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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item Incorez 397 was tested for mutagenic activity in a bacterial reverse mutation assay with 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.
The test item Incorez 397 was tested for mutagenic activity in an in vitro chromosome aberration test in Chinese hamster V79 cells. Incorez 397 tested up to cytotoxic concentrations, both with and without metabolic activation, did not induce structural chromosome aberrations in this test in Chinese hamster lung cells. Therefore, Incorez 397 is considered as not clastogenic in this system.
Incorez 397 tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency in this test in Chinese hamster ovary cells. Incorez 397 was not mutagenic in this in vitro mammalian cell gene mutation test performed with CHO-K1 cells.

Link to relevant study records

Reference

Reference 1

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.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

31 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

August 1998

Deviations:

no

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

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 / cell type:

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

Additional strain / cell type characteristics:

not specified

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)

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

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

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation

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

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

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

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

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

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

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.

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-01-27 to 2011-05-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

31 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

no target gene

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

This in vitro test is a cytogenetic test, which detects structural chromosome aberrations in somatic and/or germ cells and plays an important role in the evaluation of genotoxicity of a given item or agent (Preston et al., 1981). Structural aberrations develop due to breaks in one or both DNA strands, resulting in chromosome fragments (breaks, deletions). Faulty reunion of chromosome fragments results in formation of exchanges. These aberrations can be detected and quantified by light microscope. Extensive chromosome breaks usually cause cell death; small changes (breaks, deletions, translocations, inversions etc.) are, however, not necessarily lethal and can be regarded as an indication of molecular events, which might lead to malignant transformation.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver S 9 mix

Test concentrations with justification for top dose:

Experiment A with 3/20 h treatment/sampling time
without S9 mix: 39.1, 78.2, 156.3, 312.5, 625* µg/mL
with S9 mix: 78.2, 156.3, 312.5, 625* µg/mL

Experiment B with 20/20 h treatment/sampling time
without S9 mix: 39.1, 78.2, 156.3, 312.5, 625* µg/mL

Experiment B with 20/28 h treatment/sampling time
without S9 mix: 39.1, 78.2, 156.3, 312.5, 625* µg/mL

Experiment B with 3/28 h treatment/sampling time
with S9 mix: 78.2, 156.3, 312.5, 625* µg/mL

* This concentration was tested but not evaluated because the lower, not cytotoxic concentrations were evaluated (relative survival about or greater 50%).

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S-9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

Remarks:

with S-9 mix

Details on test system and experimental conditions:

Experiment A:
The appropriate amount of this stock solution was diluted with medium to obtain the examination concentrations. Duplicate cultures were used at each concentration and the solvent control cultures as well as the positive controls for treatment without and with S9 mix. 5E+05 cells were set up at each group. The culture medium of exponentially growing cell cultures was replaced with medium containing the test item. The exposure period was 3 hours. The exposure period followed by washing the cells with DME medium and then growth medium was added. Sampling was made at 20 hours (approximately 1.5 normal cell cycles from the beginning of treatment). For concurrent measures of cytotoxicity for all treated and negative control cultures, 5E+05 cells were set up.
Experiment B:
The test item was dissolved in DMSO for the treatment (stock solution: 50 mg/mL). The appropriate amount of this stock solution was diluted with medium to obtain the examination concentrations. In the cytogenetic Experiment B the exposure period without metabolic activation was 20 hours. The exposure period with metabolic activation was 3 hours. Experiment B, as Experiment A, included concurrent non-activated and S9-activated positive and negative controls. For each group 5E+05 cells/dish were seeded. Sampling was made at 1.5 cell cycles (20 hours, without S9 mix only) and at approximately 2 normal cell cycles (28 hours, without and with S9 mix) from the beginning of treatment to cover a potential mitotic delay.
Measurement of pH and Osmolality
The changes in pH and osmolality of the test media were determined for every treatment in Experiment A and Experiment B.
Preparation of Cultures
Cell cultures were treated with colchicine (0.2 μg/mL) 2 hours prior to harvest. Following the selection time, cells were swollen with 0.075 M KCl hypotonic solution, then washed in fixative (approx. 10 min. in 3:1 mixture of methanol: acetic-acid until the preparation became plasma free) and dropped onto slides and air-dried. The preparation was stained with 5 % Giemsa for subsequent scoring of chromosome aberration frequencies. For control of bias, all slides were coded and scored blind.
Analysis of Metaphase Cells
At least 200 metaphase cells containing 2 N ± 2 centromeres were evaluated for structural aberrations from each experimental group. Chromatid and chromosome type aberrations (gaps, deletions and exchanges) were recorded separately. Additionally the numbers of polyploid and endoreduplicated cells were scored. The nomenclature and classification of chromosome aberrations were given based upon ISCN (1985), and Savage (1976, 1983).

