4-(cyclohexylamino)butane-1-sulfonic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD TG 471): negative

In vitro chromosome aberration test (OECD TG 473): negative

Genemutation in mammalian cells (OECD TG 490): negative

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:

08 April 2019 - 26 April 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

31 May 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

S. typhimurium: Histidine gene
E. coli: Tryptophan gene

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : rat liver S9-mix induced by Aroclor 1254 (500 mg/kg bw)
- method of preparation of S9 mix: S9-mix per 10 mL: 30 mg NADP, 15.2 mg glucose-6-phosphate in 5.5 mL Milli-Q water; 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl2 solution; 1 mL 0.33 M KCl solution. The solution was filter (0.22 μm)-sterilized and 0.5 mL S9-fraction was added
- concentration of S9 in S9 mix: 5%
- quality controls of S9: Each S9 batch was characterized with the mutagens benzo-(a)-pyrene and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 μg/plate and 2.5 μg/plate, respectively

Test concentrations with justification for top dose:

Justification for top dose: 5 mg is the recommended maximum test concentration according to the guideline.

Dose-range finding test (without and with S9-mix; tester strains TA100 and WP2uvrA): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate (reported as part of experiment 1)

First experiment (without and with S9; tester strains TA1535, TA1537 and TA98): 52, 164, 512, 1600, 5000 μg/plate

Second experiment (without and with S9-mix, tester strains WP2uvrA, TA1535, TA1537, TA 98 and TA100): 52, 164, 512, 1600, 5000 μg/plate

Vehicle / solvent:

The vehicle of the test item was Milli-Q water

Untreated negative controls:

other: Not applicable

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene; ICR-191

Remarks:

For details on positive control substances, see Table 1 and Table 2

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Doses levels were tested in triplicate in each strain.

METHODS:
Direct plating assay:
The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (1E9 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test item in Milli-Q water and either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark.
Pre-incubaton assay:
The solution in the direct plating assay were pre-incubated for 30 ± 2 minutes by 70 rpm at 37 ± 1°C before added to 3 mL molten top agar

DURATION
- Exposure duration: 48 ± 4 h (in the dark at 37.0 ± 1.0 °C)

DETERMINATION OF CYTOTOXICITY
- Method: the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.
- Other: precipitation of the test item was recorded.

COLONY COUNTING
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

ACCEPTABILITY CRITERIA
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:

a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at Charles River Den Bosch.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

Rationale for test conditions:

According to guideline

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

See explanation below

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

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

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

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

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

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

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

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:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

First Experiment:
Precipitation:
Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period in any tester strain.

Cytotoxicity:
There was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.

Mutagenicity:
In the direct plate test, no increase in the number of revertants was observed upon treatment with the test item.

Second Experiment:
Precipitation:
Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period.

Cytotoxicity:
There was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.

Mutagenicity:
In the pre-incubation test, no increase in the number of revertants was observed upon treatment with the test item.

In tester strain TA1535 in the absence of S9-mix, the test item induced an up to 4.0-fold increase in the number of revertant colonies compared to the solvent control in a single experiment. The increases were not dose dependent and fell within the historical control data range. Therefore, the increases are considered to be not biologically relevant.

Acceptability criteria:
All bacterial strains showed negative responses over the entire dose-range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.
The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Details on results are included below.

Conclusions:

Based on the results of an Ames test, performed according to OECD guideline 471 and in accordance with GLP principles, 4-(cyclohexylamino)butane-1-sulfonic acid is concluded to be not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

A bacterial reverse mutation test was performed according to OECD guideline 471 and in accordance with GLP principles. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. The test item did not induce a significant dose-related increase in the number of revertant (His⁺) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp⁺) colonies in tester strain WP₂uvrA both in the absence and presence of S9 -metabolic activation when tested up to and including the recommended top concentration. These results were confirmed in a follow-up experiment. Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia colireverse 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:

10 April 2019 - 21 July 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: Cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Sex, age and number of blood donors:
Blood was collected from healthy adult, non-smoking volunteers (approximately 18 to 35 years of age). The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2018): age 24 - 33; AGT: 12.9 - 13.2h
- Whether whole blood or separated lymphocytes were used:
Blood samples were collected by venipuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin (Vacuette, Greiner Bio-One, Alphen aan den Rijn, The Netherlands). Immediately after blood collection lymphocyte cultures were started.
- Mitogen used for lymphocytes: phytohaemagglutinin

