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Genetic toxicity: in vitro

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guidelineopen allclose all
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

Test material

Constituent 1
Chemical structure
Reference substance name:
3-cyclohexylaminopropane-1-sulphonic acid
EC Number:
214-492-1
EC Name:
3-cyclohexylaminopropane-1-sulphonic acid
Cas Number:
1135-40-6
Molecular formula:
C9H19NO3S
IUPAC Name:
3-cyclohexylaminopropane-1-sulphonic acid
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): 3-(Cyclohexylamino)-propane sulfonic acid, 3-CAPS
- Storage condition of test material: room temperature (20 ± 5 °C)

Method

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
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.
Controls
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.

Results and discussion

Test results
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.

Applicant's summary and conclusion

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".