Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro: Gene mutation (Bacterial reverse mutation assay / Ames test): S. typhimurium TA97a, TA98, TA100, TA102 and TA1535: negative with and without metabolic activation (Aroclor 1254 induced rat liver S9) (according to OECD 471, GLP)

In vitro: Chromosome aberration (mammalian cell micronucleus test): Human lymphocytes: negative with and without metabolic activation (Aroclor 1254 induced rat liver S9) (according to OECD 487, GLP)

In vitro: Gene mutation (mammalian cell forward mutation test): Mouse lymphoma L5178Y cells: negative with and without metabolic activation (Aroclor 1254 induced rat liver S9) (according to OECD 490, GLP)

In vitro: Cytotoxicity: L929 Cell Line from mouse: not cytotoxic (50 mM) (growth inhibition test, Lowry assay)

In vitro: Cytotoxicity: L929 Cell Line from mouse: not cytotoxic (50 mM) (growth inhibition test, Plating efficiency)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
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)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
other: SOP 118 008 03 edition 11, valid from 01. Oct. 2014 "Bestimmung des erbgutverändernden Potentials mit dem Bacterial-Reverse-Mutation-Test (Ames-Test)"
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesamt für Umwelt, Waserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Straße 7, 55116 Mainz, Germany
Type of assay:
bacterial reverse mutation assay
Target gene:
The strains used are mutants derived from Salmonella typhimurium LT2, mutations of the strains are listed below:
hisD6610 (category: frame shift, effect: histidine deficiency) present in strain TA97a
hisD3052 (category: frame shift, effect: histidine deficiency) present in strain TA98
hisG46 (category: base pair substitution, effect: histidine deficiency) present in strains TA100 and TA1535
hisG428 (category: base pair substitution, effect: histidine deficiency) present in strain TA102
uvrB (category: deletion, effect: UV sensitivity, biotine deficiency) present in strains TA97a, TA98, TA100 and TA1535
rfa (category: deletion, effect: lipopolysaccharide side chain deficiency) present in strains TA97a, TA98, TA100, TA 102 and TA1535
pKM101 (category: plasmide, effect: ampicillin resistance) present in strains TA97a, TA98, TA100 and TA 102
pAQ1 (category: plasmide, effect: tetracyclin resistance) present in strain TA 102
Species / strain / cell type:
S. typhimurium, other: TA97a, TA98, TA100, TA102 and TA1535
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9-mix induced by Aroclor 1254
Test concentrations with justification for top dose:
First experiment: 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation
Second experiment: 313, 625, 1250, 2500 and 5000 µg/plate with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water (demin.)
- Justification for choice of solvent/vehicle:
In a non-GLP pre-test the solubility of the test item was determined: The test item is sufficiently soluble in demin. H2O (no precipitates were visible). A solution containing 50 ± 5 g/L will be prepared for the tests. In a non-GLP pre-test, possible turbidity of the resulting test item solutions in combination with phosphate buffer was tested, too. The highest test item concentration (5 mg/plate) was not turbid.
On the base of these results the preparation of the test item was the same for both experiments. On the day of the start of the respective experiment, a stock solution containing 50 g/L of the test item in demineralised water was prepared. Demin. water was chosen as vehicle, because the test item was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants. The stock solution was used to prepare the geometric series of the concentrations to be tested. Each solution was membrane filtrated to accomplish sterility.
Untreated negative controls:
other: solvent control (demin. water)
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-1,2-phenylenediamine (TA97a, TA98 & TA102); sodium azide (TA100 & TA1535); 2-amino-anthracene (TA97a, TA100, TA102 & TA1535); benzo-a-pyrene (TA98)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: Only in the 2nd experiment the materials were gently vortexed in a test tube and incubated at 37 °C for 20 minutes (in the 1st experiment they were directly poured onto the selective agar plates).
- Exposure duration: 48 h

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: Per strain and dose, three plates with and three plates without S9 mix were used; 2 experiments were performed.

DETERMINATION OF CYTOTOXICITY
- Method: Assessment for numbers of revertant colonies and examination for effects on the growth of the bacterial background lawn.