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
Culture medium consisted of RPMI 1640 medium (Life technologies), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum (Life technologies), L-glutamine (2 mM) (Life technologies), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively) (Life technologies) and 30 U/mL heparin (Sigma, Zwijndrecht, The Netherlands).
All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 25 - 95%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.1 - 38.4°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Rat S9 homogenate was obtained from Trinova Biochem GmbH, Giessen, Germany and is prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).
- method of preparation of S9 mix: S9-mix was prepared immediately before use and kept refrigerated. S9-mix components contained per mL physiological saline: 1.63 mg MgCl2.6H2O (Merck); 2.46 mg KCl (Merck); 1.7 mg glucose-6-phosphate (Roche, Mannheim, Germany); 3.4 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom); 4 μmol HEPES (Life technologies).
The above solution was filter (0.22 μm)-sterilized. To 0.5 mL S9-mix components 0.5 mL S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix.
- concentration or volume of S9 mix and S9 in the final culture medium: The concentration of the S9-fraction in the exposure medium was 1.8% (v/v).

Test concentrations with justification for top dose:

Dose range finding test:
Without S9-mix, 3/24/48hr exposure; 24/48hr fixation: 63, 125, 250, 500, 1000 and 2000 μg/mL
With S9-mix, 3hr exposure; 24hr fixation: 63, 125, 250, 500, 1000 and 2000 μg/mL
First cytogenetic test:
Without and with S9-mix, 3hr exposure time, 24hr fixation time: 500, 1000 and 2000 μg/mL
Second cytogenetic test:
Without S9-mix, 24/48hr exposure; 24/48hr fixation: 500, 1000 and 2000 μg/mL

Vehicle / solvent:

- Solvent used: RPMI 1640 culture medium
- Justification for choice of solvent: A solubility test was performed based on visual assessment. The test item formed a clear pink solution in RPMI 1640 medium (Life Technologies, Bleiswijk, The Netherlands).

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : duplicate
- Number of independent experiments : two

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 48 hr
- Exposure duration/duration of treatment: 3 hr (with and without S9-mix), 24 and 48 hr (without S9-mix)
- Harvest time after the end of treatment (sampling/recovery times): 24 and 48 hr

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): colchicine
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol /ether and cleaned with a tissue. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 6.7% (v/v) Giemsa solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded and mounted with a coverslip in an automated cover slipper.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): One hundred and fifty metaphase chromosome spreads per culture were examined in duplicate.
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Only metaphases containing 46 ± 2 centromeres (chromosomes) were analyzed.
- Determination of polyploidy: yes
- Determination of endoreplication: yes

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: The mitotic index of each culture was determined by counting the number of metaphases from at least 1000 cells (with a maximum deviation of 5%).

Evaluation criteria:

A test item is considered positive (clastogenic) in the chromosome aberration test if:
a) At least one of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
A test item is considered negative (not clastogenic) in the chromosome aberration test if:
a) None of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are inside the 95% control limits of the negative historical control data range.

Statistics:

Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA).

Key result

Species / strain:

lymphocytes: Cultured peripheral human lymphocytes

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

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH and on osmolality: The pH and osmolarity of a concentration of 2000 μg/mL were 8.1 and 265 mOsm/kg respectively (compared to 8.1 and 256 mOsm/kg in the solvent control).
- Precipitation and time of the determination: A concentration of 2000 μg/mL showed no precipitation in the culture medium. Therefore, this concentration was used as the highest concentration of the test item.

RANGE-FINDING/SCREENING STUDIES: No cytotoxicity was observed in the cultures of the 3 h, 24 h and 48 h exposure times.

STUDY RESULTS
- Concurrent vehicle negative and positive control data :
The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The mean number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database.
The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements: No cytotoxicity was observed in the duplicate cultures treated with the substance of the 3 h, 24 h and 48 h exposure times.
- Genotoxicity results (for both cell lines and lymphocytes) : Both in the absence and presence of S9-mix 4-(cyclohexylamino)butane-1-sulfonic acid did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.
No effects of the test item on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that 4-(cyclohexylamino)butane-1-sulfonic acid does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations.

