Cyclohexanaminium, N,N,N-trimethyl-, hydroxide (1:1)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames: negative

HPRT: negative

in vitro MNT: negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9-mix prepared from male wistar rats

Test concentrations with justification for top dose:

Standard plate test: 0; 33; 100; 333; 1000; 3333 and 10000 µg/plate (with and without S9 mix)
Preincubation test: 0; 33; 100; 333; 1000; 3333 and 6666 µg/plate (with and without S9 mix)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water (ultrapure water)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: 2-aminoanthracene (2-AA);N-methyl-N`-nitro-N-nitrosoguanidine (MNNG); 4-nitro-o-phenylenediamine (NOPD)

Details on test system and experimental conditions:

Standard plate test
Salmonella typhimurium
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates for ames test (heipha Dr. Müller GmbH, Eppelheim, Germany) within approx. 30 seconds. After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hinders the counting using the Image Analysis System.
Escherichia coli
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates for ames test (heipha Dr. Müller GmbH, Eppelheim, Germany) within approx. 30 seconds. After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hinders the counting using the Image Analysis System.

Preincubation Test
The experimental procedure was based on the method described by Yahagi et al. (7) and Matsushima et al. (8).
0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL S9 mix (with metabolic activation) or phosphate buffer (without metabolic activation) were incubated at 37°C for the duration of about 20 minutes using a shaker. Subsequently, 2 mL of soft agar was added and, after mixing, the samples were poured onto the agar plates within approx. 30 seconds.
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perseptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis System.

Evaluation criteria:

The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Solubility: No precipitation of the test substance was found with and without S9 mix.

Toxicity: A bacteriotoxic effect was observed depending on the strain and test conditions from about 3333 µg/plate onward.

Mutagenicity: A relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system.

According to the results of the present study, the test substance did not lead to a relevant increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in three experiments carried out independently of each other (standard plate test and preincubation assay). Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study. However, due to a technical error in the positive control plates, the 1st-Experiment with the tester strains TA 1535, TA 100 and TA 1537 was invalid, therefore the experimental part with these tester strains was not included for evaluation. In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain (see Appendix 5). In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data (see Appendix 5).

Thus, under the experimental conditions chosen here, it is concluded that Trimethylcyclohexylammonium hydroxid, aqueous solution 50 wt.% is not a mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 2017 - August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

yes

Remarks:

A series of non-GLP experiments was performed to get distinct responses of statistical significance when using the specified positive controls. Therefore, the test design (treatment, the recovery phase and harvest time) was slightly modified.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: healthy non-smoking donors not receiving medication.
- Suitability of cells: The lymphocytes of the respective donors have been shown to respond well to stimulation of proliferation with PHA and to positive control substances. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes.
- Cell cycle length, doubling time or proliferation index: Human lymphocytes were stimulated for proliferation by the addition of the mitogen PHA to the culture medium for a period of 48 hours. The cell harvest time point was approximately 2 – 2.5 x AGT (average generation time). Any specific cell cycle time delay induced by the test item was not accounted for directly.
- Sex, age and number of blood donors if applicable: For this study, blood was collected from a male donor (23 years old) for Experiment I, from a female donor (34 years old) for Experiment IIA and from a female donor (31 years old) for Experiment IIB.
- Whether whole blood or separated lymphocytes were used if applicable: whole blood

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 μg/mL), the mitogen PHA (3 μg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL). All incubations were done at 37 °C with 5.5 % CO2 in humidified air.

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Cytochalasin B (4 μg/mL) was added approximately 20 hours prior to preparation.

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

Dose selection was performed according to the current OECD Guideline for the in vitro micronucleus test. Doses used in the main experiment were 2614, 4574, and 8005 ug/ml. The highest dose corresponds to a concentration of approximately 10 mM.

