S. typhimurium TA 98 (-S9 Mix) and in TA 1537 (±S9 Mix). All of the further observed lower revertant colony counts (compared to the revertant colony numbers of the vehicle control plates) remained in the historical control data ranges: in S. typhimurium TA 98, at the concentrations of 158 and 15.8 μg/plate (+S9 Mix), in TA 100, at the concentration of 1581 μg/plate (-S9 Mix), in TA 1535, at 1581 μg/plate (-S9 Mix), at 158 and 50 μg/plate (+S9 Mix), furthermore in E. coli WP2 uvrA at 15.8 μg/plate (-S9 Mix).

The significantly higher mutation rates (above the corresponding historical control data ranges) were mostly unique values and did not follow with clear dose-related tendency. In the performed experiments the highest revertant colony number increase over the spontaneous rate of the vehicle control plates was observed in S. typhimurium TA1537 in the Confirmatory Mutation Test, at the concentration of 1581 μg/plate without metabolic activation (-S9 Mix). The highest mutation rate was: 2.64. This high rate was accompanied with dose-related tendency, however remained in the historical control data range, and far below from the biologically relevant threshold for being positive.

The revertant colony numbers of vehicle control (Dimethyl sulfoxide: DMSO) plates with and without S9 Mix were within the corresponding historical control data ranges in both experiments (Initial and Confirmatory Mutation Test). The reference mutagen treatments (positive controls) showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in all tester strains.

The revertant colony numbers of the untreated and Distilled water control plates in the different experimental phases were slightly higher or lower than the DMSO control plates. The higher or lower revertant counts of these controls remained in the historical control data ranges.

Applicant's summary and conclusion

Conclusions:

The test item Incorez 397 was tested for mutagenic activity in a bacterial reverse mutation assay wit five strains of S. typhimurium and E. coli with and without metabolic activation. The reported data of this mutagenicity assay shows, that under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the strains used. Therefore, the test item Incorez 397 is considered non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

Five bacterial strains, Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2 uvrA were used to investigate the mutagenic potential of Incorez 397 in two independent experiments, in a plate incorporation test (experiment I, Initial Mutation Test) and in a pre-incubation test (experiment II, Confirmatory Mutation Test). Each assay was conducted with and without metabolic activation (S9 Mix). The concentrations, including the controls, were tested in triplicate.

In the performed experiments positive and negative (vehicle) controls were run concurrently. The revertant colony numbers of vehicle control plates with and without S9 Mix were within the historical control data range. The reference mutagens showed a distinct increase of induced revertant colonies. In the performed experimental phases there were at least five analyzable concentrations and a minimum of three non-toxic dose levels at each tester strain. The validity criteria of the study were fulfilled.

Substantially increased (increases above the corresponding historical control data ranges) revertant colony numbers were observed in the Initial Mutation Test in S. typhimurium TA 98 and E. coli WP2 uvrA and in the Confirmatory Mutation Test in E. coli WP2 uvrA. These increases were mostly unique and did not accompany with clear dose-relationship, furthermore all of the increases remained far below the genotoxicological threshold for being positive.

In the Confirmation Mutation Test (pre-incubation test) unequivocal inhibitory toxic effect of the test item was observed (indicated by lower revertant colony numbers than the revertant colony numbers of the vehicle control plates, below the corresponding historical control data ranges and/or reduced or slightly reduced background lawn development) in all examined bacterial strains at the highest concentration level, at 5000 μg/plate (±S9 Mix). Inhibitory effect of the test item was noticed in case of S. typhimurium TA 1535 down to and including the concentration level of 500 μg/plate (+S9 Mix). No revertant growth and reduced (or slightly reduced) background lawn development was obtained at 5000 μg/plate in S. typhimurium TA 98 (-S9 Mix) and in TA 1537 (±S9 Mix).

Conclusion:

The reported data of this mutagenicity assay shows, that under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the strains used. Therefore, Incorez 397 is considered non-mutagenic in this bacterial reverse mutation assay.