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:
3 hours exposure time 24 hours exposure time 48 hours exposure time
Gaps included Gaps excluded Gaps included Gaps excluded Gaps included Gaps excluded
+ S9-mix - S9-mix + S9-mix - S9-mix - S9-mix - S9-mix - S9-mix - S9-mix
Mean number of aberrant cells per 100 cells 21.63 24.16 20.78 23.65 26.76 26.09 33.65 32.92
SD 10.45 12.83 10.26 13.04 14.43 14.41 15.28 15.26
n 128 130 128 130 124 124 120 120
Upper control limit
(95% control limits) 39.56 44.96 38.16 44.70 51.64 50.92 63.20 62.08
Lower control limit
(95% control limits) 3.70 3.35 3.39 2.60 1.89 1.25 4.10 3.76
SD = Standard deviation
n = Number of observations
Distribution historical positive control data from experiments performed between September 2015 and September 2018.
- Negative (solvent/vehicle) historical control data:
3 hours exposure time 24 hours exposure time 48 hours exposure time
Gaps included Gaps excluded Gaps included Gaps excluded Gaps included Gaps excluded
+ S9-mix - S9-mix + S9-mix - S9-mix - S9-mix - S9-mix - S9-mix - S9-mix
Mean number of aberrant cells per 100 cells 0.40 0.50 0.31 0.42 0.44 0.37 0.87 0.69
SD 0.53 0.73 0.45 0.68 0.65 0.63 2.44 2.28
n 130 130 130 130 126 126 122 122
Upper control limit
(95% control limits) 1.68 2.32 1.39 1.95 1.92 1.71 3.92 3.13
Lower control limit
(95% control limits) -0.89 -1.32 -0.77 -1.10 -1.03 -0.96 -2.17 -1.74
SD = Standard deviation
n = Number of observations
Distribution historical negative control data from experiments performed between September 2015 and September 2018.

Conclusions:

A chromosome aberration study with 4-(cyclohexylamino)butane-1-sulfonic acid was performed according to OECD 473 guideline and GLP principles, in cultured peripheral human lymphocytes in two independent experiments. It is concluded that 4-(cyclohexylamino)butane-1-sulfonic acid is not clastogenic in human lymphocytes.

Executive summary:

A chromosome aberration study with 4-(cyclohexylamino)butane-1-sulfonic acid was performed according to OECD guideline 473 and GLP principles, in cultured peripheral human lymphocytes in two independent experiments.

No toxicity and no precipitation was observed at the highest tested concentration of 2000 μg/mL. Both in the absence and presence of S9-mix 4-(cyclohexylamino)butane-1-sulfonic acid did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.

No effects of 4-(cyclohexylamino)butane-1-sulfonic acid on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that 4-(cyclohexylamino)butane-1-sulfonic acid does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations.

Based on the results it can be concluded that 4-(cyclohexylamino)butane-1-sulfonic acid is not clastogenic in human lymphocytes.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 September 2019 - 03 December 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

Thymidine kinase locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: L5178Y/TK+/- -3.7.2C mouse lymphoma cells from American Type Culture Collection, (ATCC, Manassas, USA), (2001)
- Suitability of cells: recommended test system in international guidelines

For cell lines:
- Absence of Mycoplasma contamination: Yes
- Methods for maintenance in cell culture: stock cultures of the cells were stored in liquid nitrogen (- 196°C).
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
Basic medium:
RPMI 1640 Hepes buffered medium (Dutch modification) (Life Technologies) containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively) (Life Technologies), 1 mM sodium pyruvate (Sigma, Zwijndrecht, The Netherlands) and 2 mM L-glutamin (Life Technologies).
Growth medium:
Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
Exposure medium:
For 3 hour exposure:
Cells were exposed to the test item in basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium).
For 24 hour exposure:
Cells were exposed to the test item in basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium).
Selective medium:
Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium) and 5 μg/mL trifluorothymidine (TFT) (Sigma).
Non-selective medium:
Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium).
Environmental conditions:
All incubations were carried out in a humid atmosphere (80 - 100%, actual range 55 - 95%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 34.5 - 37.4 °C).

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and was prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg body weight).
- method of preparation of S9 mix: S9-mix was prepared immediately before use and kept refrigerated. S9-mix components contained per mL physiological saline: 1.63 mg MgCl2.6H2O (Merck); 2.46 mg KCl (Merck); 1.7 mg glucose-6-phosphate (Roche, Mannheim, Germany); 3.4 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom); 4 μmol HEPES (Life technologies). The above solution was filter (0.22 μm)-sterilized. To 0.5 mL S9-mix components 0.5 mL S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix.
- concentration or volume of S9 mix and S9 in the final culture medium: The concentration of the S9-fraction in the exposure medium was 4% (v/v).