Vehicle / solvent:

Deionized water was used as vehicle.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Demecolcin

Details on test system and experimental conditions:

PULSE EXPOSURE
About 48 hrs after seeding 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test item concentration. The culture medium was replaced with serum-free medium containing the test item. For the treatment with metabolic activation the culture medium was supplemented with approx. 2.5 % S9 fraction (50 μL S9 mix/mL culture medium). After 4 hrs the cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded and the cells were resuspended in and washed with "saline G" (pH 7.2, containing 8000 mg/L NaCl, 400 mg/L KCl, 1100 mg/L glucose •H2O, 192 mg/L Na2HPO4 • 2 H2O and 150 mg/L KH2PO4). The washing procedure was repeated once as described. The cells were resuspended in complete culture medium with 10 % FBS (v/v) and cultured for a 16-hour recovery period. After this period Cytochalasin B (4 μg/mL) was added and the cells were cultured another approximately 20 hours until preparation.

CONTINUOUS EXPOSURE (without S9 mix)
About 48 hrs after seeding 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test item concentration. The culture medium was replaced with complete medium (with 10 % FBS) containing the test item. After 20 hours the cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded and the cells were re-suspended in and washed with "saline G". The washing procedure was repeated once as described. After washing the cells were re-suspended in complete culture medium containing 10 % FBS (v/v). Cytochalasin B (4 μg/mL) was added and the cells were cultured another approximately 20 hours until preparation.

PREPARATION OF CELLS
The cultures were harvested by centrifugation 40 hrs after beginning of treatment. The cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded and the cells were re-suspended in approximately 5 mL "saline G" and spun down once again by centrifugation for 5 minutes. Then the cells were resuspended in 5 mL KCl solution (0.0375 M) and incubated at 37 °C for 20 minutes. 1 mL of ice-cold fixative mixture of methanol and glacial acetic acid (19 parts plus 1 part, respectively) was added to the hypotonic solution and the cells were resuspended carefully. After removal of the solution by centrifugation the cells were resuspended for 2 x 20 minutes in fixative and kept cold. The slides were prepared by dropping the cell suspension in fresh fixative onto a clean microscope slide. The cells were stained with Giemsa.

EVALUATION OF CYTOTOXICITY AND CYTOGENETIC DAMAGE
Evaluation of the slides was performed using microscopes with 40 x objectives. The micronuclei were counted in cells showing a clearly visible cytoplasm area. The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976). The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus. At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. The frequency of micronucleated cells was reported as % micronucleated cells. To describe a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is expressed as % cytostasis. A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis.

Evaluation criteria:

The micronucleus assay will be considered acceptable if it meets the following criteria:
− The concurrent solvent control will normally be within the laboratory historical solvent control data range (95% control limit realized as 95% confidence interval).
− The concurrent positive controls should produce a statistically significant increase in the micronucleus frequency and should be within the laboratory historical positive control data range.
− Cell proliferation criteria in the solvent control are considered to be acceptable.
− All experimental conditions described in section 5.6.3 were tested unless one exposure condition resulted in a clearly positive result.
− The quality of the slides must allow the evaluation of an adequate number of cells and concentrations.

Statistics:

Statistical significance was confirmed by the Chi square test (α < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

cytotoxicity was observed in one experiment without S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: pH was adjusted to physiological values
- Effects of osmolality: No relevant influence on osmolarity was observed.
- Precipitation: no


ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No relevant cytotoxicity, indicated by reduced CBPI and described as cytostasis could be observed in Experiment I and IIB up to the highest applied and evaluated concentration. In Experiment IIA in the absence of S9 mix, cytotoxicity indicated as cytostasis was observed at the highest applied and evaluated concentration.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Version / remarks:

29 Jul 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(from the competent authority) Landesamt für Umwelt Rheinland-Pfalz

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: CXJ2015
- Expiration date of the batch: Dec 2018
- Purity: not indicated
- Content: 19.9 g/100 g
- Molecular weight: 159.267 g/mol
- Physical state, appearance: Liquid, colorless, clear