OTHER:
- Origin and Culture
Salmonella typhimurium (all strains used) were obtained from TRINOVA BioChem (batch numbers of all strains: TA97a: 4488D, TA98: 4789D, TA100: 4569D, TA102: 4712D, TA1535: 4717D) and were stored as lyophilisates in the fridge at 2-8 °C. The lyophilisates were used to prepare permanent cultures which were filled into vials and stored at < -75°C. One day before the start of each experiment, one vial per strain to be used was taken from the deep freezer. The surface was scraped with an inoculation loop and the aliquot was put into a culture vessel containing nutrient broth. After incubation over night at 37°C, the cultures were used in the experiment. During the test, the cultures were stored at room temperature as to prevent changes in the titre.
Evaluation criteria:
For a substance to be considered positive in this test system, it should have induced a dose-related and statistically significant increase in the revertant count in one or more strains of bacteria in the presence and/or absence of S9 in both experiments at sub-toxic dose levels. To be considered negative, the number of revertants at each dose level should be less than twofold to that of the vehicle control frequency.
Key result
Species / strain:
S. typhimurium, other: TA97a, TA98, TA100, TA102 and TA1535
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
with and without metabolic activation
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
other: see vehicle control
Positive controls validity:
valid
Additional information on results:
The test item did not show mutagenic effects in both experiments. The number of revertant colonies was not increased in comparison with the spontaneous revertants (solvent only). Cytotoxicity of the test item was not detected. The bacterial background lawn was visible and the number of revertants was not significantly decreased. Therefore it can be stated, that under the test conditions, the test item 3-(Cyclohexylamino)-propane sulfonic acid is not mutagenic in the Bacterial Reverse Mutation Test using Salmonella typhimurium, strains TA97a, TA98, TA100, TA102 and TA1535.
The confirmation tests of the genotype didn't show any irregularities. The control of the titre was above the demanded value. The numbers of revertant colonies of the positive controls were within the range of the historical data of the laboratory (see Attachment 1) and were definitely increased in comparison with the negative controls, as well as showing mutagenic potential of the diagnostic mutagenes.
Spontaneous revertants were not all within the normal range in comparison with the historical data of the laboratory. The values, which lay outside of the historical data do not affect the validity of the study and the deviations are marginal. For these reasons, the result of the test is considered valid.
Conclusions:
The study was performed according to the OECD TG471 and EU Method B.13/14 (GLP) without deviations and therefore considered to be of the highest quality (reliability Klimisch 1). The validity criteria of the test system are fulfilled. In both experiments no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. The test material was therefore considered to be non-mutagenic under the conditions of this test.
Executive summary:

Two valid experiments were performed according to the OECD 471 resp. EU Method B.13/14 (GLP):

First Experiment

Five concentrations of the test item, dissolved in demineralised water (ranging from 50 to 5000 µg/plate) were used. Five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 (genetically manipulated) and TA1535) were exposed to the test item both in the presence and in the absence of a metabolic activation system (S9-mix, rat liver S9-mix induced by Aroclor 1254) for 48 hours, using the plate incorporation method. None of the concentrations caused a significant increase in the number of revertant colonies in the tested strains. The test item did not show any mutagenic effects in the first experiment. The test item showed no precipitates on the plates in all tested concentrations. No signs of toxicity towards the bacteria could be observed. The sterility control and the determination of the titre did not show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range. All positive controls showed mutagenic effects with and without metabolic activation.

Second Experiment

To verify the results of the first experiment, a second experiment was performed, using five concentrations of the test item (ranging from 313 to 5000 µg/plate) and a modification in study performance (pre-incubation method). The test item did not show mutagenic effects in the second experiment, either. The test item showed no precipitates on the plates in all tested concentrations. No signs of toxicity towards the bacteria could be observed. The sterility control and the determination of the titre did not show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range. All positive controls showed mutagenic effects with and without metabolic activation.

Under the conditions of the test, the test item didn't show mutagenic effects towards Salmonella typhimurium, strains TA97a, TA98, TA100, TA102 and TA1535. Therefore, no concentration-effect relationship could be determined and the test item 3-(Cyclohexylamino)-propane sulfonic acid is considered as "not mutagenic under the conditions of the test".