Test concentrations with justification for top dose:

- Dose-range finding test, with S9 (3h) and without S9 (3h): 125, 250, 500, 1000 and 2000 μg/mL
- Dose-range finding test, without S9 (24h): 125, 250, 500, 1000 and 2000 μg/mL
- Experiment 1, with and without S9 (3h): 16, 31, 63, 125, 250, 500, 1000 and 2000 μg/mL
- Experiment 2, without S9 (24h): 13, 31, 63, 125, 250, 500, 1000 and 2000 μg/mL

Vehicle / solvent:

- Vehicle used: culture medium (exposure medium (R5) Hepes buffered medium (Dutch modification))
- Justification for choice of vehicle: A solubility test was performed based on visual assessment. The test item formed a clear pink solution in culture medium. Test item concentrations were used within 1 hours after
preparation. The pH and the osmolarity of the culture medium containing the highest, nonprecipitating concentration were recorded.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) :
test concentrations: 1 per test concentration; positive control: 1; solvent control: 2
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): below 1 x 10^6 cells/mL
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment:
Short term treatment: 3 hours (with and without S9-mix)
Prolonged treatment: 24 hours (without S9-mix)

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 2 days in which at least 4 x 10^6 cells were subcultured every day
- Selection time (if incubation with a selective agent): 11 or 12 days
- Method used: microwell plates
- Selective agent is used: 5 μg/mL trifluorothymidine (TFT)
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: For determination of the CEday2 the cell suspensions were diluted and seeded in wells of a 96-well dish. One cell was added per well (2 x 96-well microtiter plates/concentration) in non-selective medium. For determination of the mutation frequency (MF) a total number of 9.6 x 105 cells per concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFTselection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 105 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection). The microtiter plates for CEday2 and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFT-selection were stained for 1.5-2 hours, by adding 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma) to each well. The plates for the CE day2 and MF were scored with the naked eye or with the microscope.
- Criteria for small (slow growing) and large (fast growing) colonies: The small colonies are morphologically dense colonies with a sharp contour and with a diameter less than a quarter of a well. The large colonies are morphologically less dense colonies with a hazy contour and with a diameter larger than a quarter of a well. A well containing more than one small colony is classified as one small colony. A well containing more than one large colony is classified as one large colony. A well containing one small and one large colony is classified as one large colony.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative total growth (see 'Any other information on materials and methods incl. tables' for details on calculations)

Evaluation criteria:

The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
- A test item is considered positive (mutagenic) in the mutation assay if it induces a mutation frequency of more than the mutation frequency in the controls + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
- A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
- A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of the mutation frequency in the controls + 126.
ACCEPTABILITY CRITERIA:
a) The absolute cloning efficiency of the solvent controls is between 65 and 120% in order to have an acceptable number of surviving cells analyzed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors.
c) The suspension growth over the 2-day expression period for the solvent controls should be between 8 and 32 for the 3 hour treatment, and between 32 and 180 for the 24 hour treatment.
d) The positive control should demonstrate an absolute increase in the total mutation frequency, that is, an increase above the spontaneous background MF (an induced MF (IMF)) of at least 300 x 10^-6.
At least 40% of the IMF should be reflected in the small colony MF. And/or, the positive control has an increase in the small colony MF of at least 150 x 10^-6 above that seen in the concurrent solvent control (a small colony IMF of 150 x 10^-6).

Statistics:

Not performed.

Key result

Species / strain:

mouse lymphoma L5178Y cells

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 examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH and osmolarity: The pH and osmolarity at a concentration of 2000 μg/mL were 7.25 and 0.314 Osm/kg respectively (compared to 7.24 and 0.310 Osm/kg in the solvent control).
- Precipitation and time of the determination: The test item did not precipitate in the exposure medium up to and including the recommended concentration of 2000 μg/mL.

RANGE-FINDING/SCREENING STUDIES (if applicable):
- In the 3-hour exposure period, no toxicity in the suspension growth was observed up to and including the highest test item concentration of 2000 μg/mL compared to the suspension growth of the solvent control both
in the absence of S9-mix and presence of S9-mix.
- In the 24-hour exposure period (without S9-mix), no toxicity in the suspension growth was observed up to and including the highest test item concentration of 2000 μg/mL compared to the suspension growth of the solvent control.

STUDY RESULTS
- Concurrent vehicle negative and positive control data :
The mean mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database.
Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned
properly.

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements: No significant toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix in both experiments.