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Solubility and stability of the test substance in the solvent/vehicle: Due to the good solubility of the test substance in water, the aqueous culture medium (Ham's F12) was selected as vehicle.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: In culture medium, no test substance precipitation occurred up to the highest concentration at the end of exposure period in the absence and presence of S9 mix.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The substance was dissolved in culture medium (Ham's F12). The test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution. Due to the pH shift observed at the test substance stock solution in culture medium in the pretest, the test substance stock solution in the main experiments of this study was adjusted case-by-case to a physiological value using small amounts of 32 % (w/v) HCl. To achieve a solution of the test substance in the vehicle, the test substance preparation was pipetted thoroughly. The further concentrations were diluted from according to the planned doses. All test substance solutions were prepared immediately before administration.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : solution

OTHER SPECIFICS:
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is added: In the pretest the pH value was not influenced by the addition of the test substance preparation to the culture medium at the concentrations measured. However, a slight pH shift was observed at the highest required concentration prior to testing. Therefore, the pH of the stock solution was adjusted to a physiological value prior to application using small amounts of 32 % (w/v) HCl. In culture medium, no test substance precipitation occurred up to the highest applied concentration at the end of exposure period in the absence and presence of S9 mix.
- other information: After 4 hours treatment in the absence and presence of S9 mix, cytotoxicity was not observed as indicated by a reduced RS of about or below 20 % of control up to the highest applied concentration.

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Suitability of cells: The CHO (Chinese hamster ovary) cell line is a permanent cell lie derived from the Chinese hamster and has a high proliferation rate (doubling time of about 12 - 16 hours), high plating efficiency (about 90 %) and a karyotype with a modal number of 20 chromosomes.
- Doubling time: 12 - 16 hours

For cell lines:
- Number of passages if applicable: at least 2 passages were performed before cells were taken for the experiment.
- Methods for maintenance in cell culture: For cell cultivation, deep-frozen cell suspensions were thawed at 37 °C in a water bath, and volumes of 0.5 mL were transferred into 25 cm² plastic flasks containing about 5 mL Ham's F12 medium including 10 % (v/v) FCS. Cells were grown with 5 % (v/v) CO2 at 37 °C and ≥ 90 % relative humidity up to approximate confluence and subcultured twice weekly (routine passage in 75 cm² plastic flasks).
Routine passage (preparation of a single cell suspension):
• Cell medium was removed and cells were washed with 5 mL PBS or HBSS (both Ca-Mg-free).
• Cells were trypsinized with 2 mL HBSS (Hanks balanced salt solution; Ca-Mg-free) and 2 mL trypsin (0.25 % [w/v]) to remove the cells from the bottom of the plastic flasks.
• This reaction was stopped by adding 6 mL culture medium incl. 10 % (v/v) FCS.
• Cells were pipetted up and down to separate them and to prepare a homogeneous single cell suspension.
• Cells were counted in a counting chamber or using a cell counter.
• Cell suspensions were diluted with complete culture medium to the desired cell count.
- Cell cycle length, doubling time or proliferation index: 12 - 16 hours
- Modal number of chromosomes: 20

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: all media are supplemented with 1 % (v/v) penicillin / streptomycin (stock solution: 10000 IU / 10000 µg/mL) and 1 % (v/v) amphotericine B (stock solution: 250 µg/mL)
• Culture medium: Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with 10 % (v/v) fetal calf serum (FCS).
• Treatment medium (without S9 mix): Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with 10 % (v/v) FCS.
• Treatment medium (with S9 mix): Ham's F12 medium containing stable glutamine and hypoxanthine.
• Pretreatment medium ("HAT" medium): Ham's F12 medium supplemented with hypoxanthine (13.6 x 10^(-3) mg/mL), aminopterin (0.18 x 10^(-3) mg/mL), thymidine (3.88 x 10^(-3) mg/mL) and 10 % (v/v) FCS.
• Selection medium ("TG" medium): Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with 6-thioguanine (10 µg/mL) and 10 % (v/v) FCS

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : phenobarbital and ß-naphthoflavone
- method of preparation of S9 mix: The S9 mix was prepared freshly prior to each experiment. For this purpose, a sufficient amount of S9 fraction was thawed at room temperature; 1 part S9 fraction was mixed with 9 parts S9 supplement (cofactors) in the pre-experiment and main experiments. This preparation, the S9 mix (10 % S9 fraction), was kept on ice until used.
- concentration or volume of S9 mix and S9 in the final culture medium : 8 mL (40 mL total volume)