Evaluation criteria:

The criteria for determining a positive result are:
– a concentration-related increase or a reproducible increase in the number of cells with aberrations.
– biological relevance of the results should be considered first, however, for the interpretation of the data both biological and statistical significance should be considered together.
– an increase in the number of polyploid cells may indicate that the test item has the potential to inhibit mitotic processes and to induce numerical chromosome aberrations.
– an increase in the number of cells with endoreduplicated chromosomes may indicate that the test item has the potential to inhibit cell cycle progression.

A test item for which the results do not meet the above criteria is considered as non mutagenic in this system

Statistics:

NA

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

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

Additional information on results:

The cytotoxicity at the highest concentrations was adequate in the studies (Experiments A and B) as indicated by a reduction of % cell survival for at least 50 %..
In Experiment A, Incorez 397 did not induce an increase in the number of cells with aberrations at any examined concentration, either in the absence or in the presence of metabolic activation, up to and including cytotoxic concentrations. There were no statistically significant differences between treatment and control groups and no dose response relationship was noted.

In Experiment B, Incorez 397 was examined without S9 mix, over a long treatment period and the sampling was made at approximately 1.5 cell cycles (20 hours after treatment start). The cells with structural chromosome aberrations without gaps did not show significant alterations compared to the concurrent solvent controls. There was no increase in the number of cells with aberrations without S9 mix following exposure over a long treatment period of 20 hours and sampling at approximately 2 cell cycles (28 hours after treatment start).
A 3-hour treatment in the presence of S9 mix with 28-hour harvest from the beginning of treatment did not cause an increase in the number of cells with structural chromosome aberrations without gaps.
As in Experiment A, in Experiment B no statistically significant differences between treatment and control groups and no dose-response relationships were noted. The observed chromosome aberration rates were within the ranges of historical control data.
Tables show the occurrence of polyploidy and endoreduplicated metaphases. No increase in the rate of polyploid and endoreduplicated metaphases was found after treatment with the different concentrations of Incorez 397.
pH and osmolality values of control and treatment solutions were measured. In Experiments A and B no significant differences between treatment and control groups were observed.
In the concurrent solvent control group the percentage of cells with structural aberration(s) without gap was equal or less than 5 %, proving the suitability of the cell line used.
The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 µL/mL) and N-Nitrosodimethylamine (1.0 µL/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations. Thus, the study is considered as valid.

Conclusions:

The test item Incorez 397 was tested for mutagenic activity in an in vitro chromosome aberration test in Chinese hamster V79 cells. Incorez 397 tested up to cytotoxic concentrations, both with and without metabolic activation, did not induce structural chromosome aberrations in this test in Chinese hamster lung cells. Therefore, Incorez 397 is considered as not clastogenic in this system.

Executive summary:

The test item, Incorez 397 was tested in a Chromosome Aberration Assay in V79 cells. The test item was dissolved in DMSO and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (with and without metabolic activation using S9 mix). In two independent experiments (both run in duplicate) at least 200 well spread metaphase cells were analysed at concentrations and incubation/expression intervals given below, ranging from little to maximum ( < 50 % survival) toxicity:

Experiment A with 3/20 h treatment/sampling time

without S9 mix: 39.1, 78.2, 156.3, 312.5, 625* µg/mL

with S9 mix: 78.2, 156.3, 312.5, 625* µg/mL

Experiment B with 20/20 h treatment/sampling time

without S9 mix:39.1, 78.2, 156.3, 312.5, 625* µg/mL

Experiment B with 20/28 h treatment/sampling time

without S9 mix:39.1, 78.2, 156.3, 312.5, 625* µg/mL

Experiment B with 3/28 h treatment/sampling time

with S9 mix: 78.2, 156.3, 312.5, 625* µg/mL

* This concentration was tested but not evaluated because the lower, not cytotoxic concentrations were evaluated (relative survival about or greater 50%).