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23. Sep. 2015 - 30. Nov. 2015 (experimental phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well-documented GLP OECD 487 guideline study without deviations on the registered substance itself.
Qualifier:
according to guideline
Guideline:
other: OECD TG 487 “In Vitro Mammalian Cell Micronucleus Test“ adopted 26. Sep. 2014
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Commission Regulation (EU) No. 640/2012 of 06. July 2012, amending Regulation (EC) No. 440/2008, EU Method B.49: “In Vitro Mammalian Cell Micronucleus Test”
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesamt für Umwelt, Waserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Straße 7, 55116 Mainz, Germany
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
n/a
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
Blood samples were obtained from healthy donors who neither smoke nor receive medication.
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9
Test concentrations with justification for top dose:
0, 31, 63, 125, 250, 500, 1000, 2000 µg/mL (Pre-Experiment/Experiment I)
0, 250, 500, 1000, 2000 µg/mL (Experiment II)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: A stock solution containing 20 mg/mL of the test item in medium without FCS was prepared.
- Justification for choice of solvent/vehicle: Medium without FCS was chosen as solvent, because this solvent has no effects on the viability of cells, does not show genetic toxicity and the test item was completely soluble.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
other: not applicable
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Colchicine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4h or 22.5h
- Expression time (cells in growth medium): 17h or none after exposure
- Fixation time (start of exposure up to fixation or harvest of cells): 21h or 22.5h

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B
STAIN (for cytogenetic assays): 10% solution of Giemsa

NUMBER OF REPLICATIONS: duplicates

NUMBER OF CELLS EVALUATED: In all replicates, the cytokinesis-block proliferation index (using at least 500 cells per culture) was determined in order to assess the cytotoxicity of the test item. At least 1000 binucleate cells per culture resp. 2000 cells per treatment were scored for micronuclei.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
Evaluation criteria:
Classification
The test item is considered to have no genotoxic effects if:
- Neither a statistically significant nor a concentration-related increase of the number of micronucleate cells in the evaluated test concentrations is observed.
- The obtained results lie within the range of the historical laboratory control data for solvent controls.
The test item is considered to have genotoxic effects if:
- At least one test concentration shows a statistically significant increase of micronucleate cells compared to the concurrent solvent control.
- In at least one experimental condition a dose-related increase of micronucleate cells can be observed.
- Any of the results lies outside the range of the historical laboratory control data for solvent controls.
Statistics:
Statistical significance at the one (p < 0.01) resp. 5 % level (p <0.05) was evaluated by means of Fisher´s exact test resp. chi-square-test.
Key result
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
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none stated
- Effects of osmolality: none stated
- Evaporation from medium: none stated, unlikely due to the chemical structure of the test item and its vapour pressure
- Water solubility: soluble
- Precipitation: none observed
- Other confounding effects: none stated

RANGE-FINDING/SCREENING STUDIES:
see entry fields below for tables

COMPARISON WITH HISTORICAL CONTROL DATA:
In experiment I, controls were within historical data.
In experiment II, no cytotoxic effect at all was observed.
The ratio of binucleate cells with micronuclei lay above the range of the historical data for solvent controls at all evaluated concentrations of the test item, but it was in no case statistically significant increased compared with the concurrent solvent control. The value for the solvent control medium without FCS itself lay also in the upper range of the historical control data as well as the values for the positive controls. Therefore, it is assumed, that the background ratio of micronuclei was generally slightly increased in this experiment, possibly due to a specific reaction of the blood donor´s lymphocytes.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In experiment I, no relevant cytotoxicity could be observed. In the experimental part with metabolic activation, a slight decrease of growth could be observed at 2 concentrations of the test item (1000 µg/mL: 8.1%; 250 µg/mL: 11.1%). But these observations probably rep-resent the normal range of fluctuation in biological systems and do not indicate relevant cytotoxicity.
In experiment II, no cytotoxic effect at all was observed.
Conclusions:
The study was conducted under GLP according to OECD guideline 487 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the potential of 3-(Cyclohexylamino)-propane sulfonic acid to induce micronuclei in human lymphocytes.
Neither a statistically significant nor a biologically relevant increase in the number of binucleate cells containing micronuclei at the evaluated concentrations were observed.
In conclusion, under the experimental conditions reported, 3-(Cyclohexylamino)-propane sulfonic acid does not induce the formation of micronuclei in human lymphocytes in vitro, and is considered as “not genotoxic under the conditions of the test”.
Executive summary:

This GLP OECD 487 guideline study was performed to assess the genotoxic potential of 3-(Cyclohexylamino)-propane sulfonic acid to induce formation of micronuclei in human lymphocytes cultured in vitro in the absence and the presence of an exogenous metabolic activation system (liver S9 mix from male rats, treated with Aroclor 1254).