Test concentrations with justification for top dose:

Based on the data and the observations from the pretest and taking into account the current guidelines, the following doses were selected in this study.
- 1st Experiment (with and without S9 mix): 500.00; 1000.00; 2000.00; 4000.00; 8000.00 µg/mL
- 2nd Experiment (without S9 mix): 2000.00; 4000.00; 5500.00; 6500.00; 8000.00 µg/mL
- 2nd Experiment (with S9 mix): 750.00; 1500.00; 3000.00; 6000.00; 8000.00 µg/mL
- 3rd Experiment (without S9 mix): 2000.00; 4000.00; 5000.00; 6000.00; 8000.00 µg/mL

The top concentration of 8000.00 µg/mL was chosen due to the purity of 19.9 % of the test substance and corresponds to 2000 µg/mL active ingredient.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: aqueous solvents (culture medium Ham's F12)

- Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in water, the aqueous culture medium (Ham's F12) was selected as vehicle.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

vehicle control

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of independent experiments : 3

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 20 x 10^6 cells in 40 mL
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Attachment period, if applicable: test substance incubation approx. 20 - 24 hours after seeding
- Exposure duration/duration of treatment: 4 hours

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 7 - 9 days
- Selection time (if incubation with a selective agent): 6 - 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 13 - 16 days
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure.: Selection medium ("TG" medium); Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with 6-thioguanine (10 µg/mL) and 10 % (v/v) FCS; At the end of the expression period two 175 cm² flasks with 2 x 10^6 cells each from every treatment group, if possible, were seeded in 20 mL selection medium ("TG" medium). The flasks were returned to the incubator for about 6 - 7 days.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 20 x 10^6 cells in 40 mL were seeded in 300 cm² flasks; mutants were selected by treatment with "TG" medium. Only the cells resistant to 6-thioguanine that were assumed to be deficient of HPRT survived.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: cloning efficiency; relative survival (RS)

METHODS FOR MEASUREMENTS OF GENOTOXICIY : mutant frequency

- OTHER: check or determination of further parameters: pH, osmolality, solubility, cell morphology

Rationale for test conditions:

The CHO/HPRT assay detects forward mutations of the X-linked hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary (CHO) cells.
HPRT catalyzes not only the conversion of hypoxanthine or guanine to the corresponding nucleotides (salvage pathway) but also the conversion of, e.g. the nontoxic purine analogue 6-thioguanine (6TG) to its ribophosphorylated derivative, which is lethal to cells. Therefore, a loss of the enzyme or its activity leads to 6TG resistance, i.e. cells are unable to form lethal nucleotides and are therefore able to grow and form colonies in the presence of the purine analogue.
Many substances are not mutagenic or carcinogenic themselves, acquire such a potential following metabolic transformation, which is catalyzed mainly by liver enzymes in vivo. Therefore, tests are not only carried out directly, but also in the presence of an exogeneous metabolic activation system. The most commonly used system is a cofactor-supplemented postmitochondrial fraction (S9 mix) obtained from rat livers treated with enzyme-inducing agents.
The aim of the present study was to assess the potential of the test substance or its metabolite(s) to induce gene mutations in CHO cells.

Evaluation criteria:

A test substance is considered to be clearly positive if all following criteria are met:
- A statistically significant increase in mutant frequencies is obtained.
- A dose-related increase in mutant frequencies is observed.
- The corrected mutation frequencies (MF(corr.)) exceeds both the concurrent negative control value and the range of the laboratory's historical negative control data (95 % control limit).
Isolated increases of mutant frequencies above the historical negative control range or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.
A test substance is considered to be clearly negative if the following criteria are met:
- Neither a statistically significant nor dose-related increase in the corrected mutation frequencies is observed under any experimental condition.
- The corrected mutation frequencies in all treated test groups is close to the concurrent vehicle control value and within the range of the laboratory's historical negative control data (95% control limit).