- Genotoxicity results:
No biologically relevant increase in the mutation frequency at the TK locus was observed after treatment with the test item either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

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:
Mutation frequency per 10^6 survivors
- S9-mix + S9-mix
3 hour treatment 24 hour treatment 3 hour treatment
Mean 803 6 95 1545
SD 253 223 887
n 142 132 151
Upper control limit
(95% control limits) 1533 1270 3954
Lower control limit
(95% control limits) 72 119 864
SD = Standard deviation
n = Number of observations
Distribution historical positive control data from experiments performed between September 2015 and September 2018.

- Negative (solvent/vehicle) historical control data:
Mutation frequency per 10^6 survivors
- S9-mix + S9-mix
3 hour treatment 24 hour treatment 3 hour treatment
Mean 101 98 100
SD 30 31 30
n 279 262 293
Upper control limit
(95% control limits) 170 162 165
Lower control limit
(95% control limits) 31 34 36
SD = Standard deviation
n = Number of observations
Distribution historical negative control data from experiments performed between September 2015 and September 2018

Conclusions:

4-(cyclohexylamino)butane-1-sulfonic acid is not mutagenic in the TK mutation test system.

Executive summary:

A gene mutation test in mammalian cells was performed according to OECD guideline 490 and GLP principles, to assess the potential of 4-(cyclohexylamino)butane-1-sulfonic acid to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells.

In the first experiment, the test item was tested up to concentrations of 2000 μg/mL in the absence and presence S9-mix, respectively. The incubation time was 3 hours. No toxicity was observed at this dose level in the absence and presence of S9-mix. This is the highest concentration recommended in the guidelines.

In the second experiment, the test item was again tested up to concentrations of 2000 μg/mL in the absence of S9-mix. The incubation time was 24 hours. No toxicity was observed at this dose level. This is the highest concentration recommended in the guidelines. Reliable negative and positive controls were included in both experiments.

In the absence and presence of S9 -mix, 4-(cyclohexylamino)butane-1-sulfonic acid did not induce a biologically relevant increase in the mutation frequency. In conclusion, 4-(cyclohexylamino)butane-1-sulfonic acid is not mutagenic in the mouse lymphoma L5178Y test system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test:

A bacterial reverse mutation test was performed according to OECD guideline 471 and in accordance with GLP principles. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. The test item did not induce a significant dose-related increase in the number of revertant (His⁺) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp⁺) colonies in tester strain WP₂uvrA both in the absence and presence of S9 -metabolic activation when tested up to and including the recommended top concentration.These results were confirmed in a follow-up experiment. Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia colireverse mutation assay. 

Chromosome aberration test:

A chromosome aberration study with 4-(cyclohexylamino)butane-1-sulfonic acid was performed according to OECD guideline 473 and GLP principles, in cultured peripheral human lymphocytes in two independent experiments.

No toxicity and no precipitation was observed at the highest tested concentration of 2000 μg/mL. Both in the absence and presence of S9-mix 4-(cyclohexylamino)butane-1-sulfonic acid did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.

No effects of 4-(cyclohexylamino)butane-1-sulfonic acid on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that 4-(cyclohexylamino)butane-1-sulfonic acid does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations.

Based on the results it can be concluded that 4-(cyclohexylamino)butane-1-sulfonic acid is not clastogenic in human lymphocytes.

Mouse lymphoma test:

A gene mutation test in mammalian cells was performed according to OECD guideline 490 and GLP principles, to assess the potential of 4-(cyclohexylamino)butane-1-sulfonic acid to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells.

In the first experiment, the test item was tested up to concentrations of 2000 μg/mL in the absence and presence S9-mix, respectively. The incubation time was 3 hours. No toxicity was observed at this dose level in the absence and presence of S9-mix. This is the highest concentration recommended in the guidelines.

In the second experiment, the test item was again tested up to concentrations of 2000 μg/mL in the absence of S9-mix. The incubation time was 24 hours. No toxicity was observed at this dose level. This is the highest concentration recommended in the guidelines. Reliable negative and positive controls were included in both experiments.

In the absence and presence of S9 -mix, 4-(cyclohexylamino)butane-1-sulfonic acid did not induce a biologically relevant increase in the mutation frequency. In conclusion, 4-(cyclohexylamino)butane-1-sulfonic acid is not mutagenic in the mouse lymphoma L5178Y test system.

Justification for classification or non-classification

Based on the results of the Ames test, the in vitro chromosome aberration test and the mouse lymphoma test, the substance 4-(cyclohexylamino)butane-1-sulfonic acid does not have to be classified for genotoxicity according to Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).