In a non-GLP pre-test, the solubility of the test item was determined. The test item was sufficiently soluble in RPMI 1640 medium without FCS. Therefore, on the day of the respective experiment a stock solution containing 20 mg/mL was prepared. This stock solution was used to prepare a geometric series of dilutions.

Human lymphocytes, on whole blood culture, were stimulated to divide by addition of phytohaemagglutinin and exposed to medium control, solvent control, test item (up to 2000 µg/ml) and positive control.

After the culture harvest time, the cells were harvested and slides were prepared. After staining, the proportion of cells containing micronuclei was determined.

2 independent experiments were performed. In each experiment, all cell cultures were set up in duplicates. In order to assess the toxicity of the test item to cultured human lymphocytes, the cytokinesis-block proliferation index was calculated for all cultures treated with medium control, solvent control, positive control and test item. On the basis of the data of the cytokinesis-block proliferation index, the concentrations indicated in the table above were selected for micronuclei scoring.

In both independent experiments, the test item did not show cytotoxicity. In the 1st experiment with metabolic activation, a slight decrease of growth could be observed at 2 concentrations of the test item (1000 µg/mL: 8.1%; 250 µg/mL: 11.1%). But these observations probably represent the normal range of fluctuation in biological systems and do not indicate relevant cytotoxicity.

Neither a statistically significant nor a biologically relevant increase in the number of binucleate cells containing micronuclei at the evaluated concentrations were observed.

All positive control compounds caused statistically significant increases in the proportion of binucleate cells with micronuclei, demonstrating the sensitivity of the test system.

In conclusion, under the experimental conditions reported, 3-(Cyclohexylamino)-propane sulfonic acid does not induce the formation of micronuclei in human lymphocytes in vitro.

The test item 3-(Cyclohexylamino)-propane sulfonic acid is considered as “not genotoxic under the conditions of the test”.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-05-10 - 2016-07-18 (experimental phase)
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:
OECD Guideline for the Testing of Chemicals, Part 490, adopted 28. Jul. 2015 ”In vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene“
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
EU-Method B.17 of the Commission Regulation (EC) No. 440/2008, adopted 30 May 2008: “Mutagenicity – In vitro Mammalian Cell Gene Mutation Test”
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
by Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Straße 7, 55116 Mainz
Type of assay:
other: in vitro mammalian cell forward mutation test
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test item was stored in the test facility dry at 10.1 - 25.8 °C protected from light and humidity.
- Solubility and stability of the test substance in the solvent/vehicle: In a non-GLP pre-test, the solubility of the test item was determined. The test item was sufficiently soluble in RPMI 1640 medium.
Target gene:
Tk+/-
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: The cells were purchased by ATCC (Wesel, Germany) and were sold under the name L5178Y TK+/- clone (3.7.2C) [TK+/- (clone 3.7.2C)] (ATCC® CRL-9518™).
- Suitability of cells: The L5178Y is a murine T-cell lymphoma cell line, which grows as single or aggregated round cells in suspension. This cell line is characterized by a high sensitivity to chemical mutagens, by a high proliferation rate (doubling time 10-12 h in stock cultures), a high cloning efficiency (CE) and a stable spontaneous mutant frequency. The L5178Y consists of a stable karyotype and shows a diploid chromosome number (40 ± 2).
- Cell cycle length, doubling time or proliferation index: 10-12 h
- Methods for maintenance in cell culture if applicable: Cleansed and for mycoplasma contamination screened stocks of cells were stored in liquid nitrogen in the cell bank of the laboratory to allow a continuous stock of cells, which guarantees similar parameters of the experiment and reproducible characteristics of the cells.
Cells were thawed 6 d prior treatment and cultivated in RPMI 1640 complete culture medi-um with 5 % HS in cell culture flasks at 37.0 ± 1.0 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2.
- Modal number of chromosomes: 40 ± 2
- Normal (negative control) cell cycle time: 10-12 h