In Experiment A, there were no biologically significant increases in the number of cells showing structural chromosome aberrations, either in the absence or in the presence of metabolic activation, up to and including cytotoxic concentrations. There were no statistical differences between treatment and concurrent solvent control groups and no dose-response relationships were noted. In Experiment B, the frequency of the cells with structural chromosome aberrations did not show significant alterations compared to concurrent controls, up to cytotoxic concentrations without S9 mix over a prolonged treatment period of 20 hours with harvest at 20 or 28 hours following treatment start. Further, a 3-hour treatment up to cytotoxic concentrations in the presence of S9 mix with 28-hour harvest from the beginning of treatment did not cause an increase in the number of cells with structural chromosome aberrations. In both experiments, no statistically significant differences between treatment and concurrent solvent control groups and no dose-response relationships were noted. The observed chromosome aberration rates were within the ranges of historical control data. There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation. The validity of the test was shown using ethyl methanesulphonate (0.4 and1.0 µL/mL) and N-nitrosodimethylamine (1.0 µL/mL) as concurrent positive controls. Incorez 397 tested up to cytotoxic concentrations, both with and without metabolic activation, did not induce structural chromosome aberrations in this test in Chinese Hamster lung cells. Therefore, Incorez 397 is considered as not clastogenic in this system.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-06-28 to 2011-07-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

21 July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

hypoxanthine-guanine phosphoribosyl transferase enzyme locus (hprt) in cultured Chinese hamster cells

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

The CHO cell line was originally derived from the ovary of a female Chinese hamster (Puck and Kao, 1967). The CHO KI is a sub-line of CHO cell line. The CHO KI cell line was purchased from ECACC (European Collection of Cells Cultures) (ECACC). The cell stocks are kept in liquid nitrogen. Each batch of frozen cells was purged (GEN 013) of HPRT mutants and was free for mycoplasma infections, tested by Mezőgazdasági Szakigazgatási Hivatal Központ Állategészségügyi Diagnosztikai Igazgatóság Budapest, Hungary; results will be fully documented within the raw data file.
For each experiment, one or more vials will be thawed rapidly, the cells diluted in Ham's F12 medium containing 10 % foetal bovine serum and incubated at 37 °C in a humidified atmosphere of 5 % CO2 in air. When cells are growing well, subcultures will be established in an appropriate number of flasks. The CHO KI cells for this study are grown in Ham's F12 medium (F12-10) supplemented with 1 % of Antibiotic-antimycotic solution (containing 10000 U/ml penicillin, 10 mg/ml streptomycin and 25 μg/ml amphoptericin-B) and heat-inactivated bovine serum (final concentration 10 %). During the 5 and 20 hour treatments with the test item, solvent (negative control) and positive controls, the serum content is reduced to 5 % (F12-5). The selection medium for TG resistant mutants contains 10μM/ml of thioguanine (6-TG) (EX-CELL® CD CHO Serum-Free Medium for CHO Cells-SEL).

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix phenobarbital (PB) and β-naphthoflavone (BNF) induced

Test concentrations with justification for top dose:

Experiment 1, 5-hour treatment period without S9 mix:
150, 200, 250, 300, 350, 400 and 450* µg/mL
Experiment 1, 5-hour treatment period with S9 mix:
50, 100, 150, 200, 250, 300, 350, 400 and 450 µg/mL
Experiment 2, 20-hour treatment period without S9 mix:
150, 200, 250, 300, 350, 400* and 450* µg/mL
Experiment 2, 5-hour treatment period with S9 mix:
50, 100, 150, 200, 250, 300, 350, 400 and 450 µg/mL
* These concentrations were very toxic and there was not enough cells start the phenotypic expression period after the treatment.

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S-9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with S-9 mix

Details on test system and experimental conditions:

The Main Mutation Assay will be conducted in two independent experiments in the presence and in the absence of S9 mix.
For the 5-hour treatment, 10E06 cells will be placed in each of a series of sterile dishes (diameter approx. 10 mm) and incubated for approximately 24 hours before treatment at 37 °C in a humidified atmosphere of 5 % CO2.
Duplicate cultures will be used at each concentration and the solvent control cultures as well as the positive controls for treatment without and with S9 mix. On the day of treatment the culture medium of exponentially growing cell cultures will be replaced with medium (Ham's F12-5) containing the test item. The exposure period will be 5 hours. Following the exposure period the cells will be washed with Ham's F12-0 medium and incubated in fresh Ham's F12-10 medium for 19 hours. After the 19-hour incubation period, cells will be washed twice with Ham's F12-0 medium and detached with trypsin-EDTA solution and counted using a Bürker chamber. Solubility of the test item in the cultures will be assessed by the naked eye, at the beginning and end of treatment. In samples where sufficient cells survive, cell number will be adjusted to 105 cells/mL. Cells will be transferred to dishes for growth through the expression period or diluted to be plated for survival.
Five hours treatment in the presence of S9 and 20-hour treatment in the absence of S9 will be performed. Duplicate cultures (10E06 cells/dish) will be used for each treatment. 24-hours after the beginning of treatment the cells will be washed with Ham's F12-0 tissue culture medium, dissociated with trypsin-EDTA solution and counted. After this, the cells will be subcultured for phenotypic expression and to assess cytotoxicity.