Statistics:

An appropriate statistical trend test (MS EXCEL function RGP) was performed to assess a possible dose-related increase of mutant frequencies. The used model is one of the proposed models of the International Workshop on Genotoxicity Test procedures Workgroup Report. The dependent variable was the corrected mutant frequency and the independent variable was the concentratio. The trend was judged as statistically significant whenever the one-sided p-value (probability value) was below 0.05 and the slope was greater than 0.
In addition, a pair-wise comparison of each test group with the vehicle control group was carried out using one-sided Fisher's exact test with Bonferroni-Holm correction. The calculation was performed using R.
If the results of these tests were statistically significant compared with the respective vehicle control, labels (p ≤ 0.05) are printd in the tables.
However, both, biological and statistical significance are considered together.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: The pH value of the test substance preparation (stock solution) was adjusted by adding small amounts of HCl. pH value was not influenced by test substance treatment.
- Data on osmolality: Osmolality was not influenced by test substance treatment.
- Water solubility: The test substance is soluble in water.
- Precipitation and time of the determination: In this study, in the absence and the presence of S9 mix, no precipitation in culture medium was observed macroscopically up to the highest applied test substance concentration.
- Cell morphology: After 4 hours treatment either in the absence of presence of metabolic activation, the cell morphology and attachment of the cells was not adversely influenced (grade > 2) in any group tested for gene mutations.

RANGE-FINDING/SCREENING STUDIES (if applicable): In the pretest for cytotoxicity based on the purity of 19.9 g/100 g and the molecular weight of 159.267 g/mol of the test substance 8000.0 µg/mL was used as top concentration both with and without S9 mix at 4-hour exposure time. The pretest was performed following the method described for the main experiment. The cloning efficiency 1 (survival) was determined as a toxicity indicator for dose selection and various parameters were checked for all, or at least some, selected doses. In the pretest the pH value was not influenced by the addition of the test substance preparation to the culture medium at the concentrations measured. However, a slight pH shift was observed at the highest required concentration prior to testing. Therefore, the pH of the stock solution was adjusted to a physiological value prior to application using small amounts of 32 % (w/v) HCl. In culture medium, no test substance precipitation occurred up to the highest applied concentration at the end of exposure period in the absence and presence of S9 mix. After 4 hours treatment in the absence and presence of S9 mix, cytotoxicity was not observed as indicated by a reduced RS of about or below 20 % of control up to the highest applied concentration.

STUDY RESULTS
- Concurrent vehicle negative and positive control data :
The positive control substances EMS (without S9 mix; 400 µg/mL) ad DMBA (with S9 mix; 1.25 µg/mL) induced a clear increase in mutation frequencies, as expected. The values of the corrected mutant frequencies (without S9 mix: MF(corr.): 106.27 - 223.96 per 10^6 cells; with S9 mix: MF(corr.): 104.78 - 131.03 per 10^6 cells) were clearly within the historical positive control data range.
Negative control value 1st Experiment without S9 mix: 1.27 per 10^6 cells
Negative control value 1st Experiment with S9 mix: 4.55 per 10^6 cells
Negative control value 2nd Experiment: 4.75 - 8.00 per 10^6 cells
Negative control value 3rd Experiment: 2.35 per 10^6 cells

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible : In the 1st Experiment in the absence of S9 mix there was dose related increase in the mutant frequencies (MF(corr.): 2.62 - 8.33 per 10^6 cells) which was statistically significant at 4000.00 µg/mL (MF(corr.): 4.30 per 10^6 cells) and 8000.00 µg/mL (MF(corr.): 8.33 per 10^6 cells). The value for the corrected mutant frequency at 8000.00 µg/mL (MF(corr.): 8.33 per 10^6 cells) exceeded the range of the 95 % control limit and the range of the historical negative control data (MF(corr.): 0.00 - 7.09 per 10^6 cells).