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 complete culture medium with 5 % HS in cell culture flasks at 37.0 ± 1.0 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9
Test concentrations with justification for top dose:
0, 63, 125, 250, 500, 1000, 2000 µg/mL, top dose as stipulated in the guideline
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: none; stock solution of the test item was prepared in RPMI 1640 medium
Untreated negative controls:
yes
Remarks:
RPMI 1640 medium without supplements
Negative solvent / vehicle controls:
yes
Remarks:
Medium resp. .9% NaCl was used as solvent control for the positive control cyclophosphamide (CPA)
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 1*10exp7 cells were exposed to each concentration of the test item

DURATION
- Exposure duration: 4h ± S9, 24h - S9
- Expression time (cells in growth medium): 46h
- Selection time (if incubation with a selection agent): 10-12d
- Fixation time (start of exposure up to fixation or harvest of cells): 13d

SELECTION AGENT (mutation assays): Trifluorothymidine

NUMBER OF REPLICATIONS: 2 experiments, 2 replicates per culture

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Rationale for test conditions:
As desribed in OECD TG 490. Because the results of experiment I did not give an indication for a mutagenic effect of the test item at the tested concentrations, a subsequent experiment (without metabolic activation) was performed in order to verify these results.
Evaluation criteria:
Classification
The test item is considered to have mutagenic effects if:
- the induced mutant frequency reproducibly exceeds a threshold of 126 colonies per 106 cells above the corresponding solvent control.
- the relative increase of the mutant frequency shows a dose relationship.

A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.
Results of test groups are generally rejected if the relative total growth is less than 10 % of the solvent control.
The biological relevance of the results was always considered first. Appropriate statistical methods were used as an aid in evaluating the test results. However, the results of statistical testing were assessed with respect to dose-response relationship. Reproducibility and historical data was also taken into consideration.
Statistical significance was confirmed by means of the non-parametric χ2 test. However, both biological and statistical significance were considered together.
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:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Evaporation from medium: none stated
- Water solubility: Test item was soluble.
Conclusions:
The study was conducted under GLP according to OECD guideline 490 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the potential of 3-(Cyclohexylamino)-propane sulfonic acid to induce gene mutations in mouse lymphoma cells.
In all evaluated concentrations of the test item no substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item. The mutation frequency did not reach or exceed the threshold of 126 above the corresponding solvent control.
In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the L5178Y Tk+/- cell line in the absence and the presence of metabolic activation.
The recorded data in this study declare the test item 3-(Cyclohexylamino)-propane sulfonic acid as a non-mutagen.
Executive summary:

In a mammalian cell gene mutation assay (TK+/-) (OECD 490), L5178Y Tk+/- cells cultured in vitro were exposed to 3-(Cyclohexylamino)-propane sulfonic acid at concentrations of 0, 63, 125, 250, 500, 1000, 2000 µg/mL in the presence and absence of mammalian metabolic activation (Aroclor 1254 induced rat liver S9).

3-(Cyclohexylamino)-propane sulfonic acid was tested up to limit concentrations (i.e., 2000 µg/mL). The positive controls did induce the appropriate response. There was no evidence or a concentration related positive response of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 490 for in vitro mutagenicity (mammalian forward gene mutation) data.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

There are several data, all assessed with Klimisch 1, available sufficiently covering all required endpoints, i.e. gene mutation in both bacteria and mammalian cells, and chromosome aberrations (micronucleus induction) in mammalian cells. Those in vitro genetic toxicity tests are designed, i.a. via addition of additional enzymes (metabolic activation; Aroclor 1254 induced rat liver S9), to mimic the exposure of humans to a potential genotoxic agent very close. They are accepted surrogates for human data, and no indication is given, that the obtained results are not relevant for humans / risk assessment. Hence, the data base is of good quality, sufficient to exclude that any risk with regard to genotoxic effects may arise for humans from 3-(Cyclohexylamino)-propane sulfonic acid.

All available tests gave consistently negative results, which makes a mode of action analysis impossible, as no conclusions from any events can be drawn.

Additional information

Justification for classification or non-classification

All available test results for gene mutations or chromosome aberrations (micronucleus induction) are consistently negative, and no need for classification as mutagen or directly genotoxic carcinogen was identified.