Evaluation criteria:

The test item will be considered to be mutagenic in this assay if all the following criteria are met:
• The assay is valid.
• The mutant frequency at one or more doses is significantly greater than that of the relevant control.
• Increase of the mutant frequency is reproducible in the Experiment 2.
• There is a dose-response relationship.

Statistics:

Statistical analysis will be done with SPSS PC+ software for the following data:
- mutant frequency between the negative (solvent) and the test item or positive control item treated groups.
The heterogeneity of variance between groups will be checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity is detected, a one-way analysis of variance will be carried out. If the obtained result is positive, Duncan's Multiple Range test will be used to assess the significance of inter-group differences.
Where significant heterogeneity is found, the normal distribution of data will be examined by Kolmogorov-Smirnov test. In case of a none-normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance will be used. If there is a positive result, the inter-group comparisons are performed using the Mann-Whitney U-test.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- pH and osmolarity:
The pH and osmolality of control and treatment solutions were measured. In osmolality and pH no significant differences between treatment and control groups were observed Experiments 1 and 2.

- Precipitation:
Incorez 397 was dissolved in DMSO. A clear solution was obtained up to a concentration of 50 mg/mL. For all test item concentrations examined, no precipitation in the medium was noted.

RANGE-FINDING/SCREENING STUDIES:
The dose selection cytotoxicity assay was performed as part of this study to establish an appropriate concentration range for the Main Mutation Assays, both in the absence and in the presence of a metabolic activation system (rodent S9 mix). Toxicity was determined by comparing the colony forming ability of the treated groups to the negative (solvent) control and results were noted as percentage of cells in relation to the negative control. The results obtained were used for dose selection of the test item in the Main Mutation Assays. In Experiment 1 and in Experiment 2 in the absence and in the presence of metabolic activation the upper examined test item dose level selected was 450 µg Incorez 397 /mL.

COMPARISON WITH HISTORICAL CONTROL DATA:
The sensitivity of the tests and the efficacy of the S9 mix were demonstrated by large increases in mutation frequency in the positive control cultures. The mutation frequencies of the positive and negative control cultures were consistent with the historical control data from the previous studies performed at this laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
There was very clear evidence of toxicity with the test item in both absence and presence of metabolic activation (S9 mix) when compared to the negative (solvent) controls both in the absence and in the presence of the metabolic activation, confirming the response seen in the dose selection cytotoxicity assays. The Day 8 cloning efficiency data indicate that in general the cells had recovered during the expression period.

Conclusions:

The test item Incorez 397 was not mutagenic in this in vitro mammalian cell gene mutation test performed with CHO-K1 (Chinese hamster ovary) cells.

Executive summary:

The test item, Incorez 397 was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The test item was dissolved in DMSO and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (without and with metabolic activation using S9 mix).

Two independent main experiments (both run in duplicate) were performed at the concentrations and treatment intervals given below:

Experiment 1, 5-hour treatment period without S9 mix:

150, 200, 250, 300, 350, 400 and 450* µg/mL

Experiment 1, 5-hour treatment period with S9 mix:

50, 100, 150, 200, 250, 300, 350, 400 and 450 µg/mL

Experiment 2, 20-hour treatment period without S9 mix:

150, 200, 250, 300, 350, 400* and 450* µg/mL

Experiment 2, 5-hour treatment period with S9 mix:

50, 100, 150, 200, 250, 300, 350, 400 and 450 µg/mL

* These concentrations were very toxic and there was not enough cells start the phenotypic expression period after the treatment.

In Experiment 1, there were no biologically or statistically significant increases in mutation frequency at any concentration tested, either in the absence or in the presence of metabolic activation. There were no statistical differences between treatment and control groups and no dose-response relationships were noted.

In Experiment 2, the mutant frequency of the cells did not show significant alterations compared to the concurrent control, when the test item was tested without S9 mix over a prolonged treatment period (20 hours). Furthermore, a five-hour treatment with in the presence of S9 mix did not cause significant increases in mutant frequency, further indicating that the findings in Experiment 1 were within the normal biological variation.

As in Experiment 1, in Experiment 2 no statistical differences between treatment and solvent control groups and no dose response relationships were noted. The sensitivity of the tests and the efficacy of the S9 mix were demonstrated by large increases in mutation frequency in the positive control cultures.

Incorez 397 tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency in this test in Chinese hamster ovary cells. Incorez 397 was not mutagenic in this in vitro mammalian cell gene mutation test performed with CHO-K1 cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames-Test:

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).

