Sodium hydroxyquinoline propanesulfonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Sep 2015 - Jan 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Sodium rac-1-hydroxy-3-(quinolin-6-yl)propanesulfonate

Cas Number:

1609934-65-7

Molecular formula:

C12H12NO4SNa

IUPAC Name:

Sodium rac-1-hydroxy-3-(quinolin-6-yl)propanesulfonate

Test material form:

solid: particulate/powder

Method

Target gene:

The objective of this study was to evaluate the test item for its ability to induce reverse mutations in a gene of histidine-requiring Salmonella typhimurium bacterial strains resulting in histidine-independent strains.
The Salmonella typhimurium strains used in this study were TA1535, TA97, TA98, TA100 and TA102

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA1535, TA97, TA98, TA100 and TA102

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

INC280-C1 was tested in tester strain TA100 with concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate in the absence and presence of S9-mix. Based on the results of the dose range finding test, the following dose range was selected for the first mutation assay with the tester strains, TA1535, TA98, TA102 and TA97 in the absence and presence of S9-mix: 52, 164, 512, 1600 and 5000 µg/plate.
To obtain more information about the possible mutagenicity of INC280-C1, a second mutation experiment was performed in the absence of S9-mix and in the presence of 10% (v/v) S9-mix. Based on the results of the first mutation assay, INC280-C1 was tested up to the dose level of 5000 µg/plate in strains TA1535, TA97, TA98, TA100 and TA102.

Vehicle / solvent:

Milli-Q water

Controls

Untreated negative controls:

yes

Remarks:

Milli-Q water

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191, tert-butyl hydroperoxide (TBH), 2-aminoanthracene

Details on test system and experimental conditions:

Test system Salmonella typhimurium bacteria
Rationale Recommended test system in international guidelines (e.g. OECD and EC).
Source Trinova Biochem GmbH, Germany (Master culture from Dr. Bruce N. Ames) (TA1535: 2006, TA97: 1982, TA98: 2006, TA100: 2006 and TA102:2013)
The characteristics of the different Salmonella typhimurium strains were as follows:
Strain Histidine mutation M utation type
TA97 hisD6610/ R-factor* Frameshift
TA102 hisG428/ R-factor** Transitions/transversions
TA98 hisD3052/R-factor* Frameshift
TA1535 hisG46 Base-pair substitutions
TA100 hisG46/R-factor* Base-pair substitutions
*: R-factor = plasmid pKM101 (increases error-prone DNA repair)
**: R-factor = pKM101 and pAQ1

Each tester strain contained the following additional mutations:
rfa : deep rough (defective lipopolysaccharide cellcoat)
gal : mutation in the galactose metabolism
chl : mutation in nitrate reductase
bio : defective biotin synthesis
uvrB : loss of the excision repair system (deletion of the ultraviolet-repair B gene)

The Salmonella typhimurium strains were regularly checked to confirm their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA97, TA98, TA100 and TA102), tetracycline resistance (TA102), UV-sensitivity and the number of spontaneous revertants.

Stock cultures of the five strains were stored in liquid nitrogen (-196°C).

Preparation of bacterial cultures
Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, Hampshire, England) and incubated in a shaking incubator (37 ± 1°C, 150 spm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (109 cells/ml). Freshly grown cultures of each strain were used for a test.

Agar plates
Agar plates (ø 9 cm) contained 25 ml glucose agar medium. Glucose agar medium contained per liter: 18 g purified agar (Merck) in Vogel-Bonner Medium E, 20 g glucose (Fresenius Kabi, Bad Homburg, Germany). The agar plates for the test with the Salmonella typhimurium strains also contained 12.5 µg/plate biotin (Merck) and 15 µg/plate histidine (Sigma).
Top agar
Milli-Q water containing 0.6% (w/v) bacteriological agar (Oxoid LTD) and 0.5% (w/v) sodium chloride (Merck) was heated to dissolve the agar. Samples of 3 ml top agar were transferred into 10 ml glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 ± 3°C.
Environmental conditions
All incubations were carried out in a controlled environment at a temperature of 37.0 ± 1.0°C (actual range 34.9 – 38.7°C). The temperature was continuously monitored throughout the experiment. Due to addition of plates (which were at room temperature) to the incubator or due to opening and closing the incubator door, temporary deviations from the temperature may occur. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Rationale for test conditions:

At least five different doses (increasing with approximately half-log steps) of the test item were tested in triplicate in each strain. The above mentioned dose range finding study with tester strain TA100 is reported as a part of the first mutation experiment. In the second part of this experiment, INC280-C1 was tested both in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA97, TA98 and TA102. In an independent repeat of the assay with additional parameters, the test item was tested both in the absence and presence of 10% (v/v) S9-mix in all tester strains.
Final Report
INC280-C1 WIL Research Project 510616
- Page 11 -
The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.
Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture (109 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 at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+)) for Salmonella typhimurium bacteria were counted.

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100, TA97 and TA102 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535 and TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one independently repeated experiment.
A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100, TA97 and TA102 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535 and TA98 is greater than three (3) times the concurrent vehicle 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 independently repeated experiment.

Statistics:

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

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 97

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the absence of S9-mix, INC280-C1 induced dose related increases in tester strain TA97. The increases observed were above the laboratory historical control data range, in two independently repeated experiments, but was only more than two-fold the concurrent vehicle control in the second experiment: 1.9- and 3.2-fold increases in the first and second experiment, respectively.
In the presence of S9-mix, INC280-C1 induced dose related increases in tester strain TA97. The increases observed were above the laboratory historical control data range, in two independently repeated experiments, but was only more than two-fold the concurrent vehicle control in the first experiment: 2.8- and 1.9-fold increases in the first and second experiment, respectively.
The other bacterial strains showed negative responses over the entire dose range, i.e. no dose-related, increase in the number of revertants.
The negative control values were within the laboratory historical control data ranges, except the response for TA102 in the absence of S9-mix, first experiment. However since the mean number of revertant colonies showed a characteristic number of revertant colonies (239 revertant colonies) when compared against relevant historical control data (248 relevant colonies), the validity of the test was considered to be not affected.
The 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, except the response for TA102 in the first and second experiment in the absence of S9-mix. The purpose of the positive control is as a reference for the test system, where a positive response is required to check if the test system functions correctly. Since the value was more than 2 times greater than the concurrent solvent control values, this deviation in the mean plate count of the positive control had no effect on the results of the study.

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it is concluded that INC280-C1 is mutagenic in the Salmonella typhimurium reverse mutation assay.