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
o Relative survival (RS) and cloning efficiency: No cytotoxic effects, as indicated by clearly reduced cloning efficiencies of about or below 20 % of the respective negative control values were observed in all experiments either in the absence of presence of S9 mix. In the 3rd Experiment a dose independent increase was observed in the relative survival rates of the test substance treated cultures.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data:
without S9 mix: MF(corr.): 42.47 - 419.90 per 10^6 cells
with S9 mix: MF(corr.): 21.52 - 270.48 per 10^6 cells
- Negative (solvent/vehicle) historical control data: MF(corr.):
without S9 mix: 0.00 - 6.49 per 10^6 cells
with S9 mix: 0.00 - 7.09 per 10^6 cells

Discussion

According to the results of the present in vitro study, in three experiments performed independently of each other the test substance did not lead to a biologically relevant increase in the number of mutant colonies, either without S9 mix or after the addition of a metabolizing system. The mutant frequencies at any concentration were close to or within the range of the concurrent negative control values and within or slightly above the 95 % control limit of the historical negative control data.

The results obtained in the 1st Experiment could not be confirmed in the 2nd and 3rd Experiment. Therefore, this finding is regarded as biologically irrelevant. Additionally, no statistically significant dose-dependent increase in mutant colonies was observed in any other remaining experimental part of this study after 4 hours treatment either in the absence or presence of metabolic activation.

The mutation frequencies of the negative control groups were within the historical negative control data range (95 % control limit) and, thus, fulfilled the acceptance criteria of this study. The proficiency of the laboratory to perform the HPRT assay in CHO cells was demonstrated by the laboratory's historical control database on vehicle and positive controls and by X-bar chart to identify the variability of the vehicle control data.

The increase in the frequencies of mutant colonies induced by the positive control substances EMS and DMBA clearly demonstrated the sensitivity of the test method and / or of the metabolic activitiy of the S9 mix employed. The values were within the range of the historical positive control data and, thus, fulfilled the acceptance criteria of this study.

Table 1: Pretest (4 -hour exposure, without S9 mix)

Test groups Doses	pH value	Osmolality	Solubility				Relative Survival [%]
		mOsm	Veh Ma	Culture medium
				0 h	3 – 4 h
				Ma	Ma	Mi
Negative control 31.3 µg/mL 62.5 µg/mL 125.0 µg/mL 250.0 µg/mL 500.0 µg/mL 1000.0 µg/mL 2000.0 µg/mL 4000.0 µg/mL 8000.0 µg/mL	7.3 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 7.3	289 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 313	n.d. S S S S S S S S S	n.d. S S S S S S S S S	n.d. S S S S S S S S S	n.d. S S S S S S S S S	100.0 100.0 98.5 102.7 99.9 95.6 103.7 98.2 94.3 81.5

n.d. not determined

Veh vehicle

mOsm milliosmole

Ma macroscopically

Mi microscopically

S solution

Table 2: Pretest (4 -hour exposure, with S9 mix)

Test groups Doses	pH value	Osmolality	Solubility				Relative Survival [%]
		mOsm	Veh Ma	Culture medium
				0 h	3 – 4 h
				Ma	Ma	Mi
Negative control 31.3 µg/mL 62.5 µg/mL 125.0 µg/mL 250.0 µg/mL 500.0 µg/mL 1000.0 µg/mL 2000.0 µg/mL 4000.0 µg/mL 8000.0 µg/mL	7.1 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 7.1	256 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 285	n.d. S S S S S S S S S	n.d. S S S S S S S S S	n.d. S S S S S S S S S	n.d. S S S S S S S S S	100.0 91.5 95.4 84.8 80.2 85.3 86.5 81.2 86.0 91.8