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.

 

Chromosome Aberration Test:

The test item, Incorez 397 was tested in a Chromosome Aberration Assay in V79 cells. The test item was dissolved in DMSO and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (with and without metabolic activation using S9 mix). In two independent experiments (both run in duplicate) at least 200 well spread metaphase cells were analysed at concentrations and incubation/expression intervals given below, ranging from little to maximum ( < 50 % survival) toxicity:

Experiment A with 3/20 h treatment/sampling time

without S9 mix: 39.1, 78.2, 156.3, 312.5, 625* µg/mL

with S9 mix: 78.2, 156.3, 312.5, 625* µg/mL

Experiment B with 20/20 h treatment/sampling time

without S9 mix:39.1, 78.2, 156.3, 312.5, 625* µg/mL

Experiment B with 20/28 h treatment/sampling time

without S9 mix:39.1, 78.2, 156.3, 312.5, 625* µg/mL

Experiment B with 3/28 h treatment/sampling time

with S9 mix: 78.2, 156.3, 312.5, 625* µg/mL

* This concentration was tested but not evaluated because the lower, not cytotoxic concentrations were evaluated (relative survival about or greater 50%).

In Experiment A, there were no biologically significant increases in the number of cells showing structural chromosome aberrations, either in the absence or in the presence of metabolic activation, up to and including cytotoxic concentrations. There were no statistical differences between treatment and concurrent solvent control groups and no dose-response relationships were noted. In Experiment B, the frequency of the cells with structural chromosome aberrations did not show significant alterations compared to concurrent controls, up to cytotoxic concentrations without S9 mix over a prolonged treatment period of 20 hours with harvest at 20 or 28 hours following treatment start. Further, a 3-hour treatment up to cytotoxic concentrations in the presence of S9 mix with 28-hour harvest from the beginning of treatment did not cause an increase in the number of cells with structural chromosome aberrations. In both experiments, no statistically significant differences between treatment and concurrent solvent control groups and no dose-response relationships were noted. The observed chromosome aberration rates were within the ranges of historical control data. There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation. The validity of the test was shown using ethyl methanesulphonate (0.4 and1.0 µL/mL) and N-nitrosodimethylamine (1.0 µL/mL) as concurrent positive controls. Incorez 397 tested up to cytotoxic concentrations, both with and without metabolic activation, did not induce structural chromosome aberrations in this test in Chinese Hamster lung cells. Therefore, Incorez 397 is considered as not clastogenic in this system.

 

HPRT test

The test item, Incorez 397 was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The test item was dissolved in DMSO and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (without and with metabolic activation using S9 mix). Two independent main experiments (both run in duplicate) were performed at the concentrations and treatment intervals given below:

 

Experiment 1, 5-hour treatment period without S9 mix:

150, 200, 250, 300, 350, 400 and 450* µg/mL

Experiment 1, 5-hour treatment period with S9 mix:

50, 100, 150, 200, 250, 300, 350, 400 and 450 µg/mL

Experiment 2, 20-hour treatment period without S9 mix:

150, 200, 250, 300, 350, 400* and 450* µg/mL

Experiment 2, 5-hour treatment period with S9 mix:

50, 100, 150, 200, 250, 300, 350, 400 and 450 µg/mL

* These concentrations were very toxic and there was not enough cells start the phenotypic expression period after the treatment.

 

In Experiment 1, there were no biologically or statistically significant increases in mutation frequency at any concentration tested, either in the absence or in the presence of metabolic activation. There were no statistical differences between treatment and control groups and no dose-response relationships were noted.

In Experiment 2, the mutant frequency of the cells did not show significant alterations compared to the concurrent control, when the test item was tested without S9 mix over a prolonged treatment period (20 hours). Furthermore, a five-hour treatment with in the presence of S9 mix did not cause significant increases in mutant frequency, further indicating that the findings in Experiment 1 were within the normal biological variation.

As in Experiment 1, in Experiment 2 no statistical differences between treatment and solvent control groups and no dose response relationships were noted. The sensitivity of the tests and the efficacy of the S9 mix were demonstrated by large increases in mutation frequency in the positive control cultures.

Incorez 397 tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency in this test in Chinese hamster ovary cells. Incorez 397 was not mutagenic in this in vitro mammalian cell gene mutation test performed with CHO-K1 cells.

Justification for classification or non-classification

Based on the available in vitro studies, there is no indication for a mutagenic potential of the test substance. Thus, the test substance has not to be classified with regard to mutagenicity according to  Regulation (EC) No 1272/2008 (CLP).