n.d. not determined

Veh vehicle

mOsm milliosmole

Ma macroscopically

Mi microscopically

S solution

Table 3: Summary of results - experimental parts without S9 mix

Exp.	Exposure period [h]	Test groups [µg/mL]	S9 mix	Prec.*	Genotoxicity** MFcorr. [per 10^6cells]	Cytotoxicity***
						RS [%]	CE2 [%]
1	4	Negative control^1 500.00 1000.00 2000.00 4000.00 8000.00 Positive control²	- - - - - - -	n.d. - - - - - n.d.	1.27 n.c. 3.07 2.62 4.30^S 8.33^S 209.09^S	100.0 108.0 105.2 107.9 103.7 106.0 81.1	100.0 n.c. 113.3 120.9 117.7 113.9 66.1
2	4	Negative control^1 2000.00 4000.00 5500.00 6500.00 8000.00 Positive control²	- - - - - - -	n.d. - - - - - n.d.	8.00 0.72 2.13 5.53 2.43 3.45 223.96^S	100.0 125.5 105.7 95.2 102.0 97.8 84.8	100.0 100.4 102.5 92.0 89.8 94.9 78.9
3	4	Negative control^1 2000.00 4000.00 5000.00 6000.00 8000.00 Positive control²	- - - - - - -	n.d. - - - - - n.d.	2.35 0.98 1.58 3.83 1.33 3.85 106.27^S	100.0 172.4 148.4 193.9 187.8 167.5 147.7	100.0 102.7 84.9 105.0 101.0 87.2 85.6

* Precipitation in culture medium at the end of exposure period

** Mutant frequency MF_corr.: mutant colonies per 10⁶cells corrected with the CE₂value

*** Cloning efficiency related to the respective vehicle control

^SMutant frequency statistically significant higher than corresponding control values (p ≤ 0.05)

n.c. Culture was not continued since a minimum of only four analyzable concentrations is required

n.d. Not determined

¹Medium                   ² EMS 400 µg/mL                   ³ DMBA 1.25 µg/mL

Table 4: Summary of results - experimental parts with S9 mix

Exp.	Exposure period [h]	Test groups [µg/mL]	S9 mix	Prec.*	Genotoxicity** MFcorr. [per 10^6cells]	Cytotoxicity***
						RS [%]	CE2 [%]
1	4	Negative control^1 500.00 1000.00 2000.00 4000.00 8000.00 Positive control³	+ + + + + + +	n.d. - - - - - n.d.	4.55 n.c. 3.08 0.36 2.09 4.82 131.03^S	100.0 113.7 128.0 135.2 139.5 114.0 72.9	100.0 n.c. 88.5 84.2 87.0 75.5 70.3
2	4	Negative control^1 750.00 1500.00 3000.00 6000.00 8000.00 Positive control³	+ + + + + + +	n.d. - - - - - n.d.	4.75 n.c. 2.05 4.85 4.00 4.24 104.78^S	100.0 94.3 88.5 74.7 80.2 78.4 74.2	100.0 n.c. 92.7 104.4 87.0 104.4 79.4

*Precipitation in culture medium at the end of exposure period

** Mutant frequency MF_corr.: mutant colonies per 10⁶cells corrected with the CE₂value

*** Cloning efficiency related to the respective vehicle control

^SMutant frequency statistically significant higher than corresponding control values (p ≤ 0.05)

n.c. Culture was not continued since a minimum of only four analyzable concentrations is required

n.d. Not determined

¹Medium                   ² EMS 400 µg/mL                   ³ DMBA 1.25 µg/mL

Conclusions:

In the absence and the presence of metabolic activation, the test substance is not a mutagenic substance in the HPRT locus assay using CHO cells under the experimental conditions chosen.

Executive summary:

The test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Three independent experiments were carried out, with and without the addition of liver S9 mix from phenobarbital- ad ß-naphthoflavone induced rats (exogeneous metabolic activation).

According to an initial range-finding cytotoxicity test for the determination of the experimental doses and based on the content (19.9 %) and the molecular weight (159.267 g/mol) of the test substance the following concentrations were tested. Test groups printed in bold type were evaluated for gene mutations:

1st Experiment

without S9 mix

0; 500.00; 1000.00; 2000.00; 4000.00; 8000.00 µg/mL

with S9 mix

0; 500.00; 1000.00; 2000.00; 4000.00; 8000.00 µg/mL

2nd Experiment

without S9 mix

0; 2000.00; 4000.00; 5500.00; 6500.00; 8000.00 µg/mL

with S9 mix

0; 750.00; 1500.00; 3000.00; 6000.00; 8000.00 µg/mL

3rd Experiment

without S9 mix
0; 2000.00; 4000.00; 5000.00; 6000.00; 8000.00 µg/mL

Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in 6 -thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted.

The obtained negative controls gave mutant frequencies within the acceptable range for the CHO cell line. Both positive control substances, ethyl methanesulfonate (EMS) and 7,12 -dimethylbenz[a]anthracene (DMBA), led to the expected statistically significant increase in the frequencies of forward mutations.

In this study, in all experiments in the absence and the presence of metabolic activation no cytotoxicity was observed up to the highest concentrations evaluated for gene mutations.

Based on the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in all experiments performed independently of each other.

Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Additional information from genetic toxicity in vitro:

Ames Test

The test substance Trimethylcyclohexylammonium hydroxid, aqueous solution 50 wt.% was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay. The test strains are TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA. The dose range are 300 µg - 10000 µg/plate in the SPT (Standard plate test) and 33 µg - 6666 µg/plate in the PIT (preincubation test) both with and without metabolic activation (liver S9 mix from induced rats). No precipitation of the test substance was found with and without S9 mix. A bacteriotoxic effect was observed depending on the strain and test conditions from about 3 333 μg/plate onward. A relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system. Thus, under the experimental conditions of this study, the test substance Trimethylcyclohexylammonium hydroxid, aqueous solution 50 wt.% is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

MNT Test in vitro

The test item Cyclohexanaminium, N,N,N-trimethyl-, hydroxide, dissolved in deionized water, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in three independent experiments.

In each experimental group two parallel cultures were analyzed. Per culture 1000 binucleated cells were evaluated for cytogenetic damage. The highest applied concentration in this study (8005 μg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight and the content (19.9 g/100 g) of the test item and with respect to the current OECD Guideline 487. In Experiment I and IIB, in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied and evaluated concentrations. In Experiment IIA in the absence of S9 mix, cytotoxicity indicated as cytostasis was observed at the highest applied and evaluated concentration. In all three independent experiments, neither a statistically significant nor a biologically relevant increase in the number of micronucleated cells was observed after treatment with the test item. In Experiment IIA in the absence of S9 mix, however, the value of the solvent control (1.10 % micronucleated cells) slightly exceeded the historical 95% control limit (0.12 – 1.03 %).This value is clearly within the min-max range of the historical control data (0.05 – 1.35 %) and therefore classified as biological irrelevant.

Cyclohexanaminium, N,N,N-trimethyl-, hydroxide is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required concentration.

HPRT Test

The test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Three independent experiments were carried out, with and without the addition of liver S9 mix from phenobarbital- ad ß-naphthoflavone induced rats (exogeneous metabolic activation).

According to an initial range-finding cytotoxicity test for the determination of the experimental doses and based on the content (19.9 %) and the molecular weight (159.267 g/mol) of the test substance the following concentrations were tested. Test groups printed in bold type were evaluated for gene mutations:

1st Experiment

without S9 mix

0; 500.00;1000.00; 2000.00; 4000.00; 8000.00 µg/mL

with S9 mix

0; 500.00;1000.00; 2000.00; 4000.00; 8000.00 µg/mL

2nd Experiment

without S9 mix

0; 2000.00; 4000.00; 5500.00; 6500.00; 8000.00 µg/mL

with S9 mix

0; 750.00;1500.00; 3000.00; 6000.00; 8000.00 µg/mL

3rd Experiment

without S9 mix
0; 2000.00; 4000.00; 5000.00; 6000.00; 8000.00 µg/mL

Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in 6 -thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted.

The obtained negative controls gave mutant frequencies within the acceptable range for the CHO cell line. Both positive control substances, ethyl methanesulfonate (EMS) and 7,12 -dimethylbenz[a]anthracene (DMBA), led to the expected statistically significant increase in the frequencies of forward mutations.

In this study, in all experiments in the absence and the presence of metabolic activation no cytotoxicity was observed up to the highest concentrations evaluated for gene mutations.

Based on the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in all experiments performed independently of each other.

Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation

Justification for classification or non-classification

Based on the results, the test item is no subject to classification and labelling according Regulation (EC) No 1272/2008 (CLP).